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 50 μg/mL,TBHQ+PAN处理组培养液中加入10 nmol/L TBHQ以及PAN 50 μg/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)40 μmol/L,姜黄素+GO组给予姜黄素40 μmol/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TB-HQ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蛋白表达降低,差异均有统计学意义(P＜0.05);空白对照组与姜黄素组TRPC6/Transgelin及线粒体调控蛋白表达比较,差异均无统计学意义(P＞0.05).TRPC6通道序列存在多个m6A修饰位点,均具有被甲基转移酶(METTL)3、METTL14、肾母细胞肿瘤1相关蛋白(WTAP)和去甲基化酶ALKB同源蛋白(ALKBH)、m6A去甲基化酶(FTO)调控的潜在可能.对12个m6A调节基因进行表达分析,发现m6A调节基因的表达在PAN诱导足细胞损伤中发生显著差异.结论 TRPC6介导钙离子内流可被氧化应激激活参与足细胞损伤,激活Nrf2可以减少钙过负荷所致线粒体损伤而保护足细胞.TRPC6序列中存在多个高m6A修饰靶点,肾病综合征发病机理可能通过m6A修饰足细胞TRPC6离子通道,m6A相关调控基因在肾病足细胞损伤中发生明显变化.
Abstract
Objective To preliminarily investigate the role and potential mechanism of N6-methyladenosine(m6A)epigenetic modifica-tion 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,puromy-cin aminonucleoside(PAN)treatment group,and TBHQ+PAN treatment group.The control group was treated with normal complete culture me-dium,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 cul-ture 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 col-lected 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 recep-tor 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,curcu-min+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 bioinformat-ics analysis was performed on GSE124622,a public database of PAN-induced podocyte injury models.Results There was no statistically signif-icant difference between the control group and the TBHQ group in current(P＞0.05).Compared with the control group,the current of PAN treated group increased,while the current of TBHQ+PAN group decreased,the differences were statistically significant(P＜0.05).Compared with the blank control group,the expression of Nrf2,NQO-1,TRPC6,and Transgelin proteins in the GO group increased,the differences were statistically significant(P＜0.05);compared with the GO group,the expression of Nrf2,NQO-1,TRPC6,and Transgelin proteins in the cur-cumin+GO group decreased,the differences were statistically significant(P＜0.05).Compared with the blank control group,the expression of mitochondrial regulatory proteins Mfn2 and Opa1 in the GO group decreased,while the expression of Drp1 protein increased,the differences were statistically significant(P＜0.05);compared with the GO group,the expression of granule regulatory proteins Mfn2 and Opa1 in the curcumin+GO group increased,while the expression of Drp1 protein decreased,the differences were statistically significant(P＜0.05);there were no statistically significant differences in the expression of TRPC6/Transgelin and mitochondrial regulatory proteins between the blank control group and the curcumin group(P＞0.05).There were multiple m6A modification sites in the TRPC6 channel sequence,all of which are potentially regula-ted by methyltransferase(METTL)3,METTL14,Wilms tumor 1 associated protein(WTAP),and demethylases Alk B homologue(ALKBH),fat mass and obesity-associated protein(FTO).The expression of 12 m6A regulatory genes was analyzed,and it was found that the expression of m6A regulatory genes was significantly different in PAN-induced podocyte injury.Conclusion TRPC6 mediated Ca2+influx can be activated by oxidative stress and participate in podocyte injury.Activation of Nrf2 can reduce mitochondrial damage caused by calcium overload and protect podocyte.There are several high m6A modification targets in TRPC6 sequence.The pathogenesis of nephrotic syndrome may be mediated by m6A modification of TRPC6 ion channel in podocyte,and m6A related regulatory genes have significant changes in nephrotic podocyte injury.
NETL
NSTL
维普
万方数据