DEHP通过抑制Fto表达诱导睾丸间质细胞铁死亡  

DEHP induces ferroptosis in testicular interstitial cells by inhibiting Fto expression

作　　者：孙凤琼 张国伟[2] 王灵巧[2] 徐桂勇 郭成威 孙燕 杨芮 张露 杨光红[1] 周紫垣[2] 游明丹 SUN Fengqiong;ZHANG Guowei;WANG Lingqiao;XU Guiyong;GUO Chengwei;SUN Yan;YANG Rui;ZHANG Lu;YANG Guanghong;ZHOU Ziyuan;YOU Mingdan(School of Public Health,Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education,Guizhou Medical University,Guiyang,Guizhou Province,561113;Department of Environmental Health,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵阳561113 [2]陆军军医大学(第三军医大学)军事预防医学系环境卫生学教研室,重庆400038

出　　处：《陆军军医大学学报》2024年第21期2369-2382,共14页Journal of Army Medical University

基　　金：国家重点研发计划(2022YFC2702902)。

摘　　要：目的探讨邻苯二甲酸二(2-乙基己基)酯(di-2-ethylhexyl phthalate,DEHP)诱导睾丸间质细胞铁死亡中RNA去甲基化酶脂肪量和肥胖相关蛋白(fat mass and obesity-associated protein,FTO)的作用及机制。方法40只3周龄C57BL/6雄性小鼠通过随机数字表法分为对照组(玉米油)和3组DEHP染毒组(5、250、500 mg/kg体质量),连续灌胃35 d;TM3小鼠睾丸间质细胞以0、100、200、400μmol/L邻苯二甲酸单(2-乙基己基)酯(mono-2-ethylhexyl phthalate,MEHP)处理24 h,质粒转染构建Fto过表达TM3细胞。ELISA检测血清睾酮水平,免疫组化检测睾丸组织中蛋白表达,比色法检测睾丸中Fe 2+、丙二醛和脂质过氧化物水平。甲基化RNA免疫共沉淀、RT-PCR和Western blot检测N6-甲基腺嘌呤(N6-methyladenosine,m6A)修饰水平。结果250、500 mg/kg DEHP染毒组小鼠的血清睾酮水平显著降低(P<0.01),睾丸组织Fe 2+、丙二醛、脂质过氧化物水平显著升高(P<0.01),RNA去甲基化酶FTO、铁死亡相关分子铁蛋白重链1(ferritin heavy chain 1,FTH1)和谷胱甘肽过氧化酶4(glutathione peroxidase 4,GPX4)蛋白水平显著下调(P<0.05),转铁蛋白受体(transferrin receptor,TFRC)、膜铁转运蛋白(ferroportin,FPN)、环氧合酶-2(cyclooxygenase-2,COX-2)和酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain family member 4,ACSL4)蛋白水平显著上调(P<0.05)。MEHP处理TM3细胞24 h后,细胞活力下降、胞内活性氧(ROS)含量升高,线粒体膜电位(mitochondrial membrane potential,MMP)显著降低(P<0.01),Fto的mRNA和蛋白水平均显著下调(P<0.01),其余铁死亡相关蛋白的变化也与睾丸组织中趋势一致,提示睾丸间质细胞发生了铁死亡。以铁死亡抑制剂Fer-1干预或过表达Fto均能显著抑制MEHP诱导的TM3细胞毒性和铁死亡(P<0.05),同时过表达Fto使Gpx4和Fth1 mRNA的m6A修饰水平降低(P<0.05)。结论FTO表达抑制引起Gpx4、Fth1的m6A修饰异常可能是DEHP诱导睾丸间质细胞铁死亡的机制。Objective To explore the role and mechanism of RNA demethylase fat mass and obesity-associated protein(FTO)in the ferroptosis in testicular interstitial cells induced by di(2-ethylhexyl)phthalate(DEHP).Methods Forty 3-week-old C57BL/6 male mice were randomly divided into a control group(corn oil)and 3 dosed DEHP treatment groups(5,250 and 500 mg/kg),and received an intragastric infusion of corresponding agents for 35 d,respectively.After mouse testicular interstitial TM3 cells was treated with 0,100,200 and 400μmol/L mono-2-ethylhexyl phthalate(MEHP)for 24 h,corresponding plasmids were transfected to construct Fto overexpressing TM3 cells.Serum testosterone level was detected by ELISA,expression of testicular proteins was detected with immunohistochemical assay,and contents of Fe 2+,malondialdehyde(MDA)and lipid peroxides in the testicle were detected by colorimetry.Methylated RNA immunoprecipitation,RT-PCR,and Western blotting were used to detect the level of N6-methyladenosine(m6A)modification.Results In the mice exposed to 250 and 500 mg/kg DEHP,the serum testosterone level was significantly reduced(P<0.01),contents of Fe 2+,MAD and lipid peroxides in testicular tissue were obviously increased(P<0.01),and protein levels of RNA demethylase FTO,and ferroptosis related molecules ferritin heavy chain 1(FTH1)and glutathione peroxidase 4(GPX4)were significantly down-regulated(P<0.05),while those of transferrin receptor(TFRC),ferroportin(FPN),cyclooxygenase-2(COX-2),and acyl-CoA synthetase long-chain family member 4(ACSL4)were notably up-regulated(P<0.05).MEHP treatment for 24 h resulted in remarkably decreased cell viability in the TM3 cells,increased production of intracellular reactive oxygen species(ROS),reduced mitochondrial membrane potential(MMP)(P<0.01),down-regulated mRNA and protein levels of Fto(P<0.01),and the changes in other ferroptosis related proteins were consistent with the trend in testicular tissue,indicating ferroptosis in testicular interstitial cells.Intervention with ferroptosis inhibitor Fer

关 键 词：邻苯二甲酸二(2-乙基己基)酯 铁死亡 肥胖相关基因 睾丸 间质细胞 

分 类 号：R322.64[医药卫生—人体解剖和组织胚胎学] R992[医药卫生—基础医学] R994.6