孕期邻苯二甲酸二丁酯暴露抑制Hedgehog信号导致后代肾脏自噬  

Dibutyl phthalate induces autophagy of kidney in offspring by inhibiting hedgehog signaling pathway

作　　者：谢志文 赵圣[1] 华山 蒋君涛[1] 李安国 Xie Zhiwen;Zhao Sheng;Hua Shan;Jiang Juntao;Li Anguo(Department of Urology,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China;Department of Urology,the Fifth People′s Hospital of Zunyi,Guizhou 563099,China)

机构地区：[1]上海交通大学附属第一人民医院泌尿外科,200080 [2]遵义市第五人民医院泌尿外科,563099

出　　处：《中华实验外科杂志》2021年第12期2378-2380,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81771564);遵义市科技计划课题(2018-192)。

摘　　要：目的观察孕期邻苯二甲酸二丁酯(DBP)暴露对后代肾脏的毒理作用,并探讨其机制。方法将6只孕鼠在孕期14~18 d分别随机予以850 mg/(kg·d)的DBP处理3只孕鼠为染毒组,另外3只孕鼠予以等剂量的玉米油处理为对照组。在产后第1天分别随机收集各组6只子代鼠的肾脏组织,采用免疫组织化学(IHC)染色检测肾脏自噬水平,蛋白质印迹法(Western blot)及定量分析验证自噬相关基因Beclin-1在肾脏中的表达。用Western blot及实时定量反转录聚合酶链反应(RT-qPCR)检测肾脏组织中Hedgehog信号通路上的相关标志物的表达水平。在体外实验中,分别予以Hedgehog信号通路的抑制剂索尼德吉Sonidegib及激动剂重组音刺猬蛋白(SHH)处理NRK52E肾小管上皮细胞,采用Western blot分析各组的自噬水平。组间比较采用独立t检验。结果IHC提示染毒组Beclin-1的表达高于对照组(2.37±0.12比1.00±0.10,t=15.190,P<0.05),蛋白印记及其定量分析显示染毒组Beclin-1蛋白表达量高于对照组(1.20±0.104比1.00±0.01,t=3.317,P<0.01)。同时,Western blot及其定量分析表明,暴露组Hedgehog信号通路标记物Gli1和Ptch1表达低于对照组(1.00±0.15比0.43±0.09,t=5.644,P<0.01;1.00±0.01比0.35±0.10,t=11.20,P<0.01)。RT-qPCR也提示暴露组Gli1和Ptch1表达低于对照组(1.00±0.18比0.51±0.10,t=4.122,P<0.01;1.00±0.10比0.62±0.08,t=5.140,P<0.01)。体外研究证实当加入Hedgehog抑制剂Sonidegib后,实验组的NRK52E细胞自噬指数(LC3Ⅱ/LC3Ⅰ)高于对照组(0.49±0.11比1.25±0.05,t=10.890,P<0.01),而当加入其激动剂SHH后,实验组的自噬指数低于对照组(0.49±0.11比0.36±0.09,t=11.260,P<0.01)。结论孕期DBP暴露通过抑制Hedgehog信号引起后代肾脏发生自噬,从而可能引起肾脏相关疾病。Objective To study the role of prenatal exposure to dibutyl phthalate on kidney in offspring and explore the mechanism.Methods Sixpregnant rats were randomly assigned into the control anddibutyl phthalate(DBP)-exposure group.On the 14-18 days during gestation,DBP-exposure group ratswerefed with DBP at a dose of 850 mg/(kg·d),while control group rats were treated with equivalent corn il.Six offspring rats were randomly acquired from each group and their kidney was collect on the postnatal day 1.Immunohistochemical staining was used to test the expression of autophagy-related genefor renal tissues.We used Western blotting and quantification to measure the level of Beclin-1.Western blotting and reverse transcription-quantitative polymerase chain reaction(RT-qPCR)were used to detect the relevant indicators of Hedgehog signaling pathwayin kidney.In vitro,we used renal tubule cells NRK52E to test the level of autophagy after treating with agonist and antagonist of Hedgehog signal by Western blotting and quantification.Groups were compared by independent t test.Results Immunohistochemical staining showed that the biomarker of autophagy,Beclin-1,was higher in DBP-treated group than that in control group(2.37±0.12 vs.1.00±0.10,t=15.190,P<0.05).Meanwhile,Western blotting demonstrated that the expression of biomarkers for Hedgehog signaling pathway such as Gli1 and Ptch1 in DBP-exposure group were lower than that in control group(1.00±0.15 vs.0.43±0.09,t=5.644,P<0.01;1.00±0.01 vs.0.35±0.10,t=11.20,P<0.01).RT-qPCR also demonstrated the expression of Gli1 and Ptch1 in DBP-exposure group were lower than that in control group(1.00±0.18 vs.0.51±0.10,t=4.122,P<0.01;1.00±0.10 vs.0.62±0.08,t=5.140,P<0.01).In vitro,when adding antagonist Sonidegib of Hedgehog signaling pathway into the NRK52E cells,the ratio of LC3Ⅱ/LCⅠin treated group is higher than that in control group(0.49±0.11 vs.1.25±0.05,t=10.890,P<0.01).When adding agonist SHH into the NRK52E cells,the ratio of LC3Ⅱ/LCⅠin treated group is lower than that

关 键 词：邻苯二甲酸二丁酯 肾小管上皮细胞 Hedgehog信号 自噬 

分 类 号：R114[医药卫生—卫生毒理学]