线粒体氧化应激及m6A表观遗传调控TRPC6钙通道在肾病综合征发病中的作用研究  

Role of mitochondrial oxidative stress and m6A epigenetic regulation of TRPC6 calcium channel in nephrotic syndrome

作　　者：姜丽娜[1] 孔玮晶 丁瑛雪[1] JIANG Li-na;KONG Wei-jing;DING Ying-xue(Department of Pediatrics,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China)

机构地区：[1]首都医科大学附属北京友谊医院儿科,北京100050

出　　处：《临床和实验医学杂志》2024年第8期785-789,共5页Journal of Clinical and Experimental Medicine

基　　金：国家自然科学基金资助项目(编号:81400718);国家自然科学基金资助项目(编号:82202059);首都医科大学校培育基金项目(编号:PYZ21054)。

摘　　要：目的 初步探讨N6-甲基腺嘌呤(m6A)表观遗传修饰瞬时受体电位阳离子通道6(TRPC6)通道失调在肾病综合征发病中的作用及潜在机理。方法 按照随机数字表法将小鼠足细胞分为4组:对照组、叔丁基对苯二酚(TBHQ)组、嘌呤霉素氨基核苷(PAN)处理组、TBHQ+PAN处理组。对照组为正常完全培养液,TBHQ组培养液中加入10 nmol/L TBHQ,PAN处理组培养液中加入PAN 50μg/mL,TBHQ+PAN处理组培养液中加入10 nmol/L TBHQ以及PAN 50μg/mL,刺激24 h收集细胞。应用膜片钳证实PAN损伤诱导TRPC6通道激活机理及1,4,5-肌醇三磷酸(IP3)受体拮抗剂TBHQ对电流的影响,检测对照组、PAN处理组、TBHQ组和TBHQ+PAN处理组细胞TRPC6通道电流变化。通过葡萄糖氧化酶(GO)建立足细胞氧化应激模型。另外按照随机数字表法将足细胞分为4组:空白对照组、GO组、姜黄素组、姜黄素+GO组。GO组给予GO 3.5 kU/L,姜黄素组给予Nrf2激动剂(姜黄素)40μmol/L,姜黄素+GO组给予姜黄素40μmol/L和GO 3.5 kU/L处理,给药处理8~12 h后收集细胞。检测各组Nrf2和特异性调控蛋白NAD(P)H:醌氧化还原酶1(NQO-1)、TRPC6及Transgelin蛋白和线粒体调控蛋白表达变化。通过SRAMP对TRPC6通道m6A位点进行精准预测,对PAN诱导足细胞损伤模型公共数据库GSE124622进行2次生物信息学分析。结果 对照组与TBHQ组电流比较,差异无统计学意义(P>0.05);与对照组比较,PAN处理组电流升高,而TBHQ+PAN组电流减小,差异均有统计学意义(P<0.05)。与空白对照组比较,GO组Nrf2、NQO-1、TRPC6及Transgelin蛋白表达均升高,差异均有统计学意义(P<0.01);与GO组比较,姜黄素+GO组Nrf2、NQO-1、TRPC6及Transgelin蛋白表达均降低,差异均有统计学意义(P<0.05)。与空白对照组比较,GO组线粒体调控蛋白Mfn2、Opa1蛋白表达均降低,Drp1蛋白表达升高,差异均有统计学意义(P<0.05);与GO组比较,姜黄素+GO组粒体调控蛋白Mfn2、Opa1蛋白表达升高,Drp1蛋白Objective To preliminarily investigate the role and potential mechanism of N6-methyladenosine(m6A) epigenetic modification of transient receptor potential cation channel 6(TRPC6) dysregulation in the pathogenesis of nephrotic syndrome.Methods According to the random number table method,the mice podocytes were divided into four groups:control group,tert-butylhydroquinone(TBHQ) group,puromycin aminonucleoside(PAN) treatment group,and TBHQ+PAN treatment group.The control group was treated with normal complete culture medium,the TBHQ group was added to 10 nmol/L TBHQ of the culture medium,the PAN treatment group was added to 50 μg/mL PAN of the culture medium,and the TBHQ+PAN treatment group was added to 10 nmol/L TBHQ and 50 μg/mL PAN of the culture medium,cells were collected after 24 hours of stimulation.Patch clamp was used to confirm the activation mechanism of TRPC6 channel induced by PAN damage and the effect of inositol 1,4,5-trisphosphate(IP3) TBHQ on current.TRPC6 channel current changes in control group,PAN treated group,IP3 receptor antagonist group and TBHQ+PAN treated group were detected.The glucose oxidase(GO) oxidative stress model of podocyte was established.According to the random number table method,the podocytes were divided into four groups:blank control group,GO group,curcumin group,and curcumin+GO group.The GO group was given GO 3.5 kU/L,while the curcumin group was given Nrf2 agonist(curcumin) 40 μmol/L,curcumin+GO group given curcumin 40 μmol/L and GO 3.5 kU/L,cells were collected after treatment with for 8-12 hours.And the contents of Nrf2 and specific regulatory protein NAD(P)H:quinone oxidoreductase 1(NQO-1),mitochondrial regulatory proteins,TRPC6 and Transgelin were detected after co-treatment with Nrf2 agonist.The m6A site of TRPC6 channel was accurately predicted by SRAMP,and a secondary bioinformatics analysis was performed on GSE124622,a public database of PAN-induced podocyte injury models.Results There was no statistically significant difference between the control group and the

关 键 词：肾病综合征 嘌呤霉素 氨基核苷 瞬时受体电位阳离子通道6 线粒体功能异常 N6-甲基腺嘌呤 m6A转移酶样3抑制剂 

分 类 号：R692[医药卫生—泌尿科学]