Medical School, Hunan University of Chinese Medicine, No.300 Xueshi Road, Hanpu Science & Education District, Changsha, Hunan province, 410208, China.
J Ethnopharmacol. 2021 Mar 25;268:113556. doi: 10.1016/j.jep.2020.113556. Epub 2020 Nov 4.
Astragaloside IV (AST IV) is the active component of Astragalus membranaceus (Fisch.) Bunge, which regulates lipid and carbohydrate metabolism and improves insulin resistance. In this study, we investigated the effects of AST IV on insulin resistant cells and a non-alcoholic fatty liver disease (NAFLD) model induced by high-concentration insulin or oleic acid (OA) in HepG2 cells, as well as the associated regulatory markers.
First, the target of AST IV was predicted via pharmacophore model matching and molecular docking. Then, enzyme kinetics experiments were conducted in vitro to determine the effect of AST IV on the target protein. Next, AST IV's toxicity was tested on HepG2 cells in vitro, through an insulin resistance model and an NAFLD model, by high-concentration insulin or OA, respectively. To explore the effects of AST IV on insulin resistance and lipid metabolism, we detected the related indexes of glucose and lipid metabolism through commercially available kits. Relevant proteins were also detected by Western blot to provide future direction for study.
Our preliminary results of pharmacophore model matching and molecular docking suggested that AST IV and protein tyrosine phosphatase 1B (PTP1B) can be well-combined through hydrogen bonding. Further, the enzyme kinetics experiment showed that AST IV was an effective and specific inhibitor to PTP1B. We found that the protein level of PTP1B in HepG2 cells was significantly increased after treating with high-concentration insulin or OA. Additionally, the intervention of AST IV significantly increased glucose consumption in an insulin resistance model and reduced the content of triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) in the NAFLD model. Moreover, the 2-N-(7-nitrobenze-2-oxa-1, 3 diazol-4-yl) (2-NBDG) uptake rate in the NAFLD model was also greatly improved. These results validated the effects of AST IV on improving insulin resistance and lipid accumulation. Furthermore, Western blot results illustrated that AST IV suppressed PTP1B and increased levels of phosphorylated insulin receptor (p-IR) and phosphorylated insulin receptor substrate-1 (p-IRS-1) in insulin-resistant HepG2 cells, while also decreasing protein levels of PTP1B and sterol element regulatory binding protein-1c (SREBP-1c) in the NAFLD model.
This study demonstrated that AST IV inhibited PTP1B and effectively improved insulin resistance in insulin-resistant HepG2 cells and triglyceride accumulation in OA-treated HepG2 cells.
黄芪甲苷（AST IV）是黄芪（膜荚黄芪）的活性成分，可调节脂质和碳水化合物代谢，改善胰岛素抵抗。在这项研究中，我们研究了 AST IV 对高浓度胰岛素或油酸（OA）诱导的 HepG2 细胞胰岛素抵抗和非酒精性脂肪性肝病（NAFLD）模型的影响，以及相关的调节标记物。
首先，通过药效团模型匹配和分子对接预测 AST IV 的靶标。然后，通过体外酶动力学实验确定 AST IV 对靶蛋白的作用。接下来，通过高浓度胰岛素或 OA 分别在胰岛素抵抗模型和 NAFLD 模型中检测 AST IV 在 HepG2 细胞中的毒性。为了探讨 AST IV 对胰岛素抵抗和脂质代谢的影响，我们通过商业试剂盒检测了葡萄糖和脂质代谢的相关指标。还通过 Western blot 检测了相关蛋白，为进一步研究提供了方向。
药效团模型匹配和分子对接的初步结果表明，AST IV 和蛋白酪氨酸磷酸酶 1B（PTP1B）可以通过氢键很好地结合。此外，酶动力学实验表明 AST IV 是 PTP1B 的有效和特异性抑制剂。我们发现，用高浓度胰岛素或 OA 处理后 HepG2 细胞中 PTP1B 的蛋白水平显著增加。此外，AST IV 的干预显著增加了胰岛素抵抗模型中的葡萄糖消耗，并降低了 NAFLD 模型中的甘油三酯（TG）、总胆固醇（TC）和游离脂肪酸（FFA）含量。此外，NAFLD 模型中的 2-N-（7-硝基苯并-2-氧代-1,3-二唑-4-基）（2-NBDG）摄取率也大大提高。这些结果验证了 AST IV 改善胰岛素抵抗和脂质堆积的作用。此外，Western blot 结果表明，AST IV 抑制了 PTP1B，增加了胰岛素抵抗 HepG2 细胞中磷酸化胰岛素受体（p-IR）和磷酸化胰岛素受体底物-1（p-IRS-1）的水平，同时降低了 NAFLD 模型中 PTP1B 和固醇元件调节结合蛋白-1c（SREBP-1c）的蛋白水平。
本研究表明，AST IV 抑制了 PTP1B，有效改善了胰岛素抵抗 HepG2 细胞中的胰岛素抵抗和 OA 处理的 HepG2 细胞中的甘油三酯堆积。
