本研究探讨葛根芩连汤(GQD)通过缓解内质网应激(ERS)改善糖代谢的体内外作用机制。分子对接用于预测GQD的主要有效血浆成分与ERS相关靶标之间的结合亲和力。肝组织样本取自正常大鼠,高脂诱导的糖尿病大鼠,用二甲双胍治疗的大鼠,和用GQD治疗的大鼠。提取RNA和蛋白质。qPCR用于测量ERS标记葡萄糖调节蛋白78(GRP78)的mRNA表达,和未折叠的蛋白质反应(UPR)基因肌醇需要酶1(Ire1),激活转录因子6(Atf6),Atf4,C/EBP同源蛋白(Chop),和caspase-12。Westernblot检测GRP78、IRE1、蛋白激酶R样ER激酶(PERK)的蛋白表达,ATF6,X盒结合蛋白1(XBP1),ATF4,CHOP,caspase-12、caspase-9和caspase-3。通过比色法测定肝组织中的钙离子含量。衣霉素诱导6小时体外建立ERS-HepG2细胞模型,和2.5%,5%,和含10%GQD的血清给药9小时。葡萄糖氧化酶法用于测量细胞外葡萄糖水平,流式细胞术检测细胞凋亡,糖原染色测量细胞糖原含量,和免疫荧光检测GRP78的表达。通过比色测定法测量细胞内钙离子含量。而Westernblot用于检测GRP78和ERS诱导的IRE1,PERK,ATF6和真核翻译起始因子2α(eIF2α)磷酸化。此外,胰岛素受体底物1(IRS1)的磷酸化水平,磷脂酰肌醇3-激酶调节亚基p85(PI3Kp85),和蛋白激酶B(Akt),参与胰岛素信号通路,也被测量了。此外,c-JunN末端激酶(JNKs)的磷酸化水平,它们参与了ERS和胰岛素信号通路,通过蛋白质印迹测量。分子对接结果表明,GRP78、IRE1、PERK、ATF4和各种化合物,如黄芩素,小檗碱,Daidzein,jateorhizine,甘草苷,巴马汀,葛根素和沃果诺甙有很强的结合亲和力,表明GQD可能干扰ERS诱导的UPR。体内结果显示GQD下调糖尿病大鼠Ire1、Atf6、Atf4、Grp78、caspase-12和Chop的mRNA转录,并下调GRP78,IRE1,PERK,以及ERS诱导的凋亡因子ATF4和CHOP,caspase-12,caspase-9和caspase-3,同时上调XBP1以增强适应性UPR。此外,GQD增加肝组织中钙离子含量,这促进了正确的蛋白质折叠。体外结果表明,GQD增加了ERS诱导的HepG2细胞的葡萄糖消耗,而没有显着影响细胞活力,肝糖原合成增加,下调ATF6和p-eIF2α(Ser51),并下调IRE1,PERK,和GRP78,以及p-IRS1(Ser312)和p-JNKs(Thr183/Tyr185),同时上调p-PI3Kp85(Tyr607)和p-Akt(Ser473)。这些发现表明GQD减轻了肝脏中过度的ERS,减少胰岛素抵抗,并改善体内和体外肝脏葡萄糖代谢。
This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.