四君子汤对IEC-6细胞增殖相关指标及大鼠胃肠黏膜c-Myc表达的影响  

Effects of Sijunzi Decoction on IEC-6 Cell Proliferation Related Index and c-Myc Expression in Gastrointestinal Mucosa of Rats

作　　者：涂小华 杨欣 杨光勇 徐萌萌 邓颖 张庚鑫 杜海洋 王慧 TU Xiaohua;YANG Xin;YANG Guangyong;XU Mengmeng;DENG Ying;ZHANG Gengxin;DU Haiyang;WANG Hui(Faculty of Basic Medical Sciences,Guizhou University of Traditional Chinese Medicine,Guiyang 550025 Guizhou,China)

机构地区：[1]贵州中医药大学基础医学院,贵州贵阳550025

出　　处：《中药新药与临床药理》2022年第2期143-150,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81760864);贵州省教育厅青年科技人才成长项目(黔教合KY字[2017]173);贵州中医药大学博士启动基金项目(20160035)。

摘　　要：目的观察四君子汤对IEC-6细胞(大鼠小肠上皮细胞)增殖,多胺调控生长相关基因细胞周期检测点激酶2(Checkpoint kinase 2,Chk2)、c-Myc表达,以及对大鼠胃肠黏膜c-Myc蛋白表达的影响。方法采用SD大鼠制备空白血清及四君子汤含药血清;无负荷细胞实验设空白对照组、腐胺组(10μmol·L^(-1))及四君子汤含药血清低、中、高(5%、10%、20%)剂量组;α-二氟甲基鸟氨酸(α-difluoromethylornithine,DFMO)负荷细胞实验设空白对照组、DFMO组(2.5 mmol·L^(-1))、腐胺组(10μmol·L;)及四君子汤含药血清低、中、高(5%、10%、20%)剂量组;动物实验将SD大鼠随机分为空白对照组、模型组(吲哚美辛组)及四君子汤低、高(5、15 g·kg^(-1))剂量组。采用MTT法检测IEC-6细胞增殖情况;qPCR法检测细胞Chk2、c-Myc mRNA表达;Western Blot法检测细胞Chk2、p-Chk2、c-Myc蛋白表达和大鼠胃肠黏膜的c-Myc蛋白表达。结果 (1)细胞实验:与空白对照组比较,10%、20%四君子汤含药血清可促进给药24 h后IEC-6细胞的增殖(P<0.01),提高p-Chk2蛋白及c-Myc m RNA/蛋白表达水平(P<0.05,P<0.01);5%、10%、20%四君子汤含药血清可促进给药48、72 h后细胞增殖(P<0.01),促进Chk2 mRNA/蛋白表达(P<0.05,P<0.01)。与DFMO组比较,5%、10%、20%四君子汤含药血清可促进给药48 h后细胞增殖(P<0.01),提高c-Myc mRNA/蛋白表达(P<0.05,P<0.01);10%、20%四君子汤含药血清可促进给药72 h后细胞增殖(P<0.01),促进Chk2及p-Chk2蛋白表达(P<0.05,P<0.01);20%四君子汤含药血清可促进Chk2 mRNA表达(P<0.05)。(2)动物实验:与模型组比较,四君子汤5、15 g·kg^(-1)可上调模型大鼠胃黏膜及小肠黏膜c-Myc蛋白表达(P<0.05,P<0.01)。结论四君子汤可促进IEC-6细胞增殖,其作用机制可能与影响多胺信号通路,上调生长相关基因Chk2、c-Myc表达有关。四君子汤可提高胃肠黏膜损伤模型大鼠胃及小肠黏膜c-Myc蛋白表达水平。Objective To observe the effects of Sijunzi decoction on the proliferation of small intestinal epithelial cells(IEC-6),the expression of genes related to growth regulated by polyamine,including cell cycle checkpoint kinase 2(Chk2)and c-Myc,and c-Myc protein expression in gastrointestinal mucosa of rats.Methods The blank serum and serum containing Sijunzi decoction(SJZDS)were prepared from SD rats.The blank control group,putrescine group(PUT group,10μmol·L^(-1)),SJZDS 5%,10%and 20%groups were set up in the unloaded cell experiment.The blank control group,α-difluoromethylornithine(DFMO)group,PUT group(10μmol·L^(-1)),SJZDS 5%,10%and 20%groups were set up in DFMO loaded cell experiment.In animal experiment,SD rats were randomly divided into blank control group,indomethacin model group,Sijunzi decoction 5 g·kg;and 15 g·kg^(-1)groups.Proliferation of IEC-6 cells was detected by MTT assay,the mRNA expression of Chk2 and c-Myc was detected by real-time quantitative PCR;and Western Blot was used to detect the protein expression of Chk2,phosphorylated Chk2 and c-Myc.Results In the cell experiments,compared with blank control group,10%,20%SJZDS promoted cell proliferation at 24 h after administration(P<0.01) and increased the phosphorylated Chk2 protein level and the mRNA and protein levels of c-Myc(P<0.05,P<0.01);5%,10%and 20%SJZDS promoted cell proliferation at 48 h and 72 h after administration(P<0.01),and enhanced the mRNA and protein expression of Chk2(P<0.05,P<0.01).Compared with DFMO group,5%,10%and 20%SJZDS increased cell proliferation at48 h after administration(P<0.01),enhanced c-Myc mRNA and protein levels(P<0.05,P<0.01),10%and20%SJZDS promoted cell proliferation at 72 h after administration(P<0.01)and improved the protein expression of Chk2 and phosphorylated Chk2(P<0.05,P<0.01),20%SJZDS increased Chk2 mRNA expression(P<0.05).In animal experiments,compared with indomethacin model group,Sijunzi decoction(5 g·kg^(-1)and 15 g·kg^(-1))increased the protein expression of c-Myc in gastric mucosa and small int

关 键 词：四君子汤 胃肠黏膜损伤修复 细胞增殖 多胺 细胞周期检测点激酶2 c-Myc IEC-6细胞 大鼠 

分 类 号：R285.5[医药卫生—中药学]