PKC-mTOR通路对糖尿病大鼠胃平滑肌细胞凋亡的影响及作用机制  

Effect of PKC-mTOR pathway on apoptosis of gastric smooth muscle cells in diabetic rats and its mechanism

作　　者：范秀娟 蔡英兰 FAN Xiujuan;CAI Yinglan(Department of Histology and Embryology,Medical College of Yanbian University,Yanji 133000,China)

机构地区：[1]延边大学医学院组织学与胚胎学教研室,吉林延吉133000

出　　处：《中国病理生理杂志》2024年第10期1797-1805,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82160110);吉林省教育厅科学技术研究项目(No.JJKH20220544KJ)。

摘　　要：目的:探讨蛋白激酶C(PKC)-哺乳动物雷帕霉素靶蛋白(mTOR)通路在糖尿病(diabetes)大鼠胃平滑肌细胞凋亡的影响及作用机制。方法:将72只雌雄不分的SPF级4周龄SD大鼠随机分为正常对照(control)组、糖尿病组、diabetes+0.1μmol/L佛波酯(PMA)组、diabetes+0.2μmol/L PMA组、diabetes+0.5μmol/L PMA组、diabetes+2μmol/L双吲哚马来酰亚胺I(GF109203X)组、diabetes+5μmol/LGF109203X组和diabetes+10μmol/L GF109203X组,每组各9只。体外高糖环境下培养大鼠原代胃平滑肌细胞分为对照(CK)组、0.1μmol/L PMA处理组、0.2μmol/L PMA处理组、0.5μmol/L PMA处理组、2μmol/L GF109203X处理组、5μmol/L GF109203X处理组和10μmol/L GF109203X处理组。采用离体肌条实验技术检测胃窦平滑肌的自发性收缩,HE染色和流式细胞术检测大鼠胃平滑肌的病理变化及细胞凋亡,Western blot检测SCF、c-Kit、mTOR、p-mTOR、p70S6K、p-p70S6K、4E-BP1、p-4E-BP1、caspase-3、Bax和Bcl-2的蛋白表达变化。结果:(1)与control组相比,diabetes组胃窦平滑肌收缩性下降(P<0.05),胃平滑肌萎缩,细胞凋亡率增加(P<0.01),SCF、c-Kit和p-mTOR表达均下降(P<0.05或P<0.01)。(2)与diabetes组相比,diabetes+0.1μmol/L PMA组胃窦平滑肌自发性收缩无显著差异,diabetes+0.2μmol/L PMA组(P<0.05)和diabetes+0.5μmol/L PMA组(P<0.01)胃窦平滑肌自发性收缩显著增强,diabetes+0.2μmol/L PMA组和diabetes+0.5μmol/L PMA组mTOR、p70S6K(T421/S424)和p70S6K(T389)的磷酸化水平升高(P<0.05),diabetes+0.5μmol/L PMA组4E-BP1的磷酸化水平升高(P<0.05)。(3)与diabetes组相比,diabetes+2μmol/L GF109203X组胃窦平滑肌自发性收缩无显著差异,diabetes+5μmol/L GF109203X组(P<0.05)和diabetes+10μmol/L GF109203X组(P<0.01)胃窦平滑肌自发性收缩显著减弱,diabetes+5μmol/L GF109203X组和diabetes+10μmol/L GF109203X组mTOR、p70S6K(T389)的磷酸化水平下降(P<0.05),diabetes+10μmol/L GF109203X组p70S6K(T421/S424)的磷酸化水平下降(P<0.05),diabetes+AIM:To investigate the effect of protein kinase C(PKC)-mammalian target of rapamycin(mTOR)pathway on the apoptosis of gastric smooth muscle cells in diabetic rats,and to explore the related mechanism.METHODS:Male and female 4-week-old specific pathogen-free Sprague-Dawley rats were randomly allocated to control,diabetes,diabetes+0.1μmol/L phorbol 12,13-myristate acetate(PMA),diabetes+0.2μmol/L PMA,diabetes+0.5μmol/L PMA,diabetes+2μmol/L bisindolylmaleimide I(GF109203X),diabetes+5μmol/L GF109203X,and diabetes+10μmol/L GF109203X groups(n=9).Primary rat gastric smooth muscle cells cultured in a high-glucose environment in vitro were treated with 0.1μmol/L PMA,0.2μmol/L PMA,0.5μmol/L PMA,2μmol/L GF109203X,5μmol/L GF109203X,10μmol/L GF109203X,or vehicle.The isolated muscle technique was used to evaluate the spontaneous contraction of gastric antral smooth muscle.The pathologic changes and apoptosis of rat gastric smooth muscle were identified using HE staining and flow cytometry,respectively.The expression levels of stem cell factor(SCF),c-Kit,mTOR,phosphorylated(p)-mTOR,p70 ribosomal protein S6 kinase(p70S6K),p-p70S6K,eIF4E-binding protein 1(4E-BP1),p-4E-BP1,caspase-3,B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)in gastric smooth muscle were measured by Western blot.RESULTS:The contractility of the gastric antral smooth muscle was lower(P<0.05),gastric smooth muscle atrophy and the apoptosis rate was higher(P<0.01),and the expression of SCF,c-Kit,and p-mTOR was lower(P<0.05 or P<0.01)in the diabetes group than in the control group.There was no significant difference in the spontaneous contraction of the gastric antral smooth muscle between the diabetes and diabetes+0.1μmol/L PMA groups,but it was significantly greater in the diabetes+0.2μmol/L PMA and diabetes+0.5μmol/L PMA groups(P<0.05 or P<0.01).The phosphorylation levels of mTOR,p70S6K(T421/S424),and p70S6K(T389)were also higher in the diabetes+0.2μmol/L PMA and diabetes+0.5μmol/L PMA groups than in diabetes group(P<0.01).Furthermor

关 键 词：胃平滑肌 糖尿病胃动力障碍 PKC-mTOR信号通路 干细胞因子 细胞凋亡 

分 类 号：R587.2[医药卫生—内分泌] Q255[医药卫生—内科学] R363.2[医药卫生—临床医学]