Formulation and technology of oxymatrine-astragaloside IV coloaded liposomes based on quality by design / 药学学报

To optimize the formulation and technology of oxymatrine-astragaloside IV coloaded liposomes (Om-As-Lip) based on quality by design (QbD) principles, and further to verify the feasibility of its amplification process, Om-As-Lip was prepared by ethanol injection combined with pH gradient method. The critical material attributions of Om-As-Lip were evaluated by dual-risk analysis tools and Plackett-Burman design (PBD). The formulation of Om-As-Lip was further optimized with the Box-Behnken design (BBD). The design space was also established based on the contour plots of BBD. In order to further investigate the amplification process of Om-As-Lip, the critical process parameters of high-pressure homogenization (HPH) were optimized by single-factor test, and the quality of the final product was also evaluated. The results of risk analysis and PBD confirmed that the astragaloside concentration, cholesterol concentration, and phospholipid ratio (HSPC∶SPC) were the ctitical material attributes. The model established by BBD had a good predictability, and the optimized mass ratio of As to phospholipids was 1∶40, cholesterol to phospholipids was 1∶10, HSPC to SPC was 51∶9. The design space of Om-As-Lip was as follows: the ratio of cholesterol to phospholipids was 1∶12-1∶5 and HSPC to SPC was 1∶7-17∶3. The optimized high-pressure homogenization pressure was 600 bar, temperature was 4 ℃, and cycle times was 6 times for HPH-Om-As-Lip. The quality of Om-As-Lip prepared based on the QbD concept can meet the expected CQAs, and the formulation and technology established can provide a reliable experimental basis for its future development and applications.

Astragaloside IV inhibits Ang H-induced cell proliferation and collagen expression in cardiac fibroblasts by activating short-chain acyl-CoA dehydrogenase / 中国药理学通报

Aim To explore the effect of astragaloside IV (AS-IV) on cell proliferation and collagen expression in cardiac fibroblasts (CFs) of rats induced with angiotensin II (Ang II) and its mechanism. Methods CFs were pretreated with short-chain acyl-CoA dehydrogenase (SCAD) siRNA1186 for 12 h and then co-treated with Ang TJ and AS-IV for 36 h. The expressions of SCAD, α-SMA, collagen I and collagen III in CFs were detected by Western blot. mRNA expression levels of SCAD, a-SMA, collagen I and collagen III in CFs were detected by quantitative real-time PCR. The SCAD enzymatic activity, the content of ATP, hydroxyproline and free fatty acid were measured by detection kits. Results The expression of α-SMA, collagen I and collagen III were up-regulated (all P < 0. 01) in CFs induced by Ang II compared with the control cells, and the expression and enzymatic activity of SCAD significantly decreased (P < 0. 01, P< 0. 05). The content of ATP decreased (P < 0.01), and the content of hydroxyproline and free fatty acids increased (all P < 0.01). Compared with Ang II group, SCAD expression and enzymatic activity, and ATP content were significantly increased (all P < 0.01) in Ang II + AS-TV group, but the content of hydroxyproline and free fatty acids, and the expression of α-SMA, collagen I and collagen III significantly decreased (all P < 0.01). However, compared with the Ang II + NC group, there was no significant difference in all indices in the Ang II + SiRNA1186 + AS-TV group. The protective effect of AS-TV on Ang II -induced cell proliferation and collagen expression in CFs was eliminated by the interference of SCAD SiRNA1186. Conclusions AS-IV may inhibit Ang II-induced cell proliferation and collagen expression in CFs by activating SCAD.

Astragaloside IV improves podocyte injury and mitochondrial dysfunction induced by high glucose and inhibits Notch pathway activation / 西安交通大学学报(医学版)

【Objective】 To investigate the protection of astragaloside IV from high glucose induced podocyte injury and mitochondrial dysfunction and its molecular mechanisms. 【Methods】 The model of podocyte injury induced by high glucose (30 mmol/L glucose) was established, and the model cells were treated with low, medium and high doses of astragaloside IV respectively; cell activity was detected by CCK-8. Apoptosis was detected by TUNEL staining. Mitochondrial membrane potential was detected by JC-1 fluorescence probe. ATP content was detected by the kit. The expression levels of apoptosis and podocyte injury related proteins and Notch pathway related proteins were detected by Western blotting. 【Results】 Compared with the control group, cell activity was decreased, apoptosis level was increased (P<0.05), anti-apoptotic protein (Bcl2) expression was decreased, and apoptosis protein (Bax, cleaved-caspase 9, cleaved-caspase 3) expressions were increased (all P<0.05) in HG group. Compared with HG group, HG+AS-IV improved cell activity and apoptosis level induced by high glucose (P<0.05), increased expression of anti-apoptotic protein (Bcl2), and decreased expressions of apoptotic protein (Bax, cleaved-caspase 9, and cleaved-caspase 3) (all P<0.05). Compared with the control group, mitochondrial dysfunction occurred in HG group, JC-1 monomer content increased, and ATP content decreased (all P<0.05). Compared with HG group, HG+AS-IV improved mitochondrial dysfunction, increased JC-1 polymer content and ATP content (P<0.05). In addition, compared with the control group, the expression of Notch pathway-related protein was decreased in HG group (P<0.05). Compared with HG group, Notch pathway-related protein expression was increased in HG+AS-IV group (all P<0.05). Molecular docking results showed that AS-IV could bind Notch1. 【Conclusion】 Astragaloside IV can improve podocyte injury and mitochondrial dysfunction induced by high glucose, possibly by inhibiting Notch pathway activation.

Astragaloside IV alleviates lung inflammation in Klebsiella pneumonia rats by suppressing TGF-β1/Smad pathway

Astragaloside IV is a biologically active substance derived from the traditional Chinese medicine Astragalus mambranaceus Bunge, and has antioxidant, anti-inflammatory, and anti-apoptotic properties. In this study, we aimed to investigate the effects of astragaloside IV on Klebsiella pneumonia rats and the underlying mechanisms. Klebsiella pneumoniae (K. pneumoniae) rats were treated with different dosages of astragaloside IV (5, 10, and 20 mg/kg) by intragastric administration. The levels of pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) were determined. Pathological changes of lung tissue were inspected by HE staining. The expression of transforming growth factor (TGF)-β1 in lung tissue was determined with immunohistochemistry, and the expression levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3, IκBα/p-IκBα, and p65/p-p65 in lung tissue were determined by western blot. The mechanism was further investigated with TGF-β1 inhibitor SB-431542. Astragaloside IV reduced the elevated levels of pro-inflammatory cytokines caused by K. pneumoniae and improved lung tissue damage in a dose-dependent manner. Astragaloside IV also decreased the expression of TGF-β1/Smad signaling pathway-related proteins and decreased the protein levels of inflammation-related p-IκBα and p65 in lung tissues induced by K. pneumoniae. Additionally, it was found that the effects of 20 mg/kg astragaloside IV were similar to SB-431542, which could improve pulmonary fibrosis induced by K. pneumoniae, decrease the levels of TGF-β1/Smad signaling pathway-related proteins in lung, and reduce inflammation at the same time. Astragaloside IV could alleviate the inflammation of rat pneumonia induced by K. pneumoniae through suppressing the TGF-β1/Smad pathway.

Astragaloside IV for Heart Failure: Preclinical Evidence and Possible Mechanisms, A Systematic Review and Meta-Analysis / 中国结合医学杂志

OBJECTIVE@#To explore the cardioprotective effects of astragaloside IV (AS-IV) in heart failure (HF).@*METHODS@#PubMed, Excerpta Medica Database (EMBASE), Cochrane Library, Web of Science, Wanfang Database, Chinese Bio-medical Literature and Retrieval System (SinoMed), China Science and Technology Journal Database (VIP), and China National Knowledge Infrastructure (CNKI) were searched from inception to November 1, 2021 for animal experiments to explore AS-IV in treating HF in rats or mice. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular weight-to-body weight (LVW/BW) and B-type brain natriuretic peptide (BNP) were recorded. The qualities of included studies were assessed by the risk of bias according to the Cochrane handbook. Meta-analysis was performed using Stata 13.0.@*RESULTS@#Twenty-one articles involving 558 animals were considered. Compared with the control group, AS-IV improved cardiac function, specifically by increasing LVEF (mean difference (MD)=6.97, 95% confidence interval (CI)=5.92 to 8.03, P<0.05; fixed effects model) and LVFS (MD=7.01, 95% CI=5.84 to 8.81, P<0.05; fixed effects model), and decreasing LVEDD (MD=-4.24, 95% CI=-4.74 to -3.76, P<0.05; random effects model) and LVESD (MD=-4.18, 95% CI=-5.26 to -3.10, P<0.05; fixed effects model). In addition, the BNP and LVW/BW levels were decreased in the AS-IV treatment group (MD=-9.18, 95% CI=-14.13 to -4.22, P<0.05; random effects model; MD=-1.91, 95% CI=-2.42 to -1.39, P<0.05; random effects model).@*CONCLUSIONS@#AS-IV is a promising therapeutic agent for HF. However, this conclusion needs to be clinically validated in the future.

Astragaloside IV Attenuates Polymicrobial Sepsis-Induced Cardiac Dysfunction in Rats via IKK/NF-κB Pathway / 中国结合医学杂志

OBJECTIVE@#To evaluate the protective effects of Astragaloside IV (AST) in a rat model of myocardial injury induced by cecal ligation and puncture (CLP).@*METHODS@#The model of sepsis-induced cardiac dysfunction was induced by CLP. Using a random number table, 50 specific pathogen free grade of Sprague Dawley rats were randomized into 5 groups: the sham group (sham), the model group (CLP, 18 h/72 h) and AST group (18 h/72 h). Except the sham group, the rats in other groups received CLP surgery to induce sepsis. CLP groups received intragastric administration with normal saline after CLP. AST groups received intragastric administration with AST solution (40 mg/kg) once a day. The levels of inflammatory mediators and oxidative stress markers in the serum of the septic rats were determined via enzyme-linked immunosorbent assay (ELISA) at different time point, such as interleukin 6 (IL-6), IL-10, high mobility group box-1 protein B1 (HMGB-1), superoxide dismutase (SOD), and malondialdehyde (MDA). Cardiac function was determined by echocardiography. Moreover, changes in myocardial pathology were evaluated using hematoxylin and eosin staining. The levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were analysed to determine the status of CLP-induced myocardium. In addition, the apotosis of myocardial cells was analysed by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). The protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), IκB kinase α (IKKα), nuclear factor kappa B p65 (NF-κB p65) were detected by Western blot analysis. Moreover, survival rate was investigated.@*RESULTS@#AST improved the survival rate of CLP-induced rats by up to 33.3% (P<0.05). The cardioprotective effect of AST was observed by increased ejection fraction, fractional shortening and left ventricular internal diameter in diastole respectively (P<0.01 or P<0.05). Subsequently, AST attenuated CLP-induced myocardial apoptosis and the ratio of Bcl-2/Bax in the myocardium, as well as the histological alterations of myocardium (P<0.01 or P<0.05); the generation of inflammatory cytokines (IL-6, IL-10, HMGB-1) and oxidative stress markers (SOD, MDA) in the serum was significantly alleviated (P<0.01 or P<0.05). On the other hand, AST markedly suppressed CLP-induced accumulation of IKK-α and NF-κB p65 subunit phosphorylation (P<0.01 or P<0.05).@*CONCLUSIONS@#AST plays a significant protective role in sepsis-induced cardiac dysfunction and survival outcome. The possible mechanism of cardioprotection is dependent on the activation of the IKK/NF-κB pathway in cardiomyocytes.

Astragaloside IV alleviating chronic intermittent hypoxia-induced cardiac injury by enhancing autophagy / 解剖学报

Objective To investigate the protective effects of astragaloside IV (AS IV) on chronic intermittent hypoxia (CIH) -induced cardiac injury. Methods Twenty-four male adult Sprague Dawley rats were randomly assigned to control, CIH, CIH+ASIV, and ASIV group, 6 rats in each group. Circular nitrogen and oxygen were filled to make oxygen concentration change between 9%-21% for the CIH treated rats. The exposure cycle was repeated every 3 minutes, 8 hours/ day for 35 days. ASIV was given by intragastric administration daily before intermittent hypoxia exposure in the CIH+ASIV group and AS IV group. The control group and CIH group were given normal saline of the same quantity. Echocardiography was used to analyse cardiac function. Myocardial structure was assessed by HE and wheat germ agglutinin staining. The apoptosis of cardiomyocytes was detected by TUNEL assay. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in heart were detected by commercial kits. Western blotting was used to evaluate the levels of Bcl-2, Bax, LC3, Beclinl, P62, and mammalian target of rapamycin (mTOR). Results In the CIH group, the left ventricular ejection fraction (LVEF) and left ventricular internal diameter at end-systole (LVIDs) were inhibited, the myocyte cells showed disordered arranged, enlarged diameters and higher apoptosis rate. The MDA content was significantly elevated and the SOD activity decreased in CIH group when compared with those of control. What's more, the expression level of Beclin 1 decreased while the P62 expression and the p-mTOR/mTOR ratio increased in the CIH group. Compared with the model group, the LVEF, LVIDs, SOD activity, LC3 H / I ratio, and Beclinl expression of rats in the CIH + AS IV group increased. The cardiomyocytes in the rats of CIH + ASIV group showed normal arrangement and diameters. The apoptosis rate, MDA content, P62 expression and the p-mTOR/mTOR ratio decreased in the CIH+ASIV group when compared with the CIH group. Conclusion AS IV can alleviate CIH-induced cardiac injury by promoting autophagy via mTOR.

Protective effect of astragaloside on renal injury induced by radiation through toll-like receptor 4/nuclear factor kappa B pathway / 解剖学报

Objective To explore whether astragaloside IV (AS-IV) can protect radiation-induced kidney injury through toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB)-mediated signaling pathway. Methods The mice were randomly divided into normal control group, DMSO solvent group, irradiation group (IR), IR + AS-IV 20 mg/kg group and IR + AS-IV 40 mg/kg group. One month after intraperitoneal injection of AS-IV, the mice were irradiated with 8 Gy 60Coγ and to the content of serum creatinine (Cr) and uric acid (BUA) in serum were determined, HE and immunohistochemical staining, and expression of TLR4/NF-κB signaling pathway related protein in kidney were performed. Results Compared with the control group, the levels of Cr and BUA in the serum of the radiation group increased significantly (P<0.001), glomerular atrophy and tubular expansion, TLR4 and myeloid differentiation factor 88(MyD88) positive expression increased significantly (P< 0.001), and the expression of TLR4/NF-κB signaling pathway-related proteins[ TLR4, MyD88, NF-κB, interleukin-1 β (IL-Iβ), tumor necrosis factor α (TNF-α)] increased significantly (P< 0.01); AS-IV pretreatment can reduce the content of Cr and BUA in serum (P<0.05, P<0.01 or P<0.001), significantly improve the pathological response caused by radiation, and reduce the expression of TLR4/NF-κB signaling pathway-related proteins (P<0.05 or P<0.01). Conclusion AS-IV may down-regulate the release of inflammatory factors through the TLR4/NF-κB signaling pathway, thereby improving radiation-induced kidney injury in mice and playing a protective role.

Suppression of OCT2/MRP2 decreases kidney injury and enhances the chemosensitivity of co-administration of cisplatin and astragaloside IV / 药学学报

Kidney injury and decreased chemosensitivity of tumor cells are obstacles with cisplatin (CDDP) chemotherapy. Down-regulation of the organic cation transporter 2 (OCT2) and multidrug resistance-associated protein 2 (MRP2) is a key means to alleviate CDDP-induced kidney injury and increase chemosensitivity. Astragaloside IV (AS IV) is obtained from the well-known traditional Chinese herb Astragalus membranaceus. This study explored the role of AS IV in preventing kidney injury and enhancing the antitumor effect of CDDP by suppressing OCT2 expression in kidney and MRP2 in tumors. This project was reviewed and approved by the Animal Ethics Committee of the First Hospital of Jilin University. The effects of AS IV on CDDP inhibition of tumor growth and promotion of apoptosis were assessed in Lewis lung tumor (LLC)-bearing mice by H&E and TUNEL staining. Kidney injury was assessed by serum biochemical parameters and H&E staining. We used Western blotting and immunohistochemistry assays to detect OCT2 and MRP2 expression in kidney and tumor. The concentration of CDDP in kidney and tumor was measured by HPLC-MS/MS. AS IV enhanced CDDP chemosensitivity by increasing tumor cell apoptosis and slowing tumor growth, and decreased kidney injury as evidenced by lower blood creatinine (Cr) and blood urea nitrogen (BUN). Co-administration of AS IV suppressed MRP2 overexpression induced by CDDP in tumor tissues and may be an important mechanism for enhancing CDDP chemosensitivity. Moreover, AS IV reduced CDDP-induced kidney injury in mice along with suppression of OCT2 expression in kidney. The concentration of CDDP was increased in tumor but decreased in kidney. In total, AS IV not only enhanced the antitumor effect of CDDP by suppressing MRP2 expression in tumor cells, but also decreased kidney injury induced by CDDP. The results provide new insight into the combined use of a chemotherapy drug and natural ingredients to treat cancer.

Effect of astragaloside IV combined with glucocorticoids on puromycin aminonucleoside-induced rat nephropathy model and the associated mechanism / 中华肾脏病杂志

Objective:To investigate the effect and mechanism of astragaloside IV (AS-IV) combined with glucocorticoids in the treatment of puromycin aminonucleoside (PAN) rat nephropathy model.Methods:Forty specific pathogen-free healthy male Wistar rats (150-180 g) were randomly divided into 5 groups: control group, PAN group, AS-IV treatment group (PAN+AS-IV group), methylprednisone (MP) treatment group (PAN+MP group), and AS-IV+MP treatment group (PAN+AS-IV+MP group). The model was established by a single tail vein injection of PAN (50 mg/kg body weight). The treatment groups were given 40 mg·kg -1·d -1 AS-IV by intragastric administration and 15 mg·kg -1·d -1 MP by intraperitoneal injection for 10 consecutive days at the same time of modeling. Urine sample was collected on the 11th day of the experiment. The urine protein, urine creatinine and blood albumin were detected by biochemical analyzer. The changes of nephrin and synaptopodin in renal tissues were detected by immunofluorescence assay, and the expressions of nephrin, RhoA and Rac/Cdc42 proteins were detected by Western blotting. Results:Compared with the control group, urine protein creatinine ratio (uPCR) was significantly increased, serum albumin (Alb) was significantly decreased in the PAN group, nephrin expression was significantly down-regulated, and the expressions of RhoA and Rac/Cdc42 were significantly up-regulated in the renal tissue of the PAN group (all P<0.01). Compared with PAN group, serum Alb levels in PAN+AS-IV group and PAN+AS-IV+MP group were significantly increased (both P<0.01), and the uPCR levels in PAN+MP group ( P<0.05) and PAN+AS-IV+MP group ( P<0.01) were significantly decreased (all P<0.05). Compared with the PAN group, the relative expressions of nephrin in renal tissue of all drug intervention group (PAN+AS-IV group, PAN+MP group and PAN+AS-IV+MP group) were significantly increased, while the relative expressions of RhoA and Rac/Cdc42 were significantly decreased (all P<0.01). The immunofluorescence results suggested that the expressions of nephrin and synaptopodin in renal tissue of PAN group were significantly down-regulated compared with the control group, which were reversed in all treatment groups, and the reversion was most pronounced in the PAN+AS-IV+MP group. Conclusion:Both AS-IV and glucocorticoid can improve PAN-induced podocyte injury, and the combination of the two has synergistic action, which may be related to inhibiting the activation of Rho family signaling pathway.

Effects of astragaloside IV-mediated endothelial progenitor cells derived exosomes on the biological function of human endothelial cells damaged by high glucose / 中国医师杂志

Objective:To investigate the effect of Astragaloside Ⅳ-mediated Endothelial progenitor cells derived exosomes (EPC-Exos) on the biological function of EPC-Exos damaged by high glicose.Methods:EPCs from human umbilical cord blood were isolated and cultured in vitro. the EPC-Exos secreted by EPCs were extracted by ultracentrifugation combined with ultrafiltration, and identified by specific markers CD9, CD63 and CD81, respectively. After the cells were cultured for 24 hours with AS-IV at 100 mg/L and PBS at the same volume, the morphological characteristics of EPC-Exos were observed by transmission electron microscope. Human endothelial cells were isolated, cultured and identified in vitro. The identified endothelial cells were pretreated with 30 mmol/L glucose for 120 h and randomly divided into experimental group and control group, at the same time set the normal group. The cells were cultured for 24 hours, the effects of EPC-Exos on proliferation, adhesion, migration and angiogenesis of endothelial cells damaged by high glucose were observed by using cell counting kit-8 (CCK-8) Cell Proliferation Assay Kit, cell scratch test, adhesion assay and in vitro angiogenesis assay by Matrigel. Results:Compared with the normal group, the proliferation, migration, adhesion and tubulogenesis of human endothelial cells in the control group were significantly lower ( t=24.35, 6.80, 10.65, 9.62, P<0.05). Compared with the control group, the proliferation, adhesion, migration and tubulogenesis of human endothelial cells in the experimental group were significantly enhanced ( t=30.68, 5.99, 5.40, 8.25, P<0.05). Conclusions:EPC-Exos mediated by AS-Ⅳ can significantly improve the biological function of human endothelial cells damaged by high glucose and has the potential to modulate endothelial neovascularization in diabetic rats.

Astragaloside IV suppresses post-ischemic natural killer cell infiltration and activation in the brain: involvement of histone deacetylase inhibition / 医学前沿

Natural killer (NK) cells, a type of cytotoxic lymphocytes, can infiltrate into ischemic brain and exacerbate neuronal cell death. Astragaloside IV (ASIV) is the major bioactive ingredient of Astragalus membranaceus, a Chinese herbal medicine, and possesses potent immunomodulatory and neuroprotective properties. This study investigated the effects of ASIV on post-ischemic brain infiltration and activation of NK cells. ASIV reduced brain infarction and alleviated functional deficits in MCAO rats, and these beneficial effects persisted for at least 7 days. Abundant NK cells infiltrated into the ischemic hemisphere on day 1 after brain ischemia, and this infiltration was suppressed by ASIV. Strikingly, ASIV reversed NK cell deficiency in the spleen and blood after brain ischemia. ASIV inhibited astrocyte-derived CCL2 upregulation and reduced CCR2

Effects and mechanism research of astragaloside IV improving insulin resistance of HepG2 cells based on pharmacophore and molecular docking / 中草药

Objective: To research the effects of astragaloside IV (AST IV) on improving insulin resistance in HepG2 cells, and predict and verify the AST IV possible targets based on pharmacophore model matching and molecular docking. Methods: HepG2 cells insulin resistance model was induced with high concentration insulin. After being interfered by AST IV, the glucose consumption was characterized by glucose test, AST IV possible targets were predicted by pharmacophore model matching and molecular docking, the expressions of related pathway protein were detected by Western blotting. Results: AST IV significantly increased the glucose consumption in insulin-resistant HepG2 cells, the possible target of AST IV may be related to tyrosine phosphotase 1B (PTP1B) based on pharmacophore model matching and molecular docking. The Western blotting results showed that, the level of PTP1B was significantly increased and the levels of p-IR and p-IRS-1 were significantly decreased in insulin-resistant HepG2 cells. The intervention of AST IV decreased the levels of PTP1B, and increased the levels of p-IR and p-IRS-1. Conclusion: AST IV can significantly improve insulin resistance of insulin induced HepG2 cells, and its mechanism is related to inhibiting PTP1B and activating insulin signaling pathways.

In vitro study of astragaloside IV on reversing multidrug resistance of human breast cancer cell MDA-MB-231 to doxorubicin / 中草药

Objective: To investigate the reversal effect of astragaloside IV on multidrug resistance of MDA-MB-231/DOX in breast cancer cells. Methods: The cytotoxicity of astragaloside IV and sensitivity or drug resistance of breast cancer cells to doxorubicin (DOX) before and after treatment were determined by MTT assay. Liposome co-delivery system containing doxorubicin and astragaloside IV (LPs-DOX/AS) was constructed by ethanol injection-ammonium sulfate gradient method. The reversal effect of LPs-DOX/AS on multidrug resistance of breast cancer cells was determined by MTT method. The effect of LPs-DOX/AS on apoptosis was determined by flow cytometry. Results: Astragaloside IV had no significant cytotoxicity to breast cancer cells in the experimental concentration range. After combined with astragaloside IV, the IC50 values of DOX on MDA-MB-231 and MDA-MB-231/DOX cells decreased (P < 0.05), and the intervention effect on drug-resistant cells was more significant (P < 0.01). Compared with free DOX/AS-IV, the IC50 values of LPs-DOX/AS-IV on both breast cancer cells decreased (P < 0.05), and the effect on drug-resistant strains was more significant (P < 0.01). The apoptosis rate of drug-resistant strains treated with LPs-DOX/AS-IV was also significantly higher than that of free drug group (P < 0.05). Conclusion: Astragaloside IV has reversal effect on multidrug resistance of human breast cancer cell MDA-MB-231 to doxorubicin. The combination of astragaloside IV and doxorubicin and its liposome co-delivery system can effectively reverse or sensitize multidrug resistance in breast cancer.

Neuroprotective effect of borneol combined with astragaloside IV and Panax notoginseng saponins in cerebral ischemia reperfusion injury rat model through Notch signaling pathway / 中草药

Objective: To observe the neuroprotective effect of borneol combined with astragaloside IV (AST IV) and Panax notoginseng saponins (PNS) on cerebral ischemia reperfusion injury (CIRI) rat model through Notch signaling pathway. Methods: SD rats were randomly divided into sham group, model group, borneol (7.5 mg/kg) group, AST IV (25 mg/kg) group, PNS (10 mg/kg) group, AST IV (10 mg/kg) + PNS (25 mg/kg) group, borneol (7.5 mg/kg) + AST IV (25 mg/kg) + PNS (10 mg/kg) low dose group, borneol (15 mg/kg) + AST IV (20 mg/kg) + PNS (50 mg/kg) high dose group and edaravone (4 mg/kg) group. Rats in sham group and model group were ig 0.5% CMC-Na, edaravone group was ip drug, and the other groups were ig corresponding drugs, twice a day with an interval of 12 h. The right middle cerebral artery of rat was blocked by a suture method 2 h after last administration to establish a CIRI model. After 2 h of ischemia and 22 h of reperfusion, the eurological function scores were scored and pathological changes of ischemic cortex in brain tissues of rats were observed by HE staining. The expressions of neuron specific nuclear (NeuN) and endothelial barrier antigen (EBA) in ischemic cortex of brain tissue were detected by immunohistochemistry. The expressions of vascular endothelial growth factor (VEGF), Notch1, and intracellular domain of Notch (NICD) in ischemic cortex of brain tissue were detected by Western blotting. Results: The score of neural dysfunction and cell damage rate in model group were significantly increased (P < 0.01); The score of nerve function defect and rate of cell damage in each administration group were significantly reduced (P < 0.05, 0.01), the effect of borneol + AST IV + PNS group was better than that of single drug and AST IV + PNS group (P < 0.05, 0.01). NeuN and EBA protein expressions were significantly decreased in the ischemic cortex of model group (P < 0.01), while NeuN and EBA protein expressions were significantly enhanced in each administration group (P < 0.05, 0.01), and the effect of borneol + AST IV + PNS group was better than that of single drug and AST IV + PNS group (P < 0.05, 0.01). In model group, VEGF protein expression was increased significantly (P < 0.05), while NICD and Notch1 protein expression had no significant change. The expression of VEGF, NICD and Notch1 protein were significantly up-regulated in borneol + AST IV + PNS group (P < 0.01), and the effect of combination of three drugs was better than that of single drug and AST IV + PNS (P < 0.05, 0.01). Conclusion: Borneol, AST IV, and PNS have the effects of preventing neuronal and cerebral microvascular damage after CIRI, and the effect of combination of three drugs was better than that of single drug and AST IV + PNS, which may be related to the activation of the Notch signaling pathway and up-regulation of VEGF expression, thereby, exerting protective effects on ischemic brain tissue.

Optimization of infiltration process of Astragali Radix based on HPLC-DAD- ELSD and response surface design / 中草药

Objective: To optimize the infiltration process of Astragalus (Astragalus membranaceus var. mongholicus) medicinal materials by Box-Behnken response surface method. Methods: Based on the HPLC-DAD-ELSD and response surface design method, the qualified rate of decoction pieces, the content of index components and bending inspection were used as comprehensive inspection indicators, and the three factors of infiltration were selected for response surface experimental design to optimize the infiltration process of Astragalus medicinal materials parameter. Results: The best infiltration process was as following: infiltration temperature was 20 ℃, with water addition of 1:0.988 for 6 h. Under this process, the qualified rate of Astragalus pieces was 95.81%, the content of calycosin-7-glucoside was 0.072%, and the content of astragaloside IV was 0.276 %. Combining fingerprint analysis and heat map analysis, the material basis of A. membranaceus var. mongholicus changed during the infiltration process. The infiltration parameters should be strictly controlled during the infiltration process to ensure uniform quality of the pieces. Conclusion: The optimized Astragalus medicinal material infiltration process is stable and feasible with good reproducibility, which can provide a reference for the mass production process development of Astragalus medicinal slices.

Astragaloside IV ameliorates hypoxia/reoxygenation injury via regulating mitochondrial homeostasis in rat cardiomyocytes / 药学学报

This study was designed to investigate the effect and mechanism of astragaloside IV (ASIV) on mitochondrial morphology and function of rat cardiomyocytes under hypoxia/reoxygenation injury. H9c2 cells were divided into control group, hypoxia/reoxygenation (H/R) group, and H/R + ASIV group. Cell viability and lactate dehydrogenase (LDH) leakage were measured by cell counting kit-8 (CCK-8) and LDH assay kit, respectively. Oxidative stress levels, such as superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA), were analyzed by commercial kits. Intracellular and mitochondrial reactive oxygen species (ROS) levels were detected by dihydroethidium (DHE) and MitoSOX. Changes of the mitochondrial membrane potential were detected using the fluorescent probe JC-1. Opening of mitochondrial permeability transition pore was examined via calcein acetoxymethyl ester (calcein-AM). Apoptosis was assessed using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay kit. To detect protein expression of dynamin-related protein 1 (Drp1), mitofusin1 (Mfn1), Mfn2, Bax, B-cell lymphoma-2 (Bcl-2), and cleaved cysteine-aspartic protease (caspase)-3, Western blot analysis was carried out. Compared with the control group, ASIV (100 μmol·L-1) significantly improved H/R induced cell injury, LDH leakage, decrease of SOD activity, and GSH content, increase of MDA content and ROS content, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, ROS production activation, mitochondrial fission/fusion imbalance, and cell apoptosis. In addition, the effect of ASIV against H/R injury was also verified on primary rat cardiomyocytes. The animal welfare and experimental process follow the rules of Animal Ethics Committee of Zhejiang Chinese Medical University. In conclusion, ASIV may play a protective role in mitochondria by regulating morphological dynamic stability and mitochondrial function, inhibiting excessive synthesis of ROS, improving the internal environment of oxidative stress, reducing cell apoptosis, and thereby protecting against cardiomyocytes’ hypoxia/reoxygenation injury.

Study of astragaloside IV alleviating oxidatIVe stress injury of pc12 cells induced by oxygen-glucose deprIVation/reoxygenation through regulating autophagy / 中国药理学通报

Aim To observe the effects of astragaloside IV on autophagy and oxidative stress induced by oxy gen-glucose deprivation/reoxygenation in PCI2 cells. Methods PCI2 cells were divided into normal group, model group (ox-ygen-glucose deprivation/reoxygenation group) , astragaloside IV group, and autophagy inhibitor + astragaloside IV group. CCK-8 was used to detect cell viability, and ELISA to detect MDA, SOD and GSH-Px. Transmission electron microscope and MDC fluorescent staining were employed to observe the changes of au-tophagosome, and Western blot to detect the protein expression of Beclinl. Results Compared with normal group, the cell activity in model group decreased (P <0.05), MDA content increased, SOD and CSH-Px activity decreased (P < 0.05), and autophagosomes could be seen and the protein expression of Beclinl increased ( P < 0.05). Compared with model group, the cell activity in astragaloside IV group increased ( P < 0. 05), MDA content decreased, SOD and GSH-Px activity increased (P < 0.05), the number of autophagosomes and the protein expression of Beclinl increased (P<0.05). When autophagy inhibitor was given at the same time, the autophagy inhibitor could obviously antagonize the antioxidant effect of astragaloside IV while alleviating autophagy. Conclusions Astragaloside IV can protect PC 12 cells from oxidative stress injury induced by oxygen-glucose deprivation/reoxygenation by up-regulating autophagy.

Main active components combination of Astragalus membranaceus and Angelica sinensis promotes proliferation of aging hematopoietic stem cells / 中草药

Objective To explore the effects of the combination of main active components of Astragalus membranaceus and Angelica sinensis such as astragaloside IV, formononetin, calycosin, campanulin, ferulic acid on aging hematopoietic stem cells (HSCs), and clarify its mechanism through cell cycle regulation. Methods The aging model of HSCs in mice was established with three butyl hydrogen peroxide (t-BHP) to research the effects of five active components of different concentrations on the senescence and the proliferation of HSCs, and seek the main active components which could promote cell proliferation. Finally, HSCs aging model was used to prepare the drug-containing plasm of A. membranaceus combined with A. sinensis at 1:1 ratio. Furthermore, blank control group, model group, blank plasma group, ferulic acid group, astragaloside IV group, formononetin group, calycosin group, calycosin glycoside group, combination group of main active components, drug-containing plasma group of A. membranaceus combined with A. sinensis at 1:1 ratio were acted on aging cells, HSCs senescence rate was tested by SA-β-galactosidase staining and cell proliferation rate was measured by CCK-8 method, cell cycle distribution was determined by flow cytometry, and the protein expression of Cyclin D1 and cyclin dependent kinase 4 (CDK4) were detected by Western blotting. Results Ferulic acid, astragaloside IV, and formononetin significantly promoted the proliferation of aging HSCs and decreased the positive rate of senescent cells, but the effects of calycosin and calycosin glycoside on HSCs proliferation and the positive rate of senescent cells were not significant. The orthogonal experiment showed that the combination of five active components that ferulic acid, formononetin, astragaloside IV were taken as basic factors, and calycosin and calycosin glycoside were taken as secondary factors, had the strongest effect on promoting cell proliferation and decreasing the positive rate of senescent cells. Ferulic acid, astragaloside IV, formononetin, active component combination and drug-containing plasma decreased the positive rate of senescent cells, down-regulated G0/G1 phase cells while up-regulated G2/M + S phase cells, and increased the expression of Cyclin D1 and CDK4 proteins. The above effects in the active component combination group and the drug-containing plasma group were the best. Conclusion The main active components of A. membranaceus and A. sinensis such as ferulic acid, astragaloside IV, and formononetin can promote the proliferation and improve the senescent of aging HSCs, however, calycosin and calycosin glycoside have no obvious effect. The effect of promoting the proliferation is the strongest on aging HSCs when five active components are combined, and the combination can improve HSCs senescence, enhance the transformation of HSCs from static stage to proliferative stage. The main active components and the combination of A. membranaceus and A. sinensis can promote HSCs proliferation and antagonize HSCs senescent, which may be related to regulating the expression of cell cycle related proteins and promoting the transformation of cell cycle.

Study on borneol combined with astragaloside IV and Panax notoginseng saponins promoting bioactive components into brain in cerebral ischemia/reperfusion model of rats / 中草药

Objective To investigate whether borneol can promote the bioactive components of the combination of astragaloside IV (AST IV) and Panax notoginseng saponins (PNS) into the blood-brain barrier of rats with middle cerebral artery occlusion (MCAO)/reperfusion. Methods Using the model of MCAO/reperfusion, rats were randomly divided into sham-operation group, model group, borneol group, AST IV group, PNS group, AST IV + PNS group and borneol + AST IV + PNS group, and the content of AST IV and the bioactive components of PNS (ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1) in the cerebral cortex and the cerebellum of the affected side and the healthy side were determined by liquid chromatography-mass spectrometry (LC-MS/MS). Results AST IV, whether used alone or combined with PNS and borneol, was mainly distributed in the cerebral cortex after oral administration, especially in the affected cerebral cortex. Borneol combined with AST IV and PNS significantly increased the content of AST IV in the affected and the healthy cerebral cortex. The bioactive components of PNS such as ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 was mainly distributed in the affected side of the cerebellum when PNS was used alone. Borneol combined with AST IV + PNS significantly increased the content of ginsenoside Rb1 in the cerebral cortex, especially in the affected cortex, increased the content of Rg1 in the healthy and the affected cortex, and increased the content of notoginsenoside R1 in the cerebral cortex, especially in the affected cortex, as well as in the cerebellum. Conclusion AST IV and the bioactive components of PNS such as ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 have a certain distribution in the cerebral cortex and the cerebellum after cerebral ischemia-reperfusion in rats. AST IV was mainly distributed in the cerebral cortex when it was used alone, ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 were mainly distributed in the cerebellum when PNS was used alone. The combination of borneol combined with AST IV and PNS can promote the gather of AST IV, ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 to the cerebral cortex, especially to the cortex of the ischemia-reperfusion side; Moreover, it can promote the absorption of AST IV, ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 in the cerebral cortex to varying degrees, especially in the affected cortex.