Astragaloside IV improves the quality of in vitro postovulatory aged oocytes by decreasing oxidative stress in mice.

Astragaloside IV regulates the ferroptosis signaling pathway via the Nrf2/SLC7A11/GPX4 axis to inhibit PM2.5-mediated lung injury in mice

Over-accumulation of reactive oxygen species (ROS) causes hepatocyte dysfunction and apoptosis that might lead to the progression of liver damage. Sirtuin-3 (SIRT3), the main NAD+-dependent deacetylase located in mitochondria, has a critical role in regulation of mitochondrial function and ROS production as well as in the mitochondrial antioxidant mechanism. This study explores the roles of astragaloside IV (AST-IV) and formononetin (FMR) in connection with SIRT3 for potential antioxidative effects. It was shown that the condition of combined pre- and post-treatment with AST-IV or FMR at all concentrations statistically increased and rescued cell proliferation. ROS levels were not affected by pre-or post-treatment individually with AST-IV or pre-treatment with FMR; however, post-treatment with FMR resulted in significant increases in ROS in all groups. Significant decreases in ROS levels were seen when pre- and post-treatment with AST-IV were combined at 5 and 10 μM, or FMR at 5 and 20 μM. In the condition of combined pre- and post-treatment with 10 μM AST-IV, there was a significant increase in SOD activity, and the transcriptional levels of Sod2, Cat, and GPX1 in all treatment groups, which is indicative of reactive oxygen species detoxification. Furthermore, AST-IV and FMR activated PGC-1α and AMPK as well as SIRT3 expression in AML12 hepatocytes exposed to t-BHP-induced oxidative stress, especially at high concentrations of FMR. This study presents a novel mechanism whereby AST-IV and FMR yield an antioxidant effect through induction of SIRT3 protein expression and activation of an antioxidant mechanism as well as mitochondrial biogenesis and mitochondrial content and potential. The findings suggest these agents can be used as SIRT3 modulators in treating oxidative-injury hepatocytes.

Oxidative stress serves a role in endothelial dysfunction exhibited by patients with diabetes mellitus. Astragaloside IV (AS-IV) is a major active ingredient of Radix Astragali, which is considered to exhibit vasoprotective effects through unknown mechanisms. Thus, the current study was performed to investigate the protective effects of AS-IV in streptozotocin (STZ)-induced endothelial dysfunction and to explore whether antioxidant mechanisms were involved. The protective effects of AS-IV on the endothelium-dependent relaxation and contraction of aortic rings were determined by isometric tension recordings. NADPH subunits and endothelial nitric oxide synthase (eNOS) expression was identified via western blotting. Superoxide dismutase and malondialdehyde levels were assayed using ELISA. Furthermore, the generation of reactive oxygen species (ROS) and nitric oxide (NO) was detected via dihydroethidium and 4,5-diaminofluorescein diacetate staining, respectively. The results revealed that STZ-injected mice exhibited increased aortic endothelium-dependent vasoconstriction and decreased vasorelaxation to acetylcholine. However, AS-IV treatment reversed these effects. NG-nitro-L-arginine was subsequently used to completely inhibit impaired relaxation. Accordingly, impaired NO generation was restored following AS-IV treatment by increasing eNOS phosphorylation levels. Furthermore, ROS formation was also depressed following AS-IV treatment compared with that in STZ-injected mice. AS-IV also decreased the expression of various NADPH subunits, including human neutrophil cytochrome b light chain, neutrophil cytosolic factor 1, NADPH oxidase (NOX)2, NOX4 and Rac-1. The results of the current study may provide novel evidence that diabetes-induced vascular injury arises from either the inhibition of eNOS or the activation of NOX-derived ROS generation. In addition, the results warrant further investigation into the application of AS-IV treatment, leading to the improvement of oxidative stress, in patients with diabetes exhibiting endothelial dysfunction.

Astragaloside IV (AS-IV) is an active component extracted from the traditional Chinese herbal medicine. AS-IV is a neuroprotective component in cerebral ischemic models. However, roles of AS-IV in cerebral ischemia-reperfusion (I/R) injury and the underlying mechanisms are rarely investigated. The role of AS-IV in oxygen – glucose deprivation reoxygenation (OGD/R)-induced cell proliferation and apoptosis assays were analyzed by Cell Counting Kit-8 and Flow cytometric. Western Blot assays were performed to measure the related expression levels in SH-SY5Y cells. Meanwhile, activities of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) in OGD/R-induced cells were determined by relative commercial kits. AS-IV was also used in cerebral I/R rat model, aimed to investigate the effects on cerebral infarct. The results indicated that OGD/R suppressed viability, enhanced apoptosis, which could be reversed by AS-IV treatment. Compared with the control group, the expression of p-JAK2 and p-STAT3 was significantly increased by AS-IV (60 μg/mL) under the OGD/R condition. Furthermore, AS-IV (60 μg/mL) treatment markedly increased SOD activity, whereas significantly decreased MDA activity and production of ROS in OGD/R-induced cells. The protective effects of AS-IV mentioned above were weaken or abolished while adding JAK2 inhibitor AG490. In addition, the effects of AS-IV on Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in cerebral I/R injury were also verified in vivo. AS-IV protected against cerebral I/R injury and reversed by AG490. Therefore, in vitro and in vivo analyses suggested that AS-IV may protect against cerebral I/R injury partly mediated by JAK2/STAT3 signaling pathway and antioxidative effects. AS-IV may serve as a novel therapeutic regimen for cerebral I/R injury.

Ultraviolet (UV) radiation is a key risk factor of environment to contribute photoaging and skin cancer through production of reactive oxygen species (ROS) and inflammatory responses. Astragaloside IV (AS-IV) is an active component from Astragalus membranaceus, and shows various pharmacological effects on inflammation, oxidative stress and apoptosis. However, whether AS-IV shows protective effect on UVB-induced injury in epidermal keratinocytes remain unknown. To explored the effects of AS-IV on UVB-induced oxidative injury and inflammatory response in human epidermal keratinocytes. HaCaT keratinocytes were exposed to UVB irradiation, followed by AS-IV incubation. The cell viability, intracellular ROS level, oxidative stress, and apoptosis were determined. The regulatory effects of AS-IV on toll-like receptor 4 (TLR4) pathway in UVB-exposed HaCaT cells were also investigated. Astragaloside IV pretreatment (10, 25, 50, 100 and 150 μM) increased cell viability in UVB-exposed HaCaT cells. AS-IV (50 μM) significantly reduced intracellular ROS level and lipid oxidation product malondialdehyde (MDA) content, and increased a ROS-scavenging enzyme superoxide dismutase (SOD) in HaCaT cells with UVB irradiation. In addition, AS-IV pretreatment suppressed apoptosis, increased Bax protein, caspase-3 and 9, and decreased BCL-2 protein in contrast to HaCaT cells with UVB-irradiation. AS-IV suppressed proinflammatory cytokine production, inhibited TLR4 and its downstream signaling molecules NF-κB, iNOS and cyclooxygenase-2 (COX-2) protein expression. We also found that the effects of AS-IV on cell viability and TLR4 expression was reversed by NAC. The protective of AS-IV on UVB-induced damage and TLR4 expression was dependent on ROS, as the increase in viability and decrease in TLR4 protein by AS-IV was significantly attenuated by ROS scavenger NAC (1 mM). Astragaloside IV prevent UVB-induced oxidative damage and inflammation by inhibiting TLR4 expression.

The mitochondrial SIRT3 is a major deacetylase protein that regulates important mitochondrial biology, including metabolic pathways, reactive oxygen species detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response. We found that astragalus aqueous extract increased the activity of SIRT3 in vitro. We further explore the potential antioxidative effects of Astragaloside IV (AST‐IV) and Formononetin (FMR) on induced oxidative stressed AML12 (alpha mouse liver 12) cells. The AML12 cell line was established from healthy hepatocytes from a mouse (CD1 Strain, Line MT42) transgenic for human TFG alpha.We first tested whether AST‐IV or formononetin increases SIRT3 expression in cell model. We cultured AML12 and treated with various concentrations of AST‐IV or formononetin in parallel with untreated control, negative control, and positive control. We found that AST‐IV and formononetin induced SIRT3 expression through Western Blotting (WB) analysis.In addition, we assessed rescue effects of AST‐IV and formononetin on t‐BHP‐induced oxidative injury in AML12 hepatocytes under three separate conditions: pre‐treatment with AST‐IV or formononetin, post‐treatment with AST‐IV or formononetin, and both pre‐treatment and post‐treatment with AST‐IV and formononetin. Through CCK‐8 analysis, we identified that both pre‐treatment and post‐treatment significantly increased cell proliferation on induced‐oxidative stress AML12 cells compared to no treatment and control. Pre‐treatment of AML12 cells before induction of oxidative stress with AST‐IV and formononetin also showed cell proliferation but was less significant compared to AML12 cells that received both pre‐treatment and post‐treatment with AST‐IV or formononetin. Post‐treatment AST‐IV or formononetin after induction of oxidative stress on AML12 cells did not significantly show any difference in cell proliferation compared to non‐treatment and control. Significantly AST‐IV and formononetin showed induction of the SIRT3 protein on tert‐butyl hydroperoxide (t‐BHP) induced oxidative stressed AML12 hepatocytes by WB.Taken together, the increase in cell proliferation is indicative of the antioxidative effects of AST‐IV and formononetin through induction of SIRT3 protein. Further investigation is currently conducted to provide evidence of AST‐IV and formononetin's mechanistic effect on inducing the expression of the SIRT3 protein‐linked downstream pathways for their antioxidative benefit to hepatocytes. The ultimate goal is to utilize these SIRT3 modulators as pharmacologic agents in treating oxidative injury hepatocytes.

Effect of astragaloside IV and salvianolic acid B on antioxidant stress and vascular endothelial protection in the treatment of atherosclerosis based on metabonomics.

Astragaloside IV: a natural shield against ochratoxin A-induced hepatotoxicity in chicks by targeting the NRF2/NLRP3 signaling pathway.

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

Background: Cyclophosphamide (CTX) can lead to hepatotoxicity and low immunity. Astragaloside IV (AS-IV) can enhance the body's immune function. Objectives: This study aimed to investigate whether AS-IV can improve CTX-induced liver injury in mice through the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathways and to explore whether its mechanism is related to immunosuppression and oxidative stress. Methods: Forty-eight BALB/C mice aged 6 - 8 weeks were randomly divided into control groups, CTX group, AS-IV group, CTX+AS-IV low, medium, and high dose treatment groups, CTX+LY294002 group, and CTX+AS-IV-H+LY294002 group (n = 6 mice in each group). Mice in the control group and CTX group were given 1% starch paste by gavage daily. Mice in the AS-IV group and CTX+AS-IV groups were intragastrically administered AS-IV every day. Mice in the LY294002 group were intraperitoneally injected with LY294002 every 2 days. After 14 days, CTX was intraperitoneally injected for 2 consecutive days to induce a mouse liver injury model. The immune function of the mice was evaluated using HE staining and an Enzyme-Linked Immunosorbent Assay (ELISA) kit. The degree of oxidative stress and liver injury was detected by DHE fluorescent probe and ELISA kit. The PI3K/Akt axis protein expressions were detected using Western blot. Results: Compared with the CTX group, AS-IV significantly increased the cytokines and immunoglobulin levels (P < 0.05) and reduced the levels of reactive oxygen species (ROS). Malondialdehyde (MDA) levels reduced from 0.788 nmol/mg to 0.475 nmol/mg, and liver injury indices increased. Superoxide dismutase (SOD) levels increased from 5.393 U/mg to 9.867 U/mg, and catalase (CAT) levels increased from 4.617 U/mg to 8.248 U/mg, restoring the integrity and clarity of liver cell structure (P < 0.05). The AS-IV also significantly increased protein levels; p-PI3K increased from 0.526 to 0.880, and p-Akt increased from 0.263 to 0.720. After LY294002 was applied on the basis of AS-IV intervention, CTX-caused liver damage was aggravated again. The cytokines, immunoglobulin, SOD, and CAT levels were significantly decreased, and the levels of liver injury indicators were significantly increased (P < 0.05). Conclusions: The AS-IV improved CTX-induced immunosuppression and oxidative damage in mice by activating the PI3K/Akt axis and played a hepatoprotective role.

Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 µg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg−1·d−1) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation.

Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of cardiovascular disorders. Astragaloside IV (AsIV) possesses potent antioxidant properties against oxidative stress through undefined mechanism(s). We sought to investigate whether AsIV protects human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide (H2O2) induced oxidative stress focusing on eNOS uncoupling and the NADPH oxidase - ROS - NF-κB pathway. Compared with HUVECs incubated with H2O2 alone, pretreatment with AsIV significantly increased the viability of HUVECs, which was accompanied with apparent increase in nitric oxide (NO) production and decrease in intracellular superoxide anion production. Furthermore, pretreatment with AsIV increased endothelial nitric oxide synthase (eNOS) dimer/monomer ratio and its critical cofactor tetrahydrobiopterin (BH4) content, decreased Nox4 protein expression (the most abundant Nox isoform in HUVECs), inhibited translocation of NF-κB p65 subunit into nuclear fraction while enhanced the protein expression of IκB-α (the inhibitor of NF-κB p65), reduced the levels of IL-1β, IL-6, and TNF-α in HUVECs medium, and decreased iNOS protein expression. These results suggest that AsIV may protect HUVECs from H2O2-induced oxidative stress via inhibiting NADPH oxidase - ROS - NF-κB pathway and eNOS uncoupling.

We investigated the role of astragaloside IV (AS-IV) in preventing glucocorticoid-induced avascular necrosis of the femoral head (ANFH) and the underlying molecular mechanisms. Network pharmacology was used to predict the molecular targets of AS-IV. Molecular dynamic simulations were performed to explore the binding mechanism and interaction mode between AS-IV and Akt. Rat models of glucocorticoid-induced ANFH with AS-IV intervention were established, and osteogenesis, angiogenesis, apoptosis and oxidative stress were evaluated before and after blocking the PI3K/Akt pathway with LY294002. The effects of glucocorticoid and AS-IV on bone marrow mesenchymal stem cells and human umbilical vein endothelial cells incubated with and without LY294002 were determined. Downregulated p-Akt expression could be detected in the femoral heads of glucocorticoid-induced ANFH patients and rats. AS-IV increased trabecular bone integrity and vessel density of the femoral head in the model rats. AS-IV increased Akt phosphorylation and upregulated osteogenesis-, angiogenesis-, apoptosis- and oxidative stress-related proteins and mRNA and downregulated Bax, cleaved caspase-3 and cytochrome c levels. AS-IV promoted human umbilical vein endothelial cell migration, proliferation and tube formation ability; bone marrow mesenchymal stem cell proliferation; and osteogenic differentiation under glucocorticoid influence. AS-IV inhibited apoptosis. LY294002 inhibited these effects. AS-IV prevented glucocorticoid-induced ANFH by promoting osteogenesis and angiogenesis via the Akt/Runx2 and Akt/HIF-1α/VEGF pathways, respectively, and suppressing apoptosis and oxidative stress via the Akt/Bad/Bcl-2 and Akt/Nrf2/HO-1 pathways, respectively.

Tacrolimus-induced chronic nephrotoxicity (TIN) hinders its long-term use in patients. However, there are no drugs available in the clinic to relieve it at present. Astragaloside IV (AS-IV) is a saponin extract of the Astragalus which is widely used in the treatment of kidney disease. This study aimed to investigate the effect of AS-IV on TIN and its underlying mechanism. Herein, C57BL/6 mice were treated with tacrolimus and/or AS-IV for 4 weeks, and then the renal function, fibrosis, oxidative stress and p62-Keap1-Nrf2 pathway were evaluated to ascertain the contribution of AS-IV and p62-Keap1-Nrf2 pathway to TIN. Our results demonstrated that AS-IV significantly improved renal function and alleviated tubulointerstitial fibrosis compared with the model group. The expression of fibrosis-related proteins, including TGF-β1, Collagen I and α-SMA, were also decreased by AS-IV. Furthermore, AS-IV relieved the inhibition of tacrolimus on antioxidant enzymes. The data in HK-2 cells also proved that AS-IV reduced tacrolimus-induced cell death and oxidative stress. Mechanistically, AS-IV markedly promoted the nuclear translocation of Nrf2 and the renal protective effects of AS-IV were abolished by Nrf2 inhibitor. Further researches showed that phosphorylated p62 was significantly increased after AS-IV pretreatment. Moreover, AS-IV failed to increase nuclear translocation of Nrf2 and subsequent anti-oxidative stress in HK-2 cells transfected with p62 siRNA. Collectively, these findings indicate that AS-IV relieve TIN by enhancing p62 phosphorylation, thereby increasing Nrf2 nuclear translocation, and then alleviating ROS accumulation and renal fibrosis.

Astragaloside IV-induced Nrf2 nuclear translocation ameliorates lead-related cognitive impairments in mice