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

作　　者：于嘉祥 张瀚文[1] 王列[1] 石岩[1] 于睿[1] 戴俭宇[1] 曲超 马贤德[1] 韩雪莹 王智民[2] 安继仁 程玥凤 纪泓楷 张文顺 YU Jiaxiang;ZHANG Hanwen;WANG Lie;SHI Yan;YU Rui;DAI Jianyu;QU Chao;MA Xiande;HAN Xueying;WANG Zhimin;AN Jiren;CHENG Yuefeng;JI Hongkai;ZHANG Wenshun(Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;Affiliated Hospital of Liaoning University of Traditional Chinese Medicine(TCM),Shenyang 110000,China;Liaoning Academy of TCM/The Second Affiliated Hospital of Liaoning University of TCM,Shenyang 110034,China)

机构地区：[1]辽宁中医药大学,沈阳110847 [2]辽宁中医药大学附属医院,沈阳110000 [3]辽宁省中医药研究院/辽宁中医药大学附属第二医院,沈阳110034

出　　处：《中国实验方剂学杂志》2023年第13期54-64,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家重点基础研究发展计划(973计划)项目(2013CB532004);国家自然科学基金项目(82274494);辽宁省“兴辽英才计划”青年拔尖人才项目(XLYC2007121);辽宁省科技厅博士科研启动课题(2021-BS-183,2023-BS-137,2023-BS-141);中医脏象理论及应用教育部重点实验室2021年度开放基金项目(zyzx2109)。

摘　　要：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�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 determin

关 键 词：益糖康 胰岛素抵抗 糖基化终末产物(AGE)/糖基化终末产物受体(RAGE)信号通路 改善 机制 

分 类 号：R242[医药卫生—中医临床基础] R2-0[医药卫生—中医学] R2-031R285R825.6R56