双酚A通过上调Apoa 1基因的表达抑制TM3细胞睾酮合成  

Bisphenol A Inhibits Testosterone Synthesis in TM3 Cells by Upregulating Apoa 1 Gene Expression

作　　者：赵彤 杨文哲 潘飞龙 赵树臣[1,2] 刘克祥 吕占军[1,2] 赵立佳 ZHAO Tong;YANG Wenzhe;PAN Feilong;ZHAO Shuchen;LIU Kexiang;L Zhanjun;ZHAO Lijia(College of Animal Medicine,Northeast Agricultural University,Harbin 150030,China;Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and Treatment,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]黑龙江省教育厅普通疾病防治重点实验室,哈尔滨150030

出　　处：《畜牧兽医学报》2024年第8期3516-3525,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：黑龙江省自然科学基金优秀青年基金项目(YQ2022C018)。

摘　　要：旨在从脂质代谢的角度探讨载脂蛋白A1(apolipoprotein A1,Apoa 1)是否介导了双酚A(bisphenol A,BPA)暴露所致小鼠睾丸间质细胞株(TM3)睾酮合成的降低。将TM3细胞随机分为不同浓度的BPA暴露剂量(0、5、10、20、40、60、80μmol·L^(-1))组,0μmol·L^(-1)BPA为对照组(CON)。给予相应剂量处理24 h后,运用CCK-8法检测TM3细胞活力,确定BPA最适染毒剂量;通过ELISA检测TM3细胞培养上清液睾酮(testosterone,T)含量;利用RT-qPCR检测TM3细胞脂质代谢相关基因Apoa1、Apoa 2(apolipoprotein A2)、Apoc 3(apolipoprotein C3)的mRNA表达水平;运用Western blot和免疫荧光方法检测APOA1蛋白表达水平;采用油红O染色观察细胞内脂滴累积情况。结果表明,20μmol·L^(-1)BPA处理24 h对TM3细胞活力无显著影响,40μmol·L^(-1)BPA处理24 h后,TM3细胞活力受到极显著抑制(P<0.01);此外,20μmol·L^(-1)BPA处理TM3细胞24 h后,培养上清液中睾酮含量极显著低于对照组(P<0.01),Apoa 1基因的mRNA表达水平及蛋白表达量极显著升高(P<0.001),但Apoa 2和Apoc 3基因的mRNA表达水平无显著变化;与对照组相比,20μmol·L^(-1)BPA处理24 h,TM3细胞的脂滴累积量极显著降低(P<0.0001)。综上,BPA可通过上调Apoa 1基因的表达水平,增强胆固醇逆向转运(reverse cholesterol transport,RCT),引起TM3细胞内的脂滴含量减少,导致TM3细胞的睾酮合成分泌降低。The aim of the study was to investigate whether apolipoprotein A1(apolipoprotein A1,Apoa 1)mediates the reduction of testosterone synthesis in mouse Leydig cells line(TM3)induced by bisphenol A(BPA)exposure from the perspective of lipid metabolism.TM3 cells were randomly divided into 7 groups with different concentrations(0,5,10,20,40,60,and 80μmol·L^(-1)),and 0μmol·L^(-1)BPA was the control group(CON);After 24 h of treatment with the different concentrations,cell viability was detected using the by CCK-8 method to determine the optimal dose of BPA.Testosterone(testosterone,T)synthesis content in TM3 cell supernatant was detected by ELISA;mRNA expression levels of lipid metabolism-related genes Apoa1,Apoa 2(apolipoprotein A2)and Apoc 3(apolipoprotein C3)genes were measured in TM3 cells using by RT-qPCR.APOA1 protein expression level was detected using by Western blot and immunofluorescence method.Intracellular lipid droplet accumulation was observed using by oil red O staining.The results showed that 20μmol·L^(-1)BPA treatment for 24 h had no significant effect on the viability of TM3 cells;However,an extremely significant inhibition of TM3 cell viability was observed following in treatment with 40μmol·L^(-1)BPA for 24 h(P<0.01);In addition,after 20μmol·L^(-1)BPA treatment on TM3 cells for 24 h,the testosterone content in the culture supernatant was extremely significantly lower than that in the CON group(P<0.01),the mRNA expression level and protein expression of Apoa 1 gene were extremely significantly elevated(P<0.001),but there was no significant change in the mRNA expression level of Apoa 2 and or Apoc 3 genes;The accumulation of lipid droplets in TM3 cells was extremely significantly reduced by 20μmol·L^(-1)BPA treatment for 24 h compared with the CON group(P<0.0001).In conclusion,BPA can reduce the lipid droplets accumulation in TM3 cells by up-regulating Apoa 1 expression levels,enhancing reverse cholesterol transport(Reverse cholesterol transport,RCT),leading to a decrease in testosterone syn

关 键 词：双酚A(BPA) 载脂蛋白A1(Apoa 1) 小鼠睾丸间质细胞(TM3) 睾酮(T) 

分 类 号：S814[农业科学—畜牧学]