双益方剂及其有效成分对胰岛素抵抗小鼠胰岛β细胞株胰岛素分泌、增殖、凋亡的影响

摘要

目的 观察双益方及其有效成分对胰岛素抵抗的小鼠胰岛β细胞株Min6胰岛素分泌水平和细胞增殖、凋亡的影响,并探讨其作用机制.方法 培养Min6细胞并分为正常组、IR模型组、二甲双胍组、双益方组、双益方组方单味中药组(山茱萸、黄芪、黄连、葛根、桑白皮、佩兰)及双益方有效成分组(1-脱氧野尻霉素、小檗碱、黄芪甲苷、熊果酸、马钱苷、毛蕊异黄酮葡萄糖苷、葛根素).正常组加入无酚红培养液正常培养;其余各组加入葡萄糖和棕榈酸制作胰岛素抵抗的Min6细胞模型.二甲双胍组加入100μg/L的二甲双胍.双益方组、山茱萸组、黄芪组、黄连组、葛根组、桑白皮组、佩兰组、葛根素组、1-脱氧野尻霉素组、小檗碱组、黄芪甲苷组、马钱苷组、熊果酸组、毛蕊异黄酮葡萄糖苷组分别加入50、100、200μg/L的相应药物.各组于给药24 h后进行胰岛素分泌实验,检测胰岛素分泌水平.各组分别于给药24、48 h采用CCK-8试剂盒检测细胞增殖能力.于给药24 h后采用双染法检测正常组、IR模型组、二甲双胍组、双益方组的凋亡细胞,计算细胞凋亡率;采用Western blotting法检测细胞中的ERK1/2、p-ERK1/2、MEK1/2蛋白.结果 IR模型组细胞高糖、低糖刺激下胰岛素分泌水平均低于正常组(P均<0.05).二甲双胍组、黄芪组、葛根组、双益方组、黄芪甲苷组、山茱萸组、1-脱氧野尻霉素组、葛根素组、小檗碱组胰岛素分泌水平高于IR模型组(P均<0.05).IR模型组细胞增殖能力低于正常组(P均<0.05);二甲双胍组、双益方组、黄芪组、黄芪甲苷组、1-脱氧野尻霉素组、山茱萸组细胞增殖能力高于IR模型组(P均<0.05).IR模型组细胞凋亡率高于正常组,双益方各组细胞凋亡率均低于IR模型组(P均<0.05).IR模型组细胞中MEK1/2、ERK1/2、p-ERK1/2蛋白相对表达量低于正常组(P均<0.05);二甲双胍组及双益方组细胞中MEK1/2、ERK1/2、p-ERK1/2蛋白相对表达量高于IR模型组(P均<0.05).结论 双益方及其黄芪、葛根、黄芪甲苷、山茱萸、1-脱氧野尻霉素、葛根素、小檗碱等成分可有效提高胰岛素抵抗的Min6细胞的胰岛素分泌水平,促进细胞增殖并抑制其凋亡,作用机制可能与ERK1/2、p-ERK1/2、MEK1/2蛋白表达上调有关.%Objective To observe the effects of Shuangyi decoction (SY)and its effective ingredients on the insulin secretion,cell proliferation,and apoptosis of islet β cells Min 6 in insulin resistant (IR)mice,and to analyze the action mechanism. Methods We cultured Min6 cells and then divided them into the normal group,IR model group,metformin group,SY group,single herb of SY group [Cornus officinalis (Shanzhuyu),Astragalus membranaceus (Huangqi),Coptis chinensis (Huanglian),Puerarin (Gegen),Cortex mori radicis (Sangbaipi),and Eupatorium (Peilan)],and SY effec-tive ingredient group (1-deoxynojirimycin,berberine,astragaloside,ursolic acid,loganin,calycosin glucoside,and puera-rin). The normal group was cultured with phenol red free medium;the other groups were added with glucose and palmitic acid in order to make the Min6 cell IR models. Metformin group was added with 100 g/ L metformin. SY group,Shanzhuyu group,Huangqi group,Huanglian group,Gegen group,Sangbaipi group,Peilan group,1-deoxynojirimycin,berberine, astragaloside,ursolic acid,loganin,calycosin glucoside,puerarin groups were added with 50,100,and 200 g/ L the cor-responding drugs. Insulin secretion test was performed at 24 h after administration,and the level of insulin secretion was detected. The proliferation ability of cells at 24 h and 48 h after administration was detected by CCK-8 kit. Apoptotic cells were detected at 24 h after administration by double staining in the normal group,IR model group,metformin group,and SY group;the apoptotic rate was also calculated;the protein exoression of ERK1 / 2,p-ERK1 / 2,MEK1 / 2 was detected by Western blotting. Results Under the condition of high glucose and low sugar,the insulin secretion of the IR model group was lower than that of normal group (P < 0. 05). The insulin secretion levels of the metformin group,Huangqi group and Gegen group,SY group,astragaloside group,Shanzhuyu group,1-deoxynojirimycin group,puerarin group,and berberine group were higher than that of the IR model group (all P < 0. 05). The cell proliferation ability of IR model group was low-er than that of the normal group (P < 0. 05);the cell proliferation abilities of the metformin group,SY group,Huangqi group,astragaloside group,1- deoxynojirimycin group,and Shanzhuyu group were higher than that of IR model group (P <0. 05). The apoptosis rate of the IR model group was higher than that of the normal group,and the apoptosis rate of the SY group was lower than that of the IR model group (P < 0. 05). The expression levels of MEK1 / 2,ERK1 / 2,and p-ERK1 /2 proteins were lower in the IR model group than in the normal group (P < 0. 05);the expression levels of MEK1 / 2, ERK1 / 2,and p-ERK1 / 2 proteins were higher in the metformin group and SY group than in the IR model group (all P <0. 05). Conclusions SY,and its effective ingredients Huangqi,Gegen,astragaloside,Shanzhuyu,1-deoxynojirimycin, puerarin,and berberine can effectively improve the IR Min6 cell insulin secretion,promote Min6 cell proliferation and in-hibit the apoptosis by up-regulating the expression of ERK1 / 2,p-ERK1 / 2 and MEK 1 / 2 protein.