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Introduction: Si-Ni-San (SNS), a traditional Chinese medicine, is effective in treating liver fibrosis with an unclear mechanism. Although disturbance of intestinal flora and the subsequent secretion of short-chain fatty acids (SCFAs) is suggested to be involved in the progression of liver fibrosis, whether SNS produces the anti-fibrosis effect through the regulation of intestinal flora and SCFAs remains unclear. Methods: In the current study, carbon tetrachloride (CCl4)-treated mice were dosed with SNS to examine the anti-fibrotic effects and the involved mechanism. Biochemical parameters, histological staining, and analyses of fibrotic gene expression were used to evaluate the anti-fibrotic effect of SNS, while intestinal flora and SCFA content were determined by 16S rRNA and LC–MS to evaluate the mechanism. Results: In vivo results showed that SNS improved liver function, reduced hepatocyte apoptosis and FFAR2/3 expression, and restored intestinal dysbiosis and reduced PA, BA, and IsA levels. In vitro experiments showed that PA, BA, and IsA exacerbated TNF-α-induced HepG2 apoptosis. Notably, the protective effects of SNS were compromised in pseudo-sterile mice. Discussion: In conclusion, our experimental results suggest that the disturbance in intestinal flora results in elevated SCFA levels, which further exacerbates hepatocyte apoptosis in liver fibrosis, while SNS suppresses CCl4-induced liver fibrosis at least partially by reinstating intestinal flora homeostasis and reducing SCFA levels. [ABSTRACT FROM AUTHOR]

Introduction: Si-Ni-San (SNS), a traditional Chinese medicine, is effective in treating liver fibrosis with an unclear mechanism. Although disturbance of intestinal flora and the subsequent secretion of short-chain fatty acids (SCFAs) is suggested to be involved in the progression of liver fibrosis, whether SNS produces the anti-fibrosis effect through the regulation of intestinal flora and SCFAs remains unclear. Methods: In the current study, carbon tetrachloride (CCl4)-treated mice were dosed with SNS to examine the anti-fibrotic effects and the involved mechanism. Biochemical parameters, histological staining, and analyses of fibrotic gene expression were used to evaluate the anti-fibrotic effect of SNS, while intestinal flora and SCFA content were determined by 16S rRNA and LC-MS to evaluate the mechanism. Results: In vivo results showed that SNS improved liver function, reduced hepatocyte apoptosis and FFAR2/3 expression, and restored intestinal dysbiosis and reduced PA, BA, and IsA levels. In vitro experiments showed that PA, BA, and IsA exacerbated TNF-α-induced HepG2 apoptosis. Notably, the protective effects of SNS were compromised in pseudo-sterile mice. Discussion: In conclusion, our experimental results suggest that the disturbance in intestinal flora results in elevated SCFA levels, which further exacerbates hepatocyte apoptosis in liver fibrosis, while SNS suppresses CCl4-induced liver fibrosis at least partially by reinstating intestinal flora homeostasis and reducing SCFA levels. [ABSTRACT FROM AUTHOR]

Background: An accumulating body of research indicates that the pons is related to the occurrence of depression. Si-Ni-San (SNS) is a well-known Chinese herbal formula that is used to treat depression. Chinese herbal formulae have multiple therapeutic characteristics. Although it has been proven that SNS can exert antidepressant effects by improving changes in the limbic system, it is currently unclear whether SNS has therapeutic targets in the pons. This study aimed to explore the therapeutic targets of SNS in the pons for depression treatment. Materials and methods: Two experiments were conducted. In Experiment 1, 32 rats were divided into four groups: (1) a Control (C) group that received distilled water as a vehicle; (2) a Model (M) group that received the chronic unpredictable mild stress (CUMS) procedure and was administered distilled water; (3) a Stress + SNS (MS) group that received the CUMS procedure and was administered SNS dissolved in distilled water; and (4) a Stress + Fluoxetine (MF) group that received the CUMS procedure and was administered fluoxetine dissolved in distilled water. The open field test (OFT), the sucrose preference test (SPT), and the novel object recognition test (NOR) were performed to test the antidepressant effects of SNS. High-throughput mRNA sequencing (RNA-seq) was used to explore possible gene targets of SNS in the pons, and quantitative real-time PCR was performed to verify the results. High-performance liquid chromatography was used to detect neurotransmitters. Finally, correlation analyses were conducted between behaviors, genes expression, and neurotransmitters. In Experiment 2, 18 rats were divided into the same three groups as in Experiment 1: (1) C, (2) M, and (3) MS. fMRI was used to confirm whether SNS altered the pons in a rat model of depression. Results: SNS significantly improved sucrose preference in the SPT and TN-TO in the NOR compared to the M group (P < 0.05). RNA-seq filtered 49 differentially expressed genes(DEGs) that SNS could reverse in the pons of the CUMS depression model. Real-time PCR detected six genes, including Complexin2 (Cplx2), Serpinf1, Neuregulin1 (Nrg1), Annexin A1 (Anxa1), β-arrestin 1 (Arrb1) and presenilin 1 (Psen1). SNS significantly reversed changes in the expression of Anxa1, Nrg1, and Psen1 caused by CUMS (P < 0.05), which is consistent with the DEGs results. Additionally, SNS significantly reversed norepinephrine (NE) changes in the pons. There were 18 noteworthy correlations between behavior, genes, and neurotransmitters (P < 0.05). fMRI showed that SNS can decrease the amplitude of low-frequency fluctuations (ALFF) in the pons of living depressed rats. Conclusion: The pons is an important target brain region for SNS to exert its antidepressant effects. SNS may improve pontine NE levels by regulating the Anxa1, Nrg1, and Psen1 genes, thereby exerting antidepressant effects and improving cognitive function. [ABSTRACT FROM AUTHOR]

IntroductionSi-Ni-San (SNS), a traditional Chinese medicine, is effective in treating liver fibrosis with an unclear mechanism. Although disturbance of intestinal flora and the subsequent secretion of short-chain fatty acids (SCFAs) is suggested to be involved in the progression of liver fibrosis, whether SNS produces the anti-fibrosis effect through the regulation of intestinal flora and SCFAs remains unclear.MethodsIn the current study, carbon tetrachloride (CCl4)-treated mice were dosed with SNS to examine the anti-fibrotic effects and the involved mechanism. Biochemical parameters, histological staining, and analyses of fibrotic gene expression were used to evaluate the anti-fibrotic effect of SNS, while intestinal flora and SCFA content were determined by 16S rRNA and LC–MS to evaluate the mechanism.ResultsIn vivo results showed that SNS improved liver function, reduced hepatocyte apoptosis and FFAR2/3 expression, and restored intestinal dysbiosis and reduced PA, BA, and IsA levels. In vitro experiments showed that PA, BA, and IsA exacerbated TNF-α-induced HepG2 apoptosis. Notably, the protective effects of SNS were compromised in pseudo-sterile mice.DiscussionIn conclusion, our experimental results suggest that the disturbance in intestinal flora results in elevated SCFA levels, which further exacerbates hepatocyte apoptosis in liver fibrosis, while SNS suppresses CCl4-induced liver fibrosis at least partially by reinstating intestinal flora homeostasis and reducing SCFA levels.

Ning Zhang,1,&ast; Tong Liu,1,&ast; Jianan Wang,2,&ast; Yingying Xiao,1 Ying Zhang,1 Jun Dai,1 Zhihong Ma,1,3 Donglai Ma4 1School of Basic Medicine, Hebei University of Chinese Medicine, Shijiazhuang, Hebei, 050200, People’s Republic of China; 2Graduate School, Hebei University of Chinese Medicine, Shijiazhuang, Hebei, 050200, People’s Republic of China; 3Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, Shijiazhuang, Hebei, 050200, People’s Republic of China; 4School of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang, Hebei, 050200, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhihong Ma; Donglai Ma, Hebei University of Chinese Medicine, Xingyuan Road 3, Shijiazhuang, Hebei, 050200, People’s Republic of China, Tel +86 31189926238, Email mazh1969@163.com; mdl_hebei@aliyun.comBackground: Metabolic associated fatty liver disease (MAFLD) is a chronic disease characterized by excessive lipid deposition in the liver without alcohol or other clear liver-damaging factors. AMP-activated protein kinase (AMPK)/silencing information regulator (SIRT)1 signaling pathway plays an important role in MAFLD development. Si-Ni-San (SNS), a traditional Chinese medicine, has shown reducing hepatic lipid deposition in MAFLD rats, however, the underlying mechanisms of SNS are barely understood.Purpose: The aim of this research was to investigate the mechanisms of SNS in reducing hepatic lipid deposition in MAFLD rats by regulating AMPK/SIRT1 signaling pathways.Methods: The components of SNS were determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) analysis. MAFLD rats were induced by high-fat and high-cholesterol diet (HFHCD), and treated by SNS. SNS-containing serum and Compound C (AMPK inhibitor) were used to treat palmitic acid (PA)-induced HepG2 cells. To elucidate the potential mechanism, lipid synthesis-related proteins (SREBP-1c and FAS), fatty acid oxidation (PPARα and CPT-1), and AMPK/SIRT1 signaling pathway (p-AMPK and SIRT1) were assessed by Western blot.Results: SNS improved serum lipid levels, liver function and reduced hepatic lipid deposition in MAFLD rats. SNS-containing serum reduced lipid deposition in PA-induced HepG2 cells. SNS could up-regulate protein expressions of PPARα, CPT-1, p-AMPK and SIRT1, and down-regulate protein expressions of SREBP-1c and FAS. Similar effects of SNS-containing serum were observed in PA-induced HepG2 cells. Meanwhile, Compound C weakened the therapeutic effects of SNS-containing serum on lipid deposition.Conclusion: SNS could reduce hepatic lipid deposition by inhibiting lipid synthesis and promoting fatty acid oxidation, which might be related with activating the AMPK/SIRT1 signaling pathway. This study could provide a theoretical basis for the clinical use of SNS to treat MAFLD.Graphical Abstract: Keywords: Si-Ni-San, MAFLD, lipid deposition, lipid synthesis, fatty acid oxidation, AMPK

Background: Early life stress (ELS) is a major risk factor for depression in adolescents. The nucleus accumbens (NAc) is a key center of the reward system, and spine remodeling in the NAc contributes to the development of depression. The Si-Ni-San formula (SNS) is a fundamental prescription for treating depression in traditional Chinese medicine. However, little is known about the effects of SNS on behavioral abnormalities and spine plasticity in the NAc induced by ELS. Purpose: This study aimed to investigate the therapeutic effect and the modulatory mechanism of SNS on abnormal behaviors and spine plasticity in the NAc caused by ELS. Methods: We utilized a model of ELS that involved maternal separation with early weaning to explore the protective effects of SNS on adolescent depression. Depressive-like behaviors were evaluated by the sucrose preference test, the tail suspension test, and the forced swimming test; anxiety-like behaviors were monitored by the open field test and the elevated plus maze. A laser scanning confocal microscope was used to analyze dendritic spine remodeling in the NAc. The activity of Rac1 was detected by pull-down and Western blot tests. Viralmediated gene transfer of Rac1 was used to investigate its role in ELS-induced depression-like behaviors in adolescence. Results: ELS induced depression-like behaviors but not anxiety-like behaviors in adolescent mice, accompanied by an increase in stubby spine density, a decrease in mushroom spine density, and decreased Rac1 activity in the NAc. Overexpression of constitutively active Rac1 in the NAc reversed depressionrelated behaviors, leading to a decrease in stubby spine density and an increase in mushroom spine density. Moreover, SNS attenuated depression-like behavior in dolescent mice and counteracted the spine abnormalities in the NAc induced by ELS. Additionally, SNS increased NAc Rac1 activity, and the inhibition of Rac1 activity weakened the antidepressant effect of SNS. Conclusion: These results suggest that SNS may exert its antidepressant effects by modulating Rac1 activity and associated spine plasticity in the NAc. [ABSTRACT FROM AUTHOR]

Introduction: Chronic psychological stress is a well-established risk factor for breast cancer development. Si-Ni-San (SNS) is a classical traditional Chinese medicine formula prescribed to psychological disorder patients. However, its action effects, molecular mechanisms, and bioactive phytochemicals against breast cancer are not yet clear. Objectives: This study aimed to explore the modulatory mechanism and bioactive compound of SNS in regulating estrogen metabolism during breast cancer development induced by chronic psychological stress. Methods: Mouse breast cancer xenograft was used to determine the effect of SNS on breast cancer growth and metastasis. Metabolomics analysis was conducted to discover the impact of SNS on metabolic profile changes in vivo. Multiple molecular biology experiments and breast cancer xenografts were applied to verify the anti-metastatic potentials of the screened bioactive compound. Results: SNS remarkably inhibited chronic psychological stress-induced breast cancer growth and metastasis in the mouse breast cancer xenograft. Meanwhile, chronic psychological stress increased the level of cholic acid, accompanied by the elevation of estradiol. Mechanistic investigation demonstrated that cholic acid activated farnesoid X receptor (FXR) expression, which inhibited hepatocyte nuclear factor 4α (HNF4α)-mediated estrogen sulfotransferase (EST) transcription in hepatocytes, and finally resulting in estradiol elevation. Notably, SNS inhibited breast cancer growth by suppressing estradiol level via modulating FXR/EST signaling. Furthermore, luciferase-reporting gene assay screened naringenin as the most bioactive compound in SNS for triggering EST activity in hepatocytes. Interestingly, pharmacokinetic study revealed that naringenin had the highest absorption in the liver tissue. Following in vivo and in vitro studies demonstrated that naringenin inhibited stress-induced breast cancer growth and metastasis by promoting estradiol metabolism via FXR/EST signaling. Conclusion: This study not only highlights FXR/EST signaling as a crucial target in mediating stress-induced breast cancer development, but also provides naringenin as a potential candidate for breast cancer endocrine therapy via promoting estradiol metabolism.

[Display omitted] • Chronic psychological stress promotes breast cancer growth and metastasis. • Chronic psychological stress results in elevation of cholic acid and estradiol via FXR/HNF4α/EST signaling. • SNS promotes estradiol metabolized into estradiol-3-O-sulfate. • Naringenin is identified as a natural EST agonist with liver targeting ability. • Naringenin inhibits breast cancer growth and metastasis via modulating FXR/HNF4α/EST signaling. Chronic psychological stress is a well-established risk factor for breast cancer development. Si-Ni-San (SNS) is a classical traditional Chinese medicine formula prescribed to psychological disorder patients. However, its action effects, molecular mechanisms, and bioactive phytochemicals against breast cancer are not yet clear. This study aimed to explore the modulatory mechanism and bioactive compound of SNS in regulating estrogen metabolism during breast cancer development induced by chronic psychological stress. Mouse breast cancer xenograft was used to determine the effect of SNS on breast cancer growth and metastasis. Metabolomics analysis was conducted to discover the impact of SNS on metabolic profile changes in vivo. Multiple molecular biology experiments and breast cancer xenografts were applied to verify the anti-metastatic potentials of the screened bioactive compound. SNS remarkably inhibited chronic psychological stress-induced breast cancer growth and metastasis in the mouse breast cancer xenograft. Meanwhile, chronic psychological stress increased the level of cholic acid, accompanied by the elevation of estradiol. Mechanistic investigation demonstrated that cholic acid activated farnesoid X receptor (FXR) expression, which inhibited hepatocyte nuclear factor 4α (HNF4α)-mediated estrogen sulfotransferase (EST) transcription in hepatocytes, and finally resulting in estradiol elevation. Notably, SNS inhibited breast cancer growth by suppressing estradiol level via modulating FXR/EST signaling. Furthermore, luciferase-reporting gene assay screened naringenin as the most bioactive compound in SNS for triggering EST activity in hepatocytes. Interestingly, pharmacokinetic study revealed that naringenin had the highest absorption in the liver tissue. Following in vivo and in vitro studies demonstrated that naringenin inhibited stress-induced breast cancer growth and metastasis by promoting estradiol metabolism via FXR/EST signaling. This study not only highlights FXR/EST signaling as a crucial target in mediating stress-induced breast cancer development, but also provides naringenin as a potential candidate for breast cancer endocrine therapy via promoting estradiol metabolism. [ABSTRACT FROM AUTHOR]

Background and Objective: Based on comprehensive network-pharmacology and molecular docking analysis, this study was intended to unveil the multiple mechanisms of Si-Ni-San (SNS) in treating anxious insomnia., Methods: The compounds of SNS were meticulously analyzed, selected and standardized with references to their pharmacological attributes. The components included chaihu (Bupleurum chinense DC.), baishao (Paeonia lactiflora Pall.), zhishi (Citrus aurantium L.) and gancao (Glycyrrhiza uralensis Fisch. ex DC.). We used the Traditional Chinese Medicine System Pharmacology (TCMSP) Database, Traditional Chinese Medicines Integrated Database (TCMID), GeneCards database, therapeutic target database (TTD) and comparative toxicogenomic database (CTD) to construct the components-compounds-targets networks and used Cytoscape 3.9.1 software to visualize the outcome. Afterwards, the STRING database and Cytoscape 3.9.1 software were utilized to construct and visualize the protein-protein interaction (PPI) network analysis. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also conducted through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The molecular docking program was carried out using AutoDock 4.2 software to understand interactions between target receptors and compound ligands selected for study., Results: We thoroughly sorted and filtered 31 pharmacologically active compounds from SNS. Subsequently, several potential target genes were predicted, of which there were 59 target genes distinctly associated with anxious insomnia. The PPI analysis indicated that the core target proteins included AKT1, IL6, TNF, SLC6A4, MAOA and GABRA2. The results of our study indicated that SNS potentially remediates anxious insomnia by reducing inflammation, neurodegeneration, and cell apoptosis of neurons. In addition, GO and KEGG enrichment analysis results indicated that SNS could modulate multiple aspects of anxious insomnia through mechanisms related to pathways of neuroactive ligand-receptor interaction. These pathways include various kinds of synaptic transmission pathways, and anti-inflammatory activity associated with response pathways. When we compared the components-compounds-targets networks and the compounds-targets-synaptic pathways networks, the five active compounds, including beta-Sitosterol, Kaempferol, Tetramethoxyluteolin, Isorhamnetin and Shinpterocarpin, were selected to conduct molecular docking experiments. Eleven target proteins, (AKT1, SLC6A4, ADRB2, MAOA, ACHE, ESR1, CYP3A4, CHRNA7, GABRA2, HTR2A and NOS3), which also play significant roles in regulating serotonergic, cholinergic, dopaminergic and GABAergic systems in the PPI network, were selected to act as receptors in molecular docking trials. The results showed that docking pairs isorhamnetin-AKT1, isorhamnetin-SLC6A4, β-sitosterol-MAOA, β- sitosterol-ACHE, isorhamnetin-CHRNA7 and shinpterocarpin-GABRA2 provided the most stable conformations of ligand-receptor binding between key compounds and core target proteins in the SNS., Conclusion: In the study, we offer a computational result, revealing that SNS may alleviate sleep disorders associated with anxiety through a "multi-compounds, multi-targets, and multi-pathways" mechanism. The network-pharmacology and molecular docking outcomes could theoretically confirm the anti-anxiety and anti-insomnia effects of SNS. Although this research is purely statistical and systematic without empirical validation, it serves as a stepping stone and cornerstone for subsequent experimental investigations., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Chronic stress-induced diarrhea is a common clinical condition, characterized by an abnormal bowel movement and loose stools, which lacks effective treatment in the clinic. Si-Ni-San (SNS) is a compound traditional Chinese medicine extensively used in China for stress-related diarrhea. However, the mechanism is unclear. Methods: Male Wistar rats (200 ± 20 g) were placed in a restraint cylinder and fixed horizontally for 3 h once daily for 21 consecutive days to establish a chronic restraint stress (CRS) rat model. SNS (0.6944 g/kg or 1.3888 g/kg) was given by gavage 1 h before the restraint once daily for 21 consecutive days. We examined the fecal score, dopamine ß hydroxylase (DßH), and c-fos expression in locus coeruleus, norepinephrine (NE) content in ileum and plasma, expression of a1 adrenergic receptors, MLCK, MLC, and p-MLC in the colon and mesenteric arteries, contraction of isolated mesenteric arteries, The expression of subunit d of ATP synthase (ATP5D) in intestinal tissues, ATP, ADP, and AMP content in the ileum and colon, occludin expression between ileum epithelial cells, the number of enterochromaffin cells (ECs) and mast cells (MCs) in the ileum, and 5-hydroxytryptamine (5-HT) content in the ileum and plasma. Results: After SNS treatment, the fecal score was improved. The increased expression of DßH and c-fos in locus coeruleus was inhibited. SNS suppressed the increased NE content in the ileum and plasma, down-regulated a1 adrenergic receptors in mesenteric arteries and MLCK, MLC, p-MLC in the colon and mesenteric arteries, and inhibited the contraction of mesenteric arteries. SNS also increased the ATP content in the ileum and colon, inhibited low expression of ATP5D in intestinal tissues, inhibited the decrease of ATP/ADP in the ileum and ATP/AMP in the colon, and up-regulated the occludin expression between ileum epithelial cells. In addition, SNS inhibited the increase of ECs and MCs in the ileum and the increase of 5-HT content in the ileum and plasma. Conclusion: This study demonstrated that SNS could improve CRS-induced abnormal feces in rats. This effect was related to the inhibition of CRS-induced increased expression of DßH and c-fos in the locus coeruleus, NE content in the ileum and plasma, and the contraction of isolated mesenteric arteries; inhibition of energy metabolism abnormality and decreased occludin expression; inhibition of increased ECs and MCs in the ileum, and 5-HT content in the ileum and plasma. [ABSTRACT FROM AUTHOR]

Autoimmune thyroid diseases (AITD), a group of prevalent and persistent immune-mediated disorders affecting the endocrine system, can progressively result in total thyroid failure, thereby drastically impacting metabolic processes. Given the inadequacies of current clinical approaches to managing AITD, The exigency to investigate novel therapeutic strategies demands immediate attention, given the limitations and potential resistances associated with conventional approaches. Si-Ni-San (SNS), first chronicled in the esteemed Eastern Han Dynasty medical text " Treatise on Cold Damage and Miscellaneous Diseases" circa 200-210 AD, is a time-honored remedy known for its harmonizing effects on the liver and invigorating properties for the spleen. Research indicates that saikosaponins and peony glycosides, two primary constituents of SNS, possess anti-inflammatory properties and can ameliorate immune dysfunction in the treatment of AITD. Despite initial insights, a comprehensive exploration of the underlying mechanisms by which SNS alleviates AITD symptoms requires further in-depth investigation to decipher their intricate interplay. This study aimed to identify the key therapeutic components of SNS for the treatment of AITD and to elucidate the underlying molecular mechanisms, revealing potential targets. We initially screened prospective components of SNS for AITD therapy through comprehensive database exploration, followed by an evaluation of the results via PPI networks. To illuminate the therapeutic mechanisms of SNS in AITD, we employed GO enrichment analysis and surveyed the KEGG pathways. Employing UHPLC-QE-MS, we conducted an in-depth analysis of SNS's principal elements, complemented by molecular docking studies to unravel their interaction dynamics. Finally, we substantiated the central therapeutic pathway of SNS in the treatment of AITD using an experimental autoimmune thyroiditis (EAT) mouse model, validated meticulously through in vivo experimentation. Network pharmacology analysis revealed 32 common targets from the overlap between SNS and AITD-related targets. Based on subsequent PPI network and KEGG analysis, we focused on the IL-6/JAK2/STAT3/IL-17 pathway, which drives the differentiation of Th17 cells, as a central therapeutic target of SNS in AITD. Crucially, our in vivo findings, substantiated through immunohistochemical, Western blot, RT-qPCR analyses and Flow cytometry analysis, reveal SNS's therapeutic potential in AITD. It effectively dampens IL-6 production, inhibits IL-6/JAK2/STAT3/IL-17 pathway activation, and rebalances the Th17/Treg cell ratio, thus elucidating its anti-inflammatory mechanism. The protective effect of SNS against AITD is likely mediated through the modulation of the IL-6/JAK2/STAT3/IL-17 pathway and the restoration of balance within the Th17/Treg ratio. This suggests that SNS may exert its therapeutic effects on AITD by targeting these key molecular mechanisms, thereby providing a novel perspective for the treatment of AITD. [Display omitted] • Si-Ni-San（SNS） can significantly improve thyroid autoimmune injury in EAT mice. • SNS can reduce the lymphocyte infiltration in thyroid tissue and the content of inflammatory cytokines in blood of EAT mice. • SNS restored the balance between Th17/Treg cells. • SNS inhibited the activation of IL-6/JAK2/STAT3/Th17 pathway and reduced the differentiation of Th17 cell. [ABSTRACT FROM AUTHOR]

Current treatment options for cholestatic liver diseases are limited, and addressing impaired intestinal barrier has emerged as a promising therapeutic approach. Si-Ni-San (SNS) is a Traditional Chinese Medicine (TCM) formula commonly utilized in the management of chronic liver diseases. Our previous studies have indicated that SNS effectively enhanced intestinal barrier function through the modulation of gut microbiota. This study aims to verify the therapeutic effects of SNS on cholestatic liver injury, focusing on elucidating the underlying mechanism involving the gut-liver axis. The 16s RNA gene sequencing, non-targeted metabolomics were used to investigate the effects of SNS on the gut microbiota dysbiosis. Fecal microbiota transplantation (FMT) was conducted to identify potential beneficial probiotics underlying the therapeutic effects of SNS. Our results demonstrated that SNS significantly ameliorated cholestatic liver injury induced by partial bile duct ligation (pBDL). Additionally, SNS effectively suppressed cholestasis-induced inflammation and barrier dysfunction in both the small intestine and colon. While SNS did not impact the intestinal FXR-FGF15-hepatic CYP7A1 axis, it notably improved gut microbiota dysbiosis and modulated the profile of microbial metabolites, including beneficial secondary bile acids and tryptophan derivatives. Furthermore, gut microbiota depletion experiments and FMT confirmed that the therapeutic benefits of SNS in cholestatic liver disease are dependent on gut microbiota modulation, particularly through the promotion of the growth of potential probiotic P. goldsteinii. Moreover, a synergistic improvement in cholestatic liver injury was observed with the co-administration of P. goldsteinii and SNS. Our study underscores that SNS effectively alleviates cholestatic liver injury by addressing gut microbiota dysbiosis and enhancing intestinal barrier function, supporting its rational clinical utilization. Furthermore, we highlight P. goldsteinii as a promising probiotic candidate for the management of cholestatic liver diseases. • SNS protects partial bile duct ligation-induced cholestasis. • SNS modulates gut microbiota dysbiosis and protects intestinal barrier. • P. goldsteinii is the target of SNS and a probiotic for cholestatic liver diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Si-Ni-San (SNS), a traditional Chinese medicinal formula derived from Treatise on Febrile Diseases , is considered effective in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice. However, the bioactive ingredients and underlying mechanisms are still unclear and need further investigation. This study aimed to evaluate the effect, explore the bioactive ingredients and the underlying mechanisms of SNS in ameliorating ulcerative colitis (UC) and associated liver injury in dextran sodium sulphate (DSS)-induced mouse colitis models. The effect of SNS (1.5, 3, 6 g/kg) on 3% DSS-induced acute murine colitis was evaluated by disease activity index (DAI), colon length, inflammatory cytokines, hematoxylin-eosin (H&E) staining, tight junction proteins expression, ALT, AST, and oxidative stress indicators. HPLC-ESI-IT/TOF MS was used to analyze the chemical components of SNS and the main xenobiotics in the colon of UC mice after oral administration of SNS. Network pharmacological study was then conducted based on the main xenobiotics. Flow cytometry and immunohistochemistry techniques were used to demonstrate the inhibitory effect of SNS on Th17 cells differentiation and the amelioration of Th17/Treg cell imbalance. LC-MS/MS, Real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting techniques were performed to investigate the oxysterol-Liver X receptor (LXRs) signaling activity in colon. Targeted bile acids metabolomics was conducted to reveal the change of the two major pathways of bile acid synthesis in the liver, and the expression of key metabolic enzymes of bile acids synthesis was characterized by RT-qPCR and western blotting techniques. SNS (1.5, 3, 6 g/kg) decreased the DAI scores, protected intestinal mucosa barrier, suppressed the production of pro-inflammatory cytokines, improved hepatic and splenic enlargement and alleviated liver injury in a dose-dependent manner. A total of 22 components were identified in the colon of SNS (6 g/kg) treated colitis mice, and the top 10 components ranked by relative content were regarded as the potential effective chemical components of SNS, and used to conduct network pharmacology research. The efficacy of SNS was mediated by a reduction of Th17 cell differentiation, restoration of Th17/Treg cell homeostasis in the colon and spleen, and the experimental results were consistent with our hypothesis and the biological mechanism predicted by network pharmacology. Mechanistically, SNS regulated the concentration of 25-OHC and 27-OHC by up-regulated CH25H, CYP27A1 protein expression in colon, thus affected the expression and activity of LXR, ultimately impacted Th17 differentiation and Th17/Treg balance. It was also found that SNS repressed the increase of hepatic cholesterol and reversed the shift of BA synthesis to the acidic pathway in UC mice, which decreased the proportion of non-12-OH BAs in total bile acids (TBAs) and further ameliorated colitis and concomitant liver injury. This study set the stage for considering SNS as a multi-organ benefited anti-colitis prescription based on the significant effect of ameliorating intestinal and liver damage, and revealed that derivatives of cholesterol, namely oxysterols and bile acids, were closely involved in the mechanism of SNS anti-colitis effect. [Display omitted] • SNS effectively alleviates acute colitis and concomitant liver injury in mice. • Network pharmacology based on xenobiotics analysis in UC mice was conducted. • SNS regulated colonic "oxysterol-LXRs" signaling and restored Th17/Treg homeostasis. • SNS restored perturbed BA synthesis in the liver of colitis mice. [ABSTRACT FROM AUTHOR]

Introduction: Si-Ni-San (SNS), a traditional Chinese medicine, is effective in treating liver fibrosis with an unclear mechanism. Although disturbance of intestinal flora and the subsequent secretion of short-chain fatty acids (SCFAs) is suggested to be involved in the progression of liver fibrosis, whether SNS produces the anti-fibrosis effect through the regulation of intestinal flora and SCFAs remains unclear., Methods: In the current study, carbon tetrachloride (CCl 4 )-treated mice were dosed with SNS to examine the anti-fibrotic effects and the involved mechanism. Biochemical parameters, histological staining, and analyses of fibrotic gene expression were used to evaluate the anti-fibrotic effect of SNS, while intestinal flora and SCFA content were determined by 16S rRNA and LC-MS to evaluate the mechanism., Results: In vivo results showed that SNS improved liver function, reduced hepatocyte apoptosis and FFAR2/3 expression, and restored intestinal dysbiosis and reduced PA, BA, and IsA levels. In vitro experiments showed that PA, BA, and IsA exacerbated TNF-α-induced HepG2 apoptosis. Notably, the protective effects of SNS were compromised in pseudo-sterile mice., Discussion: In conclusion, our experimental results suggest that the disturbance in intestinal flora results in elevated SCFA levels, which further exacerbates hepatocyte apoptosis in liver fibrosis, while SNS suppresses CCl 4 -induced liver fibrosis at least partially by reinstating intestinal flora homeostasis and reducing SCFA levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wu, Zhu, Yao, Chen, Ding, Su and Ge.)

Background: Si-Ni-San (SNS) is the formula prescription of Traditional Chinese Medicine (TCM) with anti-depression properties, but its underlying mechanisms remain unclear., Objective: This study provides novel approaches for the study of Traditional Chinese Medicine (TCM) and offers new opportunities for exploring the pharmacological properties of SNS., Methods: The ingredients in SNS implicated in the treatment of depression were identified and studied using network pharmacology. SwissTargetPrediction and molecular docking were used to study the interaction of SNS ingredients and their targets. The protective effect of these ingredients and their cocktail in rat pheochromocytoma cells (PC12) exposed to corticosterone (Cor) were evaluated using the CCK-8 assay, Hoechst 33342 staining, 2',7'-dichlorodihydro fluorescein diacetate (H2DCFDA) staining, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and in-cell Western analysis., Results: The network pharmacology study showed that the HIF-1 signaling pathway was the most crucial pathway implicated in the anti-depressive property of SNS. MAPK1 (ERK2), MAPK3 (ERK1), AKT1, VEGFA, STAT3, and EGF were identified as hub target proteins in the HIF-1 signaling pathway. Quercetin, naringenin, licochalcone A, and kaempferol from SNS, which targeted the six proteins mentioned above, were used to create a cocktail. This cocktail exerted protective properties, decreased the oxidative stress in PC12 exposed to Cor, and successfully regulated the expressions of AKT1, p-AKT1, ERK1, ERK2, p-ERK1/2, STAT3, p- STAT3, and VEGFA induced by Cor exposure. The SwissTargetPrediction and molecular docking study showed that the cocktail may regulate the HIF-1 signaling pathway by directly binding with AKT1 and MAPK1., Conclusion: The cocktail from SNS comprised of quercetin, naringenin, licochalcone A, and kaempferol exerts anti-depression potentiality by modulating the HIF-1 signaling pathway via direct interactions with AKT1 and MAPK1., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: An accumulating body of research indicates that the pons is related to the occurrence of depression. Si-Ni-San (SNS) is a well-known Chinese herbal formula that is used to treat depression. Chinese herbal formulae have multiple therapeutic characteristics. Although it has been proven that SNS can exert antidepressant effects by improving changes in the limbic system, it is currently unclear whether SNS has therapeutic targets in the pons. This study aimed to explore the therapeutic targets of SNS in the pons for depression treatment., Materials and Methods: Two experiments were conducted. In Experiment 1, 32 rats were divided into four groups: (1) a Control (C) group that received distilled water as a vehicle; (2) a Model (M) group that received the chronic unpredictable mild stress (CUMS) procedure and was administered distilled water; (3) a Stress + SNS (MS) group that received the CUMS procedure and was administered SNS dissolved in distilled water; and (4) a Stress + Fluoxetine (MF) group that received the CUMS procedure and was administered fluoxetine dissolved in distilled water. The open field test (OFT), the sucrose preference test (SPT), and the novel object recognition test (NOR) were performed to test the antidepressant effects of SNS. High-throughput mRNA sequencing (RNA-seq) was used to explore possible gene targets of SNS in the pons, and quantitative real-time PCR was performed to verify the results. High-performance liquid chromatography was used to detect neurotransmitters. Finally, correlation analyses were conducted between behaviors, genes expression, and neurotransmitters. In Experiment 2, 18 rats were divided into the same three groups as in Experiment 1: (1) C, (2) M, and (3) MS. fMRI was used to confirm whether SNS altered the pons in a rat model of depression., Results: SNS significantly improved sucrose preference in the SPT and T N -T O in the NOR compared to the M group ( P < 0.05). RNA-seq filtered 49 differentially expressed genes(DEGs) that SNS could reverse in the pons of the CUMS depression model. Real-time PCR detected six genes, including Complexin2 (Cplx2), Serpinf1, Neuregulin1 (Nrg1), Annexin A1 (Anxa1), β-arrestin 1 (Arrb1) and presenilin 1 (Psen1). SNS significantly reversed changes in the expression of Anxa1, Nrg1, and Psen1 caused by CUMS ( P < 0.05), which is consistent with the DEGs results. Additionally, SNS significantly reversed norepinephrine (NE) changes in the pons. There were 18 noteworthy correlations between behavior, genes, and neurotransmitters ( P < 0.05). fMRI showed that SNS can decrease the amplitude of low-frequency fluctuations (ALFF) in the pons of living depressed rats., Conclusion: The pons is an important target brain region for SNS to exert its antidepressant effects. SNS may improve pontine NE levels by regulating the Anxa1, Nrg1, and Psen1 genes, thereby exerting antidepressant effects and improving cognitive function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Zhang, Li, Nie, Qi, Chen, Chen, Liu, Li and Li.)

Background: Si-Ni-San (SNS), a commonly used traditional Chinese medicine (TCM) formula, has potency against liver diseases, such as hepatitis and non-alcoholic fatty liver disease (NAFLD). However, the therapeutic efficacy and pharmacological mechanisms of action of SNS against liver fibrosis remain largely unclear.Methods: A carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was adopted for the first time to investigate the beneficial effects of SNS on liver fibrosis. The potential mechanisms of action of SNS were explored using the network pharmacology-based strategy and validated with the aid of diverse assays.Results: SNS treatment reduced collagen and ECM deposition, downregulated fibrosis-related factor (hyaluronic acid and laminin) contents in serum, maintained the morphological structure of liver tissue, and improved liver function in the liver fibrosis model. Based on network pharmacology results, apoptosis, inflammation and angiogenesis, together with the associated pathways (including VEGF, TNF, caspase, PPAR-γ and NF-κB), were identified as the mechanisms underlying the effects of SNS on liver fibrosis. Further in vivo experiments validated the significant mitigatory effects of SNS on inflammatory infiltration and pro-inflammatory cytokine contents (IFNγ, IL-1β and TGF-β1) in liver tissues of mice with liver fibrosis. SNS suppressed pathologic neovascularization as well as levels of VEGFR1, VEGF and VEGFR2 in liver tissues. SNS treatment additionally inhibited hepatic parenchyma cell apoptosis in liver tissues of mice with liver fibrosis and regulated apoptin expression while protecting L02 cells against apoptosis induced by TNF-α and Act D in vitro. Activation of hepatic stellate cells was suppressed and the balance between MMP13 and TIMP1 maintained in vitro by SNS. These activities may be associated with SNS-induced NF-κB suppression and PPAR-γ activation.Conclusion: SNS effectively impedes liver fibrosis progression through alleviating inflammation, ECM accumulation, aberrant angiogenesis and apoptosis of hepatic parenchymal cells along with inhibiting activation of hepatic stellate cells through effects on multiple targets and may thus serve as a novel therapeutic regimen for this condition.

Background: Si-Ni-San (SNS), a commonly used traditional Chinese medicine (TCM) formula, has potency against liver diseases, such as hepatitis and non-alcoholic fatty liver disease (NAFLD). However, the therapeutic efficacy and pharmacological mechanisms of action of SNS against liver fibrosis remain largely unclear. Methods: A carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was adopted for the first time to investigate the beneficial effects of SNS on liver fibrosis. The potential mechanisms of action of SNS were explored using the network pharmacology-based strategy and validated with the aid of diverse assays. Results: SNS treatment reduced collagen and ECM deposition, downregulated fibrosis-related factor (hyaluronic acid and laminin) contents in serum, maintained the morphological structure of liver tissue, and improved liver function in the liver fibrosis model. Based on network pharmacology results, apoptosis, inflammation and angiogenesis, together with the associated pathways (including VEGF, TNF, caspase, PPAR-γ and NF-κB), were identified as the mechanisms underlying the effects of SNS on liver fibrosis. Further in vivo experiments validated the significant mitigatory effects of SNS on inflammatory infiltration and pro-inflammatory cytokine contents (IFNγ, IL-1β and TGF-β1) in liver tissues of mice with liver fibrosis. SNS suppressed pathologic neovascularization as well as levels of VEGFR1, VEGF and VEGFR2 in liver tissues. SNS treatment additionally inhibited hepatic parenchyma cell apoptosis in liver tissues of mice with liver fibrosis and regulated apoptin expression while protecting L02 cells against apoptosis induced by TNF-α and Act D in vitro. Activation of hepatic stellate cells was suppressed and the balance between MMP13 and TIMP1 maintained in vitro by SNS. These activities may be associated with SNS-induced NF-κB suppression and PPAR-γ activation. Conclusion: SNS effectively impedes liver fibrosis progression through alleviating inflammation, ECM accumulation, aberrant angiogenesis and apoptosis of hepatic parenchymal cells along with inhibiting activation of hepatic stellate cells through effects on multiple targets and may thus serve as a novel therapeutic regimen for this condition. [ABSTRACT FROM AUTHOR]

The efficacy of clinical treatments for various liver diseases is intricately tied to the liver's regenerative capacity. Insufficient or failed liver regeneration is a direct cause of mortality following fulminant hepatic failure and extensive hepatectomy. Si–Ni-San (SNS), a renowned traditional Chinese medicine prescription for harmonizing liver and spleen functions, has shown clinical efficacy in the alleviation of liver injury for thousands of years. However, the precise molecular pharmacological mechanisms underlying its effects remain unclear. This study aimed to investigate the effects of SNS on liver regeneration and elucidate the underlying mechanisms. A mouse model of 70% partial hepatectomy (PHx) was used to analyze the effects of SNS on liver regeneration. Aquaporin-9 knockout mice (AQP9 −/−) were used to demonstrate that SNS-mediated enhancement of liver regeneration was AQP9-targeted. A tandem dimer-Tomato-tagged AQP9 transgenic mouse line (AQP9-RFP) was utilized to determine the expression pattern of AQP9 protein in hepatocytes. Immunoblotting, quantitative real-time PCR, staining techniques, and biochemical assays were used to further explore the underlying mechanisms of SNS. SNS treatment significantly enhanced liver regeneration and increased AQP9 protein expression in hepatocytes of wild-type mice (AQP9 +/+) post 70% PHx, but had no significant effects on AQP9 −/− mice. Following 70% PHx, SNS helped maintain hepatic oxidative equilibrium by increasing the levels of reactive oxygen species scavengers glutathione and superoxide dismutase and reducing the levels of oxidative stress molecules H 2 O 2 and malondialdehyde in liver tissues, thereby preserving this crucial process for hepatocyte proliferation. Simultaneously, SNS augmented glycerol uptake by hepatocytes, stimulated gluconeogenesis, and maintained glucose/lipid metabolism homeostasis, ensuring the energy supply required for liver regeneration. This study provides the first evidence that SNS maintains liver oxidative equilibrium and glucose/lipid metabolism homeostasis by upregulating AQP9 expression in hepatocytes, thereby promoting liver regeneration. These findings offer novel insights into the molecular pharmacological mechanisms of SNS in promoting liver regeneration and provide guidance for its clinical application and optimization in liver disease treatment. [Display omitted] • SNS upregulates hepatocyte AQP9 expression. • SNS promotes liver regeneration following 70% partial hepatectomy. • SNS reduces hepatocyte oxidative stress in regenerating liver. • SNS improves glucose/lipid metabolism in regenerating liver. • In AQP9 knockout mice, SNS lacks beneficial effects on liver regeneration. [ABSTRACT FROM AUTHOR]

1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism. [ABSTRACT FROM AUTHOR]

Depression is becoming a major public health concern worldwide. Si-Ni-San (SNS) is a famous formula in Traditional Chinese Medicine (TCM) with potent antidepressant effects. However, the antidepressant mechanism of SNS has not been clearly elucidated. This study was performed to verify whether the antidepressant effects of SNS were related to its anti-inflammatory effects, the levels of brain-derived neurotrophic factor (BDNF) and Cytochrome P450 (CYP450) enzymatic activity. In our study, behavioral tests such as the forced swim test, sucrose preference test and open-field test were evaluated to ensure the establishment of depressive rats. The levels of IL-1β, IL-6, and TNF-α in the serum, liver, and hippocampus of rats were measured by enzyme-linked immunosorbent assays (ELISA). Furthermore, the key proteins NF-κB, BDNF, and TrkB were analyzed by Western blot (WB) analysis in the hippocampus. In addition, CYP450 enzymatic activity analysis was performed using LC-MS/MS in conjunction with drug and statistics (DAS 3.0) after oral administration of six probe drugs. Our results showed that SNS attenuated reserpine-induced increases in IL-1β, IL-6, and TNF-α expression in the serum, liver, and hippocampus. The levels of NF-κB, BDNF, and TrkB in the hippocampus of depressive rats were also altered. According to the pharmacokinetic parameters, SNS had moderate inhibitory effects in the reserpine-induced depression model on CYP1A2, CYP2D1, CYP2E1, and CYP3A2, but no significant metabolic changes to CYP2C6 and CYP2D2. These findings suggested that SNS has a protective effect on reserpine-induced depressive rats, which may be related to the improvement of the inflammatory factors, the level of BDNF and the activity of CYP450 enzymes.

Depression is becoming a major public health concern worldwide. Si-Ni-San (SNS) is a famous formula in Traditional Chinese Medicine (TCM) with potent antidepressant effects. However, the antidepressant mechanism of SNS has not been clearly elucidated. This study was performed to verify whether the antidepressant effects of SNS were related to its anti-inflammatory effects, the levels of brain-derived neurotrophic factor (BDNF) and Cytochrome P450 (CYP450) enzymatic activity. In our study, behavioral tests such as the forced swim test, sucrose preference test and open-field test were evaluated to ensure the establishment of depressive rats. The levels of IL-1β, IL-6, and TNF-α in the serum, liver, and hippocampus of rats were measured by enzyme-linked immunosorbent assays (ELISA). Furthermore, the key proteins NF-κB, BDNF, and TrkB were analyzed by Western blot (WB) analysis in the hippocampus. In addition, CYP450 enzymatic activity analysis was performed using LC-MS/MS in conjunction with drug and statistics (DAS 3.0) after oral administration of six probe drugs. Our results showed that SNS attenuated reserpine-induced increases in IL-1β, IL-6, and TNF-α expression in the serum, liver, and hippocampus. The levels of NF-κB, BDNF, and TrkB in the hippocampus of depressive rats were also altered. According to the pharmacokinetic parameters, SNS had moderate inhibitory effects in the reserpine-induced depression model on CYP1A2, CYP2D1, CYP2E1, and CYP3A2, but no significant metabolic changes to CYP2C6 and CYP2D2. These findings suggested that SNS has a protective effect on reserpine-induced depressive rats, which may be related to the improvement of the inflammatory factors, the level of BDNF and the activity of CYP450 enzymes. [ABSTRACT FROM AUTHOR]

Soothing the liver and regulating qi is one of the core ideas of traditional Chinese medicine (TCM) in the treatment of fatty liver. Si-Ni-San (SNS) is a well-known herbal formula in TCM for liver soothing and qi regulation in fatty liver treatment. However, its efficacy lacks modern scientific evidence. This study was aimed to investigate the impact of SNS on metabolic associated fatty liver disease (MAFLD) in mice and explore the underlying molecular mechanisms, particularly its effects on lipid metabolism in hepatocytes. The therapeutic effect of SNS was evaluated using in vivo and in vitro models of high-fat/high-cholesterol (HFHC) diet-induced mice and palmitic acid (PA)-induced hepatocytes, respectively. Molecular biological techniques such as RNA-sequencing (RNA-seq), co-immunoprecipitation (co-IP), and western blotting were employed to elucidate the molecular mechanism of SNS in regulating lipid metabolism in hepatocytes. Our findings revealed that SNS effectively reduced lipid accumulation in the livers of HFHC diet-induced mice and PA-induced hepatocytes. RNA-seq analysis demonstrated that SNS significantly down-regulated the expression of fatty acid synthase (Fasn) in the livers of HFHC-fed mice. Mechanistically, SNS inhibited Fasn expression and lipid accumulation by activating adenosine monophosphate (AMP)-activated protein kinase (AMPK). Activation of AMPK suppressed the activity of the transcriptional coactivator p300 and modulated the protein stability of sterol regulatory element-binding protein-1c (SREBP-1c). Importantly, p300 was required for the inhibition of Fasn expression and lipid accumulation by SNS. Furthermore, SNS activated AMPK by decreasing adenosine triphosphate (ATP) production in hepatocytes. This study provided novel evidence on the regulatory mechanisms underlying the effects of SNS on Fasn expression. Our findings demonstrate, for the first time, that SNS exerts suppressive effects on Fasn expression through modulation of the AMPK/p300/SREBP-1c axis. Consequently, this regulatory pathway mitigates excessive lipid accumulation and ameliorates MAFLD in mice. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract A UHPLC-MS/MS untargeted serum metabonomic method combined with quantitative analysis of five potential biomarkers in rat serum was developed and validated, to further understand the anti-liver injury effect of Si-Ni-San and its mechanism on liver injury rats in this study. The metabolites were separated and identified on BEH C 18 column (100 mm × 2.1 mm, 1.7 μm) using the ACQUITY UHPLC-MS system (Waters Corp., Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential biomarkers including phenylalanine, tryptophan, Glycochenodeoxycholic acid (GCDCA) and hysophosphatidylcholine (LPC), which were related to amino acid metabolism, lipid metabolism, bile acid biosynthesis and oxidation-antioxidation balance, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode. The proposed method was linear for each analyte with correlation coefficients over 0.99. The intra- and inter-day precision values (relative standard deviation, RSD) were less than 13.1% and accuracy (relative error, RE) was from −9.5% to 10.3% at all quality control (QC) levels. The validated method was successfully applied to study the serum samples of control group, model group, positive control group (silymarin group) and Si-Ni-San group in rats. Graphical abstract Image 1 Highlights • A UHPLC-MS/MS untargeted metabonomic method combined with quantitative analysis. • The validated method was applied to control group, model group and Si-Ni-San group. • The changes of potential biomarkers and relevant metabolic pathways were demonstrated. • Understand the effect and mechanism of Si-Ni-San on liver injury rats. [ABSTRACT FROM AUTHOR]

Background: Depression is becoming an urgent mental health problem. Si-Ni-San has been widely used to treat depression, yet its underlying pharmacological mechanism is poorly understood. Thus, we aim to explore the antidepressant mechanism of Si-Ni-San by chemical analysis and in-silico methods., Methods: Compounds in Si-Ni-San were determined by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Then, bioactive compounds were obtained from Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform and SwissADME, and the potential targets of which were acquired from SwissTargetPrediction. Depression-related targets were collected from GeneCards. The intersection between compound-related targets and depression-related targets were screened out, and the overlapped targets were further performed protein-protein interaction, biological functional and pathway enrichment analysis. Finally, networks of Si-Ni-San against depression were constructed and visualized by Cytoscape., Results: One hundred nineteen compounds in Si-Ni-San were determined, of which 24 bioactive compounds were obtained. Then, 137 overlapped targets of Si-Ni-San against depression were collected. AKT1, PIK3R1, PIK3CA, mTOR, MAPK1 and MAPK8 were the key targets. Furthermore, PI3K-Akt signalling pathway, serotonergic synapse, MAPK signalling pathway and neurotrophin signalling pathway were involved in the antidepressant mechanism of Si-Ni-San. It showed that components like sinensetin, hesperetin, liquiritigenin, naringenin, quercetin, albiflorin and paeoniflorin were the mainly key active compounds for the antidepressant effect of Si-Ni-San., Conclusions: This study demonstrated the key components, key targets and potential pharmacological mechanisms of Si-Ni-San against depression. These results indicate that Si-Ni-San is a promising therapeutic approach for treatment of depression, and may provide evidence for the research and development of drugs for treating depression., Competing Interests: The authors declare that they have no conflict of interest.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article, (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Early life stress (ELS) is a major risk factor for depression in adolescents. The nucleus accumbens (NAc) is a key center of the reward system, and spine remodeling in the NAc contributes to the development of depression. The Si-Ni-San formula (SNS) is a fundamental prescription for treating depression in traditional Chinese medicine. However, little is known about the effects of SNS on behavioral abnormalities and spine plasticity in the NAc induced by ELS. Purpose: This study aimed to investigate the therapeutic effect and the modulatory mechanism of SNS on abnormal behaviors and spine plasticity in the NAc caused by ELS. Methods: We utilized a model of ELS that involved maternal separation with early weaning to explore the protective effects of SNS on adolescent depression. Depressive-like behaviors were evaluated by the sucrose preference test, the tail suspension test, and the forced swimming test; anxiety-like behaviors were monitored by the open field test and the elevated plus maze. A laser scanning confocal microscope was used to analyze dendritic spine remodeling in the NAc. The activity of Rac1 was detected by pull-down and Western blot tests. Viral-mediated gene transfer of Rac1 was used to investigate its role in ELS-induced depression-like behaviors in adolescence. Results: ELS induced depression-like behaviors but not anxiety-like behaviors in adolescent mice, accompanied by an increase in stubby spine density, a decrease in mushroom spine density, and decreased Rac1 activity in the NAc. Overexpression of constitutively active Rac1 in the NAc reversed depression-related behaviors, leading to a decrease in stubby spine density and an increase in mushroom spine density. Moreover, SNS attenuated depression-like behavior in adolescent mice and counteracted the spine abnormalities in the NAc induced by ELS. Additionally, SNS increased NAc Rac1 activity, and the inhibition of Rac1 activity weakened the antidepressant effect of SNS. Conclusion: These results suggest that SNS may exert its antidepressant effects by modulating Rac1 activity and associated spine plasticity in the NAc., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ye, Wu, Liu, Deng, Bai, Yang, Xuan, Liu, Shi, Liu, Zhang and Zhao.)

Ethnopharmacological relevance Si-Ni-San (SNS) is a well-known decoction in traditional Chinese medicine. Although studies have indicated that the anti-inflammatory and anti-allergic properties of SNS and its components can account for their therapeutic effects, the role and mechanism of SNS in treating skin dysfunction remain unclear. Aim of the study Atopic dermatitis (AD), a disorder known for its prevalence in infants and adults, severely influences the quality of life of affected patients. In this study, we aimed to investigate the anti-inflammatory and immune response modulations of SNS in 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin dysfunction. Materials and methods Dermatitis was induced in Kunming mice by the topical application of DNCB. SNS or dexamethasone (positive control) was topically applied every day over the course of the 21-day study. The following were assessed: dermatitis severity scores; ear and dorsal skin haematoxylin and eosin staining; interleukin (IL)− 1α, IL-1β, IL-2, IL-4, IL-6, and tumour necrosis factor (TNF)-α cytokine levels in the serum; spleen index; spleen CD4 + /CD8 + T lymphocyte ratio; and phosphorylation levels of mitogen-activated protein kinases (MAPKs- p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)), IκB-α, and nuclear factor (NF)-κB (p65) in skin lesions. Results SNS significantly alleviated the symptoms of AD-like lesions induced by DNCB, decreased the infiltration of inflammatory cells in the ear and dorsal tissues, suppressed the increased cytokine levels in the serum, reduced the CD4 + /CD8 +T lymphocyte ratio in the spleen, and downregulated the activation of MAPKs, IκB-α, and NF-κB (p65) in the dorsal skin. The effects were similar to those of dexamethasone. Conclusions SNS alleviated the DNCB-induced AD-like skin dysfunction in mice through anti-inflammatory and immune system modulation, indicating that SNS shows potential for AD treatment in clinical settings. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most common chronic liver disease worldwide. However, its complex pathogenesis and lack of effective drugs for treating it present significant challenges. Si-Ni-San (SNS) is one of the representative formulas for treating patients with MAFLD in traditional Chinese medicine (TCM) clinics. According to our previous work, SNS reduces lipid droplet (LD) deposition in livers of mice with MAFLD. To elucidate the mechanism of SNS in reducing LD deposition in MAFLD. First, LD areas were detected with Oil red O staining in HepG2 cells induced by oleic acid (OA). Cell Counting Kit-8 (CCK-8) assay was used to test cell viability after treatment with different concentrations of SNS serum. The expression of Yes-associated protein 1 (YAP1) was monitored by Western blot. Second, C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks and gavaged with SNS decoction during the 11th and 12th weeks. Then, the weight of the body and the liver was examined. LD numbers and their locations in the liver were detected by triglyceride (TG) assay and hematoxylin and eosin staining (H&E). The expression levels of YAP1 and perilipin2 (PLIN2) were detected using Western blot and immunohistochemistry (IHC) in liver tissues. Finally, active ingredients of SNS decoction and SNS serum were identified by liquid chromatography-mass spectrometry (LC-MS). Finally, molecular docking was performed between the compounds in SNS and YAP1 to analyze their active interaction. Cellular experiments showed that SNS serum reduced LD vacuoles and YAP1 expression in OA-induced HepG2 cells. Animal experiments confirmed that LD vacuoles, PLIN2 expression (3.16-fold), and YAP1 expression (2.50-fold) were increased in the HFD group compared with the normal diet (ND) group. SNS reduced LD vacuoles, TG content (0.84-fold), PLIN2 expression (0.33-fold), and YAP1 expression (0.27-fold) compared with the normal saline (NS) group in Yap1 Flox mice with MAFLD. In SNS, baicalein-6-glucuronide, desoxylimonin, galangin-7-glucoside, glycyrrhizic-acid, licoricesaponin-K2, and nobiletin showed a high binding effect with YAP1. Knockout of hepatocyte YAP1 reduced LD vacuoles, TG content (0.40-fold), and PLIN2 expression (0.62-fold) in mice. Meanwhile, SNS reduced LD vacuoles, TG content (0.70-fold), and PLIN2 expression (0.19-fold) in Yap1 LKO mice with MAFLD. The effect of SNS in reducing TG and PLIN2 was diminished in Yap1 LKO mice compared with Yap1 Flox mice. SNS reduced LD deposition and YAP1 expression in MAFLD liver cells both in vivo and in vitro. YAP1 was highly expressed in livers with MAFLD, and knockout of hepatocellular YAP1 reduced LD deposition in mice. SNS reduced LD deposition associated with decreased YAP1 in MAFLD liver cells. [Display omitted] [ABSTRACT FROM AUTHOR]

Ulcerative colitis (UC) is widely believed to be a leading risk factor of colorectal cancer. Gut microbiota is a known vital player in the progression of UC. Si–Ni-San (SNS) has been considered to effectively treat colitis in clinical practice during thousands of years, yet whether SNS ameliorated acute colitis mouse model by modulating intestinal flora has not been distinctly elucidated. Our study aimed to elucidate the effect of SNS against acute murine colitis and focused on the underlying mechanisms of SNS targeting gut microbiota. 16S RNA sequencing, molecular biological analysis, and fecal microbiota transplants (FMT) were conducted to reveal the mechanisms of SNS in regulating gut microbiota. In our study, SNS dramatically inhibited DSS-induced acute inflammatory responses by improving gut microbiota dysbiosis, as evidenced by decreased abundance proinflammatory species, upregulated abundance of anti-inflammatory species and potentially altered microbiota metabolite metabolism. Additionally, intestinal flora knockout and FMT experiments confirmed that the therapeutic effect of SNS on colitis was dependent on gut microbiota, and specifically on favoring the growth of potential probiotics, Akkermansia genus. Furthermore, we found that SNS alone and SNS combined with Akkermansia muciniphila (A. muciniphila) increased Mucin 2 (MUC2) production, thus enhancing the competitive edge of A. muciniphila among pathogenic gut microbiota. Our study shed lights on the underlying mechanism of SNS in attenuating acute murine colitis from the perspective of intestinal flora and provides novel insights into the discovery of adjacent therapeutic strategy against colitis based on SNS and probiotics. Gastro-intestinal system [Display omitted] • SNS ameliorated acute colitis by restructuring gut microbiota. • The abundance of Akkermansia was increased by SNS. • SNS enhanced the competitive edge of A. muciniphila. [ABSTRACT FROM AUTHOR]

Objective: To observe the therapeutic effect of Si-Ni-San (SNS) on interstitial cystitis/bladder pain syndrome (IC/BPS) in rats, and explore the possible regulatory mechanism of SNS on IC/BPS combined with transcriptome analysis., Methods: An IC/BPS model of Sprague-Dawley (SD) rats was established with cyclophosphamide (CYP), and the SNS was extracted for treatment. The rats were divided into 4 groups (n = 10 in each group): Control group (blank), cyclophosphamide group (CYP group, CYP injection + normal saline gavage), lower-dose SNS group (LSNS group, CYP injection + 6 g/kg SNS gavage), and higher-dose SNS group (HSNS group, CYP injection + 12 g/kg SNS gavage). Urination, pain, and histological changes were observed in the rats after the experiment, and Western blotting (WB) and transcriptome analysis were performed on bladder tissues., Results: Compared with the CYP group, the urination, pain and inflammation symptoms of the IC/BPS model rats in the SNS treatment groups (LSNS and HSNS) were significantly improved ( p < 0.05). WB results showed that the expressions of inflammation-related proteins interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the SNS treatment groups were significantly decreased compared with those in the CYP group. Transcriptome results showed that SNS can affect the expression of inflammation-related genes and inflammatory signaling pathways., Conclusions: SNS can significantly alleviate the symptoms of inflammation and pain in IC/BPS rats, and its mechanism may be related to the down-regulation of inflammatory factors IL-6 and TNF-α through messenger RNA (mRNA) and long non-coding RNA (LncRNA) pathways., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s).)

Si–Ni-San (SNS) is a famous Chinese herbal formula used in China for thousands of years. It has clinical effects on a variety of lipid metabolism disorders, but the ameliorating effects of SNS on obesity and underlying mechanisms remained poorly elucidated. Aim of the study: This study aims to explore the therapeutic effect and mechanism of SNS on obesity from multiple perspectives in vitro and in vivo. The high-fat diet (HFD)-induced obesity mouse model was established to evaluate the effect of SNS. Then network pharmacologic methods were performed to predict underlying mechanisms, and the core pathways were verified in animal and cell studies. Our results demonstrated that SNS significantly reduced body weight, body fat content, white adipose tissue (WAT) expansion in obese mice, and lipid accumulation in primary mouse embryonic fibroblasts (MEFs) cells. Network pharmacologic analysis identified 66 potential therapeutic targets, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these genes revealed that the most important signaling pathway includes AMP-activated protein kinase (AMPK) signaling pathway, regulation of lipolysis in adipocytes, lipid and atherosclerosis. Western blot assay confirmed that SNS activated hormone-sensitive triglyceride lipase (HSL) and adipose triglyceride lipase (ATGL) activity and promoted lipolysis through AMPK signaling pathway. The results confirmed that SNS improves lipid accumulation through AKT/AMPK/HSL axis mediated lipolysis, which opens a new option for clinical treatment of obesity and associated complications. [Display omitted] • The therapeutic effect of SNS on obesity was clarified. • Network pharmacological prediction combined with multi-angle validation of in vitro and in vivo experiment. • SNS promotes adipose tissue lipolysis by activating AMPK signaling pathway to improve obesity. [ABSTRACT FROM AUTHOR]

1. The pharmacokinetic differences of paeoniflorin, naringin, naringenin and glycyrrhetinic acid (GA) following oral administration of pure compounds, single herbs and Si-Ni-San (SNS) decoction to rats were studied. Blood samples were analyzed with a validated UPLC-MS/MS method. Student's t-test was used for the statistical comparison. 2. The Cmax and AUC0-∞ were 1470 ± 434 ng/mL and 4663 ± 916 ng h/mL for paeoniflorin, 64.29 ± 59.21 ng/mL and 311.8 ± 131.8 ng h/mL for naringin, 244.2 ± 138.8 ng/mL and 4761 ± 3167 ng h/mL for naringenin, and 1183 ± 294 ng/mL and 38 994 ± 14 377 ng h/mL for GA after oral administration of paeoniflorin, naringin and glycyrrhizic acid. The Cmax and AUC0-∞ were 812.6 ± 259.6 ng/mL and 2489 ± 817 ng h/mL for paeoniflorin, 344.3 ± 234.9 ng/mL and 1479 ± 531 ng h/mL for naringin, 981.9 ± 465.4 ng/mL and 12 284 ± 6378 ng h/mL for naringenin, and 3164 ± 742 ng/mL and 78 817 ± 16 707 ng h/mL for GA after oral administration of SNS decoction. 3. There were significant differences between the pharmacokinetic behavior after oral administration of SNS decoction compared with pure components or herbs. The results indicated that some components in the other herbs of SNS had a pharmacokinetic interaction with paeoniflorin, naringin, naringenin and GA. [ABSTRACT FROM AUTHOR]

A simple and accurate HPLC–UV method was developed for simultaneous determination of seven constituents in Si-Ni-San decoction. Separation was performed on a Hypersil C18column and detection was set with gradient wavelength at 240 nm (0–26 min) and 210 nm (26–30 min). The mobile phase consisted of 0.03% phosphoric acid in water (v/v), and acetonitrile was used with a flow rate of 1.0 − 1. This method provides good linearity (r>0.9992), precision (RSD < 1.9%), repeatability (RSD < 2.9%), stability (RSD < 2.9%) and recovery (97.6–102.0%), which has been successfully applied to quantitative determination of the seven constituents in Si-Ni-San decoction and different compatibility groups. [ABSTRACT FROM PUBLISHER]

ABSTRACT In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of seven bioactive components including paeoniflorin, nobiletin, tangeretin, liquiritigenin, isoliquiritigenin, liquiritin and formononetin in rat plasma and tissues after oral administration of Si-Ni-San extract using astragaloside IV as internal standard (IS). The plasma and tissue samples were extracted by solid-phase extraction. Chromatographic separation was accomplished on a C18 column with a multiple-step gradient elution. The quantification was obtained by scanning with multiple reaction monitoring via an electrospray ionization source that was operated by switching between the positive and negative modes in two MS/MS scan segments. Full validation of the assay was implemented. In conclusion, this method demonstrated good linearity and specificity. The lower limits of quantification for the analytes were <7.5 ng/mL. Intra- and inter-day precisions (RSD) were <12.5% and accuracy (RE) ranged from −10.2 to 7.3%. The average recoveries of the analytes from rat plasma and tissues were >65.2% and 58.6%, respectively. The validated method was further applied to the determination of actual rat plasma and tissues after oral administration of Si-Ni-San extract. The results provided a meaningful basis for the clinical application of this prescription. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: The traditional Chinese medicine formula Si-Ni-San has well therapeutic applications in improvement of mental diseases including depression. However, the neuropharmacological and neuroendocrine mechanisms of the formula on antidepressantlike action have not been reported. Objective: Herein, we explored the antidepressant-like effect and its mechanism of Si- Ni-San. Materials and Methods: Acute effect of Si-Ni- San on the immobility time was assessed in the mouse forced swim test (FST) and tail suspension test (TST). Moreover, we investigated the neurochemical, neuroendocrine, and neurotrophin systems involved in the antidepressant-like effect of this formula. Results: Si- Ni-San significantly decreased the immobility time after acute treatment in the mouse TST (1300 mg/kg) but not in the FST compared with the control group. In addition, pretreatment of mice with PCPA or AMPT prevented the anti-immobility effect of Si-Ni-San (1300 mg/ kg) in the TST. Moreover, acute Si-Ni-San (1300 mg/kg) decreased serum corticosterone levels, elevated serotonin (5-HT), norepinephrine (NE), and dopamine (DA) levels without affecting brain-derived neurotrophic factor (BDNF) levels in the whole brain exposed to TST. Conclusion: The acute antidepressant-like action of Si-Ni-San is mediated by the monoaminergic and neuroendocrine systems although underlying mechanism still remains to be further elucidated, and this formula should be further investigated as an alternative therapeutic approach for the treatment of depression. [ABSTRACT FROM AUTHOR]

Background: Chronic stress-induced diarrhea is a common clinical condition, characterized by an abnormal bowel movement and loose stools, which lacks effective treatment in the clinic. Si-Ni-San (SNS) is a compound traditional Chinese medicine extensively used in China for stress-related diarrhea. However, the mechanism is unclear. Methods: Male Wistar rats (200 ± 20 g) were placed in a restraint cylinder and fixed horizontally for 3 h once daily for 21 consecutive days to establish a chronic restraint stress (CRS) rat model. SNS (0.6944 g/kg or 1.3888 g/kg) was given by gavage 1 h before the restraint once daily for 21 consecutive days. We examined the fecal score, dopamine β hydroxylase (DβH), and c-fos expression in locus coeruleus, norepinephrine (NE) content in ileum and plasma, expression of α1 adrenergic receptors, MLCK, MLC, and p-MLC in the colon and mesenteric arteries, contraction of isolated mesenteric arteries, The expression of subunit δ of ATP synthase (ATP5D) in intestinal tissues, ATP, ADP, and AMP content in the ileum and colon, occludin expression between ileum epithelial cells, the number of enterochromaffin cells (ECs) and mast cells (MCs) in the ileum, and 5-hydroxytryptamine (5-HT) content in the ileum and plasma. Results: After SNS treatment, the fecal score was improved. The increased expression of DβH and c-fos in locus coeruleus was inhibited. SNS suppressed the increased NE content in the ileum and plasma, down-regulated α1 adrenergic receptors in mesenteric arteries and MLCK, MLC, p-MLC in the colon and mesenteric arteries, and inhibited the contraction of mesenteric arteries. SNS also increased the ATP content in the ileum and colon, inhibited low expression of ATP5D in intestinal tissues, inhibited the decrease of ATP/ADP in the ileum and ATP/AMP in the colon, and up-regulated the occludin expression between ileum epithelial cells. In addition, SNS inhibited the increase of ECs and MCs in the ileum and the increase of 5-HT content in the ileum and plasma. Conclusion: This study demonstrated that SNS could improve CRS-induced abnormal feces in rats. This effect was related to the inhibition of CRS-induced increased expression of DβH and c-fos in the locus coeruleus, NE content in the ileum and plasma, and the contraction of isolated mesenteric arteries; inhibition of energy metabolism abnormality and decreased occludin expression; inhibition of increased ECs and MCs in the ileum, and 5-HT content in the ileum and plasma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Liu, Weng, Yan, Pan, Sun, Guo, Wang, Anwaier, Jiao, Li and Han.)

Abstract: Si-Ni-San (SNS) is a widely used traditional Chinese medicine formula (TCMF) in treating various diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, a liquid chromatography coupled with diode array detection and triple–quadrupole spectrometry (LC-DAD–MS/MS) method was developed for detection and identification of SNS metabolites in rat plasma and urine at a normal clinical dosage. Accurate structural elucidation was performed using MS/MS, UV data and n-octanol/water partition coefficient. Based on the proposed strategy, 36 absorbed compounds and 29 metabolites in plasma and 33 metabolites in urine were detected by a highly sensitive MRM method. Our results indicated that phase II reactions (e.g., methylation, glucuronidation and sulfation) were the main metabolic pathways of gallic acid and flavanones, while phase I reactions (e.g., hydroxylation) were the major metabolic reaction for triterpenoid saponins. The metabolite profile analysis of SNS provided a comprehensive understanding of the in vivo metabolic fates of constituents in SNS. Moreover, the results in this work demonstrated the present strategy based on the combination of chromatographic, spectrophotometric, mass-spectrometric, and software prediction to detect and identify metabolites was effective and reliable. And such a strategy may also be extended to investigate the metabolism of other TCMF. [Copyright &y& Elsevier]

Abstract: Si-Ni-San, a traditional Chinese medicinal formula, exerts an important function in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice, but the underlying mechanism is still unclear. In this study, we investigated the therapeutic potential of Si-Ni-San and its ingredient glycyrrhizin in trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis in mice, a well-characterized murine model for Crohn''s disease. Si-Ni-San and glycyrrhizin significantly ameliorated TNBS-induced colitis with reduced mortality and recovery of body weights. In addition, Si-Ni-San and glycyrrhizin dose-dependently decreased macroscopic inflammation scores, microscopic histoligical scores, and myeloperoxidase activity. Furthermore, Si-Ni-San and glycyrrhizin caused a decrease in pro-inflammatory cytokines including IFN-γ, IL-12, TNF-α and IL-17 and an increase in regulatory cytokine IL-10 in colon of the mice. It should be noticed the therapeutic effect of Si-Ni-San at 450mg/kg was much better than that of its contained content of glycyrrhizin at 10mg/kg. In conclusion, Si-Ni-San and glycyrrhizin significantly ameliorated TNBS-induced colitis in mice through regulating pro- and anti-inflammtory cytokine production. [Copyright &y& Elsevier]

Abstract: Aims: The roles of specific active ingredients in Chinese medicinal formulas have not been clearly elucidated. In this study, we selectively deleted and replenished paeoniflorin from Si-Ni-San, a traditional Chinese prescription, and aimed to identify the molecular basis of how paeoniflorin exerted its effect in Si-Ni-San. Main methods: Contact dermatitis was induced in mice with picryl chloride. Paeoniflorin was selectively deleted from Si-Ni-San by an immunoaffinity column. Quantitative real-time PCR, western blot, and enzyme-linked immunosorbent assay were used in this study. Key findings: Both Si-Ni-San and paeoniflorin significantly reduced ear swelling in mice while the paeoniflorin-deleted Si-Ni-San (Si-Ni-SanPF−) showed little ameliorative effect. In lipopolysaccharide-evoked macrophages, Si-Ni-San and paeoniflorin markedly inhibited tumor necrosis factor-α production, cyclooxygenase-2 activity, as well as extracellular signal-regulated kinase 1/2 phosphorylation while Si-Ni-SanPF− exhibited no or slight inhibitory effect. Furthermore, the inhibitory effect on the production of tumor necrosis factor-α reappeared when different proportions of paeoniflorin were replenished in Si-Ni-SanPF−. In addition, the expression of macrophage migration inhibitory factor in T cells, rather than macrophages, was significantly inhibited by Si-Ni-San, but not Si-Ni-SanPF−. Our data indicate paeoniflorin is the principal component of Si-Ni-San, exerting negative regulation on the function of macrophages in contact dermatitis. Significance: The present study suggests that dissecting the role of specific constituents in medicinal formulas through selective deletion and replenishment may be a useful strategy in recognizing and validating an active ingredient in traditional Chinese medicine. [Copyright &y& Elsevier]

Abstract: In the present study, we performed to selectively deplete glycyrrhizin from Si-Ni-San, a traditional Chinese prescription that consists of 4 Chinese herbs including Radix Glycyrrhizae Uralensis, and examined its influence on the suppressing activity of Si-Ni-San against contact sensitivity in mice. An immunoaffinity column was made by covalently coupling the polyclonal antibody, obtained by the immunization with glycyrrhizin–BSA conjugate, to CNBr-activated Sepharose 4B. By using this column, glycyrrhizin in Si-Ni-San was selectively and almost completely depleted from the whole extract, which was confirmed by high-performance liquid chromatography (HPLC). Both 200 mg/kg of Si-Ni-San and 10 mg/kg of glycyrrhizin, the dose corresponding to its proportion contained in Si-Ni-San, significantly reduced the ear swelling of picryl chloride (PCl)-induced ear contact sensitivity in mice and the inhibition by Si-Ni-San was stronger than that by glycyrrhizin. The adhesion activity to type IV collagen of the isolated spleen cells from PCl-sensitized mice was significantly decreased by both Si-Ni-San and glycyrrhizin. However, the glycyrrhizin-depleted sample of Si-Ni-San (Si-Ni-SanGL−) only showed a slight inhibition on the cell adhesion. Furthermore, the spleen cells from PCl-sensitized mice produced more matrix metalloproteinase (MMP)-2 and -9 than naive spleen cells did, and both Si-Ni-San and glycyrrhizin remarkably reduced MMP-2 and MMP-9 production. In contrast, Si-Ni-SanGL− only showed a slight inhibition. These results suggest that glycyrrhizin may act as one of the active constituents of Si-Ni-San in inhibiting delayed-type hypersensitivity reaction via down-regulating the MMP production and the cell adhesion to extracellular matrix. The present study also provides a new approach to recognize and validate an active constituent in traditional prescription through a selective depletion. [Copyright &y& Elsevier]

Background: Ulcerative colitis (UC) is a chronic relapsing inflammatory disease that markedly elevates the risk of colon cancers and results in disability. The disrupted immune homeostasis has been recognized as a predominant player in the pathogenesis of UC. However, the overall remission rate of current therapies based on immunoregulation is still unsatisfactory. Si-Ni-San (SNS) has been found effective in relieving UC through thousands of years of clinical practice, yet the specific mechanisms of the protective effect of SNS were not fully elucidated.Purpose: We aim to investigate the therapeutic effects of SNS against the development of chronic colitis and the underlying mechanisms.Methods: We established a DSS-induced chronic experimental colitis mouse model to evaluate the effect of SNS. RNA-sequencing, bioinformatic analysis, and in vitro studies were performed to investigate the underlying mechanisms.Results: Our data demonstrated that SNS significantly ameliorated chronic experimental colitis via inhibiting the expression of genes associated with inflammatory responses. Interestingly, SNS significantly suppressed DSS-induced type I interferon (IFN) responses instead of directly downregulating the production of pro-inflammatory cytokines, such as Il-6. In vitro study further found that SNS selectively inhibited STING and RIG-I pathway-induced type I IFN responses by modulating TBK1- and IRF3-dependent signaling transduction. SNS also suppressed the expression of IFN-stimulated genes by directly inhibiting STAT1 and STAT2 activation.Conclusion: Our study not only provides novel insights into the pathogenic role of type I IFN responses in colitis but also suggested that SNS or bioactive compounds derived from SNS may serve as novel therapeutic strategies for the treatment of UC via interfering type I IFN-mediated inflammation. [ABSTRACT FROM AUTHOR]

• 37 main compounds in SNS were precisely identified with LC-MS/MS. • The plant origin of the identified compound was assigned. • Simultaneous quantification of 37 compounds enables quality control of SNS. Mass spectrometry based precision identification of natural products requires the validation of the reference compounds. This study attempted to develop a LC-QTOF MS combined with LC-TQ MS method to precisely characterize the chemicals of Si-Ni-San (SNS), a classic traditional Chinese medicine formula, which is composed of four medicinal plants, and widely used for the treatments of liver disorders. 74 compounds in SNS were provisionally identified by acquiring MS spectra and MS/MS spectra of the possible chemical features, as well as retrieving small-molecule database. By comparing with the accurate MS/MS spectra of reference compounds, 37 compounds in SNS were precisely identified for the first time. In addition, our effort also successfully assigned the origin of each identified compounds against four medicinal plants. Furthermore, we developed a UHPLC-TQ MS based quantitative-profiling method for simultaneous determination of 37 targeted compounds in the different extracts of the raw SNS and commercial lyophilized powders, enabling to facilitate overall quality control of SNS and associated commercial products. Collectively, our finding precisely characterized the main chemicals in SNS, which also provides a new strategy with LC-MS/MS based chemical profiling to precisely identify a diversity of chemicals in Chinese medicinal plants and associated formulae. [ABSTRACT FROM AUTHOR]

Background: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide. However, to date, there is no ideal therapy for this disease., Aim: To study the effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice., Methods: Twenty-four male C57BL/6 mice were randomized into three groups of eight. The control group (CON) was allowed ad libitum access to a normal chow diet. The high fat diet group (FAT) and Si-Ni-San group (SNS) were allowed ad libitum access to a high fat diet. The SNS group was intragastrically administered Si-Ni-San freeze-dried powder (5.0 g/kg) once daily, and the CON and FAT groups were intragastrically administered distilled water. After 12 wk, body weight, liver index, visceral fat index, serum alanine aminotransferase (ALT), portal lipopoly-saccharide (LPS), liver tumor necrosis factor (TNF)-α and liver triglycerides were measured. Intestinal microbiota were analyzed using a 16S r DNA sequencing technique., Results: Compared with the FAT group, the SNS group exhibited decreased body weight, liver index, visceral fat index, serum ALT, portal LPS, liver TNF-α and liver triglycerides ( P < 0.05). Intestinal microbiota analysis showed that the SNS group had different bacterial composition and function compared with the FAT group. In particular, Oscillospira genus was a bacterial biomarker of SNS group samples., Conclusion: The beneficial effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice may be associated with its anti-inflammatory and changing intestinal microbiota effects., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest related to this study.