雷公藤甲素对小鼠睾丸支持细胞人白细胞分化抗原36蛋白表达及脂质代谢水平的影响  

Effects of triptolide on protein expression of cluster of differentiation 36 and lipid metabolism in mouse testis Sertoli cell

作　　者：罗立 卢树宏 张嘉慧 郑锦芬 沈飞海[1,2] 黄芝瑛 LUO Li;LU Shuhong;ZHANG Jiahui;ZHENG Jinfen;SHEN Feihai;HUANG Zhiying(School of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou 510006,China;School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]中山大学药学院,广东广州510006 [2]广东药科大学药学院,广东广州510006

出　　处：《药物评价研究》2022年第7期1266-1273,共8页Drug Evaluation Research

基　　金：国家自然科学基金资助项目(81773992)。

摘　　要：目的研究雷公藤甲素影响睾丸支持细胞的人白细胞分化抗原36(CD36)蛋白表达及脂质代谢的作用与分子机制。方法体外培养小鼠睾丸支持细胞系TM4细胞,CCK-8法检测雷公藤甲素(40、80、160、320、640 nmol·L^(−1))作用24 h细胞存活率;流式细胞术测定雷公藤甲素(80、160、320 nmol·L^(−1))作用24 h细胞凋亡率;氟硼二吡咯化合物(BODIPY)染色和油红O染色检测雷公藤甲素(80、160、320 nmol·L^(−1))作用24 h细胞内脂滴聚积情况;试剂盒法检测雷公藤甲素(80、160、320 nmol·L^(−1))作用24 h细胞内三酰甘油(TG)水平;TM4细胞经0.1mmol·L^(−1)三磷酸腺苷(ATP)预处理3 h后,再与160 nmol·L^(−1)雷公藤甲素共处理24 h,用CCK-8法检测TM4细胞的存活率;Western blotting法检测雷公藤甲素(40、80、160、320 nmol·L^(−1))作用24 h、或160 nmol·L^(−1)雷公藤甲素作用6、12、24、36、48 h后TM4细胞中CD36蛋白表达量;Western blotting法检测雷公藤甲素(40、80、160、320 nmol·L^(−1))作用24 h蛋白激酶1(PKD1)及其磷酸化蛋白、组蛋白去乙酰化酶7(HDAC7)和叉头框蛋白O1(FOXO1)的蛋白表达水平。结果与对照组比较,雷公藤甲素80、160、320、640 nmol·L^(−1)浓度组TM4细胞存活率显著下降(P<0.05、0.01),半数抑制浓度(IC50)为214.1 nmol·L^(−1);80、160、320 nmol·L^(−1)雷公藤甲素的细胞凋亡率分别为11.89%、23.17%、32.12%,与对照组比较差异显著(P<0.05、0.01)。BODIPY染色结果显示,与对照组比较,雷公藤甲素组细胞内的红色荧光强度显著下降(P<0.01),油红O染色也显示,雷公藤甲素160 nmol·L^(−1)组细胞内脂滴数量明显低于对照组;与对照组比较,雷公藤甲素组TM4细胞内TG水平显著下降(P<0.01)。与雷公藤甲素组比较,使用ATP协同给药显著减轻了雷公藤甲素对TM4细胞存活率的抑制(P<0.01)。与对照组比较,80、160、320 nmol·L^(−1)的雷公藤甲素作用24 h后,TM4细胞的CD36蛋白表达量显著�Objective To investigate the effect and molecular mechanism of triptolide on cluster of differentiation 36(CD36)protein expression and lipid metabolism of testicular Sertoli cells.Methods Mouse testis Sertoli cell line TM4 cells were cultured in vitro.The survival rate of TM4 cells treated with triptolide(40,80,160,320,640 nmol·L^(−1))for 24 h was determined by CCK-8 method.The apoptosis rate of triptolide treated with 80,160,320 nmol·L^(−1)for 24 h was determined by flow cytometry.The accumulation of intracellular lipid dropletswas detected by BODIPYstaining and oil-redOstaining after treatedwith triptolide(80,160,320 nmol·L^(−1))for 24 h.The level of triacylglycerol(TG)in cells treated with triptolide(80,160,320 nmol·L^(−1))for 24 h was detected by kit method.TM4 cells were pretreated with 0.1 mmol·L^(−1)adenosine triphosphate(ATP)for 3 h,and then treated with 160 nmol·L^(−1)triptolide for 24 h.The survival rate of TM4 cells was determined by CCK-8 method.Western blotting was used to detect the expression of CD36 protein in TM4 cells treated with triptolide(40,80,160,320 nmol·L^(−1))for 24 h or 160 nmol·L^(−1)for 6,12,24,36,48 h.Western blotting was used to detect the protein expression levels of protein kinase 1(PKD1)and its phosphorylated protein,histone deacetylase 7(HDAC7)and forkhead box protein O1(FOXO1)treated by triptolide(40,80,160,320 nmol·L^(−1))for 24 h.Results Compared with control group,TM4 cell survival rate was significantly decreased in triptolide 80,160,320 and 640 nmol·L^(−1)groups(P<0.05,0.01),and IC50 was 214.1 nmol·L^(−1).The apoptosis rates of triptolide at 80,160 and 320 nmol·L^(−1)were 11.89%,23.17%and 32.12%,respectively,significantly different compared with control group(P<0.05,0.01).BODIPY staining showed that the intracellular red fluorescence intensity in triptolide group was significantly decreased compared with control group(P<0.01).Oil red O staining also showed that the number of intracellular lipid droplets in triptolide 160 nmol·L^(−1)gr

关 键 词：雷公藤甲素 睾丸支持细胞 脂代谢紊乱 人白细胞分化抗原36(CD36) 三磷酸腺苷 蛋白激酶1 组蛋白去乙酰化酶7 叉头框蛋白O1 

分 类 号：R285.5[医药卫生—中药学]