摘要
目的基于小鼠肺癌细胞/小鼠成肌细胞(Lewis/C2C12)体外共培养模型研究六君子汤防治肿瘤恶病质的分子机制。方法采用Lewis/C2C12细胞共培养方法模拟肿瘤恶病质的环境,并将细胞分为模型组、六君子汤组、信号传导及转录激活蛋白3(Signal transducer and activator of transcription3,STAT3)抑制剂Stattic组、联合用药组,并以正常培养的成肌细胞(C2C12)作为空白对照。各组药物分别作用细胞48h后,采用MTT法检测各组药物对肿瘤环境下C2C12细胞生长的影响;免疫荧光法检测肿瘤环境下C2C12细胞中肌球蛋白重链(MyHC)的表达情况;酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA),检测共培养条件下C2C12培养基上清中白介素-6(interleukin-6,IL-6)的含量;蛋白质印迹法(Western Blot,WB)检测肿瘤环境下C2C12细胞中IL-6/STAT3信号通路相关蛋白表达情况的影响。结果MTT结果显示六君子汤和Stattic最佳浓度分别为1000μg/m和3μM;倒置显微镜下观察到当Lewis/C2C12共培养比例为1:1时,肌管出现萎缩、变细,MyHC蛋白表达最强;Elisa检测结果显示,与模型组比较三个药物组中IL6含量均降低;Western blot检测显示,与空白对照组比较,模型组STAT3、JAK2、MAFbx、MuRF1蛋白表达显著升高,三组药物干预后出现表达降低的改变。结论六君子汤可缓解肿瘤环境下肌肉蛋白的降解,其机制与抑制泛素蛋白酶体系统(Ubiquitin-proteasome system,UPS)活性有关,六君子汤可通过抑制IL-6/STAT3信号的活性从而关闭UPS途径,另外还可能通过调控其他信号而关闭UPS途径,从而缓解肿瘤恶病质肌肉降解。
Objective To study the molecular mechanism of Liujunzi Decoction in the prevention and treatment of cancer cachexia based on Lewis/C2C12 cell coculture model in vitro.Methods Lewis/C2C12 cells were co cultured to simulate the environment of cancer cachexia.The cells were divided into model group,Liujunzi Decoction group,STAT3 inhibitor stattic group and combination group.Normal cultured C2C12 cells were used as blank control.After 48 hours of treatment,MTT method was used for detecting the effect of each group of drugs on the growth of C2C12 cells in tumor environment;The expression of My HC protein in C2C12 cells under tumor environment was detected by immunofluorescence method;Enzyme linked immunosorbent assay(ELISA)was used for detecting the content of interleukin-6(IL-6)in the supernatant of C2C12 medium under co culture conditions;Western blot(WB)was used for detecting the expression of IL-6/STAT3 signaling pathway related proteins in C2C12 cells under tumor environment.Results MTT results showed that the optimal concentrations of Liujunzi Decoction and stattic were 1000μg/M and 3μm,respectively;Under the inverted microscope,when the Lewis/C2C12 co culture ratio was 1∶1,the myotubes were atrophied and thinned,and the expression of My HC protein was the strongest;ELISA results showed that compared with the model group,the content of IL6 in the three drug groups were decreased;Western blot showed that compared with the blank control group,the protein expression of STAT3,JAK2,MAFbx and Mu RF1 in the model group was significantly increased,and the expression of the three groups decreased after drug intervention.Conclusion Liujunzi Decoction can alleviate the degradation of muscle protein in tumor environment,and its mechanism is related to the inhibition of UPS protein degradation pathway activity.Liujunzi Decoction can close UPS pathway by inhibiting the activity of IL-6/STAT3 signal.In addition,Liujunzi Decoction may also close UPS pathway by regulating other signals,so as to alleviate muscle degradation of cancer cachexia.
作者
贺倩文
刘冬静
刘燕
蒋时红
陈玉龙
吴耀松
He Qianwen;Liu Dongjing;Liu Yan;Jiang Shihong;Chen Yulong;Wu Yaosong(Henan University of Chinese Medicine,Zhengzhou 450046,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2021年第10期3630-3637,共8页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
河南省科技厅河南省科技攻关项目(192102310160):六君子汤干预STAT3/泛素—蛋白酶体系统防治肿瘤恶病质肌肉萎缩的分子机制,负责人:吴耀松
河南省科技厅河南省科技攻关(202102310493):逍遥散对肝郁脾虚证大鼠下丘脑能量代谢干预作用及分子机制,负责人:刘燕
河南中医药大学科研创新重点项目(2019KYCX008):基于STAT3/mTOR自噬通路探讨人参对肿瘤恶病质的成肌细胞的分化影响,负责人:贺倩文。
