六君子汤改善肿瘤环境下C2C12成肌细胞分化的分子机制  

Liujunzi Decoction(六君子汤)Improves Molecular Mechanism of C2C12 Myoblast Differentiation in Tumor Environment

作　　者：郝羚伦 张艳[2] 彭梦薇 桑亚洲 陈玉龙[1] 刘燕[1] 吴耀松[1] HAO Linglun;ZHANG Yan;PENG Mengwei;SANG Yazhou;CHEN Yulong;LIU Yan;WU Yaosong(Henan Key Laboratory of Traditional Chinese Medicine Syndrome and Prescription Signaling,Henan International Joint Laboratory of Traditional Chinese Medicine Syndrome and Prescription in Signaling,Henan University of Chinese Medicine,Zhengzhou 450046,Henan,China;Tianjin University of Traditional Chinese Medicine,Tianjin 300193,China;Huazhong University of Science and Technology,Wuhan 430064,Hubei,China)

机构地区：[1]河南中医药大学河南省中医方证信号传导重点实验室,河南省中医方证信号传导国际联合重点实验室,河南郑州450046 [2]天津中医药大学,天津300193 [3]华中科技大学,湖北武汉430064

出　　处：《中华中医药学刊》2025年第1期122-127,I0016-I0018,共9页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82074313);河南省科技攻关项目(192102310160);河南省高等学校重点科研项目(23A360024);河南省中医学“双一流”创建科学研究项目(HSRP-DFCTCM-2023-1-28)。

摘　　要：目的研究六君子汤改善肿瘤环境下C2C12成肌细胞分化的分子机制,为健脾和胃法治疗肿瘤恶病质肌肉萎缩提供实验基础。方法诱导成肌细胞C2C12分化,倒置显微镜观察分化后肌管长度;成肌细胞C2C12与肺癌细胞Lewis共培养,分为空白组、模型组、叉头框蛋白O1(forkhead box protein O1,FoxO1)抑制剂组、六君子汤组、联合用药组,蛋白免疫印迹法(western blot,WB)检测C2C12细胞成肌分化抗原(myogenic differentiation antigen,MyoD)的表达及与过氧化物酶体增殖物受体γ辅助激活因子α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha,PGC-1α)/FoxO1通路相关的蛋白表达;电子显微镜观察C2C12细胞线粒体数量及超微结构的变化;流式细胞术(flow cytometry,FCM)观测C2C12细胞线粒体膜电位的变化;实时荧光定量PCR(quantitative real-time PCR,qPCR)法检测C2C12细胞PGC-1α/FoxO1通路相关分子mRNA。结果C2C12细胞于分化48h肌管横径最长;与空白组比,模型组C2C12细胞MyoD蛋白表达明显减少(P<0.05),线粒体数量减少(P<0.05),线粒体双层膜皱曲不清晰甚至呈C形或基质肿胀,线粒体嵴数量减少、排列紊乱、断裂或溶解消失成空泡状,线粒体膜电位降低,细胞凋亡率显著升高(P<0.05),线粒体转录因子A(mitochondrial transcription factor A,tFAM)的mRNA表达明显降低(P<0.05),FoxO1蛋白表达明显增高,PGC-1α、脂肪和肥胖相关基因(fat mass and obesity-associated gene,FTO)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、tFAM蛋白表达明显减少(P<0.05);与模型组比较,六君子汤组C2C12细胞MyoD蛋白表达明显增高,FoxO1抑制剂组、六君子汤组、联合用药组中C2C12细胞线粒体数量显著增多(P<0.05),线粒体双层膜结构正常,线粒体膜电位升高,细胞凋亡率显著降低(P<0.05),联合用药组C2C12细胞mTOR和tFAM mRNA表达明显增加(P<0.05),FoxO1抑制剂组、六君子汤组及联合用药组中C2C12细胞FoxO1�Objective To study the molecular mechanism of Liujunzi Decoction(六君子汤)to improve C2C12 myoblast differentiation in tumor environment,providing experimental basis for the treatment of tumor cachexia muscle atrophy with strengthening spleen and harmonizing stomach method.Methods It induced the differentiation of myoblasts C2C12 and observed the length of the differentiated myotube with an inverted microscope.Myoblasts C2C12 were co-cultured with lung cancer cell Lewis,and then were divided into blank group,model group,Forkhead box protein O1(FoxO1)inhibitor group,Liujunzi Decoction(六君子汤)group and combination group.Western Blot detected the expression of myogenic differentiation antigen(MyoD)in C2C12 cells and the expressions of proteins associated with the peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α)/FoxO1 pathway.Electron microscopy observed the changes in the number of mitochondria and ultrastructure of C2C12 cells.Flow cytometry observed the changes in mitochondrial membrane potential of C2C12 cells.Quantitative real-time PCR detected mRNA in the PGC-1α/FoxO1 pathway in C2C12 cells.Results Experiments showed that C2C12 cells had the longest transverse diameter in the muscle tube after 48 h of differentiation.Compared with those in the blank group,the MyoD protein expression and mitochondria number were significantly reduced in the model group(P<0.05).The wrinkled boundary of the mitochondrial bilayer membrane was not clear or even more C-shaped or stromal swelling,and the number of mitochondrial cristae was reduced.The arrangement was disordered,fractured or dissolution disappeared into vacuoles.The mitochondrial membrane potential was decreased.The apoptotic rate was significantly increased(P<0.05).The mRNA expression of mitochondrial transcription factor A(tFAM)was significantly reduced,and FoxO1 protein expression was significantly increased.The protein expressions of PGC-1α,FTO,mTOR and tFAM were significantly decreased(P<0.05).Compared with the model group,

关 键 词：六君子汤 PGC-1α/FoxO1 线粒体 肿瘤恶病质 肌肉萎缩 

分 类 号：R289.5[医药卫生—方剂学]