TRPC6 DNA甲基化在同型半胱氨酸致胰岛β细胞焦亡中的作用研究  

The role of TRPC6 DNA methylation in homocysteine-induced pancreatic islet β-cell pyrolysis

作　　者：揭育祯 杨慧霞 周瑜瑾 张喜文 虎小忠 柳杨 丁宁[1] 卢冠军[3] 马胜超[1] JIE Yu-zhen;YANG Hui-xia;ZHOU Yu-jin;ZHANG Xi-wen;HU Xiao-zhong;LIU Yang;DING Ning;LU Guan-jun;MA Sheng-chao(National Health Commission Key Laboratory of Metabolic Cardiovascular Disease Research,Ningxia Medical University,Yinchuan 750004,Ningxia,China;不详)

机构地区：[1]宁夏医科大学国家卫生健康委代谢性心血管疾病研究重点实验室,宁夏银川750004 [2]宁夏医科大学 [3]宁夏医科大学总医院泌尿外科,宁夏银川750004

出　　处：《广东医学》2022年第2期162-168,共7页Guangdong Medical Journal

基　　金：国家自然科学基金地区项目(82060139,81760139);国家自然科学基金青年项目(81900273);宁夏回族自治区重点研发计划一般项目(2018BEG03026);第三批宁夏青年科技人才托举工程项目(TJGC2018010);中国科学院西安分院“西部青年学者”项目(XAB2018AW07);大学生创新创业训练项目(NXCX2018122)。

摘　　要：目的探讨瞬时感受器电位通道6(TRPC6)DNA甲基化在同型半胱氨酸致胰岛β细胞焦亡中的作用。方法20只cbs^(+/-)小鼠随机分为2组,每组10只。对照组为正常普通饮食,高蛋氨酸组为高蛋氨酸饮食(普通饮食中加入1.7%蛋氨酸)。采用全自动生化分析仪检测小鼠血糖水平;Western blot检测焦亡相关蛋白NLRP3、Caspase-1、IL-1β和TRPC6的表达;qRT-PCR测定胰岛β细胞TRPC6 mRNA表达水平;qRT-PCR和Western blot检测DNMT1、DNMT3a及DNMT3b mRNA和蛋白的表达;采用巢式降落式特异性PCR(nMS-PCR)测验TRPC6 DNA甲基化水平;Pearson相关系数对胰腺组织中TRPC6甲基化水平与血糖以及细胞焦亡水平进行相关性分析。结果与对照组比较,高蛋氨酸组中cbs^(+/-)小鼠血糖水平明显升高(P<0.01);NLRP3、Caspase-1以及IL-1β表达水平显著升高(P<0.01);TRPC6的mRNA和蛋白表达水平明显升高(P<0.01)。同时,高蛋氨酸组TRPC6启动子区DNA甲基化水平降低(P<0.01),DNMT1 mRNA和蛋白表达水平明显降低(P<0.01),而DNMT3a和DNMT3b蛋白表达变化未有统计学意义(P>0.05);Pearson相关性分析发现TRPC6甲基化水平与血糖水平(r=-0.7824,P=0.0075)以及NLRP3(r=-0.7775,P=0.0081)、Caspase-1(r=-0.8992,P=0.0004)、IL-1β(r=-0.6691,P=0.0344)蛋白表达均呈负相关。结论同型半胱氨酸可以诱导胰岛β细胞焦亡的发生,而TRPC6 DNA低甲基化表达可能是Hcy诱导胰岛β细胞焦亡发生的重要机制之一。Objective To investigate the role of transient receptor potential channel 6(TRPC6) DNA methylation in homocysteine-induced pancreatic β-cell pyrolysis. Methods Twenty cbs^(+/-) mice were randomly divided into 2 groups. The cbs^(+/-) control group was fed with normal diet, and the cbs^(+/-) high methionine group was with high methionine diet. Automatic biochemical analyzer was used to assess the blood glucose level of mice. Western blot was used to detect the expression of pyrolysis-related proteins NLRP3, Caspase-1, IL-1β and TRPC6. qRT-PCR was used to determine the expression level of TRPC6 mRNA in pancreatic β cells. And qRT-PCR and Western Blot were applied to assess the DNMT1, DNMT3 a, and DNMT3 b mRNA and protein expression. Nested landing specific PCR(nMS-PCR) was used to test the TRPC6 DNA methylation level. Pearson correlation coefficient was used to determine the correlation between TRPC6 methylation level in pancreatic tissues and blood glucose. Results Compared with the normal control group, the blood glucose level of cbs^(+/-) mice in the high methionine group was significantly increased(P<0.01);the expression of NLRP3, Caspase-1 and IL-1β was significantly increased(P<0.01);the mRNA and protein expression of TRPC6 was also significantly increased(P<0.01). In the high methionine group TRPC6 promoter region DNA methylation level was significantly reduced(P<0.01), so was DNMT1 mRNA and protein expression(P<0.01);while there was no significant difference in DNMT3 a and DNMT3 b protein expression. Pearson correlation analysis showed that TRPC6 methylation and blood glucose level(r=-0.782 4, P=0.007 5), NLRP3(r=-0.777 5, P=0.008 1), Caspase1(r=-0.899 2, P=0.000 4), or IL-1β(r=-0.669 1, P=0.034 4) protein expression were significantly negatively correlated. Conclusion Homocysteine can induce pancreatic β-cell pyrolysis, and the expression of TRPC6 DNA hypomethylation may play an important role in it.

关 键 词：同型半胱氨酸 胰岛Β细胞 TRPC6 DNA甲基化 焦亡 

分 类 号：R587.1[医药卫生—内分泌] R335.6[医药卫生—内科学]