Astragaloside IV Group Research Articles

Neuroprotective Effect of Astragaloside IV on Cerebral Ischemia/Reperfusion Injury Rats Through Sirt1/Mapt Pathway

Background: Ischemic stroke is a common disease with poor prognosis, which has become one of the leading causes of morbidity and mortality worldwide. Astragaloside IV (AS-IV) is the main bioactive ingredient of Astragali Radix (which has been used for ischemic stroke for thousands of years) and has been found to have multiple bioactivities in the nervous system. In the present study, we aimed to explore the neuroprotective effects of AS-IV in rats with cerebral ischemia/reperfusion (CIR) injury targeting the Sirt1/Mapt pathway. Methods: Sprague–Dawley rats (male, 250–280 g) were randomly divided into the Sham group, middle cerebral artery occlusion/reperfusion (MCAO/R) group, AS-IV group, MCAO/R + EX527 (SIRT1-specific inhibitor) group, and AS-IV + EX527 group. Each group was further assigned into several subgroups according to ischemic time (6 h, 1 d, 3 d, and 7 days). The CIR injury was induced in MCAO/R group, AS-IV group, MCAO/R + EX527 group, and AS-IV + EX527 group by MCAO surgery in accordance with the modified Zea Longa criteria. Modified Neurological Severity Scores (mNSS) were used to evaluate the neurological deficits; TTC (2,3,5-triphenyltetrazolium chloride) staining was used to detect cerebral infarction area; Western Blot was used to assess the protein levels of SIRT1, acetylated MAPT (ac-MAPT), phosphorylated MAPT (p-MAPT), and total MAPT (t-MAPT); Real-time Quantitative Polymerase Chain Reaction (qRT-PCR) was used in the detection of Sirt1 and Mapt transcriptions. Results: Compared with the MCAO/R group, AS-IV can significantly improve the neurological dysfunction (p < 0.05), reduce the infarction area (p < 0.05), raise the expression of SIRT1 (p < 0.05), and alleviate the abnormal hyperacetylation and hyperphosphorylation of MAPT (p < 0.05). While compared with the AS-IV group, AS-IV + EX527 group showed higher mNSS scores (p < 0.05), more severe cerebral infarction (p < 0.05), lower SIRT1 expression (p < 0.01), and higher ac-MAPT and p-MAPT levels (p < 0.05). Conclusion: AS-IV can improve the neurological deficit after CIR injury in rats and reduce the cerebral infarction area, which exerts neuroprotective effects probably through the Sirt1/Mapt pathway.

Astragaloside IV attenuates hypoxia/reoxygenation injury-induced apoptosis of type II alveolar epithelial cells through miR-21-5p

ABSTRACT We aimed to explore the role of miR-21-5p in the inhibitory effects of astragaloside IV (As-IV) on hypoxia/reoxygenation injury-induced apoptosis of type II alveolar epithelial cells. Rat type II alveolar epithelial cells RLE-6TN were cultured in vitro and randomly divided into control (C), hypoxia/reoxygenation injury (H/R), As-IV and miR-21-5p-siRNA + As-IV groups (n = 6). H/R model was established by 24 h of hypoxia and 4 h of reoxygenation. As-IV group was given 1 nmol/L As-IV and incubated for 1 h before modeling. MiR-21-5p-siRNA + As-IV group was transfected with 50 nmol/L miR-21-5p-siRNA. After 48 h, they were incubated with 1 nmol/L As-IV for 1 h before modeling. Cell viability was detected by cell counting kit-8 assay, and apoptosis rate was detected by flow cytometry. The expression levels of TLR4 and NF-κB were measured by immunofluorescence assay. The targeting relationship between miR-21-5p and TLR4 was determined by luciferase assay. Compared with H/R group, the cell viability, miR-21-5p, bax and cleaved caspase-3 expressions of As-IV group increased, apoptosis rate and Bcl-2 expression decreased, and TLR4 and NF-κB expressions were down-regulated (P < 0.05). Compared with As-IV group, the cell viability, miR-21-5p, bax and cleaved caspase-3 expressions of miR-21-5p-siRNA + As-IV group decreased, apoptosis rate and Bcl-2 expression increased, and the expressions of TLR4 and NF-κB were up-regulated (P < 0.05). As-IV up-regulates miR-21-5p expression, inhibits the TLR4/NF-κB signaling pathway and suppresses the apoptosis of type II alveolar epithelial cells during hypoxia/reoxygenation injury.

Astragaloside Ⅳ inhibits inflammation after cerebral ischemia in rats through promoting microglia/macrophage M2 polarization

To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on microglia/macrophage M1/M2 polarization and inflammatory response after cerebral ischemia in rats. Forty eight male SD rats were randomly divided into sham operation control group, model control group and AS-Ⅳ group with 16 rats in each. Focal cerebral ischemia model was induced by occlusion of the right middle cerebral artery (MCAO) using the intraluminal filament. After ischemia induced, the rats in AS-Ⅳ group were intraperitoneally injected with 40 mg/kg AS-Ⅳ once a day for 3 days. The neurological functions were evaluated by the modified neurological severity score (mNSS) and the corner test on d1 and d3 after modelling. The infarct volume was measured by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining on d3 after ischemia. The expression of M1 microglia/macrophage markers CD86, inducible nitric oxide synthase (iNOS) and pro-inflammatory factors TNF-α, IL-1β, IL-6, M2 microglia/macrophages markers CD206, arginase-1 (Arg-1), chitinase-like protein (YM1/2) and anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) was detected by real-time RT-PCR. The expression of CD16/32/Iba1 and CD206/Iba1 was determined by double labeling immunefluorescence method in the peripheral area of cerebral ischemia. Compared with model control group, AS-Ⅳ treatment improved neurological function recovery and reduced infarct volume after ischemia (P<0.05 or P<0.01). The qRT-PCR results showed that AS-Ⅳ treatment down-regulated the expression of CD86, iNOS, TNF-α, IL-1β, IL-6 mRNA (all P<0.01), and up-regulated the expression of CD206, Arg-1, YM1/2, IL-10 and TGF-β mRNA (all P<0.01). Furthermore, the results of immunefluorescence labeling showed that AS-Ⅳ treatment reduced the number of CD16/32+/Iba1+ cells (P<0.05) and increased the number of CD206+/Iba1+ cells (P<0.01) after cerebral ischemia. The findings suggest that AS-Ⅳ ameliorates brain injury after cerebral ischemia in rats, which may be related to inhibiting inflammation through promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.

Transcriptomic Analysis Reveals the Protection of Astragaloside IV against Diabetic Nephropathy by Modulating Inflammation.

Background Diabetic nephropathy (DN) is one of the leading causes of end-stage kidney disease. Recently, there is no specific drug available to block the kidney damage. Astragaloside IV (AS-IV) is a major active component of Astragalus membranaceus (Fisch) Bge and has been demonstrated to benefit the kidney functions. This study explores the potential pharmacological action of AS-IV in DN of rats. Methods Male Sprague-Dawley rats were fed with high-fat diet and injected with streptozotocin to induce diabetes. The diabetic rats were randomized and treated with vehicle or AS-IV (80 mg/kg) daily by gavage for 12 weeks as the DN or AS-IV group, respectively. The normal control rats were fed with normal chow and injected with vehicles (n = 8 per group). These rats were monitored for diabetes- and kidney function-related measures. The expression profiles of gene mRNA transcripts in the kidney tissues were analyzed by RNA-seq and quantitative RT-PCR. The levels of advanced glycation end products (AGEs), IL-1β, and IL-18 in the serum samples and kidney tissues were quantified by ELISA. The levels of collagen IV (COL-4) and fibronectin (FN) expression in kidney tissues were examined by immunohistochemistry and Western blot. Results In comparison with the DN group, AS-IV treatment significantly reduced blood glucose levels, food and water consumption, 24 h urine, renal index values, 24 h urine total proteins, blood urea nitrogen (BUN) levels, and creatinine clearance rates (CCR), accompanied by minimizing the DN-induced early kidney damages, fibrosis, and microstructural changes. Furthermore, AS-IV treatment significantly modulated the DN-altered gene transcription profiles in the kidney of rats, particularly for inflammation-related genes, including the nucleotide-binding oligomerization domain-like receptor signaling, which was validated by quantitative RT-PCR. AS-IV treatment significantly decreased the levels of serum and kidney AGEs, IL-1β, and IL-18 expression and fibrosis indexes in the kidney of rats. Conclusion AS-IV treatment ameliorated the severity of DN by inhibiting inflammation-related gene expression in the kidney of rats.

Astragalus polysaccharides and astragaloside IV ameliorates cyclophosphamide-induced mouse model of overactive bladder

ObjectivePrevious studies have shown that Astragalus polysaccharides (APS) and Astragaloside IV (AS-IV) protect against inflammation-related cell damage and exhibit immune enhancement. Since urothelial injury may result in an overactive bladder (OAB), the aim of this study was to investigate the efficacy of APS and AS-IV on urothelial injury in an experimental animal model. Materials and methodsThe effects of APS and AS-IV on the proliferation and migration of primary human urothelial cells (HUCs) or primary human fibroblast cells (HFCs) were assessed using an in vitro wounding model and colorimetric thiazolyl blue assays. Sixty virgin female mice were randomized into five groups: group 1–saline-injected plus treatment with H2O, group 2–cyclophosphamide (CYP) plus treatment with H2O, group 3–CYP plus treatment with solifenacin succinate (SS; 10 mg/kg), group 4–CYP plus treatment with AS-IV (100 mg/kg), and group 5–CYP plus treatment with APS (100 mg/kg). Cystometry assessment was conducted and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured. Voiding interval values (time between voids) were assessed in mice under anesthesia. Lastly, immunohistochemistry analysis was used to confirm the location and level, respectively, of ZO-2 expression. ResultsAPS and AS-IV did not influence the cell viability but increased migration in HFCs compared with the controls. The OAB mice showed significantly lower voiding interval values. Voiding interval values were significantly higher in the CYP plus treatment with APS (100 mg/kg) and AS-IV (100 mg/kg) groups than in the CYP-induced OAB group. Additionally, the expression of ZO-2, a tight junction protein, was increased in the CYP plus treatment APS (100 mg/kg) and AS-IV (100 mg/kg) groups compared with the CYP-induced OAB group. ConclusionThese findings suggest that APS and AS-IV modulate urothelial wound healing, which ameliorates urinary frequency of mice treated with CYP. APS or AS-IV may have the potential benefit of acting as urothelial wound healing modulators.

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.

Anti-apoptotic effect of Astragaloside IV on delayed cerebral vasospasm following subarachnoid hemorrhage in rats

Objective Investigate the effect of Astragaloside Ⅳ (AS-Ⅳ) on delayed cerebral vasospasm (DCVS) following subarachnoid hemorrhage. Methods The male SD rats were divided into Sham group (A), SAH+ AS-Ⅳ group (B), SAH model group (C) and SAH+ vehicle group (D). The model of DCVS following subarachnoid hemorrhage was made by the double injection method of occipital cistern. During the 5 days after model success, rats in the B group were treated with daily AS-Ⅳ (20 mg/kg) which was dissolved in 5% alcohol; rats in the D group were injected with identical volume of physiological saline containing 5% alcohol; rats in the SAH group and the Sham group were inject with identical volume of physiological saline. Morphological changes of basilar artery was observed by hematoxylin and eosin (HE) staining. The apoptosis of basilar artery endothelial cells and vascular smooth muscle cells was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. The cleaved cysteinyl aspartate-specific protease (Caspase)-3 expression in basilar artery was detected by immunohistochemical staining. The expression of B cell lymphoma/leukemia-2 (bcl-2), bcl-2 associated X protein (bax) and cleaved Caspase-3 was detected by Western blotting. Results Compared with Sham group, the basilar artery wall became thicker and lumen cross-sectional area became smaller. The expression of bax and cleaved Caspase-3 (P<0.05) was decreased in basilar artery (P<0.05). Compared with SAH+ vehicle group and SAH group, the expression of cleaved Caspase-3 in SAH+ AS-Ⅳ group increased (P<0.05) and the expression of bax and cleaved Caspase-3 decreased after AS-Ⅳ intervention (Cleaved Caspase-3: 0.030 8±0.003 2 vs. 0.070 4±0.006 0, 0.069 0±0.005 7, P<0.05). Conclusion AS-Ⅳ can relieve delayed cerebral vasospasm and its mechanism may be related to the fact that AS-Ⅳ can interfere with the apoptosis signaling pathway mediated by bcl-2, bax and Caspase-3. Key words: Astragaloside Ⅳ; Subarachnoid hemorrhage; Delayed cerebral vasospasm; Apoptosis

Astragaloside IV alleviates myocardial ischemia-reperfusion injury in rats through regulating PI3K/AKT/GSK-3β signaling pathways.

Purpose:To investigate the effect of astragaloside IV (As-IV) on myocardial ischemia-reperfusion (I/R) injury in rats and reltaed mechanisms.Methods:Sixty rats were randomly divided into sham-operated, control I/R and 2.5, 5 and 10 mg/kg As-IV groups, 12 rats in each group. The later three groups were intragastrically administered with As-IV for 7 days, with a dose of 2.5, 5 and 10 mg/kg, respectively. The myocardial I/R injury model was constructed in later four groups. At the end of reperfusion, the cardiac function indexes, serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels, heart weight (HW)/body weight (BW) ratio and infarct size, and expressions of phosphatidylinositol-3 kinase/serine-threonine protein kinase (PI3K/AKT) and glycogen synthase kinase-3β (GSK-3β) proteins and the phosphorylated forms (p-AKT, p-GSK-3β) were determined.Results:Compared with control I/R group, in 5 and 10 mg/kg As-IV groups the left ventricular systolic pressure, fractional shortening and ejection fraction were increased, the left ventricular end-diastolic pressure was decreased, the serum LDH and CK levels were decreased, the HW/BW ratio and myocardial infarct size were decreased, and the p-Akt/Akt ratio and p-GSK-3β/GSK-3β ratio were increased (all P < 0.05).Conclusion:As-IV can alleviate the myocardial I/R injury in rats through regulating PI3K/AKT/GSK-3β signaling pathways.

Protective Effects of Astragaloside IV Combined with Budesonide in Bronchitis in Rats by Regulation of Nrf2/Keap1 Pathway.

BackgroundThis study was conducted to evaluate the effects of astragaloside IV and budesonide on bronchitis in rats and to explore the mechanism involved.Material/MethodsEighty Sprague-Dawley (SD) rats were randomly divided into 5 groups, including a Bronchitis model group (BM), a Budesonide group (BG), an Astragaloside IV group (AG), an Astragaloside IV combined with Budesonide group (CG), and a blank control group (BC). Lung tissue was stained with hematoxylin and eosin (H&E). The activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA). The nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2), Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1 (Keap1), BTB and CNC homology 1 (Bach1), B-cell lymphoma-2(Bcl-2), and BCl-2-associated X protein (Bax) mRNA and protein were examined by RT-PCR and Western blot, respectively.ResultsCompared with the Bronchitis model group, the lung tissue lesions in the Budesonide group, Astragaloside IV group, and Astragaloside IV combined with Budesonide group were effectively ameliorated and the airway resistance was significantly decreased. The activities of SOD, GSH-Px, and CAT were increased after treatment with drugs, while the content of MDA was decreased. The levels of Nrf2, Keap1, and Bcl-2 proteins were increased and the levels of Bach1 and Bax were decreased after treatment with Budesonide and Astragaloside IV.ConclusionsAstragaloside IV combined with budesonide can ameliorate the lesions caused by bronchitis in rats through activating the Nrf2/Keap1 pathway, which plays a protective role on anti-oxidative stress injury.

Astragaloside IV combating liver cirrhosis through the PI3K/Akt/mTOR signaling pathway.

Astragaloside IV (AS-IV) in improving liver cirrhosis injury in rats and its effect on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling pathway were observed. Rat model of liver cirrhosis was induced by injection of carbon tetrachloride (CCl4). A total of 36 Sprague-Dawley (SD) rats were randomly divided into three groups: the normal control group (n=10), the model control group (n=13), and the AS-IV group (n=13). The normal control group was injected with olive oil and given carboxymethyl cellulose (CMC)-Na (10 ml/kg/day), the model control group was given CMC-Na (10 ml/kg/day), and the AS-IV group underwent intragastric administration of AS-IV (20 ml/kg/day). The content of alanine transaminase (ALT) and aspartate transaminase (AST) of rats was detected. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β in serum were detected via enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (H&E) staining was applied to observe morphological changes in liver tissues. The expression of collagens in liver tissues was detected via Masson's trichrome staining. Additionally, the expression of proteins in liver tissues was detected via western blotting. Compared with those in the blank group, the levels of AST, ALT, TNF-α, IL-6 and IL-1β were higher, the expression level of collagens in liver tissues was increased, and the expression ratios of phosphorylated (p)-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR proteins were increased in the model group. Compared with the model group, AS-IV could significantly decrease the content of AST, ALT, TNF-α, IL-6 and IL-β in serum of rats, obviously inhibit the expression of collagens in liver tissues and decrease the expression ratios of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR proteins in liver tissues. AS-IV can inhibit the inflammatory response so as to reduce the expression of collagens, and its mechanism may play a key role by inhibiting the PI3K/Akt/mTOR signaling pathway.

Astragaloside IV Promotes Adult Neurogenesis in Hippocampal Dentate Gyrus of Mouse through CXCL1/CXCR2 Signaling.

Astragaloside IV (ASI) has been reported to promote neural stem cells proliferation in vitro and CXCR2 expression on neutrophils. The present study was aimed to investigate the influence of ASI on adult neurogenesis in hippocampal dentate gyrus (DGs) of mouse and to discuss the possible underlying mechanisms. Total number of proliferative cells (BrdU+), pre-mature neurons (DCX+), early proliferative cells (BrdU+/DCX+), proliferative radial gila-like cells (BrdU+/GFAP+) and newly generated neurons (BrdU+/NeuN+) after ASI or vehicle administration for two weeks were counted, respectively. The results showed that BrdU+ cells and DCX+ cells were significantly increased in DGs of mice administered with ASI. The numbers of BrdU+/DCX+, BrdU+/GFAP+ cells and BrdU+/NeuN+ cells were also elevated in the ASI group. Correspondingly, ASI increased the protein expression of hippocampal DCX, GFAP and NeuN. Further study disclosed that ASI remarkably up-regulated the mRNA and protein expressions of CXCL1 as well as that of CXCR2 in the hippocampus. The promotive effect of ASI on DCX, GFAP and NeuN protein expression was abolished by SB225002, the inhibitor of CXCR2. Our results indicated that ASI modulated the homeostasis of the CXCL1/CXCR2 signaling pathway, which might be responsible for the increased neurogenesis within the hippocampal DGs of mice.

Astragaloside IV inhibits ventricular remodeling and improves fatty acid utilization in rats with chronic heart failure.

Chronic heart failure (CHF) is the end-stage of many cardiovascular diseases and severely affects the patients’ lifespan. Inhibiting ventricular remodeling is thus a primary treatment target for CHF patients. Astragaloside IV (AS-IV) can improve cardiac function and protect myocardial cells. The study aims to investigate the effects of AS-IV on ventricular remodeling and explore its role in regulating energy metabolism using a rat CHF model. Sprague–Dawley rats were divided into five groups (n=20 per group): CHF + benazepril hydrochloride (Benazepril HCL), CHF + low-dose (30 mg.kg−1.day−1) AS-IV, CHF + high-dose (60 mg.kg−1.day−1) AS-IV, and a sham control group. After 8 weeks of treatment, the cardiac structure and functional parameters were measured. Morphological changes in the myocardial tissue in five groups were evaluated. Protein and mRNA expression of peroxisome proliferator-activated receptor α (PPARα), medium-chain acyl-CoA dehydrogenase (MCAD), and muscle carnitine palmitoyl transferase-1 (MCPT1) were also analyzed. Our results showed that the left ventricular mass index (LVMI), collagen volume fraction (CVF), and free fatty acid (FFA) concentration of CHF group rats increased when compared with sham control group, while the protein and mRNA expressions of PPARα, MCAD, and MCPT1 decreased in CHF. Importantly, treatment with AS-IV (CHF + AS-IV group) showed improved heart function and structure, increased expression of PPARα, MCAD, and MCPT1 and improved FFA utilization in comparison with CHF group. In conclusion, our study shows that AS-IV inhibits ventricular remodeling, improves cardiac function, and decreases FFA concentration of CHF model rats. Our findings suggest a therapeutic potential of using AS-IV in CHF.

Effects of astragaloside-IV on the expression of inflammatory factor and proliferation of glomerular mesangial cells induced by angiotensin Ⅱ

To study the effects of astragaloside-IV (As-IV) on the expression of inflammatory factor and proliferation of glomerular mesangial cells (GMCs) induced by angiotensin Ⅱ(AngⅡ). The in vitro model of diabetic nephropathy(DN) was mimic by angiotensin Ⅱ (10-6mol/L)inducing GMCs injury. Then the GMCs were treated with As-IV at different concentrations(25,50,100 μmol/L)for 48 hours. The proliferation of GMCs was detected by MTT. The level of reactive oxidative species (ROS) was measured by flow cytometry. The expression of monocyte chemoattractant protein-1(MCP-1) protein in supernatant was detected by enzyme- linked immunosorbent assay (ELISA). The expression of transforming growth factor-β1(TGF-β1) in GMCs was measured by Western blot. Compared with model group, the proliferation of GMCs was inhibited in As-IV group. As-IV decreased the level of intercellular ROS, down-regulated the secretion of MCP-1 and the expression of TGF-β1 proteins. As-IV could inhibit cell proliferation and inflammatory factors expression on GMCs induced by AngⅡ.

Astragaloside IV reverses MNNG-induced precancerous lesions of gastric carcinoma in rats: Regulation on glycolysis through miRNA-34a/LDHA pathway.

This study was designed to investigate the precancerous lesions of gastric carcinoma (PLGC)-reversing mechanisms of astragaloside IV (ASIV) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PLGC rats. All rats were sacrificed after 10-week treatment. Gastric tissue was analyzed by using histopathology and electron microscope. To be fully evidenced, LDHA, p53, TIGAR, MCT1, MCT4, HIF-1α, CD147, and miRNA-34a were detected by Western blotting and Real-time Quantitative polymerase chain reaction (RT-qPCR). As histopathology and electron microscope showed, it can be clearly observed that the area of dysplasia was reduced in ASIV groups, indicating that MNNG-induced PLGC was markedly reversed by ASIV. Moreover, compared with model group, a significant decrease in gene expressions of LDHA, MCT1, MCT4, HIF-1α, CD147, and TIGAR was observed whereas miRNA-34a level was increased in ASIV groups. A significant up-regulation induced by MNNG in protein levels of LDHA, MCT1, MCT4, HIF-1α, and CD147 was attenuated in rats treated with ASIV. In contrast, the decreased expression of TIGAR was restored by ASIV. Interestingly, up-regulation of p53 expression induced by MNNG was further increased in ASIV groups. In brief, these results implied that abnormal glycolysis was relieved by ASIV via regulation of the expressions of LDHA, p53, TIGAR, MCT1, MCT4, HIF-1α, CD147, and miRNA-34a.

Astragaloside Ⅳ Protects Against Aβ1-42-induced Oxidative Stress, Neuroinflammation and Cognitive Impairment in Rats.

Objective To investigate the neuroprotective action of astragaloside Ⅳ (AS-Ⅳ) on spatial learning and memory impairment induced by amyloid-beta 1-42 (Aβ1-42) in rats and elucidate its underlying molecular mechanisms. Methods Adult-male Sprague-Dawley rats (230-250 g) were divided into six groups randomly: control, Aβ1-42, AS-Ⅳ, Aβ1-42 plus 5 mg/kg·d AS-Ⅳ, Aβ1-42 plus 25 mg/kg·d AS-Ⅳ, and Aβ1-42 plus 50 mg/kg·d AS-Ⅳ groups. Aβ1-42 were delivered by intracerebroventricular injection under the guidance of a brain stereotaxic apparatus. The Morris water maze test (hidden platform test, probe trials, visible platform test) was performed one week after Aβ1-42 injection to obtain the ability of rat spatial learning and memory. AS-Ⅳ (5, 25 and 50 mg/kg·d) was administrated intraperitoneally once per day from the 8th day after Aβ1-42 injection for 5 consecutive days. Average escape latencies, distances for searching for the platform under water and the percentage of total time elapsed and distance swam in the right quadrant after removing platform were determined by behavior software system. The vision and swim speeds of rats were also determined to exclude the effect of these factors on the parameters of learning and memory. After behavioral tests, the rats were sacrificed immediately by decapitation. Hippocampus were collected. The enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) in the hippocampus obtained from different-treated rat brain were measured by following the manufacturer's instructions. The levels of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in tissue lysates were assayed with ELISA. Results The water maze test results indicated that chronic treatments with AS-Ⅳ effectively protected the rats from Aβ1-42-induced spatial learning and memory impairment. Furthermore, the activities of SOD, GSH-px and CAT decreased by Aβ1-42 were also restored by AS-Ⅳ treatment in the hippocampus of rats. In addition, AS-Ⅳ significantly decreased the levels of IL-1β and TNF-α in the hippocampus of Aβ1-42-induced amnesia's rats. Conclusion Our findings suggest that AS-Ⅳ might be a useful chemical in improving the spatial memory and relieving the oxidative stress and neuroinflammation in Alzheimer patients.

Effect of Combined Intervention of Electroacupuncture and Astragaloside IV on Myocardial Hypertrophy and TGF-β 1/Smad Signaling in Rats with Myocardial Fibrosis

To observe the effect of combined intervention of electroacupuncture (EA) and astragaloside IV(ASIV) on cardiac hypertrophy and transforming growth factor β 1 (TGF-β 1)/Smad signaling in isoproterenol (ISO) induced cardiac hypertrophy rats, so as to investigate its underlying mechanisms in improving myocardial fibrosis. A total of 50 SD rats were randomly divided into 5 groups: normal control, model (ISO), Propranolol (PRO)，ASIV and EA＋ASIV groups (n＝10 in each group). The myocardial fibrosis model was established by intraperitoneal injection (i.p.) of ISO (10 mg·kg－1·d－1), once daily for 30 days. Rats of the control group were given normal saline (i.p.), those of the PRO group given with PRO (40 mg·kg－1·d－1, gavage), and those of the ASIV and EA＋ASIV groups were treated by gavage of ASIV (40 mg·kg－1·d－1), once daily for 30 days. EA (20 Hz, 6 V) was applied to bilateral "Neiguan" (PC 6) for 10 min, once every day for 30 d. The heart mass index (HMI, whole heart weight/body weight) and left ventricular (LV) mass index (LVMI, weight of the LV/body weight) were calculated to assess the state of cardiac hypertrophy. The enzyme linked immunosorbent assay (ELISA) was used to determine the levels of procollagen I carboxy-terminal propeptide (PICP，a marker of extracellular matrix remodeling) and carboxyterminal telopeptide of type I collagen (ICTP, a metabolite of type I collagen) in serum, and Western blot was used to test protein contents of TGF- β 1, Smad 2 / 3, Smad 4, Smad 7 in the left ventricle tissue of the heart. After modeling, the HMI and LVMI, serum PICP and ICTP contents and the expression levels of myocardial TGF-β 1, Smad 2/3 and Smad 4 proteins were significantly increased in the model (ISO) group (P<0.05), suggesting a deposition of collagen and cardiac hypertrophy, and were considerably decreased in PRO, ASIV and EA＋ASIV groups after the intervention (P<0.05). The expression level of myocardial Smad 7 protein was significantly lower in the model group than in the normal control group (P<0.05), and significantly up-regulated in PRO, ASIV and EA＋ASIV groups (P<0.05). Sirius Red staining of the left ventricular myocardium showed a dense deposition of collagen and a severer myocardial fibrosis in the model group, and a relatively lighter fibrosis in the PRO, ASIV and EA＋ASIV groups. The therapeutic effects of EA＋ASIV were comparable to those of PRO, and were significantly superior to those of ASIV in down-regulating HMI, serum ICTP, and myocardial Smad 2/3 and Smad 4 expression and up-regulating Smad 7 protein (P<0.05). There were no significant differences among the PRO, ASIV and EA＋ASIV groups in LVMI, PICP and TGF-β 1 levels, and between the PRO and EA＋ ASIV groups in HMI, ICTP, Smad 2/3, Smad 4 and Smad 7 levels (P> 0.05). EA stimulation of PC 6 combined with ASIV can relieve cardiac hypertrophy and myocardial fibrosis in rats, which may be associated with its effects in regulating myocardial TGF-β 1/Smad signaling pathway.

Protective effect of Astragaloside IV on the oxygenation induced by RAW264.7 murine macrophage

Objective To investigate the protective effect and mechanism of Astragaloside Ⅳ (AS-Ⅳ) on hypoxia/re-oxygenation induced by RAW264.7 murine macrophages. Methods The hypoxia/re- oxygenation induced RAW264.7 murine macrophages served as a model of I/R injury. The cells were divided into the control group, the model group, and the AS-Ⅳ group. The cell morphological changes of each group were observed directly under inverted microscope. Inducible nitric oxide synthase (iNOS), cluster of differentiation 206 (CD206), and peroxisome proliferator activated receptor- γ (PPAR-γ) were separately examined by RT-PCR, Western blot analysis and immunofluorescence staining. Results Compared with the model group, the numbers of cell in AS-Ⅳ group significantly increased , and the cell physiological status were much better. Compared with the model group, the iNOS immunofluorescence semi quantitative (0.62 ± 0.02 vs. 1.32 ± 0.09), the expression of mRNA (1.51 ± 0.07 vs. 3.46 ± 0.39), and protein (2.30 ± 0.14 vs. 5.16 ± 0.49) significantly reduced in AS-Ⅳ group (P<0.01). The CD206 immunofluorescence semi quantitative (1.01 ± 0.03 vs. 0.61 ± 0.01), the expression of mRNA (0.91 ± 0.03 vs. 0.51 ± 0.01), and protein (0.61 ± 0.04 vs. 0.19 ± 0.01) significantly reduced in AS-Ⅳ group (P<0.01). Compared with the model group, the PPAR-γ immunofluorescence semi quantitative (0.60 ± 0.14 vs. 0.34 ± 0.03), mRNA (2.00 ± 0.14 vs. 1.04 ± 0.03), and protein (0.67 ± 0.05 vs. 0.19 ± 0.01) significantly increased (P<0.01). Conclusions The AS-Ⅳ could attenuate ischemia-reperfusion injury by altering macrophages phenotype through upregulation PPAR-γ. Key words: Astragaloside Ⅳ; Reperfusion injury; Macrophages; PPAR gamma

Effects of Astragaloside IV on tumor necrosis factor-α-induced expressions of matrix metalloproteinases in rat vascular smooth muscle cells

Objective To investigate the effect of Astragaloside IV (AS-IV) on tumor necrosis factor-α (TNF-α)-induced expressions of matrix metalloproteinases (MMPs) in a rat vascular smooth muscle cells (VSMCs) proliferation model and its mechanism. Methods VSMCs were prepared from the thoracoabdominal aorta of rats by using issue-sticking method. Morphology of cells was observed by inverted microscope, and identified by immunohistochemical methods with antibody against SM-α-actin. The model of VSMCs proliferation and migration was established by TNF-α inducer in vitro, and randomly divided into the following groups: the control group, the TNF-α group, the TNF-α+ AS-IV (0.5 μg/ml) group, the TNF-α+ AS-IV (5 μg/ml) group, the TNF-α+ AS-IV (25 μg/ml) group, and the TNF-α+ AS-IV (50 μg/ml) group. The effect of AS-IV on TNF-α-induced VSMCs proliferation activity was detected by the caerulein and cholecystokinin octapeptide (CCK-8) method. The quantitative real-time polymerase chain reaction (real-time PCR) and Western blotting were used to examine the effects of AS-IV on the VSMCs-secreted mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2), respectively. Results The proliferative activity, migratory distance and invasive capacity of VSMCs were obviously increased in TNF-α stimulation group versus in control group (all P<0.01), which suggested that TNF-α can promote VSMCs proliferation and migration, and that the rat model of VSMCs proliferation in vitro was successfully established. The results of CCK-8 tests showed that VSMCs proliferation was obvious and the optical density (OD) value was elevated (P<0.01) after a preset time incubation with TNF-α. VSMCs proliferation was inhibited in each AS-IV treatment group, and the OD value was decreased as compared with the TNF-α group. And the inhibitive effect was increased along with the increments of AS-IV concentration and the acting time, which indicated that AS-IV can inhibit TNF-α-induced VSMCs proliferation in a time- and dose-dependent manner. The results of real-time PCR and Western blotting assays indicated that TNF-α changed the ratio of MMPs to the tissue inhibitors of metalloproteinases (TIMPs) by down-regulating active MMP-2 expression without influencing proMMP-2 and TIMP-2 expressions, and thus promoted the degradation of ECM. AS-IV (0.5-50 μg/ml) inhibited VSMCs proliferation and migration by down-regulating the TNF-α-induced MMP-2 overexpression in a dose-dependent manner, up-regulating the mRNA and protein expressions of TIMP-2, and modulating the ratio of MMPs to TIMPs, thereby inhibited the degradation of ECM. Conclusions AS-IV inhibits VSMCs proliferation and migration in a time- and dose-dependent manner. AS-IV inhibits VSMCs proliferation and migration by down-regulating TNF-α-induced MMP-2 overexpression, up-regulating TIMP-2 expressions, and normalizing the ratio of MMPs to TIMPs. Therefore, AS-IV inhibits the degradation of ECM, which may play a role in the prevention and treatment of in-stents restenosis after PCI. Key words: Astragalus; Muscle, smooth, vascular; Tumor necrosis factor-alpha; Matrix

Astragaloside IV protects against polymicrobial sepsis through inhibiting inflammatory response and apoptosis of lymphocytes

BackgroundSepsis is a major clinical challenge in modern medicine, representing one of the leading causes of death in developed countries. The syndrome is a consequence of a dysregulated immune response, including early uncontrolled systemic inflammation and prolonged immunosuppression in the late phase. The present study was conducted to investigate the therapeutic effects of astragaloside IV (ASI-IV) on the cecal ligation and puncture (CLP)-induced sepsis in mice. Materials and methodsC57BL/6 mice were randomly divided into sham control + vehicle, CLP + vehicle, and CLP + ASI-IV groups. ASI-IV (3 mg/kg) was intravenously injected 1 h after CLP surgery. Survival rate, bacterial clearance, inflammatory mediators, phagocytes emigration, histopathology, and lymphocyte apoptosis were examined. The effects of ASI-IV on peritoneal macrophage activation and its underlying mechanisms were also evaluated. ResultsWe reported that treatment with ASI-IV significantly improved survival in septic mice. In agreement with this protective effect, the pathologic damage that was typically seen in lung and spleen was ameliorated; the level of bacterial burden was lessened; inflammatory cytokines and chemokines in circulation were profoundly reduced; lymphocyte apoptosis was inhibited. ASI-IV suppressed LPS-induced macrophage activation through inhibiting NF-κB and ERK1/2 signaling pathways. ConclusionsASI-IV protected mice against polymicrobial sepsis by inhibiting inflammatory response and lymphocyte apoptosis. Therefore, ASI-IV might provide a novel therapeutic approach for septic patients.

Astragaloside IV Regulates Expression of ATP-sensitive Potassium Channel Subunits after Ischemia-reperfusion in Rat Ventricular Cardiomyocytes

Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb Astragalus membranaceus, which has been widely used to treat cardiovascular disease. Recent studies have shown that AsIV can potentially protect the heart from myocardial ischemic injury, but the mechanisms of action are unknown. ATP-sensitive potassium (KATP) channels are activated during ischemia and exert a compensatory protective effect on cardiomyocytes. We therefore examined the effects of AsIV on KATP channel currents and channel expression in isolated rat ventricular cardiomyocytes after ischemia-reperfusion injury. Forty Wistar rats were divided into five groups: control group, ischemia-reperfusion (IP) group, IP + glibenclamide group, IP + pinacidil group and IP + AsIV group. The ischemia-reperfusion injury model was established in enzymatically isolated ventricular cardiomyocytes by perfusion with calcium-free Tyrode solution for 10 min, arrest for 30 min, and reperfusion for 45 min. The different drugs were applied for 10-15 min, and the KATP channel current (I(KATP)) was recorded with voltage-clamp mode by whole-cell patch-clamp technique. Protein and mRNA expression of the KATP channel subunits Kir6.1, Kir6.2, SUR2A and SUR2B was quantified using western blotting and real-time PCR. The KATP current in IP group was significantly greater than that in control group (211.45 +/- 33.67 vs 83.51 +/- 23.67 pA; P < 0.01). Glibenclamide (10 micromol/L) blocked KATP currents, whereas both AsIV (1 mg/L) and the known channel opener pinacidil (50 micromol/L) significantly increased I(KATP) (P < 0.05). Consistent with this, AsIV significantly up-regulated protein and mRNA expression of Kir6.1, Kir6.2, SUR2A, SUR2B (P < 0.01 vs IP group). The protective effects of AsIV in ischemia-reperfusion injury may be related to the up-regulation of several KATP channel subunits and facilitation of KATP currents.