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Background: Heart failure is primarily characterized by damage to the structure and function of the heart. Ferroptosis represents a form of programmed cell death, and studies indicate that it constitutes one of the primary mechanisms underlying cardiomyocyte death in heart failure. Calycosin, a natural compound derived from astragalus, exhibits various pharmacological properties, including anti-ferroptosis, antioxidant effects, and cardiovascular protection. Nonetheless, the specific role of Calycosin in the treatment of ferroptosis in heart failure remains poorly understood. Objective: This study aims to elucidate the regulatory effect of Calycosin on ferroptosis and its influence on the treatment mechanisms of heart failure through in vivo and in vitro experiments. Methods: A rat model of heart failure was induced using doxorubicin, and the cardiac function was evaluated through cardiac ultrasound examination and NT-Pro BNP detection. Myocardial injury was assessed using H&E staining and Masson staining. The extent of mitochondrial damage was evaluated through transmission electron microscopy. Concurrently, the level of ferroptosis was analyzed by measuring ferroptosis markers, including MDA, ferrous ions, the GSH/GSSG ratio, and GPX4 activity. Subsequently, the molecular mechanism by which Calycosin exerts its therapeutic effects in heart failure was investigated through immunofluorescence and Western blotting. Finally, H9c2 cardiomyocytes were treated with doxorubicin to simulate myocardial injury, and the mechanism by which Calycosin mediates its effects in the treatment of heart failure was further verified through Nrf2 gene silencing. Results: Calycosin significantly improves cardiac function in rats, reduces serum NT-Pro BNP levels, and alleviates myocardial cell damage. Additionally, it significantly decreases the levels of ferroptosis in myocardial tissue, as confirmed through transmission electron microscopy and the assessment of ferroptosis markers, including MDA, ferrous ions, GSH, and GPX4 activity. At the molecular level, Calycosin exerts its effects by activating the Nrf2/SLC7A11/GPX4 signaling pathway, evidenced by the upregulation of Nrf2, SLC7A11, GPX4, GSS, and GCL protein expression. This process substantially enhances the antioxidant capacity of rat myocardial tissue and effectively suppresses ferroptosis in myocardial cells. The results obtained from both in vivo and in vitro experiments are consistent. Notably, when Nrf2 is silenced, the protective effect of Calycosin on the myocardium is markedly diminished. Conclusion: Calycosin effectively treats doxorubicin-induced cardiac injury, and its therapeutic effect is likely closely associated with the activation of the Nrf2/SLC7A11/GPX4 signaling pathway and the inhibition of ferroptosis in myocardial cells. Consequently, Calycosin, as a promising compound against doxorubicin-induced cardiotoxicity, warrants further investigation. [ABSTRACT FROM AUTHOR]

Objective To investigate effects of calycosin (CA) on cigarette smoke (CS) induced airway epithelial cell damage in mice and the sirtuin 3/superoxide dismutase 2 (SIRT3/SOD2) signaling pathway in mice. Methods A total of 90 mice were randomly separated into the control group, the cigarette smoke (CS) group, the CA low-dose treatment group (CAL group), the CA high-dose treatment group (CA-H group) and the CA high-dose treatment plus SIRT3 inhibitor 3-TYP group (CA-H+3-TYP group), with 18 mice in each group. Tidal volume (TV) and peak expiratory flow rate (PEF) of lung function were detected by whole body plethysmography system. Serum levels of inflammatory factors [interleukin (IL) -6, tumor necrosis factor (TNF) - α] and oxidative stress indicators [reactive oxygen species (ROS), SOD] were detected by enzyme-linked immunosorbent assay (ELISA). The injury of airway epithelial cells in lung tissue was observed by HE staining. The expression levels of barrier related proteins (OCLN and ZO-1) in airway epithelial cells were detected by immunohistochemistry. Immunoblotting was applied to detect the expression of SIRT3/SOD2 signaling pathway related proteins. Results Compared with the control group, levels of TV, PEF, MAN and SOD and the expression levels of OCLN, ZO-1, SIRT3 and SOD2 were decreased in the CS group, while the levels of MLI, IL-6, TNF-α and ROS were increased (P＜0.05). Compared with the control group, the lung tissue structure was significantly damaged, the alveolar enlargement was obvious, the surrounding alveolar was accompanied by inflammatory cell infiltration, and the airway epithelial cells were obviously shed in the CS group. Different doses of CA alleviated lung tissue destruction, improved alveolar structure, reduced inflammatory cell infiltration, reduced airway epithelial cell shedding, increased levels of TV, PEF, MAN, SOD and OCLN, ZO-1, SIRT3 and SOD2, and decreased levels of MLI, IL-6, TNF- α and ROS. The effect of high dose CA was more significant than that of low dose CA (P＜0.05). SIRT3/SOD2 signaling pathway inhibitor 3-TYP partially reversed the ameliorative effect of CA on CS induced airway epithelial cell injury in mice. Conclusion CA can ameliorate CS induced airway epithelial cell damage in mice, and its mechanism is related to the activation of the SIRT3/SOD2 signaling pathway. [ABSTRACT FROM AUTHOR]

Background: To anticipate the potential molecular mechanism of Astragalus membranaceus (AM) and its monomer, Calycosin, against peritoneal fibrosis (PF) and related muscle atrophy using mRNA-seq, network pharmacology, and serum pharmacochemistry. Methods: Animal tissues were examined to evaluate a CKD-PF mice model construction. mRNA sequencing was performed to find differential targets. The core target genes of AM against PF were screened through network pharmacology analysis, and CKD-PF mice models were given high- and lowdose AM to verify common genes. Serum pharmacochemistry was conducted to clarify which components of AM can enter the blood circulation, and the selected monomer was further validated through cell experiments for the effect on PF and mesothelial mesenchymal transition (MMT) of peritoneal mesothelial cells (PMCs). Results: The CKD-PF mice models were successfully constructed. A total of 31,184 genes were detected in the blank and CKD-PF groups, and 228 transcription factors had significant differences between the groups. Combined with network pharmacology analysis, a total of 228 AM-PF-related targets were identified. Androgen receptor (AR) was the remarkable transcription factor involved in regulating transforming growth factor-β1 (TGF-β1). AM may be involved in regulating the AR/TGF-β1 signaling pathway and may alleviate peritoneal dialysis-related fibrosis and muscle atrophy in CKD-PF mice. In 3% peritoneal dialysis solution-stimulated HMrSV5 cells, AR expression levels were dramatically reduced, whereas TGF-β1/p-smads expression levels were considerably increased. Conclusion: AM could ameliorate PF and related muscle atrophy via the cotarget AR and modulated AR/TGF-β1 pathway. Calycosin, a monomer of AM, could partially reverse PMC MMT via the AR/TGF-β1/smads pathway. This study explored the traditional Chinese medicine theory of "same treatment for different diseases," and supplied the pharmacological evidence of "AM can treat flaccidity syndrome." [ABSTRACT FROM AUTHOR]

Calycosin (Caly), a flavonoid compound, demonstrates a variety of beneficial properties. However, the specific mechanisms behind Caly's anticancer effects remain largely unexplored. Network pharmacology was used to explore the potential targets of Caly in renal cancer. Additionally, RNA‐seq sequencing was used to detect changes in genes in renal cancer cells after Caly treatment. Validation was carried out through quantitative reverse transcription‐PCR and Western blot analysis. The luciferase reporter assay was applied to pinpoint the interaction site between MAZ and HAS2. Furthermore, the immunoprecipitation assay was utilized to examine the ubiquitination and degradation of MAZ. In vivo experiments using cell line‐derived xenograft mouse models were performed to assess Calycosin's impact on cancer growth. Network pharmacology research suggests Caly plays a role in promoting apoptosis and inhibiting cell adhesion in renal cancer. In vitro, Caly has been observed to suppress proliferation, colony formation, and metastasis of renal cancer cells while also triggering apoptosis. Additionally, it appears to diminish hyaluronic acid synthesis by downregulating HAS2 expression. MAZ is identified as a transcriptional regulator of HAS2 expression. Calycosin further facilitates the degradation of MAZ via the ubiquitin‐proteasome pathway. Notably, Caly demonstrates efficacy in reducing the growth of renal cell carcinoma xenograft tumors in vivo. Our findings indicate that Caly suppresses the proliferation, metastasis, and progression of renal cell carcinoma through its action on the MAZ/HAS2 signaling pathway. Thus, Caly represents a promising therapeutic candidate for the treatment of renal cell carcinoma. [ABSTRACT FROM AUTHOR]

Our former studies have identified the alleviating effect of Calycosin (CA) on spinal cord injury (SCI). In this study, our purpose is to explore the influence of CA on SCI from the perspective of promoting axon growth. The SCI animal model was constructed by spinal cord compression, wherein rat primary cortex neuronal isolation was performed, and the axonal growth restriction cell model was established via chondroitin sulfate proteoglycan (CSPG) treatment. The expressions of axon regeneration markers were measured via immunofluorescent staining and western blot, and the direct target of CA was examined using silver staining. Finally, the expression of the protein tyrosine phosphatase receptor type S (PTPRS) was assessed using western blot. CA treatment increased neuronal process outgrowth and the expressions of axon regeneration markers, such as neurofilament H (NF-H), vesicular glutamate transporter 1 (vGlut1), and synaptophysin (Syn) in both SCI model rats and CSPG-treated primary cortical neurons, and PTPRS levels were elevated after SCI induction. In addition, PTPRS was the direct target of CA, and according to in vivo findings, exposure to CA reduced the PTPRS content. Furthermore, PTPRS overexpression inhibited CA’s enhancement of axon regeneration marker content and neuronal axon lengths. CA improves SCI by increasing axon development through regulating PTPRS expression. [ABSTRACT FROM AUTHOR]

To explore the effect of calycosin (CAL) on neuronal autophagy and apoptosis in rats with spinal cord injury (SCI) by regulating PI3K/Akt signaling pathway and its mechanism. Methods A total of 32 male or female adult SD rats were randomly divided into the sham group, the SCI group, the CAL low (20 mg/kg) dose group and the CAL high (40 mg/kg) dose group with 8 rats in each group. The rat model of moderate SCI was established by modified Allen’s method. After successful modeling, rats were injected intraperitoneally immediately with different dosage of CAL or equal amount of saline once a day for 7 consecutive days. Basso, Beattie and Bresnahan (BBB) scores were used to evaluate the recovery of motor function of rats at 1, 3 and 7 d after surgery. At 7 d after surgery, Nissl staining was used to detect the surviving number of motor neurons in anterior horn of spinal cord. Western blot assay was used to assess expression levels of p62, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3B), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, Cleaved-Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) proteins. Immunofluorescence staining was used to measure the expression of LC3B in anterior neurons of spinal cord. Results Compared with the sham group, BBB scores, the surviving number of motor neurons and levels of Bcl-2, Bcl-2/Bax, p-PI3K, p-PI3K/PI3K, p-Akt and p-Akt/Akt were significantly decreased (P＜0.05), and levels of p62, Beclin-1, LC3B Ⅱ, LC3B Ⅱ/Ⅰ, Cleaved-Caspase-3 and Bax were significantly increased in the SCI group (P＜0.05). Compared with the SCI group, BBB scores, the survival of anterior horn motor neurons and levels of LC3B Ⅱ, LC3B Ⅱ/Ⅰ, Bcl-2, Bcl-2/Bax, p-PI3K, pPI3K/PI3K, p-Akt and p-Akt/Akt were increased in the CAL low dose group and CAL high dose group, and levels of p62, Cleaved-Caspase-3 and Baxcould were significantly decreased (P＜0.05). Conclusion CAL could promote autophagy and inhibit apoptosis of neurons through activating PI3K/Akt signaling pathway, thereby conferring a protective role following SCI in rats. [ABSTRACT FROM AUTHOR]

Background: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. Methods: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. Results: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1β and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. Conclusions: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI. [ABSTRACT FROM AUTHOR]

Oxidative stress is a hallmark of secondary injury of spinal cord injuries. Controlling oxidative stress is crucial for mitigating secondary injury and promoting functional recovery after spinal cord injuries. Calycosin is an O‐methylated isoflavone with antioxidant activity. To evaluate the effect of calycosin on spinal cord neurons under oxidative stress and clarify the molecular mechanism underlying the effect, we tested the neuroprotective activity of calycosin in a primary spinal cord neuron culture model. We found that calycosin protected neurons from H2O2‐induced neuronal death in a dose‐dependent manner. Further experiments revealed that calycosin decreased H2O2‐induced mitochondrial fragmentation and mitochondrial membrane potential loss, and subsequently reduced H2O2‐triggered release of mitochondrial cytochrome c into the cytoplasm. In addition, calycosin inhibited H2O2‐induced reactive oxygen species generation and activation of NF‐κB signaling in spinal cord neurons. Furthermore, the expression of several antioxidant enzymes such as HO‐1, NQO1, GCLC, GCLM, TrxR1, and Trx1 was significantly promoted by calycosin. More importantly, we revealed that the Nrf2/Keap1 signal is crucial for the effect of calycosin, because calycosin increased the amount of nuclear Nrf2 while decreasing the amount of cytoplasmic Nrf2. Nrf2 knockdown with siRNA transfection abolished the neuroprotective effect of calycosin. Taken together, this study disclosed a novel mechanism by which calycosin combats oxidative stress. Our study thus sheds light on the potential clinical application of calycosin in SCI treatment. [ABSTRACT FROM AUTHOR]

BACKGROUND: Endothelial injury is one of the causes of cardiovascular diseases. Human induced pluripotent stem cells are easy to obtain, have strong differentiation ability, and have less exclusiveness, and their endothelial differentiated cells can be used as ideal cells for cardiovascular disease research. OBJECTIVE: To investigate the effect and mechanism of calycosin on endothelial differentiation of human induced pluripotent stem cells and to provide technical support for microvascular regeneration. METHODS: Human induced pluripotent stem cells were divided into control group and calycosin group (1.25, 2.5 μg/mL), and growth factors were added to induce single-layer endothelial differentiation. After the induction of differentiation for 8 days, the positive rate of endothelial cell marker CD144 was detected by flow cytometry. Fluorescent expressions of CD144 and CD31 were detected by the immunofluorescence method. Lentivirus RNAi GFP puromycin was used to silence human-induced pluripotent stem cell Piezo1 mRNA followed by endothelial directed differentiation. After 8 days of differentiation, the positive rate of CD144 in differentiated cells was detected by flow cytometry. The mRNA expression levels of CD144, Piezo1 and MEK were detected by qPCR. RESULTS AND CONCLUSION: (1) Compared with the control group, the positive rate of CD144 was significantly increased in the 1.25 and 2.5 μg/mL calycosin groups (P < 0.05). The expressions of CD144, Piezo1, and MEK mRNA were increased in the 2.5 μg/mL calycosin group (P < 0.05). The fluorescence expressions of CD144 (P < 0.01) and CD31 (P < 0.001) were significantly increased in the 2.5 μg/mL calycosin group. (2) Compared with the shNT group, CD144 positive rate and CD144, Piezo1, MEK mRNA expressions were significantly increased in the shNT + calycosin 1.25, 2.5 μg/mL groups (P < 0.05). Compared with the shPiezo1 group, the positive rate of CD144 and mRNA expressions of CD144, Piezo1 and MEK had no significant changes in the shPiezo1+calycosin 1.25, 2.5 μg/mL groups (P > 0.05). (3) It is concluded that 2.5 μg/mL calycosin promotes the differentiation of human-induced pluripotent stem cells into endothelial lineages. Calycosin promotes the downstream MEK expression, thereby promoting the endothelial differentiation of human induced pluripotent stem cells by targeting the expression level of Piezo1. [ABSTRACT FROM AUTHOR]

Peanut is not only an important economic crop but also considered to be as a kind of functional food. However, the presence of calycosin, a bioactive isoflavonoid, in peanuts is still unknown. In this study, calycosin was firstly identified in leaves, needles and pods of four peanut cultivars examined including Yuanza 9102 (P1), Kainong 176 (P2), Zhuhua 2 (P3) and Yuhua 23 (P4). Moreover, the effects of three nitrogen fertilization levels (N0: 0 kg/ha, N1: 75 kg/ha, N2: 150 kg/ha) on calycosin accumulation in leaves, needles and pods of four different peanut cultivars at DAF30, DAF60 and DAF90 were investigated. The results showed that calycosin content varied from 0.87 to 718.83 μ g/g FW in peanut and the relatively higher calycosin content was found in leaves, followed by needles and pods. Furthermore, calycosin contents in leaves of all four peanut cultivars at DAF60 were higher than that at DAF30 and DAF90, respectively. Calycosin accumulation in needles and pods at DAF90 was more than that at DAF30 and DAF60. In addition, the optimal combination for the highest calycosin yields in the leaves, needles and pods were P1 under N1 (75 kg/ha) at DAF60, P2 under N2 (150 kg/ha) at DAF60 and P2 under N1 at DAF90, respectively. [ABSTRACT FROM AUTHOR]

Atherosclerosis, caused by lipid deposit in the arterial wall for narrowing the arteries, is an increased risk factor of developing heart failure. Presently, clinical first-line drug therapy can be found with side effects, and thus new substitute medication should be developed needfully. Calycosin is one of the most bioactive products refined from natural plant, and it exerts promising cardiovascular protective effect. However, the pharmacological mechanisms of calycosin against atherosclerosis have not been elaborated. In this study, a systematic network pharmacology combined with molecular docking analysis was used to reveal the interaction activity and biological target in calycosin against atherosclerosis. We screened all preparative targets linked to calycosin and atherosclerosis from the available public databases. These results indicated total 409 putative targets in calycosin action, 71 of which were interacted with atherosclerosis. Further biological docking analysis suggested that calycosin displayed the powerful binding affinities with target proteins, including interleukin-6 (IL6) and mitogen-activated protein kinase 3 (MAPK3) MAPK3. Then enrichment findings revealed that calycosin action to treat atherosclerosis might be related to inhibition of inflammatory reaction and oxidative stress through modulating nucleolus transcription factor for improving lipid metabolism. In conclusion, the anti-atherosclerotic targets and molecular mechanisms in calycosin action were revealed systematically through preclinical evaluation. And calycosin may be a potential natural compound for the treatment of atherosclerosis. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs), which function as chaperones, are activated in response to various environmental stressors. In addition to their role in diverse aspects of protein production, HSPs protect against harmful protein-related stressors. Calycosin exhibits numerous beneficial properties. This study aims to explore the protective effects of calycosin in the heart under heat shock and determine its underlying mechanism. H9c2 cells, western blot, TUNEL staining, flow cytometry, and immunofluorescence staining were used. The time-dependent effects of heat shock analyzed using western blot revealed increased HSP expression for up to 2 h, followed by protein degradation after 4 h. Hence, a heat shock damage duration of 4 h was chosen for subsequent investigations. Calycosin administered post-heat shock demonstrated dose-dependent recovery of cell viability. Under heat shock conditions, calycosin prevented the apoptosis of H9c2 cells by upregulating HSPs, suppressing p-JNK, enhancing Bcl-2 activation, and inhibiting cleaved caspase 3. Calycosin also inhibited Fas/FasL expression and activated cell survival markers (p-PI3K, p-ERK, p-Akt), indicating their cytoprotective properties through PI3K/Akt activation and JNK inhibition. TUNEL staining and flow cytometry confirmed that calycosin reduced apoptosis. Moreover, calycosin reversed the inhibitory effects of quercetin on HSF1 and Hsp70 expression, illustrating its role in enhancing Hsp70 expression through HSF1 activation during heat shock. Immunofluorescence staining demonstrated HSF1 translocation to the nucleus following calycosin treatment, emphasizing its cytoprotective effects. In conclusion, calycosin exhibits pronounced protective effects against heat shock-induced damages by modulating HSP expression and regulating key signaling pathways to promote cell survival in H9c2 cells. [ABSTRACT FROM AUTHOR]

Objective To establish the method of simultaneous determination of four main components of Danggui Liuhuang Decoction, including phellodendrine, palmatine, calycosin, and ferulic acid and provide reference for the quality control of Danggui Liuhuang Decoction. Methods Based on the HPLC-MS/MS analysis method, the positive ion data acquisition mode were adopted for the mass spectrometry detection and the four main components were quantified with multiple reaction monitoring mode (MRM) by ESI source. The chromatographic column was Agilent Extend-C18 (5 μm, 4.6 mm×250 mm), and gradient elution was performed with methanol and 0.5% formic acid in water. Results The linear range of phellodendrine was from 2-200 nmol/ml, and the linear range of palmatine, calycosin and ferulic acid was from 20-2000 nmol/ml. The contents of the four components in the seven batches of Danggui Liuhuang Decoction were relatively stable, among which ferulic acid was mainly found in Phellodendrine and Coptidis; Phellodendrine was only detected in cortex phellodendri; the content of calycosin in Scutellaria baicalensis and Astragalus was higher; palmatine was detected in both Phellodendron and Astragalus. Conclusion The method had high sensitivity, good specificity and sample stability, which could meet the requirements of quantitative analysis of Traditional Chinese Medicine compounds, and could provide reference for further pharmacokinetics study on the content changes of traditional Chinese medicine compounds in biological samples. [ABSTRACT FROM AUTHOR]

Background: The rate of disability and mortality associated with cerebral ischemia/reperfusion injury (CIRI) is high due to limited treatment options, making it a major challenge to clinical management. Calycosin is a biologically active compound hostile to inflammatory, neuroprotective, and tumor effects. Whether calycosin has an ischemia/reperfusion effect or mechanism is unclear. Materials and Methods: For in vivo experiments, we randomly divided rats into five groups: blank control group, middle cerebral artery occlusion/reperfusion (MCAO/R) surgical group, calycosin + MCAO/R group (5 mg/kg), calycosin + MCAO/R group (10 mg/kg), and calycosin + MCAO/R group (20 mg/kg). Molding of the middle cerebral artery was performed. Calycosin's neuroprotective effects were evaluated using the neurological deficit score, brain edema rate, and cerebral infarct volume. For in vitro experiments, we divided PC12 cells into five groups: blank control group, oxygen and glucose deprivation/reperfusion (OGD/R) group, calycosin + OGD/R group (1 × 10−6 mol/L), calycosin + OGD/R group (4 × 10−6 mol/L), and calycosin + OGD/R group (16 × 10−6 mol/L). The optimal concentration of calycosin on PC12 cells was determined using the cell counting kit-8 (CCK-8) cell activity assay. The expression of nuclear factor kappa-B (NF-κB)-related factors was detected using real-time quantitative polymerase chain reaction and Western blotting. Results: In rats, the MCAO/R model resulted in elevated neurological deficit scores, increased brain infarct volumes, and increased brain edema rates. The OGD/R model decreased rat adrenal pheochromocytoma (PC12) cell activity, and calycosin had a significant cerebral protective effect on PC12 cells under OGD/R conditions. In addition, calycosin can inhibit the activation of the NF-κB pathway, and its neuroprotective effect may be related to the NF-κB pathway. Conclusion: Calycosin can reduce focal CIRI, and the neuroprotective effect of calycosin may be related to the inhibition of the high mobility group protein 1/toll-like receptor 4 (TLR4)/NF-κB signaling pathways. [ABSTRACT FROM AUTHOR]

BackgroundHeart failure is primarily characterized by damage to the structure and function of the heart. Ferroptosis represents a form of programmed cell death, and studies indicate that it constitutes one of the primary mechanisms underlying cardiomyocyte death in heart failure. Calycosin, a natural compound derived from astragalus, exhibits various pharmacological properties, including anti-ferroptosis, antioxidant effects, and cardiovascular protection. Nonetheless, the specific role of Calycosin in the treatment of ferroptosis in heart failure remains poorly understood.ObjectiveThis study aims to elucidate the regulatory effect of Calycosin on ferroptosis and its influence on the treatment mechanisms of heart failure through in vivo and in vitro experiments.MethodsA rat model of heart failure was induced using doxorubicin, and the cardiac function was evaluated through cardiac ultrasound examination and NT-Pro BNP detection. Myocardial injury was assessed using H&E staining and Masson staining. The extent of mitochondrial damage was evaluated through transmission electron microscopy. Concurrently, the level of ferroptosis was analyzed by measuring ferroptosis markers, including MDA, ferrous ions, the GSH/GSSG ratio, and GPX4 activity. Subsequently, the molecular mechanism by which Calycosin exerts its therapeutic effects in heart failure was investigated through immunofluorescence and Western blotting. Finally, H9c2 cardiomyocytes were treated with doxorubicin to simulate myocardial injury, and the mechanism by which Calycosin mediates its effects in the treatment of heart failure was further verified through Nrf2 gene silencing.ResultsCalycosin significantly improves cardiac function in rats, reduces serum NT-Pro BNP levels, and alleviates myocardial cell damage. Additionally, it significantly decreases the levels of ferroptosis in myocardial tissue, as confirmed through transmission electron microscopy and the assessment of ferroptosis markers, including MDA, ferrous ions, GSH, and GPX4 activity. At the molecular level, Calycosin exerts its effects by activating the Nrf2/SLC7A11/GPX4 signaling pathway, evidenced by the upregulation of Nrf2, SLC7A11, GPX4, GSS, and GCL protein expression. This process substantially enhances the antioxidant capacity of rat myocardial tissue and effectively suppresses ferroptosis in myocardial cells. The results obtained from both in vivo and in vitro experiments are consistent. Notably, when Nrf2 is silenced, the protective effect of Calycosin on the myocardium is markedly diminished.ConclusionCalycosin effectively treats doxorubicin-induced cardiac injury, and its therapeutic effect is likely closely associated with the activation of the Nrf2/SLC7A11/GPX4 signaling pathway and the inhibition of ferroptosis in myocardial cells. Consequently, Calycosin, as a promising compound against doxorubicin-induced cardiotoxicity, warrants further investigation.

Two new isoflavone glycosides 1 and 2 were isolated from roots of Astragalus mongholicus Bunge (A. membranaceus Fisch. ex Bunge, Fabaceae). The compounds were calycosin 7-O-(2′′-O-β-Dglucopyranosyl)-β-D-glucopyranoside (astramonghoside A, 1) and calycosin 7-O-(2′′-O-β-D-glucopyranosyl-6′′-O-malonyl)-β-D-glucopyranoside (astramonghoside B, 2) according to UV and NMR spectroscopy and mass spectrometry. Compounds 1 and 2 inhibited fungal tyrosinase. [ABSTRACT FROM AUTHOR]

Background: Calycosin, a flavonoid compound extracted from Astragalus membranaceus, has shown anti-asthma benefits in house dust mite-induced asthma. Recent studies have suggested that innate-type cells, including group 2 innate lymphoid cells (ILC2s) and macrophages, serve as incentives for type 2 immunity and targets for drug development in asthma. This work focuses on the effects of calycosin on the dysregulated ILC2s and macrophages in allergic asthma. Methods: In vivo, the asthmatic mouse model was established with ovalbumin (OVA) sensitization and challenge, and calycosin was intraperitoneally administered at doses of 20 and 40 mg/kg. In vivo, mouse primary ILC2s were stimulated with interleukin (IL)-33 and mouse RAW264.7 macrophages were stimulated with IL-4 and IL-13 to establish the cell models. Cells were treated with calycosin at doses of 5 and 10 µM. Results: In vivo, we observed significantly reduced numbers of eosinophils, neutrophils, monocyte macrophages and lymphocytes in the bronchoalveolar lavage fluid (BALF) of OVA-exposed mice with 40 mg/kg calycosin. Histopathological assessment showed that calycosin inhibited the airway inflammation and remodeling caused by OVA. Calycosin markedly decreased the up-regulated IL-4, IL-5, IL-13, IL-33, and suppression tumorigenicity 2 (ST2) induced by OVA in BALF and/or lung tissues of asthmatic mice. Calycosin repressed the augment of arginase 1 (ARG1), IL-10, chitinase-like 3 (YM1) and mannose receptor C-type 1 (MRC1) levels in the lung tissues of asthmatic mice. In vivo, calycosin inhibited the IL-33-induced activation as well as the increase of IL-4, IL-5, IL-13 and ST2 in ILC2s. Calycosin also repressed the increase of ARG1, IL-10, YM1 and MRC1 induced by IL-4 and IL-13 in RAW264.7 macrophages. In addition, we found that these changes were more significant in 40 mg/kg calycosin treatment than 20 mg/kg calycosin. Conclusions: Collectively, this study showed that calycosin might attenuate OVA-induced airway inflammation and remodeling in asthmatic mice via preventing ILC2 activation and macrophage M2 polarization. Our study might contribute to further study of asthmatic therapy. [ABSTRACT FROM AUTHOR]

Periprosthetic osteolysis (PPO) caused by wear particles is one of the leading causes of implant failure after arthroplasty. Macrophage polarization imbalance and subsequent osteogenic inhibition play a crucial role in PPO. Calycosin (CA) is a compound with anti‐inflammatory and osteoprotective properties. This study aimed to evaluate the effects of CA on titanium (Ti) particle‐induced osteolysis, Ti particle‐induced macrophage polarization and subsequent osteogenic deficits, and explore the associated signalling pathways in a Ti particle‐stimulated calvarial osteolysis mouse model using micro‐CT, ELISA, qRT‐PCR, immunofluorescence and western blot techniques. The results showed that CA alleviated inflammation, osteogenic inhibition and osteolysis in the Ti particle‐induced calvarial osteolysis mouse model in vivo. In vitro experiments showed that CA suppressed Ti‐induced M1 macrophage polarization, promoted M2 macrophage polarization and ultimately enhanced osteogenic differentiation of MC3T3‐E1 cells. In addition, CA alleviated osteogenic deficits by regulating macrophage polarization homeostasis via the NF‐κB signalling pathway both in vivo and in vitro. All these findings suggest that CA may prove to be an effective therapeutic agent for wear particle‐induced osteolysis. [ABSTRACT FROM AUTHOR]

Recent studies have shown that calycosin possess multiple pharmacological properties, containing anti-cancer activity. Hence, this project explored the effect and possible mechanism of calycosin in pancreatic cancer. After being cultured, SW1990 cells were exposed to different concentrations of calycosin. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, plate colony formation, and transwell experiments assessed cell proliferation, colony formation, migration, and invasion. Circ_0000285 expression was assessed using quantitative reverse transcription-polymerase chain reaction experiment. E-cadherin and N-cadherin protein levels were examined using Western blot. After calycosin treatment, SW1990 cell proliferation, migration, invasion, circ_0000285 expression, N-cadherin were repressed, and E-cadherin was increased in dose-dependent way. Circ_0000285 deficiency might hinder cell proliferation, migration, and invasion. Furthermore, the upregulation of circ_0000285 might partly antagonize calycosin-mediated SW1990 cell proliferation, migration, and invasion inhibition. Calycosin treatment might block pancreatic cancer cell development through down-regulating circ_0000285. [ABSTRACT FROM AUTHOR]

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Abstract Osteoarthritis (OA) is a severe chronic inflammatory disease. As the main active component of Astragalus mongholicus Bunge, a classic traditional ethnic herb, calycosin exhibits anti-inflammatory action and its mechanism of exact targets for OA have yet to be determined. In this study, we established an anterior cruciate ligament transection (ACLT) mouse model. Mice were randomized to sham, OA, and calycosin groups. Cartilage synthesis markers type II collagen (Col-2) and SRY-Box Transcription Factor 9 (Sox-9) increased significantly after calycosin gavage. While cartilage matrix degradation index cyclooxygenase-2 (COX-2), phosphor-epidermal growth factor receptor (p-EGFR), and matrix metalloproteinase-9 (MMP9) expression were decreased. With the help of network pharmacology and molecular docking, these results were confirmed in chondrocyte ADTC5 cells. Our results indicated that the calycosin treatment significantly improved cartilage damage, this was probably attributed to reversing the imbalance between chondrocyte synthesis and catabolism.

Objective We aimed to (i) investigate the protective effect of active components of water extract of huangqi (milkvetch root) on high glucose (25 mmol/L) induced injury of human umbilical vein endothelial cells (HUVEC) and (ii) elucidate the underlying mechanism. Methods Male SD rats were continuously fed with water extract [20 g/(kg·d)] for 7 d, and blood samples were colleeted 1, 2 and 4 h after the last gavage, combined with supernatant, and performed ultra-performance liquid chromatography (UPLC-MS) analysis to screen the active components of the extract in rats. Cells were divided into normal group, model group, different active components groups treated with different concentrations, and the concerted application of active components group. Cell viability was measured by Cell Counting Kit-8 to explore the optimal concentration of the effective components of milkvetch root. Oxidative damage was measured by reactive oxygen species (ROS). The enzyme-linked immunosorbent assay measured the contents of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA); the protein expression level of nuclear factor E2 related factor (Nrf 2) and heme oxygenase 1 (HO-1) by Western blotting. Results Formononetin, daidzein, and calycosin are the active components of milkvetch root water extract.Compared with the normal group, the cell viability of the model group was increased (P<0.05). Compared with the model group, cell viability was decreased in each concentration(26, 13, 6.5 μmol/L) of formononetin group (P<0.05), a high dose of daidzein (0.11 μmol/L) could reduce cell viability (P<0.05). Compared with the normal group, the ROS levels in the model group were increased(P<0.05); compared with the model group, the ROS levels in the formononetin high and medium concentrations(26, 13 μmol/L) groups, different concentrations of daidzein (0.11, 0.06, 0.03 μmol/L), the calycosin high concentration group (7.00 μmol/L) were decreased (P<0.05). The combined active components inhibitive rate was higher than the single active components, and highest in the high concentration group. Compared with the normal group, Nrf2 and HO-1 protein expressions were decreased in the model group (P<0.05); compared with the model group, Nrf2 and HO-1 proteins were increased in the group with high concentration of concerted application group (P<0.05). Compared with the normal group, SOD, CAT were decreased (P<0.05), MDA increased (P<0.05) in the model group; compared with the model group, SOD, CAT in the high concentration concerted application group were increased (P<0.05), MDA was significantly decreased (P<0.05). Conclusion The active components of milkvetch root water extract, formononetin, daidzein, calycosin can protect HUVEC from high glucose-induced injury. The mechanism may be related to the regulation of Nrf2, HO-1, SOD, CAT, and MDA expression and their antioxidant effects. [ABSTRACT FROM AUTHOR]

Background: Calycosin may be a potential candidate regarding chemotherapeutic agent, because already some studies against multivarious cancer have been made with this natural compound. Aim: This review elucidated a brief overview of previous studies on calycosin potential effects on various cancers and its potential mechanism of action. Methodology: Data retrieved by systematic searches of Google Scholar, PubMed, Science Direct, Web of Science, and Scopus by using keywords including calycosin, cancer types, anti‐cancer mechanism, synergistic, and pharmacokinetic and commonly used tools are BioRender, ChemDraw Professional 16.0, and ADMETlab 2.0. Results: Based on our review, calycosin is available in nature and effective against around 15 different types of cancer. Generally, the anti‐cancer mechanism of this compound is mediated through a variety of processes, including regulation of apoptotic pathways, cell cycle, angiogenesis and metastasis, oncogenes, enzymatic pathways, and signal transduction process. These study conducted in various study models, including in silico, in vitro, preclinical and clinical models. The molecular framework behind the anti‐cancer effect is targeting some oncogenic and therapeutic proteins and multiple signaling cascades. Therapies based on nano‐formulated calycosin may make excellent nanocarriers for the delivery of this compound to targeted tissue as well as particular organ. This natural compound becomes very effective when combined with other natural compounds and some standard drugs. Moreover, proper use of this compound can reverse resistance to existing anti‐cancer drugs through a variety of strategies. Calycosin showed better pharmacokinetic properties with less toxicity in human bodies. Conclusion: Calycosin exhibits excellent potential as a therapeutic drug against several cancer types and should be consumed until standard chemotherapeutics are available in pharma markets. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a severe chronic inflammatory disease. As the main active component of Astragalus mongholicus Bunge, a classic traditional ethnic herb, calycosin exhibits anti-inflammatory action and its mechanism of exact targets for OA have yet to be determined. In this study, we established an anterior cruciate ligament transection (ACLT) mouse model. Mice were randomized to sham, OA, and calycosin groups. Cartilage synthesis markers type II collagen (Col-2) and SRY-Box Transcription Factor 9 (Sox-9) increased significantly after calycosin gavage. While cartilage matrix degradation index cyclooxygenase-2 (COX-2), phosphor-epidermal growth factor receptor (p-EGFR), and matrix metalloproteinase-9 (MMP9) expression were decreased. With the help of network pharmacology and molecular docking, these results were confirmed in chondrocyte ADTC5 cells. Our results indicated that the calycosin treatment significantly improved cartilage damage, this was probably attributed to reversing the imbalance between chondrocyte synthesis and catabolism. [ABSTRACT FROM AUTHOR]

Vascular calcification (VC) is a pathological condition frequently observed in cardiovascular diseases. Primary factors contributing to VC are osteogenic differentiation of vascular smooth muscle and hydroxyapatite deposition. Targeted autophagy (a lysosome-mediated mechanism for degradation/recycling of unnecessary cellular components) is a useful approach for inhibiting VC and promoting vascular cell health. Calycosin has been shown to alleviate atherosclerosis by enhancing macrophage autophagy, but its therapeutic effect on VC has not been demonstrated. Using an in vitro model (rat thoracic aortic smooth muscle cell line A7r5), we demonstrated effective inhibition of VC using calycosin (the primary flavonoid component of astragalus), based on the enhancement of autophagic flux. Calycosin treatment activated AMPK/mTOR signaling to induce initiation of autophagy and restored mTORC1-dependent autophagosome–lysosome fusion in late-stage autophagy by promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation, thereby preventing stoppage of autophagy in calcified cells. Calycosin substantially reduced degrees of both osteogenic differentiation and calcium deposition in our VC cell model by enhancing autophagy. The present findings clarify the mechanism whereby calycosin mitigates autophagy stoppage in calcified smooth muscle cells and provide a basis for effective VC treatment via autophagy enhancement. [ABSTRACT FROM AUTHOR]

The tumour microenvironment (TME) is crucial for tumour development and progression. Tumour‐associated macrophages (TAMs) in the TME can promote tumour progression and metastasis by releasing cytokines, such as IL‐6. Calycosin, a phytoestrogen that is one of the active compounds in Radix Astragali, has been shown to inhibit tumour growth and metastasis. However, the underlying mechanism by which calycosin inhibits tumour growth remains unclear. Thus, this study aimed to investigate the effect of calycosin on IL‐6 production in peripheral blood mononuclear cell (PBMC)‐ and THP‐1‐derived macrophages and explore its potential mechanisms using co‐immunoprecipitation, western blotting, immunofluorescence, chromatin immunoprecipitation and luciferase assays. We found that calycosin treatment substantially upregulated the expression of ER‐α36, a variant of the ER, and reduced IL‐6 production in macrophages. Mechanistically, ER‐α36 physically interacted with NF‐κBp65 and retained p65 in the cytoplasm to attenuate NF‐κB function as an IL‐6 transcriptional inducer. In conclusion, our result indicated that calycosin inhibited IL‐6 production by enhancing ER‐α36 expression and its interaction with p65, which attenuated NF‐κB function as an IL‐6 inducer. Therefore, calycosin can be developed as an effective agent for cancer therapy by targeting TAMs. [ABSTRACT FROM AUTHOR]

Intestinal interstitial fibrosis is a core event of inflammatory bowel disease (IBD) development. Calycosin has been recognized to carry various therapeutic bioactivities. However, the role of calycosin in intestinal interstitial fibrosis remains to be illustrated. This aim of this study was to explore the effects of calycosin on intestinal interstitial fibrosis in IBD and the underlying mechanisms. The in vitro and in vivo models were established by using TNBS-induced mouse IBD model and co-culture of intestinal epithelial cells and intestinal interstitial cells; moreover, lentivirus-mediated knockdown of NLRP3 expression was applied. The results showed that calycosin significantly improved the intestinal interstitial fibrosis of TNBS-induced IBD. Mechanistically, calycosin downregulated NLRP3 expression and inhibited the activation of IL-33/ST2 signaling in intestinal epithelial cells, which subsequently impedes intestinal interstitial cell migration and activation by regulating the secretion of IL-33/ST2 signaling-induced fibrosis mediators. Notably, combination of calycosin and NLRP3 signaling blockade improved the intestinal interstitial fibrosis extent. Altogether, this study suggests calycosin can improve intestinal interstitial fibrosis by downregulating NLRP3-IL-33/ST2 signaling, reducing inflammation and decreasing pro-fibrotic factors’ secretion, which provides a new perspective for therapeutic options of IBD.

Random-pattern skin flaps play a vital role in repairing and reconstructing soft tissue defects. Clinical utilization of skin flaps is limited due to distal necrosis. Calycosin (CAL) is an effective ingredient present in Astragalus, and is reported to possess diverse pharmacological properties. Our study aims to assess the potential benefits of CAL on random flap survival and reveal the underlying mechanisms. Our study revealed that CAL promoted the survival area of the skin flap and improved flap blood flow. CAL treatment exerted anti-apoptosis and anti-ferroptosis effects in the flap model. In our mechanistic investigation, we demonstrated that CAL could activate the AMPK-mTOR signaling pathway and increase transcription factor EB (TFEB) nucleus translocation, thereby suppressing ferroptosis and apoptosis as a result of decreased reactive oxygen species (ROS) which was triggered by increasing superoxide dismutase 1(SOD1). In summary, our findings revealed CAL may protect against flap necrosis by inhibiting ferroptosis and apoptosis via AMPK-mTOR signaling pathway modulation.

BackgroundChemotherapy with anthracyclines can cause cardiotoxicity, possibly leading to stopping treatment in some cancer patients. In cardio-oncology research, preventing and minimizing anthracycline-induced cardiotoxicity (AIC) is a hot issue. For the treatment of AIC, calycosin (CA), an isoflavone component in astragali radix (AR), has become a research focus. However, the elaborate mechanisms of calycosin treating AIC remain to be unrevealed.Aim of the studyTo explore the effects of CA on AIC through multiple dimensions concerning network pharmacology, molecular docking, and experimental evaluations.MethodsThe study evaluated calycosin's potential targets and mechanisms for treating AIC using network pharmacology and molecular docking. The candidate genes/targets of CA and AIC were screened using the online-available database. Protein-protein interactions (PPI) between the common targets were constructed using the STRING platform, and the results were then visualized using Cytoscape. Molecular docking was used to evaluate the strength of the binding force between CA and the common targets. The possible pharmacological mechanisms of CA were explained by pathway enrichment and GSEA. Subsequently, the candidate targets were identified in vitro experiments.ResultsNetwork pharmacology effectively discovered the CA's multitarget intervention in AIC, including TNF, ABCC1, TOP2A, ABCB1, and XDH. CA binds to the ATP-binding cassette subfamily B member 1(ABCB1) had the highest binding energy (−7.5 kcal/mol) according to the molecular docking analysis and was selected and visualized for subsequent analysis. In vitro experiments showed that ABCB1 exhibited significant time-curve changes under different doses of doxorubicin (DOX) compared with DMSO control experiments. The anti-AIC pharmacological mechanism of CA were revealed by highlighting the biological processes of oxidative stress (OR) and inflammation.ConclusionsWe employed a practicable bioinformatics method to connect network and molecular docking to determine the calycosin's therapeutic mechanism against AIC and identified some bioinformatics results in in vitro experiments. The results presented show that CA may represent an encouraging treatment for AIC.

Stroke seriously threatens human life and health worldwide, but only very few effective stroke medicines are currently available. Our previous studies have indicated that the phytoestrogen calycosin exerts neuroprotective effects in cerebral ischemia and reperfusion injury rats. Therefore, the objective of this study is to further explore the protective effect of calycosin on inflammatory injury in microglia after oxygen-glucose deprivation/reoxygenation (OGD/R) and to clarify whether its protective effect is related to the HMGB1/TLR4/NF-κB signaling pathway. Here, the OGD/R model of rodent microglia is established in vitro to simulate cerebral ischemia-reperfusion injury. Through the CCK-8 test, ELISA, qRT-PCR, and western blot analysis, we find that the activity of microglia is decreased, the expressions of HMGB1 and TLR4 and the phosphorylation of NF-κB (p-NF-κB) are increased, and the releases of the inflammatory factors IL-6, IL-1β, and TNF-α are increased after OGD/R. Pretreatment with calycosin could ameliorate these states, increase cell viability, reduce HMGB1, TLR4 and p-NF-κB expression, and reduce inflammatory cytokine production. In addition, the effect of calycosin is similar to that of TAK-242 (an inhibitor of TLR4), and the effect of the combined treatment is better than that of the single treatment. The results indicate that calycosin protects microglia from OGD/R injury and reduces the inflammatory response. Calycosin might alleviate cerebral ischemia-reperfusion injury by inhibiting the HMGB1/TLR4/NF-κB pathway.

Background: Ferroptosis, a pathologic state induced by lipid-driven oxidative stress, is associated with the development of human cancers. Calycosin, a natural compound with antioxidant and anti-inflammatory activities, has promising antitumor effects. However, the ferroptosis-related mechanism of calycosin in the treatment of hepatic carcinoma has not been reported. Methods: This study applied network pharmacology and bioinformatic approaches (including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis) to investigate the pharmacologic targets and mechanism of action of calycosin in the treatment of hepatic carcinoma through targeting ferroptosis. By searching online databases including The Cancer Genome Atlas, FerrDb, GeneCards, SwissTargetPrediction, SuperPred, BindingDB, TargetNet, BATMAN-TCM, and Drugbank, we identified 13 ferroptosis-related putative target genes of calycosin against hepatic carcinoma including IL-6, PTGS2, SRC, HRAS, NQO1, NOX4, PGK1, G6PD, GPI, MIF, NOS2, ALDOA, and SQSTM1. Results: Molecular docking analysis revealed that calycosin potentially binded directly with the target proteins IL-6, PTGS2, and SRC. Functional enrichment analysis of these proteins indicated that they were involved in gluconeogenesis and apoptosis through regulation of ERK1, ERK2, and MAPK activities (P

Currently, the effect and molecular mechanism of calycosin, the main active ingredient of Qinshi Simiao San, which can alleviate chronic prostatitis (CP), on CP remain unclear. This study aimed to elucidate the potential mechanism of action of calycosin in CP in a rat CP model. The prostate tissue morphology was evaluated based on hematoxylin–eosin staining. Enzyme-linked immunosorbent assay was conducted to evaluate inflammatory cytokine and immune factor levels (secretory immunoglobulin A [SIgA]; immunoglobulin G [IgG]) in prostate tissues and serum. Additionally, representative biomarkers of oxidative stress, including malondialdehyde, superoxide dismutase, and catalase were detected using detection kits, and reactive oxygen species release was evaluated using immunofluorescence staining. Furthermore, the p38 mitogen-activated protein kinase (p38MAPK)/NF-kappaB (NF-κB) signaling pathway was analyzed by western blotting. The results showed that calycosin substantially ameliorated the pathological damage to prostate tissues of the CP rats. Moreover, calycosin significantly downregulated interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha, IgG, and SIgA levels. Furthermore, we found that calycosin considerably suppressed oxidative stress and inhibited the activation of the p38MAPK/NF-κB signaling pathway in rats with CP. In summary, our findings revealed that calycosin protects against CP in rats by inhibiting the p38MAPK/NF-κB pathway.

Abstract Background High mobility group box 1 protein (HMGB1), a lethal late inflammatory mediator, contributes to the pathogenesis of diverse inflammatory and infectious diseases. Astragaloside IV and calycosin as active ingredients in Astragalus membranaceus, possess potent regulatory ability on HMGB1-induced inflammation, however, the interaction between these two phytochemicals and HMGB1 has not been elucidated yet. Methods To further investigate the interaction of astragaloside IV, calycosin with HMGB1 protein, surface plasma resonance (SPR) and a series of spectroscopic methods, including UV spectra, fluorescence spectroscopy, circular dichroism (CD), were used. Molecular docking was also carried out to predict the atomic level’s binding modes between two components and HMGB1. Results Astragaloside IV and calycosin were found to be able to bind HMGB1 directly and affect the secondary structure and environment of the chromogenic amino acids of HMGB1 to different extents. In silico, astragaloside IV and calycosin showed a synergistic effect by binding to the two independent domains B-box and A-box in HMGB1, respectively, where hydrogen and hydrophobicity bonds were regarded as the crucial forces. Conclusion These findings showed that the interaction of astragaloside IV and calycosin with HMGB1 impaired its proinflammatory cytokines function, providing a new perspective for understanding the mechanism of A. membranaceus in treating aseptic and infectious diseases.

Abstract Background Calycosin may be a potential candidate regarding chemotherapeutic agent, because already some studies against multivarious cancer have been made with this natural compound. Aim This review elucidated a brief overview of previous studies on calycosin potential effects on various cancers and its potential mechanism of action. Methodology Data retrieved by systematic searches of Google Scholar, PubMed, Science Direct, Web of Science, and Scopus by using keywords including calycosin, cancer types, anti‐cancer mechanism, synergistic, and pharmacokinetic and commonly used tools are BioRender, ChemDraw Professional 16.0, and ADMETlab 2.0. Results Based on our review, calycosin is available in nature and effective against around 15 different types of cancer. Generally, the anti‐cancer mechanism of this compound is mediated through a variety of processes, including regulation of apoptotic pathways, cell cycle, angiogenesis and metastasis, oncogenes, enzymatic pathways, and signal transduction process. These study conducted in various study models, including in silico, in vitro, preclinical and clinical models. The molecular framework behind the anti‐cancer effect is targeting some oncogenic and therapeutic proteins and multiple signaling cascades. Therapies based on nano‐formulated calycosin may make excellent nanocarriers for the delivery of this compound to targeted tissue as well as particular organ. This natural compound becomes very effective when combined with other natural compounds and some standard drugs. Moreover, proper use of this compound can reverse resistance to existing anti‐cancer drugs through a variety of strategies. Calycosin showed better pharmacokinetic properties with less toxicity in human bodies. Conclusion Calycosin exhibits excellent potential as a therapeutic drug against several cancer types and should be consumed until standard chemotherapeutics are available in pharma markets.

Objective: Septic patients are especially vulnerable to acute lung injury (ALI). Calycosin (CAL) has various promising pharmacological activities. This paper aims to expound on the role of CAL in mice with sepsis-induced ALI and the associated mechanisms. Methods: Mouse models of sepsis-induced ALI were established using lipopolysaccharide (LPS). Pulmonary histopathological changes were observed by HE staining. Cell apoptosis was assessed by TUNEL staining. Pulmonary edema was evaluated by measuring wet/dry weight. Bronchoalveolar lavage fluid (BALF) was collected to count inflammatory cells. In vitro LPS models were established using MLE-12 cells. miR-375-3p expression was determined by RT-qPCR. Cell viability and apoptosis were assessed by MTT assay and flow cytometry. Levels of inflammatory cytokines were determined by ELISA. The target relationship between miR-375-3p and ROCK2 was analyzed by the dual-luciferase assay. ROCK2 protein level was determined by Western blot. Results: miR-375-3p was weakly-expressed in mice with sepsis-induced ALI, and CAL treatment elevated miR-375-3p expression. CAL treatment mitigated pulmonary tissue damage and edema, decreased apoptosis and inflammatory cells, downregulated levels of pro-inflammatory cytokines, and upregulated levels of anti-inflammatory cytokines in mice with sepsis-induced ALI. CAL treatment increased MLE-12 cell viability and decreased apoptosis and inflammation in MLE-12 cells. Inhibition of miR-375-3p partially abrogated CAL-mediated protective action on MLE-12 cells. miR-375-3p attenuated LPS-induced MLE-12 cell injury by targeting ROCK2. Conclusion: CAL upregulates miR-375-3p to target ROCK2, thus protecting against sepsis-induced ALI in mice.

Rheumatoid arthritis poses a significant threat to the well-being of individuals as an autoimmune disorder. Calycosin is a flavonoid isolated from the root of Astragalus that may be beneficial in treating rheumatoid arthritis. The anti-inflammatory role and mechanism of calycosin were validated in this study through the utilization of collagen-induced arthritis mice models and lipopolysaccharide/interferon gamma stimulated bone marrow-derived macrophages. By applying RNA sequencing technology and the network pharmacological method, it is demonstrated in the findings that the treatment with calycosin leads to a significant alleviation of arthritis symptoms, mitigation of synovial proliferation, impediment of bone and cartilage devastation, and suppression of activated synovial macrophages in collagen-induced arthritis. Mechanistically, activation of JNK and NF-κB pathways is inhibited by calycosin in lipopolysaccharide/interferon gamma stimulated bone marrow-derived macrophages. These results confirm the potential of calycosin, a natural compound, for attenuating rheumatoid arthritis by acting as an anti-inflammatory agent through multiple pathways.

It has been documented that calycosin induces anticancer effects against a wide range of cancer cells, in vitro. However, pharmacokinetic characteristics of calycosin based on its interaction with albumin as a main carrier protein as well as corresponding anticancer mechanisms remain largely unknown. Herein, we inquired into the interaction of calycosin with human serum albumin (HSA) by a wide range collection of spectroscopic and theoretical data. Also, anticancer effects of calycosin on breast cancer cells, MDA-MB 231, were explored by cell viability, lactate dehydrogenase (LDH), caspase-3 activity, and real-time PCR assays. The findings demonstrated that the quenching process resulting from the interaction of calycosin and HSA was largely static in nature and that the calycosin-HSA system spontaneously forms. Cellular and molecular studies revealed that calycosin did not induce a significant cytotoxic effect on the viability of normal human breast epithelial cells, (MCF-10A), even at 200 μM concentration at which it was able to inhibit the proliferation of breast cancer MDA-MB 231 cells up to 50%. Then, it was disclosed that calycosin can disrupt the membrane integrity and induces apoptosis in MDA-MB 231 cells through overexpression of caspase-3 mRNA, down regulation of Bcl-2, and elevation of caspase-3 mRNA and activity mediated by downregulation of mTOR and Akt mRNA. Therefore, it was realized that in breast cancer MDA-MB 231 cells, calycosin is able to suppress cellular proliferation via apoptosis, which is controlled by the Akt/mTOR signaling cascade.

Objective: To observe effect of calycosin （CA） on central nerve cell apoptosis in mice with experimental autoimmune encephalomyelitis（ EAE）, and to explore its possible mechanisms. Methods: Ten mice were randomly selected from 45 C57BL/6 mice as sham operation group, and remaining mice were established EAE model, which were randomly divided into EAE group （10 mice）, CA group （10 mice）, combined group （11 mice） after successful modeling. Mice in combined group were injected with CA （20 mg/kg） intraperitoneally, and injected with cytochrome C （Cyt-C） agonist antimycin A （AA, 100 μg/kg） in tail vein. In CA group, CA （20 mg/kg） was injected intraperitoneally, and an equal volume of PBS was injected into tail vein. Sham operation group and EAE group were intraperitoneally injected with an equal volume of PBS, and tail vein was injected with an equal volume of PBS. Once a day for 14 days. Neurological disorder score was assessed. Content of adenosine triphosphate （ATP） in spinal cord tissue was tested. Ultrastructure of mitochondria was observed by transmission electron microscopy. Tunel staining was used to observe apoptosis of spinal cord nerve cells. Pathological changes of spinal cord were observed by HE staining. Western blot was used to detect expressions of Cyt-C and Apaf-1 proteins in spinal cord tissues. Results: Compared with EAE group, mice neurological disorder score, spinal cord neuronal apoptosis rate, Cyt-C, Apaf-1 protein expressions were decreased, and ATP level of spinal cord tissues was increased in CA group （P<0.05）. Compared with CA group, mice neurological disorder score, spinal cord neuronal apoptosis rate, Cyt-C, Apaf-1 protein expressions were increased, and ATP level of spinal cord tissue was decreased in combined group （P<0.05）. Conclusion: CA can reduce apoptosis of central nerve cells in EAE mice, promote repair of spinal cord injury and reduce mitochondrial damage, which may be achieved by inhibiting Cyt-C/Apaf-1 signaling pathway. [ABSTRACT FROM AUTHOR]

Background: High mobility group box 1 protein (HMGB1), a lethal late inflammatory mediator, contributes to the pathogenesis of diverse inflammatory and infectious diseases. Astragaloside IV and calycosin as active ingredients in Astragalus membranaceus, possess potent regulatory ability on HMGB1-induced inflammation, however, the interaction between these two phytochemicals and HMGB1 has not been elucidated yet. Methods: To further investigate the interaction of astragaloside IV, calycosin with HMGB1 protein, surface plasma resonance (SPR) and a series of spectroscopic methods, including UV spectra, fluorescence spectroscopy, circular dichroism (CD), were used. Molecular docking was also carried out to predict the atomic level's binding modes between two components and HMGB1. Results: Astragaloside IV and calycosin were found to be able to bind HMGB1 directly and affect the secondary structure and environment of the chromogenic amino acids of HMGB1 to different extents. In silico, astragaloside IV and calycosin showed a synergistic effect by binding to the two independent domains B-box and A-box in HMGB1, respectively, where hydrogen and hydrophobicity bonds were regarded as the crucial forces. Conclusion: These findings showed that the interaction of astragaloside IV and calycosin with HMGB1 impaired its proinflammatory cytokines function, providing a new perspective for understanding the mechanism of A. membranaceus in treating aseptic and infectious diseases. [ABSTRACT FROM AUTHOR]

Background: Acute liver failure (ALF) is a serious liver disease that is difficult to treat owing to its unclear pathogenesis. This study aimed to investigate the roles and molecular mechanisms of calycosin (CA) in ALF. Methods: In this study, the roles and mechanism of CA in ALF were explored using an in vitro lipopolysaccharide (LPS)‐induced ALF cell model. Additionally, 3‐(4,5‐dimethyl‐2‐thiazolyl)−2,5‐diphenyltetrazolium bromide assay was used to assess the effect of CA on the activity of LPS‐induced L02 human liver epithelial cells, and flow cytometry was used to detect apoptosis in L02 cells. Expression levels of apoptosis‐related genes, Bax and Bcl‐2, were measured using reverse transcription‐quantitative polymerase chain reaction and Western blot analysis. Expression levels of inflammatory factors in LPS‐induced L02 cells were measured using an enzyme‐linked immunosorbent assay. Additionally, the effect of CA on ALF was inhibited via transfection of a toll‐like receptor 4 (TLR4)‐plasmid to elucidate the relationship between CA and TLR4/nuclear factor (NF)‐κB signaling pathway in ALF. Results: CA had no toxic effects on L02 cells, but enhanced the activity of LPS‐induced L02 cells in a dose‐dependent manner. Apoptosis and inflammatory factor release was increased in ALF, activating the TLR4/NF‐κB signaling pathway. However, CA treatment inhibited the apoptosis and release of inflammatory factors. Further mechanistic studies revealed that the upregulation of TLR4 expression reversed the alleviating effects of CA on inflammation and apoptosis in LPS‐induced L02 cells. Conclusion: CA alleviates inflammatory damage in LPS‐induced L02 cells by inhibiting the TLR4/NF‐κB pathway and may be a promising therapeutic agent for ALF treatment. [ABSTRACT FROM AUTHOR]

Lung cancer is the most common malignant cancer worldwide. Combination therapies are urgently needed to increase patient survival. Calycosin is a phytoestrogen isoflavone that has been reported previously to inhibit tumor cell growth, although its effects on lung cancer remain unclear. The aim of this study was to investigate the effects of calycosin on cell proliferation and apoptosis of gemcitabine-resistant lung cancer cells. Using calycosin to treat human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) and examine the effects on the cells. Cultured human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) were treated with increasing concentrations of calycosin. Cell viability and apoptosis were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, flow cytometry, and TUNEL assays. Western blots were used to measure the expression levels of proliferation-related proteins and cancer stem cell proteins in CL1-0 GEMR cells. The results showed that calycosin treatment inhibited cell proliferation, decreased cell migration ability, and suppressed cancer stem cell properties in CL1-0 GEMR cells. Interestingly, in CL1-0 GEMR cells, calycosin treatment not only increased LDOC1 but also decreased GNL3L/NFκB protein levels and mRNA levels, in concentration-dependent manners. We speculate that calycosin inhibited cell proliferation of the gemcitabine-resistant cell line through regulating the LDOC1/GNL3L/NFκB pathway. [ABSTRACT FROM AUTHOR]

Bone marrow-derived mesenchymal stem cells (MSCs) show antifibrotic activity in various chronic kidney diseases. Here, we aimed to investigate whether Calycosin (CA), a phytoestrogen, could enhance the antifibrotic activity of MSCs in primary tubular epithelial cells (PTECs) induced by TGF-β1 and in a mouse model of unilateral ureteral obstruction (UUO). We found that MSCs treatment significantly inhibited fibrosis, and CA pretreatment enhanced the effects of MSCs on fibrosis in vitro. Consistent with the in vitro studies, MSCs alleviated tubular injury and renal fibrosis in mice after UUO, and CA-pretreated MSCs resulted in more significant improvements in tubular injury and renal fibrosis than MSCs after UUO. Moreover, MSCs treatment significantly inhibited necroptosis by repressing the elevation of MLKL, RIPK1, and RIPK3 in PTECs treated by TGF-β1and in mice after UUO, and CA-pretreated MSCs were superior to MSCs in alleviating necroptosis. MSCs significantly reduced TNF-α and TNFR1 expression induced by TGF-β1 in PTECs and inhibited TGF-β1, TNF-α, and TNFR1 expression induced by UUO in mice. These effects of MSCs were significantly enhanced after CA pretreatment. Therefore, our results suggest that CA pretreatment enhances the antifibrotic activity of MSCs by inhibiting TGF-β1/TNF-α/TNFR1 signaling-induced necroptosis.

Breast cancer is the most commonly diagnosed cancer worldwide. Previously, we reported that calycosin, a typical isoflavone phytoestrogen, triggers apoptosis and is associated with lncRNA HOTAIR in the estrogen receptor (ER)-positive breast cancer MCF-7-cell line. In the present study, we aim to uncover the mechanism of lncRNA HOTAIR in the inhibitory effect induced by calycosin in both ER-positive and ER-negative breast cancer cell lines. Results show that calycosin significantly inhibits proliferation and triggers apoptosis in both ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and SK-BR-3) breast cancer cell lines, accompanied by downregulation of lncRNA HOTAIR expression. Accordingly, knockdown of lncRNA HOTAIR promotes the anti-tumor effect of calycosin, while overexpression of lncRNA HOTAIR attenuates this effect. Meanwhile, the expression levels of HuR and IGF2BP1 are also reduced by calycosin. More importantly, calycosin facilitates the downregulation of HuR and IGF2BP1 caused by decreasing lncRNA HOTAIR expression, and the upregulation of HuR and IGF2BP1 caused by overexpression of lncRNA HOTAIR is weakened by calycosin. These results demonstrate that downregulating HuR and IGF2BP1 by suppressing lncRNA HOTAIR results in inhibited growth of breast cancer cells by calycosin. In addition, the binding of HuR and IGF2BP1 to lncRNA HOTAIR is detected by RIP assay, implying an interaction between these two proteins and lncRNA HOTAIR. Together, lncRNA HOTAIR may play a carcinogenic role in breast cancer development and has the potential to be a novel therapeutic target for breast cancer in the future, especially in isoflavone phytoestrogen therapy.

Arsenic (As), an environmental pollutant, is a highly poisonous metalloid. Accumulated evidence has shown the association between As exposure and elevated risk of the development of coronary heart disease (CHD). Calycosin, a beneficial flavonoid, has demonstrated cardioprotective activities, including those against CHD, in preclinical studies. The anti-As-related CHD activity and mechanism of calycosin have not yet been elucidated. Here, we aimed to determine the core biotargets and molecular mechanisms of calycosin against As-interrelated CHD via integrated bioinformatic analysis, including network pharmacology and molecular docking. The network pharmacology data demonstrated 41 intersection genes of calycosin against As-related CHD, prior to the identification of 15 core targets. Additional in silico investigation indicated that mitogen-activated protein kinase-3 (MAPK3), epidermal growth factor receptor (EGFR), and interleukin-6 (IL6) were the primary pharmacological targets of calycosin for the treatment of As-related CHD. In addition, the therapeutic effects can be realized via cardioprotection-associated signaling pathways for reducing As-induced myocardial toxicity and impairment and boosting CHD functional reparation. In summary, calycosin mediates potent pharmacological effects in As-related CHD therapy functioning via multiple targets and multiple pathways. The results may eventually aid in promoting future clinical application after experimental verification.

Acute myocardial infarction (AMI) is one of the most common ischemic heart diseases. However, lack of sufficient drug concentration (in the ischemic heart) is the major factor of treatment failure. It is urgent for researchers to engineer novel drug delivery systems to enhance the targeted delivery of cardioprotective agents. The aim of the present study was to investigate the anti-AMI ability of calycosin (CAL) and tanshinone (TAN) co-loaded; mitochondrion-targeted tetrapeptide (MTP) and cyclic arginyl-glycyl-aspartic acid (RGD) peptide co-modified nano-system.: We prepared CAL and TAN combined lipid-polymer hybrid nano-system, and RGD was modified to the system to achieve RGD-CAL/TAN NS. MTP-131 was conjugated with PEG and modified onto the nanoparticles to achieve dual ligands co-modified MTP/RGD-CAL/TAN NS. The physicochemical properties of nano-systems were characterized. The AMI therapy ability of the systems was investigated in AMI rats’ model. The size of MTP/RGD-CAL/TAN NS was 170.2 ± 5.6 nm, with a surface charge of −18.9 ± 1.9 mV. The area under the curve (AUC) and blood circulation half-life (T1/2) of MTP/RGD-CAL/TAN NS was 178.86 ± 6.62 μg·min/mL and 0.47 h, respectively. MTP/RGD-CAL/TAN NS exhibited the most significant infarct size reduction effect of 22.9%. MTP/RGD-CAL/TAN NS exhibited the highest heart accumulation and best infarct size reduction effect, which could be used as a promising system for efficient treatment of cardiovascular diseases.

Abstract Background Estrogen receptor β (ERβ) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERβ. Here, we explore the effect of overexpressed ERβ on human HSCs and the role of ERβ in pharmacological action of calycosin. Methods LX-2 cells were transfected with lentivirus to overexpress ERβ. In the presence or absence of overexpressed ERβ, the effects of ERβ and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-β1-induced LX-2 cells and the role of ERβ in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERβ or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERβ on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot. Results ERβ overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERβ or calycosin alone inhibited the TGF-β1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERβ and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERβ overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed. Conclusions ERβ mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect. Graphical Abstract

Abstract Background Acute liver failure (ALF) is a serious liver disease that is difficult to treat owing to its unclear pathogenesis. This study aimed to investigate the roles and molecular mechanisms of calycosin (CA) in ALF. Methods In this study, the roles and mechanism of CA in ALF were explored using an in vitro lipopolysaccharide (LPS)‐induced ALF cell model. Additionally, 3‐(4,5‐dimethyl‐2‐thiazolyl)−2,5‐diphenyltetrazolium bromide assay was used to assess the effect of CA on the activity of LPS‐induced L02 human liver epithelial cells, and flow cytometry was used to detect apoptosis in L02 cells. Expression levels of apoptosis‐related genes, Bax and Bcl‐2, were measured using reverse transcription‐quantitative polymerase chain reaction and Western blot analysis. Expression levels of inflammatory factors in LPS‐induced L02 cells were measured using an enzyme‐linked immunosorbent assay. Additionally, the effect of CA on ALF was inhibited via transfection of a toll‐like receptor 4 (TLR4)‐plasmid to elucidate the relationship between CA and TLR4/nuclear factor (NF)‐κB signaling pathway in ALF. Results CA had no toxic effects on L02 cells, but enhanced the activity of LPS‐induced L02 cells in a dose‐dependent manner. Apoptosis and inflammatory factor release was increased in ALF, activating the TLR4/NF‐κB signaling pathway. However, CA treatment inhibited the apoptosis and release of inflammatory factors. Further mechanistic studies revealed that the upregulation of TLR4 expression reversed the alleviating effects of CA on inflammation and apoptosis in LPS‐induced L02 cells. Conclusion CA alleviates inflammatory damage in LPS‐induced L02 cells by inhibiting the TLR4/NF‐κB pathway and may be a promising therapeutic agent for ALF treatment.

Background: Colorectal cancer (CRC) is a type of malignant gastroenteric tumors associated with a high mortality rate worldwide. Calycosin, a natural phytoestrogen, possesses potent anti-cancer properties. We structurally modified calycosin to improve its physicochemical properties, and generated a novel small molecule termed CA028. Methods: By using network pharmacology, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis and molecular docking, we aimed to predict and disclose the biological functions and mechanism of CA028 in the treatment of CRC through bioinformatic analyses. Results: By searching the online Swiss Target Prediction and TargetNet databases, we identified 150 genes shared by CA028 and CRC. Using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape software, we identified 14 hub-functional genes, namely the FYN proto-oncogene, a Src family tyrosine kinase (FYN), mitogen-activated protein kinase 1 (MAPK1), MAPK8, MAPK14, Rac family small GTPase 1 (RAC1), epidermal growth factor receptor (EGFR), protein tyrosine kinase 2 (PTK2), sphingosine-1-phosphate receptor 1 (S1PR1), S1PR2, Janus kinase 1 (JAK1), JAK2, the RELA proto-oncogene NF-κB subunit (RELA), bradykinin receptor B1 (BDKRB1), and BDKRB2. Additionally, biological docking analysis using the Autodock Vina software revealed that FYN and MAPK1 were the main pharmacological proteins of CA028 against CRC. The gene ontology analysis using R-language packages further revealed the anti-CRC functions of CA028, including biological processes, cell components, and molecular pathways. Conclusion: CA028 exhibits effective pharmacological activity against CRC by suppressing the proliferation of CRC cells and improving the tumor microenvironment. Importantly, certain predicted genes (e.g., FYN and MAPK1) may be the pharmacological targets of CA028 in the treatment of CRC.

Calycosin, an O-methylated isoflavone, has been widely reported to induce anticancer activity in different cancer cells in vitro. Nonetheless, the associated mechanism of calycosin in glioblastoma multiforme cells (U87) still remains unknown. To explore the anticancer effects, the apoptotic mechanism of calycosin via Wnt/GSK3β/β-catenin signaling was explored in U87 cells. Different assays including: cytotoxicity, free radical determination, SOD and CAT activity, GSH content, qPCR, mitochondrial membrane potential, caspase activity, and western blotting assays were performed. It was shown that calycosin mitigated cell viability in U87 cells, whereas it showed no apparent effect on BV2 microglial cells. Calycosin triggered apoptosis via upregulating the mitochondria-associated caspase pathway in U87 cells. Calycosin induced the reduction of the mitochondrial membrane potential, overexpression of Bax, downexpression of Bcl-2, and activation of caspase-9 and caspase-3. Calycosin-stimulated apoptosis was associated with the upregulation of free radical scavenging through the modulation of antioxidant enzymes, such as SOD and CAT as well as the level of GSH. The apoptotic activity of calycosin was mediated by suppression of pGSK-3βser9, β-catenin, and c-Myc at protein level. The present study suggested that calycosin triggers U87 cell death through an antioxidant effect mediated by Wnt/GSK3β/β-catenin signaling pathway.

Calycosin is an isoflavone isolated from the roots of Astragalus membranaceus. Due to its antioxidant and anti-inflammatory activities, it has been extensively used in traditional Chinese medicine formulations. Herein, we have evaluated the mechanism of action of calycosin using high glucose induced HRCEC cells as a model. The observed effects of calycosin include alleviation of oxidative stress induced by high glucose through promotion of SOD, GSH-Px and inhibition of ROS expression. Moreover, calycosin relieved HG-induced inflammation and pyroptosis by upregulating IL-1β, IL-18 and downregulating NLRP3, ASC, and cleaving caspase-1/procaspase-1. Calycosin also inhibited HMGB1/NF-κB pathway activation by promoting p-P65/P65, p-IκBα, HMGB1 and decreasing IκBα. All of these results support the notion that calycosin alleviates oxidative stress and pyroptosis of HRCEC cells induced by high glucose by inhibiting activation of the HMGB1/ NF-κB pathway. [ABSTRACT FROM AUTHOR]