益糖康通过抑制AGE/RAGE信号通路改善骨骼肌细胞凋亡的机制

Mechanism of Yitangkang in Improving Apoptosis of Skeletal Muscle Cells by Inhibiting AGE/RAGE Signaling Pathway

目的:研究益糖康通过抑制糖基化终末产物(AGE)/糖基化终末产物受体(RAGE)信号通路纠正骨骼肌细胞过度凋亡进而改善胰岛素抵抗(IR)的作用机制。方法:①体外实验,制备益糖康含药血清,将C2C12细胞设置空白组、模型组、益糖康含药血清高、中、低剂量(40、20、10 g·kg^(-1))组、RAGE抑制剂组,除空白组外,用棕榈酸诱导C2C12 IR模型细胞,然后分别采取相应的干预方式后检测各组细胞的活性及葡萄糖消耗量水平,用流式细胞术检测各组细胞凋亡率,实时荧光定量聚合酶链式反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测重要凋亡蛋白[B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、p53、胱天蛋白酶(Caspase)-3、Caspase-9]的mRNA及蛋白表达水平。②体内实验,将96只符合研究标准的Wistar大鼠设置空白组、对照组、益糖康高、中、低剂量组(40、20、10 g·kg^(-1))、吡格列酮(1.35 mg·kg^(-1))组,运用高糖高脂糖尿病模型诱导饲料联合小剂量链脲佐菌素(STZ)腹腔注射建立胰岛素抵抗动物模型并运用高胰岛素-正常血糖钳夹(HEC)实验验证,模型判定成功后对各组大鼠按要求进行灌胃给药干预。用原位末端标记法(TUNEL)检测各组大鼠骨骼肌组织阳性凋亡细胞个数,Real-time PCR及Western blot技术检测Bcl-2、Bax、p53、Caspase-3、Caspase-9的mRNA及蛋白表达水平。结果:①体外实验,与空白组比较,模型组C2C12细胞凋亡率显著升高,细胞活性、葡萄糖消耗量均显著降低(P<0.01)。与模型组比较,益糖康含药血清各剂量组和RAGE抑制剂组C2C12细胞凋亡率显著降低,细胞活性、葡萄糖消耗量均显著升高(P<0.01);与模型组比较,益糖康含药血清各剂量组和RAGE抑制剂组C2C12细胞凋亡率显著降低,细胞活性、葡萄糖消耗量均显著升高(P<0.01)。与空白组比较,模型组C2C12细胞Bcl-2 mRNA和蛋白表达水平显著降低,Bax、p53、Caspase-3、Caspase-9 mRNA和蛋白表达水平显著升高(P<0.01)。与模型组比较,益糖康含药血清各剂量组和RAGE抑制剂组C2C12细胞Bcl-2 mRNA和蛋白表达水平显著升高(P<0.01),Bax、p53、Caspase-3、Caspase-9 mRNA和蛋白表达水平明显降低(P<0.05,P<0.01)。②体内实验,与空白组比较,模型组大鼠骨骼肌组织阳性凋亡细胞个数显著升高(P<0.01)。与模型组比较,益糖康高、中、低剂量组,吡格列酮组大鼠骨骼肌组织阳性凋亡细胞个数显著降低(P<0.01)。与空白组比较,模型组大鼠骨骼肌组织Bcl-2 mRNA和蛋白表达水平显著降低,Bax、p53、Caspase-3、Caspase-9 mRNA和蛋白表达水平显著升高(P<0.01)。与模型组比较,益糖康各剂量组和吡格列酮组大鼠骨骼肌组织Bcl-2 mRNA和蛋白表达水平显著升高(P<0.01),Bax、p53、Caspase-3、Caspase-9 mRNA和蛋白表达水平明显降低(P<0.05,P<0.01)。益糖康中剂量具有RAGE抑制剂类似的功效,且疗效等同于盐酸吡格列酮。结论:益糖康可能通过抑AGE/RAGE信号通路抑制骨骼肌细胞凋亡。

Objective:To determine the mechanism of Yitangkang in correcting excessive apoptosis of skeletal muscle cells to improve insulin resistance(IR)by inhibiting the advanced glycation end product(AGE)/receptor for the advanced glycation end product(RAGE)signaling pathway.Method:①In vitro experiments.Yitangkang-medicated serum was prepared.C2C12 cells were divided into a blank group,a model group,high-,medium-,and low-dose Yitangkang-medicated serum groups(40,20,and 10 g·kg^(-1)),and a RAGE inhibitor group.The IR model was induced by palmitic acid in C2C12 cells except for those in the blank group.After the corresponding intervention methods were conducted,the cell viability and glucose consumption level of each group were determined.In addition,the apoptosis rate was determined using flow cytometry.The mRNA and protein expression levels of the important apoptotic proteins[B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),p53,cysteinyl aspartate-specific protease-3(Caspase-3),and cysteinyl aspartate-specific protease-9(Caspase-9)]were determined using Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot.②In vivo experiments.Ninety-six eligible Wistar rats were divided into a blank group,a model group,high-,medium-,and low-dose Yitangkang groups(40,20,and 10 g·kg^(-1)),and a western medicine group(pioglitazone hydrochloride,1.35 mg·kg^(-1)).The IR model was induced using highglucose and high-fat feed for diabetes combined with intraperitoneal injection of low-dose streptozotocin(STZ)in animals and verified by the hyperinsulinemic-euglycemic clamp(HEC)test.After the model was determined successfully,the rats in each group were given intragastric administration of drugs as required.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay was performed to determine the number of positive apoptotic cells in the skeletal muscle tissues of rats in each group,while Real-time polymerase chain reaction(Real-time PCR)and Western blot were performed to determine the mRNA and protein expression levels of the important apoptotic proteins Bcl-2,Bax,p53,Caspase-3,and Caspase-9.Result:①In vitro experiments.compared with the blank group,the model groups showed increased apoptosis rate of C2C12 cells and decreased cell viability and glucose consumption(P<0.01).Compared with the model group,the Yitangkang-medicated serum groups and the RAGE inhibitor group showed decreased apoptosis rate of C2C12 cells and increased cell viability and glucose consumption(P<0.01).Compared with the blank group,the model group showed decreased expression levels of Bcl-2 mRNA and protein in C2C12 cells and increased mRNA and protein expression levels of Bax,p53,Caspase-3,and Caspase-9(P<0.01).Compared with the model group,the Yitangkang-medicated serum groups and the RAGE inhibitor group showed increased expression levels of Bcl-2 mRNA and protein in C2C12 cells(P<0.01)and decreased mRNA and protein expression levels of Bax,p53,Caspase-3,and Caspase-9(P<0.05,P<0.01).②In vivo experiments.The number of positive apoptotic cells in the skeletal muscle tissues of rats in the model group significantly increased as compared with that in the blank group(P<0.01).The number of positive apoptotic cells in the skeletal muscle tissues of rats in the Yitangkang groups and the western medicine group decreased as compared with that in the model group(P<0.01).Compared with the blank group,the model group showed decreased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats and increased mRNA and protein expression levels of Bax,p53,Caspase-3,and Caspase-9(P<0.01).Compared with the model group,the Yitangkang groups and the western medicine group showed increased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats(P<0.01)and decreased mRNA and protein expression levels of Bax,p53,Caspase-3,and Caspase-9(P<0.05,P<0.01).The medium-dose Yitangkang showed a similar effect as RAGE inhibitor,and the effect was equivalent to that of pioglitazone hydrochloride.Conclusion:Yitangkang can inhibit skeletal muscle cell apoptosis by inhibiting the AGE/RAGE signaling pathway.