Effects Of Astragaloside IV Research Articles

The Role of Ca2+/PI3K/Akt/eNOS/NO Pathway in Astragaloside IV-Induced Inhibition of Endothelial Inflammation Triggered by Angiotensin II.

Inflammation induced by angiotensin II (Ang II) is a key event in the progression of numerous cardiovascular diseases. Astragaloside IV (AS-IV), a glycoside extracted from Astragalus membranaceus Bunge, has been shown to inhibit Ang II-induced inflammatory responses in vivo. However, the mechanisms underlying the beneficial effects are still unclear. This study investigated whether AS-IV attenuates endothelial inflammation induced by Ang II via the activation of endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway. Human umbilical vein endothelial cells (HUVECs) were cultured in the presence of AS-IV with or without the specific inhibitor of NOS or Ca2+- and phosphatidylinositol 3-kinase (PI3K)/Akt-dependent cascade prior to Ang II exposure. Incubation of HUVECs with AS-IV enhanced NO production and eNOSser1177 phosphorylation. These responses were abrogated by the inhibition of NOS or Ca2+- and PI3K/Akt-dependent pathway. In addition, preincubation of HUVECs with AS-IV inhibited Ang II-induced cytokine and chemokine production, adhesion molecule expression, monocyte adhesion, and nuclear factor kappa B (NF-κB) activation as evidenced by the attenuation of inhibitor of kappa B alpha phosphorylation and subsequent NF-κB DNA binding. These effects of AS-IV were abolished by the suppression of NOS or Ca2+- and PI3K/Akt-dependent cascade. Our findings indicate that AS-IV attenuates inflammatory responses triggered by Ang II possibly via the activation of Ca2+/PI3K/Akt/eNOS/NO pathway in endothelial cells.

Astragaloside IV Inhibits Rotenone-Induced α-syn Presentation and the CD4 T-Cell Immune Response.

The increased α-synuclein (α-syn)-dependent activation of CD4 T cells leads to the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN) in Parkinson's disease (PD). Astragaloside IV (AS-IV) protects DA neurons against neuroinflammation. The effects of AS-IV on CD4 T-cell-mediated immune responses in PD remain unknown. Rotenone (ROT) injected unilaterally into the substantia nigra pars compacta (SNc) of rats induced PD. AS-IV (20mg/kg) was intraperitoneally injected once a day for 14days. The limb hanging test and rotarod test were performed to evaluate the alteration of behavior at 4 and 6weeks. Total gastrointestinal transit tests were performed at 4weeks. Western blotting was used to detect the expression of proinflammatory cytokine proteins. Immunofluorescence staining was conducted to test the expression and localization of major histocompatibility complex class II (MHCII), cleaved caspase-1 and α-syn in astrocytes. Flow cytometry analysis, immunohistochemistry and immunofluorescence staining were used to measure the expression of CD4 T-cell subsets in the SN. The application of AS-IV protected against the loss of DA neurons and behavioral deficits in ROT-induced PD rat models. AS-IV administration inhibited the aggregation of α-syn in DA neurons and the expression of proinflammatory cytokines such as TNF-α, IL-18, IL-6 and IL-1β. AS-IV decreased the activation of CD4 T cells and three CD4 T-cell subsets: Tfh, Treg and Th1. AS-IV interrupted the ROT-induced interaction between astrocytes and CD4 T cells and the colocalization of MHCII and α-syn in astrocytes. AS-IV inhibited the expression of α-syn in astrocytes and the colocalization of α-syn and cleaved caspase-1 in astrocytes. AS-IV prevents the loss of DA neurons in PD by inhibiting the activation of α-syn-specific CD4 T cells, which is regulated by MHCII-mediated antigen presentation in astrocytes.

Astragaloside-IV induces the differentiation of bone marrow mesenchymal stem cells into osteoblasts through NMUR2-mediated Wnt/β-catenin pathway.

Aim: Given that astragaloside-IV (As-IV) has the ability to promote osteogenic differentiation, its mechanism is worthy of exploration. Methods: The effect of As-IV on rat tibial defects was examined by histopathological staining and MiR-CT scan. The alkaline phosphatase (ALP) content, osteogenic differentiation-related gene expressions, and mineralized nodule formation in bone marrow mesenchymal stem cells (BMSCs) were detected. Results: As-IV repaired tibial defects of rats. As-IV or neuromedin receptor 2 (NMUR2) overexpression elevated ALP content, mineralized nodules, osteogenic differentiation-related genes, β-catenin and NMUR2 levels, the effects of which were reversed by NMUR2 silencing or Wnt/β-catenin pathway inhibitors. Conclusion: As-IV regulates the Wnt/β-catenin pathway through NMUR2 to promote the repair of tibial defects in rats and the differentiation of BMSCs into osteoblasts.

Astragaloside IV alleviates sepsis-induced muscle atrophy by inhibiting the TGF-β1/Smad signaling pathway.

Muscle atrophy occurs in patients with sepsis and increases mortality and disability. Remission of muscle atrophy may improve the quality of life in patients with sepsis. Astragaloside IV (ASIV) has been shown to have excellent anti-inflammatory and anti-fibrotic effects and to reduce organ damage caused by sepsis. However, the effect of ASIV on sepsis-induced muscle atrophy has not been reported. Therefore, this study explored the pharmacological effects and mechanisms of ASIV in sepsis-induced muscle atrophy. Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis and lipopolysaccharide (LPS)-stimulated C2C12 myotubes. After administration of ASIV, the body weight, tibialis anterior (TA) and gastrocnemius muscle weight and fiber cross-sectional area of the mice were measured. The diameter of myotubes was observed by immunofluorescence staining. ELISA was used to assess inflammatory factors in plasma and cell culture supernatants. RT-PCR and Western blotting were used to detect the expression of MuRF1, Atrogin-1 and TGF-β1/Smad signaling pathway components in TA and C2C12 myotubes. Our study found that ASIV reduced serum inflammatory factors and improved survival in septic mice. ASIV alleviated muscle mass reduction, myofiber cross-sectional area reduction, and C2C12 myotube atrophy by inhibiting the expression of the E3 ubiquitin ligases MuRF1 and atrogin-1. In addition, we observed that ASIV inhibited TGF-β1/Smad signaling. Inhibition of the TGF-β1/Smad signaling pathway partly blocked the anti-muscle atrophy effect of ASIV. ASIV can alleviate sepsis-induced muscle atrophy, which may be related to the inhibition of the TGF-β1/Smad signaling pathway.

Astragaloside IV ameliorates spinal cord injury through controlling ferroptosis in H2O2-damaged PC12 cells in vitro.

Spinal cord injury (SCI) is associated with significant paralysis and high fatality. Recent research has revealed that ferroptosis participates in the pathogenesis of SCI. Astragaloside IV (AS-IV), the main active ingredient of the plant Astragalus membranaceus, has been reported to promote motor function recovery in rats with SCI. This study explored the effects of AS-IV in H2O2-treated PC12 pheochromocytoma cells. The optimal concentration and duration of AS-IV treatment in PC12 cells was assessed using the cell counting kit 8 (CCK-8) assay. Subsequently, the SCI cell model was established in PC12 cells using H2O2. The effects of AS-IV, FIN56, and transcription factor EB (TFEB) small interfering (si)RNA on cell viability and apoptosis in the SCI model were determined using the CCK-8 assay and flow cytometry, respectively. Caspase‑3 and lactate dehydrogenase (LDH) levels were measured by colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. Cellular reactive oxygen species (ROS) were detected by flow cytometry combined with dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The cellular ultrastructure was analyzed by transmission electron microscopy (TEM). The ferroptosis pathway-related proteins were confirmed using Western blot analysis. TFEB expression was confirmed by Western blot and immunofluorescence. The optimal concentration and duration of AS-IV treatment in PC12 cells was determined to be 1.0 µM and 48 h, respectively. AS-IV markedly accelerated proliferation, suppressed apoptosis, and reduced ROS and LDH accumulation. Furthermore, AS-IV enhanced TFEB expression in H2O2-damaged PC12 cells. The effects of AS-IV on SCI were inhibited by si-TFEB, and this inhibition was further reinforced by the addition of FIN56. The results of this investigation using the SCI cell model suggested that AS-IV alleviated SCI by promoting TFEB expression and subsequently mediating ferroptosis. This may represent a potential clinical treatment for SCI.

Astragaloside Ⅳ alleviates ulcerative colitis by regulating the balance of Th17/Treg cells

BackgroundRestoring immune homeostasis by targeting the Th17/Treg response is a potentially valuable therapeutic strategy for ulcerative colitis (UC). Astragaloside IV (AS-Ⅳ) is a phytochemical naturally occurring in Astragalus membranaceus that has good anti-inflammatory, anti-oxidant and anti-stress properties. However, the effects of AS-IV on the homeostasis of Th17/Treg cells in colitis mice remains unknown. PurposeTo investigate the protective effects and potential immunomodulatory mechanisms of AS-IV on UC. MethodsThis study was constructed for DSS-induced acute colitis and recurrent colitis, with AS-IV administered prophylactically and therapeutically, respectively. The balance of Th17/Treg cells was analyzed by flow cytometry, their specific nuclear transcription factors were detected by RT-PCR as well as their secreted inflammatory cytokines were detected by ELISA and RT-PCR. Notch signaling-related proteins were detected by RT-PCR and Western blotting. Oxidative stress indicators were measured by biochemical technology. ResultsIn this study, AS-IV treatment not only effectively prevented and alleviated the clinical symptoms of DSS-induced colitis mice, including weight loss, DAI soaring, colon length shortening and colon weight gain, but also significantly improved ulcer formation, inflammatory cell infiltration and index, and regulated the expression of inflammatory cytokines in colon tissues. Importantly, the efficacy of high-dose AS-IV (100 mg/kg/day) in mice with recurrent colitis in this study was comparable to that of 5-ASA. AS-IV early administration was able to reshape the homeostasis of Th17/Treg cells in mice with acute colitis; meanwhile, AS-IV inhibited Th17 cell responses and promoted Treg cell responses in mice with recurrent colitis. Moreover, AS-IV not only inhibited the activation of Notch signaling pathway in colitis mice, but also prevented and ameliorated DSS-induced oxidative stress injury. ConclusionIn conclusion, AS-IV effectively prevented and alleviated UC by reshaping Th17/Treg cell homeostasis and anti-oxidative stress.

Astragaloside IV alleviates oxidative stress-related damage via inhibiting NLRP3 inflammasome in a MAPK signaling dependent pathway in human lens epithelial cells.

Oxidative stress alters cellular microenvironment, facilitating cell apoptosis and inflammatory response, and oxidation of lens constituents may ultimately result in cataracts. Astragaloside IV (AS-IV) exhibits a variety of pharmacological activities, such as antioxidative and anti-inflammatory activity via regulating various signaling pathways. However, the effect of AS-IV on lens epithelial cells and its potential therapeutic role in cataracts remained to be investigated. In this study, AS-IV prevented H2 O2 -induced injury and inflammatory response in human lens epithelial cell line HLE-B3 through inhibiting NLRP3 inflammasome in a mitogen-activated protein kinase-dependent pathway, providing a potential novel therapeutic strategy for cataracts.

Preliminary study of astragaloside IV on oxaliplatin-induced peripheral neurotoxicity in rats

Objective: As an effective component of Astragalus membranaceus, astragaloside IV (AS-IV) has a history of thousands of years in China. Many evidences have indicated that AS-IV has a potential neuroprotective effect. In this study, we aimed to preliminarily study the effects of AS-IV on oxaliplatin-induced peripheral neurotoxicity (OIPN) in rats. Methods: Intraperitoneal injection of oxaliplatin for 4 weeks (4 mg/kg, twice a week) was used to establish peripheral neurotoxicity in rats. 40 Sprague Dawley rats were randomly divided into five groups, eight rats in each group, including control group, model group, and three AS-IV treated groups. Different doses of AS-IV (5 mg/kg, 10 mg/kg, and 20 mg/kg, daily) were orally administrated to OIPN rats once a day for 4 weeks at beginning of oxaliplatin administration. Behaviors and histologic evaluation of sciatic nerve and dorsal root ganglia (DRG) were performed to assess the changes of peripheral neurotoxicity through mechanical allodynia and cold allodynia, immunofluorescence, H&E staining, myelin staining, and Nissl staining. Results: AS-IV treatments were able to significantly reduce oxaliplatin induced mechanical and cold allodynia. Moreover, AS-IV administration could increase the levels of NGF, but decrease the levels of TNF-α and IL-6 in oxaliplatin induced rats. AS-IV suppressed the activation of Iba-1 in anterior horn of spinal cord of OIPN rats. The myelin sheath degenerations in the sciatic nerve of OIPN rats were repaired after AS-IV administration. Through observation of sciatic nerves and DRG, AS-IV treatments improved the oxaliplatin induced pathologic injuries in a dose-dependent. Conclusion: AS-IV administration was able to attenuate the oxaliplatin-induced peripheral neurotoxicity in rats.

Astragaloside IV promotes the angiogenic capacity of adipose-derived mesenchymal stem cells in a hindlimb ischemia model by FAK phosphorylation via CXCR2

BackgroundTherapeutic angiogenesis by transplantation of autologous/allogeneic adipose stem cells (ADSCs) is a potential method for the treatment of critical limb ischemia (CLI). However, the therapeutic efficiency is limited by poor viability, adhesion, migration and differentiation after cell transplantation into the target area. Astragaloside IV (AS-IV), one of the main active components of Astragalus, has been widely used in the treatment of ischemic diseases and can promote cell proliferation and angiogenesis. However, there is no report on the effect of AS-IV on ADSCs and its effect on hindlimb ischemia through cell transplantation. PurposeThe purpose of this study was to elucidate that AS-IV pretreatment enhances the therapeutic effect of ADSC on critical limb ischemia, and to characterize the underlying molecular mechanisms. MethodsADSCs were obtained and pretreated with the different concentration of AS-IV. In vitro, we analyzed the influence of AS-IV on ADSC proliferation, migration, angiogenesis and recruitment of human umbilical vein endothelial cells (HUVECs) and analyzed the relevant molecular mechanism. In vivo, we injected drug-pretreated ADSCs into a Matrigel or hindlimb ischemia model and evaluated the therapeutic effect by the laser Doppler perfusion index, immunofluorescence, and histopathology. ResultsIn vitro experiments showed that AS-IV improved ADSC migration, angiogenesis and endothelial recruitment. The molecular mechanism may be related to the upregulation of CXC receptor 2 (CXCR2) to promote the phosphorylation of focal adhesion kinase (FAK). In vivo, Matrigel plug assay showed that ADSCs pretreated with AS-IV have stronger angiogenic potential. The laser Doppler perfusion index of the hindlimbs of mice in the ADSC/AS-IV group was significantly higher than the laser Doppler perfusion index of the hindlimbs of mice of the ADSC group and the control group, and the microvessel density was significantly increased. ConclusionOur results demonstrate that AS-IV pretreatment of ADSC improves their therapeutic efficacy in ameliorating severe limb exclusion symptomology through CXCR2 induced FAK phosphorylation, which will bring new insights into the treatment of severe limb ischemia.

Mechanism of astragaloside Ⅳ alleviating PC12 cell injury by activating PI3K/AKT signaling pathway: based on network pharmacology and in vitro experiments

In this study, the molecular mechanism of astragaloside Ⅳ(AS-Ⅳ) in the treatment of Parkinson's disease(PD) was explored based on network pharmacology, and the potential value of AS-Ⅳ in alleviating neuronal injury in PD by activating the PI3 K/AKT signaling pathway was verified through molecular docking and in vitro experiments. Such databases as SwissTargetPrediction, BTMAN-TAM, and GeneCards were used to predict the targets of AS-Ⅳ for the treatment of PD. The Search Tool for the Retrieval of Interacting Genes/Proteins(STRING) was employed to analyze protein-protein interaction(PPI) and construct a PPI network, and the Database for Annotation, Visualization and Integrated Discovery(DAVID) was used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Based on the results of GO enrichment analysis and KEGG pathway analysis, the PI3 K/AKT signaling pathway was selected for further molecular docking and in vitro experiments in this study. The in vitro cell model of PD was established by MPP~+. The cell viability was measured by MTT assay and effect of AS-Ⅳ on the expression of the PI3 K/AKT signaling pathway-related genes and proteins by real-time polymerase chain reaction(RT-PCR) and Western blot. Network pharmacology revealed totally 122 targets of AS-Ⅳ for the treatment of PD, and GO enrichment analysis yielded 504 GO terms, most of which were biological processes and molecular functions. Totally 20 related signaling pathways were screened out by KEGG pathway analysis, including neuroactive ligand-receptor interaction, PI3 K/AKT signaling pathway, GABAergic synapse, and calcium signaling pathway. Molecular docking demonstrated high affinity of AS-Ⅳ to serine/threonine-protein kinases(AKT1, AKT2), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma(PIK3 CG), and phosphoinositide-3-kinase, catalytic, alpha polypeptide(PIK3 CA) on the PI3 K/AKT signaling pathway. In vitro experiments showed that AS-Ⅳ could effectively inhibit the decrease of the viability of PC12 induced by MPP~+ and up-regulate the mRNA expression levels of AKT1 and PI3 K as well as the phosphorylation levels of AKT and PI3 K. As an active component of Astragali Radix, AS-Ⅳ acts on PD through multiple targets and pathways. Furthermore, it inhibits neuronal apoptosis and protects neurons by activating the PI3 K/AKT signaling pathway, thereby providing reliable theoretical and experimental supports for the treatment of PD with AS-Ⅳ.

Protective effect of astragaloside IV on cadmium-induced spermatogenesis microenvironment damage in rats

ABSTRACT The previous study using Sertoli cells cultured in vitro has shown that the protective effects of astragaloside IV (AsIV) on cadmium (Cd)-induced damage to Sertoli cells and its membrane proteins. Yet, it is not known if AsIV has an equivalent effect on Cd-induced damage to the spermatogenesis microenvironment in rats. Using an in vivo model, Cd-induced damage to the spermatogenesis microenvironment and the protective effects of AsIV were studied. Eighteen male Sprague Dawley (SD) rats were randomly divided into three groups (n = 6/group): Cd group, Cd&AsIV group, and control group. Cd was administered to the rats in the Cd group via i.p. at 1 mg/kg body weight once daily, Cd and AsIV was administered to the rats in the Cd&AsIV group via i.p. at 1 mg/kg body weight and 10 mg/kg body weight respectively once daily, and the same volume of saline was administered to the rats in control group via i.p. once daily. The rats in the three groups were injected continuously for 5 days. Vesicular formation in the seminiferous tubules was observed in the Cd treatment group. The average optical density of claudin-11, zonal occludin-1 (ZO-1), and connexin 43 (Cx43) decreased significantly in the Cd treatment group. The ultrastructural damage of the Sertoli cells and tight junctions were also observed by electron microscopy. AsIV treatment rescued the morphologic changes of the seminiferous tubules of the testis and the ultrastructural damage of the Sertoli cells and tight junctions. The average optical density of claudin-11, ZO-1, and Cx43 also increased significantly after AsIV treatment. Cd damages the spermatogenesis microenvironment in rats, which can be rescued by AsIV treatment. These results illustrate that AsIV may also have a protective effect on Cd-induced damage to the spermatogenesis microenvironment in rats. Abbreviations: AsIV: astragaloside IV; Cd: cadmium; SD: Sprague Dawley; ZO-1: zonal occludin-1; Cx43: connexin 43; BTB: blood-testis barrier; MAPKs: mitogen-activated protein kinases; OSP: oligodendrocyte-specific protein; Cxs: connexins; GJIC: gap junctional intercellular communication; ROS: reactive oxygenspecies; MDA: malondialdehyde; TGF: tumor growth factor; PBS: phosphate buffersaline; BSA: bovine serum albumin

Astragaloside Ⅳ's Therapeutic Effect on Myocardial Infarction via Affecting Autophagy and the Mechanism Study

The purpose of this study was to investigate the protective effect of astragaloside Ⅳ (AS-Ⅳ) on neonatal rats' hypoxic/reoxygenated (H/R) injured myocardial cells and to explore its underlying mechanism. Cardiac cells were extracted from newborn rats and divided into control, H/R, H/R-low AS-Ⅳ (0.1 μmol/L AS-Ⅳ), H/R-medium AS-Ⅳ (1 μmol/L AS-Ⅳ), H/R-high AS-Ⅳ (10 μmol/L AS-Ⅳ) and H/R-high AS-Ⅳ-AKT (10 μmol/L AS-Ⅳ+5 μmol/L AKT) groups. After 48 h of treatment, the contents of LC3-Ⅱ, p62, AKT, pAKT, rapamycin (mTOR) mammalian targets and uncoordinated 51-like kinase 1 (ULK1) in cardiac myocytes were compared. Immunofluorescence staining was used to detect the expression of P62 in myocardium autophagosome. AS-Ⅳ improved the proliferative activity of cardio AS-Ⅳ improved the proliferative activity of cardiomyocytes in H/R injury in a dose-dependent manner and inhibited the level of cell autophagy. However, when AKT inhibitors were added, the effect of AS-Ⅳ was partially inhibited ( P<0.05). Gene and protein expression showed that AS-Ⅳ had no significant effect on the expression of AKT and mTOR genes ( P>0.05), but could significantly promote the phosphorylation of AKT and mTOR ( P<0.05). Immunofluorescence staining results showed that high concentrations of the AS - Ⅳ can reverse H/R injury induced the expression of autophagy body P62. AS-Ⅳ showed protection effect on H/R injured myocardial cells. The possible mechanism is by reducing the autophagy level via activating the mTOR signal in the PI3K/AKT pathway, thereby preventing H/R damage in neonatal rat cardiomyocytes.

Astragaloside IV Improves High-Fat Diet-Induced Hepatic Steatosis in Nonalcoholic Fatty Liver Disease Rats by Regulating Inflammatory Factors Level via TLR4/NF-κB Signaling Pathway.

Objective: Astragaloside IV (AS-IV) is the primary bioactive component purified from Astragalus membranaceus which is one of the traditional Chinese medicines. Research studies found that AS-IV has significant pharmacological effects on focal cerebral ischemia/reperfusion, cardiovascular disease, pulmonary disease, liver cirrhosis, and diabetic nephropathy, but little is known about the effects of AS-IV on nonalcoholic fatty liver disease (NAFLD). In this study, we investigated whether AS-IV has beneficial effects on NAFLD in rats and its potential mechanisms. Methods: Male SD rats were fed with high-fat diet (HFD) for 12 weeks to establish NAFLD rat model, and then, the rats were divided into five groups. The control group rats were fed with normal diet for 12 weeks and then were given normal saline (1.0 ml kg−1 day−1) by intragastric administration for 4 weeks. The model group rats were fed with HFD for 12 weeks and then were given normal saline (1.0 ml kg−1 day−1) by intragastric administration for 4 weeks. The AS-IV-L, AS-IV-M, and AS-IV-H groups were treated with 20, 40, and 80 mg kg−1 day−1 of AS-IV by intragastric administration for 4 weeks and given HFD diet. Then, we detected serum transaminase (ALT, AST), blood lipid (TG, TC), inflammatory cytokines (IL-6, IL-8 and TNF-α), liver histology(NAFLD activity score), TLR4/MyD88 signaling pathway in liver tissue. Results: We found AS-IV significantly reduced serum levels of AST, ALT, TG, TNF-α, IL-6, and IL-8 in NAFLD rats and downregulate the expression of TLR4 mRNA, MyD88 mRNA, NF-κB mRNA, and proteins in liver tissue. Moreover, AS-IV could significantly reduce the NAFLD activity score of NAFLD rat liver. Conclusion: In this study, we demonstrated that AS-IV have a protective effect on NAFLD by inhibiting TNF-α, IL-6 and IL-8 levels and down-regulating TLR4, MyD88 and NF-κB expression in rat liver tissues.

Influence of astragaloside IV on pharmacokinetics of triptolide in rats and its potential mechanism

Context It is common to combine two or more drugs in clinics in China. Triptolide (TP) has been used primarily for the treatment of inflammatory and autoimmune diseases. Astragaloside IV (AS-IV) has been applied with many other drugs, due to its various pharmacological effects. AS-IV and TP can be used together for the treatment of diseases in clinics in China. Objective This study investigates the effects of astragaloside IV (AS-IV) on the pharmacokinetics of TP in rats and its potential mechanism. Materials and methods The pharmacokinetics of orally administered triptolide (2 mg/kg) with or without AS-IV pre-treatment (100 mg/kg/day for 7 d) were investigated. Additionally, the effects of AS-IV on the transport of triptolide were investigated using the Caco-2 cell transwell model. Results The results indicated that when the rats were pre-treated with AS-IV, the C max of triptolide decreased from 418.78 ± 29.36 to 351.31 ± 38.88 ng/mL, and the AUC0-t decreased from 358.83 ± 19.56 to 252.23 ± 15.75 μg/h/L. The Caco-2 cell transwell experiments indicated that AS-IV could increase the efflux ratio of TP from 2.37 to 2.91 through inducing the activity of P-gp. Discussion and conclusions In conclusion, AS-IV could decrease the system exposure of triptolide when they are co-administered, and it might work through decreasing the absorption of triptolide by inducing the activity of P-gp.

Effects of astragaloside IV on activity and proliferative function of endothelial progenitor cells

Objective The objective is to probe into the effects of astragaloside Ⅳ (AS-Ⅳ) on activity and proliferative function of endothelial progenitor cells (EPCs), which lays a foundation for further study on the effects of AS-Ⅳ on vascular neovascularization mediated by endothelial progenitor cells. Methods The mononuclear cells were isolated by the density gradient centrifugation in umbilical cord blood of full-term healthy infants, and EPCs were obtained by subculture and cell identification when the cells presented spindle shapes. The obtained EPCs were randomly divided into the experimental group and the control group. In the experimental group, EPCs were cultured by AS-Ⅳ with different concentration gradients (25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L), while in the control group, they were treated with the same amount of phosphate buffer saline (PBS) solutions. The effects of AS-Ⅳ on the proliferation of endothelial progenitor cells was studied by cell counting kit-8 (CCK-8) cell proliferation experiment, and the activity rate of EPCs cells was measured at the optimum concentration of EPCs proliferation. Results EPCs were successfully obtained after confirming nuclear staining test of CD31 antibody and 4′, 6-diamidino-2-phenylindole (DAPI). Further study showed that AS-Ⅳ can promote the proliferation of EPCs, and its optimal concentration of EPCs proliferation is 100 mg/L. Compared with the normal control group, the activity rate of endothelial progenitor cells after intervention of AS-Ⅳ was 98.7%, higher than 98.12% in the control group, with significant difference (χ2=49.59, P<0.01). Conclusions AS-Ⅳ can enhance the activity of human EPCs and promote their proliferation in vitro. Key words: Astragalus membranaceus; Saponins; Endothelial progenitor cells; Cell proliferation; Cell survival; In vitro

Astragaloside Ⅳ regulates Nrf2/Bach1/HO-1 signaling pathway and inhibits H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation

Astragaloside Ⅳ(AS-Ⅳ) has protective effects against ischemia-reperfusion injury(IRI), but its mechanism of action has not yet been determined. This study aims to investigate the protective effects and mechanism of AS-Ⅳ on H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation(H/R). The H/R model of myocardial cells was established by hypoxic culture for 12 hours and then reoxygenation culture for 8 hours. After AS-Ⅳ treatment, cell viability, the reactive oxygen species(ROS) levels, as well as the content or activity of superoxide dismutase(SOD), malondialdehyde(MDA), interleukin 6(IL-6), and tumor necrosis factor alpha(TNF-α), were measured to evaluate the effect of AS-Ⅳ treatment. The effect of AS-Ⅳ on HO-1 protein expression and nuclear Nrf2 and Bach1 protein expression was determined by Western blot. Finally, siRNA was used to knock down HO-1 gene expression to observe its reversal effect on AS-Ⅳ intervention. The results showed that as compared with the H/R model group, the cell viability was significantly increased(P<0.01), ROS level in the cells, MDA, hs-CRP and TNF-α in cell supernatant and nuclear protein Bach1 expression in the cells were significantly decreased(P<0.01), while SOD content, HO-1 protein expression in cells and expression of nuclear protein Nrf2 were significantly increased(P<0.01) in H/R+AS-Ⅳ group. However, pre-transfection of HO-1 siRNA into H9c2 cells by liposome could partly reverse the above effects of AS-Ⅳ after knocking down the expression of HO-1. This study suggests that AS-Ⅳ has significant protective effect on H/R injury of H9c2 cardiomyocytes, and Nrf2/Bach1/HO-1 signaling pathway may be a key signaling pathway for the effect.

QiShenYiQi Pills® ameliorates ischemia/reperfusion-induced myocardial fibrosis involving RP S19-mediated TGFβ1/Smads signaling pathway

QiShenYiQi Pills (QSYQ) is a compound Chinese medicine widely used in China for treatment of cardiovascular disease. However, limited data are available regarding the anti-fibrotic role of QSYQ after ischemia/reperfusion (I/R) injury. This study aimed to investigate the effect of post-treatment with QSYQ on myocardial fibrosis after I/R-induced myocardium injury, and the role of different compounds of QSYQ, focusing especially on the involvement of chemokine ribosomal protein S19 (RP S19) dimer and monocyte migration. Male Sprague-Dawley rats were subjected to left anterior descending coronary artery occlusion for 30 min followed by reperfusion with or without administration of QSYQ (0.6, 1.2, or 1.8 g/kg) once daily by gavage for 6 days. Post-treatment with QSYQ diminished I/R-induced infarct size, alleviated myocardium injury, attenuated myocardial fibrosis after 6 days of reperfusion, and restored heart function and myocardial blood flow after I/R. In addition, the drug significantly inhibited monocyte infiltration and macrophage polarization towards M2, which was attributable to chemokine RP S19 dimer. Moreover, Western blots revealed that QSYQ blocked I/R-induced increase in TGFβ1 and TGFβRⅡ and reversed its relevant gene expression, such as Smad3,4,6,7, and inhibited the increase of MMP 2,9 expression. As the major components of QSYQ, astragaloside IV (AsIV), 3,4-dihydroxy-phenyl lactic acid (DLA), and notoginsenoside R1 (R1) were assessed as to the contribution of each of them to the expression of the proteins concerned. The results showed that the effect of AsIV was similar to QSYQ, while DLA and R1 only partly simulated the effect of QSYQ. The results provide evidence for the potential role of QSYQ in treating myocardial fibrosis following I/R injury. This effect may be associated with QSYQ’s inhibition effect on monocyte chemotaxis and TGFβ1/Smads signaling pathway with different component targeting distinct link (s) of the signaling.

Astragaloside IV (AS-IV) alleviates the malignant biological behavior of hepatocellular carcinoma via Wnt/β-catenin signaling pathway.

Astragaloside IV (AS-IV) is an active substance isolated from Astragalus membranaceus (Fisch.) Bungede, which has been shown to have pharmacological effects in a variety of cancers. However, the effects of AS-IV in hepatocellular carcinoma (HCC) and its related mechanisms have been poorly understood. In this study, we explored the roles of AS-IV on HCC and the underlying signaling pathway. We reported that the appropriate concentrations of AS-IV (25, 50, 100 nmol l−1) significantly suppressed the proliferation and cell cycle of HepG2 and Hep3B cell lines whilst promoting apoptosis. Besides, a trans-well and wound healing assay showed that AS-IV could markedly inhibit the migration and invasion of HepG2 and Hep3B cells, the expression of E-cadherin was up-regulation but the expression of N-cadherin and vimentin was down-regulation, and the protein levels of cleaved-caspase-3, 9 were increased markedly compared with the corresponding control. Furthermore, animal model treatment revealed that AS-IV could effectively reduce tumor formation. Moreover, AS-IV also significantly weakened the expression of Wnt, β-catenin and TCF-4 in vitro and in vivo. Taken together, these results suggested that AS-IV inhibited the biological processes of HCC via regulating of the Wnt/β-catenin pathway.

Immune regulation mechanism of Astragaloside IV on RAW264.7 cells through activating the NF-κB/MAPK signaling pathway

The present study was designed to investigate the effects of Astragaloside IV (ASIV) on the immune functions of RAW264.7 cells. Compared with control group, the concentrations of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α) and nitric oxide (NO) were higher in the 100μg/mL ASIV-treatment group. The interleukin 6 (IL-6) concentration was significantly higher in the 50 and 100μg/mL ASIV-treatment groups. The relative mRNA expression levels of IL-1β, TNF-α and inducible nitric oxide synthase (iNOS) were significantly higher in the 50 and 100μg/mL ASIV-treatment groups. The relative mRNA expression levels of IL-6 in the 100μg/mL ASIV-treatment group were significantly higher. In contrast, the relative mRNA expression levels of interleukin 4 (IL-4) and IL-6 markedly reduced in ASIV-treatment groups. Furthermore, ASIV promoted the secretion of CD40 and CD86 and increased the number of cells in G2/M phase. The apoptosis of RAW264.7 cells was decreased in ASIV-treatment groups. The protein levels of cyclin D1, CDK4 and CDK6, p50 and p-p65 increased in a dose-dependent manner. The ratio of p50/β-actin was significantly higher in the 50 and 100μg/mL ASIV-treatment groups, and p-p65/p65 was significantly higher in the 25, 50 and 100μg/mL ASIV-treatment groups. The phosphorylation levels of p38, ERK and JNK increased, and the protein expression of total p38, ERK and JNK decreased in a dose-dependent manner. These effects of ASIV were alleviated by PDTC. ASIV enhances the immune function of RAW264.7 cells by activating the NF-κB/MAPK signaling pathway.

Astragaloside IV inhibits PMA-induced EPCR shedding through MAPKs and PKC pathway

Astragaloside IV (AS-IV), a main active substance isolated from Astragalus membranaceus Bunge, has been shown to have multiple pharmacological effects. Endothelial cell protein C receptor (EPCR) is a marker of inflammation, and is also a major member of protein C (PC) anti-coagulation system. EPCR can be cut off from the cell surface by tumor necrosis factor-α converting enzyme (TACE), which is controlled through mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. To develop novel therapeutic drug for EPCR shedding, the effect of AS-IV was studied in phorbol-12-myristate 13-acetate (PMA)-induced human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism of AS-IV action was investigated. The results showed that AS-IV could significantly inhibit PMA-induced EPCR shedding. In further study, AS-IV suppressed the expression and activity of TACE. In addition, AS-IV could decrease the phosphorylation of MAPK such as janus kinase (JNK) and p38, and inhibit activation of PKC through the prevention of non-phosphorylation and phosphorylation of specific PKC isoforms in PMA-stimulated HUVECs. These findings indicate that AS-IV may be used as a natural medicine to treat EPCR-related systemic inflammation and cardiovascular diseases by targeting MAPK and PKC pathway.