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Purpose. To seek for a small interfering RNA (siRNA) sequence targeting a cynomolgus monkey inhibitor of nuclear factor kappa B α (IκBα) that can specifically and effectively suppress IκBα gene expression of cynomolgus monkey ciliary muscle (CM) cells and trabecular meshwork (TM) cells in vitro and screen for optimal siRNA transfection concentration. Methods. Three IκBα-specific double-stranded siRNAs were designed and synthesized. They were transfected into primarily cultured cynomolgus monkey CM cells and TM cells. The mRNA and protein levels of IκBα were examined by using real-time quantitative polymerase chain reaction (real-time PCR) and western blot to screen a pair of candidate valid sequences with the highest inhibitory rate. Both cells were transfected with Cy5-labeled nonspecific control-siRNA (NC-siRNA) of four different concentrations (10, 20, 50, and 100 nmol/L(nM)), and flow cytometry was used to assess transfection efficiency. Then, cells were transfected with the candidate valid IκBα -siRNA of the same four concentrations, and the cytotoxicity was detected by using Cell Counting Kit-8 (CCK8), and the inhibitory efficiency of IκBα was identified via real-time PCR to find out optimal siRNA transfection concentration. Results. The suppression effect of the siRNA targeting the GCACTTAGCCTCTATCCAT of IκBα gene was most obvious by in vitro screening. The inhibitory rate of IκBα was 82% for CM cells and 82% for TM cells on the mRNA level and 98% for CM cells and 93% for TM cells on the protein level, respectively. The results of flow cytometry showed that the transfection efficiency was the highest at 100 nM, which was 89.0% for CM cells and 48.2% for TM cells, respectively. The results of CCK8 showed that there was no statistically significant difference in cell viability after transfection of different concentrations of IκBα-siRNA. The results of real-time PCR indicated that there was no statistical difference in the inhibitory efficiency of IκBα after transfection of different concentrations of IκBα-siRNA. Conclusion. It proves that the siRNA targeting the GCACTTAGCCTCTATCCAT of IκBα gene is the valid sequence to suppress cynomolgus monkey IκBα expression of CM cells and TM cells by RNAi. 10 nM is the optimal transfection concentration. [ABSTRACT FROM AUTHOR]

Objective. Observe the protective effect of chlorogenic acid on dextran sulfate-induced ulcerative colitis in mice and explore the regulation of MAPK/ERK/JNK signaling pathway. Methods. Seventy C57BL/6 mice (half males and half females) were randomly divided into 7 groups, 10 in each group: control group (CON group), UC model group (UC group), and sulfasalazine-positive control group (SASP group), chlorogenic acid low dose group (CGA-L group), chlorogenic acid medium dose group (CGA-M group), chlorogenic acid high dose group (CGA-H group), and ERK inhibitor + chlorogenic acid group (E+CGA group). The effects of chlorogenic acid on UC were evaluated by colon mucosa damage index (CMDI), HE staining, immunohistochemistry, ELISA, and Western blot. The relationship between chlorogenic acid and MAPK/ERK/JNK signaling pathway was explored by adding ERK inhibitor. Results. The UC models were established successfully by drinking DSS water. Chlorogenic acid reduces DSS-induced colonic mucosal damage, inhibits DSS-induced inflammation, oxidative stress, and apoptosis in colon, and reduces ERK1/2, p -ERK, p38, p-p38, JNK, and p-JNK protein expression. ERK inhibitor U0126 reversed the protective effect of chlorogenic acid on colon tissue. Conclusion. Chlorogenic acid can alleviate DSS-induced ulcerative colitis in mice, which can significantly reduce tissue inflammation and apoptosis, and its mechanism is related to the MAPK/ERK/JNK signaling pathway. [ABSTRACT FROM AUTHOR]

Bronchitis is a respiratory disease characterized by a productive cough. Polygala tenuifolia Willd., commonly known as Yuan zhi , is a traditional Chinese herbal medicine used for relieving cough and removing phlegm. Despite its historical use, studies are lacking on the effectiveness of P. tenuifolia in treating bronchitis. Furthermore, the molecular mechanisms underlying the action of its bioactive compounds remain unknown. This study aims to identify the main bioactive compounds responsible for the effects of P. tenuifolia liquid extract (PLE) in treating bronchitis and to elucidate the associated molecular mechanisms. The main chemical compounds in PLE were identified and determined using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The antitussive, expectorant and anti-inflammatory activities of PLE were evaluated in an ammonia-induced mouse cough model, a tracheal phenol red excretion mouse model, and a xylene-induced ear swelling mouse model, respectively. A network pharmacology analysis was conducted to investigate the associated gene targets, gene ontology, and KEGG pathways related to the main bioactives in PLE targeting bronchitis. PLE and its five bioactive compounds were assessed for their potential anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Western blot analysis was conducted to elucidate the associated molecular mechanisms. Thirty-seven compounds in PLE were identified, and twelve main compounds were further quantified in PLE using UPLC-MS/MS. PLE oral gavage administrations (0.6 and 0.12 mg/kg) for 7 days markedly reduced cough frequency, prolonged latency period of cough, reduced phlegm and inflammation in mice. The network pharmacology analysis identified 57 gene targets of PLE against bronchitis. The PI3K/AKT and MAPK signalling pathways were the top two modulated pathways. In RAW264.7 cells, PLE (12.5–50 μg/mL) significantly reduced cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. PLE downregulated LPS-elevated protein targets in both PI3K/AKT and MAPK signaling pathways. In PLE, tenuifolin, polygalaxanthone ⅠⅠⅠ, polygalasaponin ⅩⅩⅤⅢ, tenuifoliside B, and 3,6′-Disinapoyl sucrose, were identified as the top five core components responsible for treating bronchitis. These compounds were also found to modulate the protein targets in the PI3K/AKT and MAPK signalling pathways. This study demonstrated the potential therapeutic effects of PLE on bronchitis by reducing cough, phlegm and inflammation. The anti-inflammatory action and molecular mechanisms of the 5 main bioactive compounds in PLE were partly validated through the in vitro assays. The findings provide valuable insights into the mechanisms underlying the traditional use of PLE for bronchitis. [Display omitted] • New UPLC-MS/MS method validated for PLE chemical composition. • PLE reduced cough frequency, phlegm and inflammation in mice. • PLE modulated the PI3K/AKT and MAPK pathways against bronchitis. • Five bioactive compounds identified in PLE against bronchitis. • These compounds modulated protein targets in PI3K/AKT and MAPK pathways. [ABSTRACT FROM AUTHOR]

Aidi injection (ADI) is a popular anti-tumor Chinese patent medicine, widely used in clinics for the treatment of hepatocellular carcinoma (HCC) with remarkable therapeutic effects through multiple targets and pathways. However, the scientific evidence of the synergistic role of the complex chemical component system and the potential mechanism for treating diseases are ignored and remain to be elucidated. This study aimed to elucidate and verify the cooperative association between the potential active ingredient of ADI, which is of significance to enlarge our understanding of its anti-HCC molecular mechanisms. Firstly, the anti-HCC effect of ADI was evaluated in various HCC cells and the zebrafish xenograft model. Subsequently, a variety of bioinformatic technologies, including network pharmacology, weighted gene co-expression network analysis (WGCNA), meta-analysis of gene expression profiles, and pathway enrichment analysis were performed to construct the competitive endogenous RNA (ceRNA) network of ADI intervention in HCC and to establish the relationship between the critical targets/pathways and the key corresponding components, which were involved in ADI against HCC in a synergistic way and were validated by molecular biology experiments. ADI exerted remarkable anti-HCC in vitro cells and in vivo zebrafish model, especially that the Hep 3B2.1-7 cell showed substantial sensibility to ADI. The ceRNA network revealed that the EGFR/PI3K/AKT signaling pathway was identified as the promising pathway. Furthermore, the meta-analysis also demonstrated the critical role of BIRC5 and FEN1 as key targets. Finally, the synergistic effect of ADI was revealed by discovering the inhibitory effect of cantharidin on BIRC5, formononetin on FEN1 and EGFR, as well as isofraxidin on EGFR. Our study unveiled that the incredible protective effect of ADI on HCC resulted from the synergistic inhibition effect of cantharidin, formononetin, and isofraxidin on multiple targets/pathways, including BIRC5, FEN1, and EGFR/PI3K/AKT, respectively, providing a scientific interpretation of ADI against HCC and a typical example of pharmacodynamic evaluation of other proprietary Chinese patent medicine. [Display omitted] • Systematically elucidated the role of ADI in the regulation of ncRNA in HCC. • Proved the synergistic effect of multiple components and pathways of ADI against HCC. • Established and confirmed the correspondence between components and targets in ADI. [ABSTRACT FROM AUTHOR]

Pien Tze Huang is a classic traditional Chinese medicinal product, used for inflammatory diseases as stated in Chinese Pharmacopoeia. In particular, it is effective in treating liver diseases and pro-inflammatory conditions. Acetaminophen (APAP) is a widely used analgesic drug, but its over-dose is associated with acute liver failure where the clinical approved antidote treatment is limited. Inflammation has been considered as one of the therapeutic targets against APAP-induced liver injury. We aimed to explore the therapeutic potential of Pien Tze Huang tablet (PTH) on protecting liver against APAP-induced liver injury through its strong anti-inflammatory pharmacological action. Wild-type C57BL/6 mice were given PTH (75, 150 and 300 mg/kg) by oral gavage 3 days before the APAP injection (400 mg/kg). The protective effect of PTH was assessed by aspartate aminotransferase (AST) and alanine transaminase (ALT) levels and pathological staining. The mechanisms underlying PTH's hepatoprotective effects were investigated in nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) knock-out (NLRP3−/−), over expression NLRP3 (oe-NLRP3) mice, and wild-type mice with the injection of autophagy inhibitor (3-methyladenine, 3-MA). APAP-exposed mice resulted in evident liver injury which was evidenced by hepatic necrosis and elevated levels of AST and ALT in the wild-type C57BL/6 mice. PTH dose-dependently reduced ALT, AST and upregulated autophagy activity. In addition, PTH significantly reduced elevated levels of proinflammatory cytokines and NLRP3 inflammasome. The liver protective effect of PTH (300 mg/kg) was still obvious in the oe-NLRP3 mice, however, it became insignificant in the NLRP3−/− mice. When PTH (300 mg/kg) was co-treated with 3-MA to the wild-type C57BL/6 mice, the NLRP3 inhibition were reversed when autophagy was blocked. PTH exerted a beneficial effect in protecting liver against APAP-induced liver injury. The underlying molecular mechanism was associated with the NLRP3 inflammasome inhibition which was likely driven by the upregulated autophagy activity. Our study underpins the traditional use of PTH in protecting liver through its anti-inflammatory action. [Display omitted] • Pien Tze Huang tablet (PTH) protected liver against acetaminophen-induced liver injury. • PTH suppressed liver inflammation induced by overdose acetaminophen. • The antidote mechanism is associated with nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome inhibition. • PTH's NLRP3 inhibition was driven by the upregulated autophagy. [ABSTRACT FROM AUTHOR]