肾气丸加减方对高糖高脂诱导的MIN6细胞保护作用及PI3K/AKT/GSK-3β信号通路的影响  

Effects of New Shenqi Pill on Protection of high-sugar/high-fat Induced MIN6 Cells and Influence on the PI3K/AKT/GSK-3βSignaling Pathway

作　　者：张月颖 张珊 温志歌 史佩玉 王皓朔 倪青[1] ZHANG Yueying;ZHANG Shan;WEN Zhige;SHI Peiyu;WANG Haoshuo;NI Qing(Guang’anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China)

机构地区：[1]中国中医科学院广安门医院内分泌科,北京100053

出　　处：《环球中医药》2023年第10期1945-1952,共8页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(82174354)。

摘　　要：目的利用MIN6细胞损伤模型探讨肾气丸加减方通过调控磷脂酰肌醇3-激酶/丝氨酸—苏氨酸激酶/糖原合成激酶3β(phosphoinositide3-kinase/threonine-protein kinase/glycogen synthase kinase-3β,PI3K/AKT/GSK-3β)信号通路保护胰岛β细胞功能的机制。方法将MIN6细胞分为空白组,模型组,肾气丸加减方低、中、高剂量组以及PI3K阻滞剂组(LY294002)。用CCK-8法检测各组细胞活性,胰岛素放射免疫分析试剂盒检测各组MIN6细胞上清液胰岛素含量,流式细胞术检测各组细胞凋亡率,免疫蛋白印迹法检测各组细胞中PI3K、磷酸化(phosphorylated,p)-PI3K、AKT、p-AKT、GSK-3β、p-GSK-3β及胰腺十二指肠同源盒-1(pancreatic duodenal homeobox-1,PDX-1)和肌腱膜纤维肉瘤癌基因同源物A(v-maf musculoaponeurotic fibrosarcoma oncogene homologue A,MafA)蛋白的表达。结果与空白组比较,模型组MIN6细胞活性明显下降,胰岛素分泌水平下降,细胞凋亡率明显升高(P<0.01);与模型组比较,肾气丸加减方低、中、高剂量组细胞活力升高,胰岛素分泌水平升高,细胞凋亡率均降低(P<0.05或P<0.01);各干预组间比较差异无统计学意义(P>0.05)。与空白组比较,模型组PI3K/p-PI3K、AKT/p-AKT、p-GSK-3β蛋白表达明显下调,GSK-3β蛋白表达上调(P<0.05),PDX-1和MafA蛋白表达明显下调(P<0.01);与模型组比较,肾气丸加减方中、高剂量组PI3K/p-PI3K、AKT/p-AKT、p-GSK-3β蛋白表达均明显上调(P<0.05或P<0.01),GSK-3β蛋白表达明显下调(P<0.01);PDX-1和MafA蛋白表达明显上调(P<0.01);加入通路阻滞剂LY294002后肾气丸加减方上述作用被抵消(P<0.05或P<0.01)。结论肾气丸加减方可提高糖脂毒性作用下MIN6细胞活力,减轻细胞凋亡,促进胰岛素分泌,以中、高剂量效果较佳,其机制可能与激活PI3K/AKT/GSK-3β信号通路、抑制GSK-3β的表达有关,进而升高PDX-1和MafA的蛋白表达,恢复胰岛β细胞功能。Objective To explore the effect of New Shenqi Pill on regulating phosphoinositide 3-kinase/threonine-protein kinase/glycogen synthase kinase-3β(PI3K/AKT/GSK-3β).Using the MIN6 cell injury modelβsignal pathway protection of pancreatic isletsβand the mechanism of cellular function.Methods MIN6 cells were divided into blank group,model group,low,medium,and high dose groups of New Shenqi Pill,and PI3K blocker group(LY294002).CCK-8 method was used to detect cell activity in each group,Insulin radioimmunoassay kit was used to detect insulin content in the supernatant of MIN6 cells in each group,flow cytometry was used to detect cell apoptosis rate in each group,Western blot method was used to detect PI3K,phosphorylated(p)-PI3K,Akt,p-AKT,and GSK-3β,p-GSK-3βand the expression of pancreatic duodenal homologous box(PDX-1)and tendon fibrosarcoma oncogene homologous A(MafA)protein.Results Compared with the blank group,the MIN6 cell activity was decreased in the model group,the insulin secretion level was decreased,and the cell apoptosis rate was increased(P<0.01);compared with the model group,the low,medium,and high dose groups of New Shenqi Pill showed an increase in cell viability,an increase in insulin secretion level,and a decrease in cell apoptosis rate(P<0.05 or P<0.01);there was no statistically significance difference between the intervention groups(P>0.05).Compared with the blank group,the protein expression of PI3K/p-PI3K,AKT/p-AKT,p-GSK-3βsignificant downregulation of protein expression,GSK-3βupregulation of protein expression(P<0.05),PDX-1 and MafA were significantly downregulated(P<0.01)in the model group;compared with the model group,the middle and high dose groups of New Shenqi Pill PI3K/p-PI3K,AKT/p-AKT,p-GSK-3β.The protein expression was significantly upregulated(P<0.05 or P<0.01),GSK-3β.The protein expression was significantly downregulated(P<0.01);the expression of PDX-1 and MafA proteins was significantly upregulated(P<0.01);after adding pathway blocker LY294002,the above effects of the modified

关 键 词：肾气丸加减方 高糖高脂毒性 MIN6细胞 脂酰肌醇3-激酶/丝氨酸—苏氨酸激酶/糖原合成激酶3β 胰腺十二指肠同源盒-1/肌腱膜纤维肉瘤癌基因同源物A 

分 类 号：R285.5[医药卫生—中药学]