靶向抑制m6A甲基化对肾间质纤维化的影响  

Improvement effect of inhibiting m6A methylation on renal interstitial fibrosis

作　　者：杨云巧 田倩婷 潘婷 朱佳美 王子名 王旭艳 陈腾祥 金帮明[1,2,3] YANG Yunqiao;TIAN Qianting;PAN Ting;ZHU Jiamei;WANG Ziming;WANG Xuyan;CHEN Tengxiang;JIN Bangming(Department of Physiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 561113,Guizhou,China;Guizhou Institute of Precision Medicine,the Affiliated Hospital of Guizhou Medical University,Guiyang 550003,Guizhou,China;Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,Guizhou Medical University,Guiyang 561113,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院生理教研室,贵州贵阳561113 [2]贵州医科大学附属医院贵州精准医学研究院,贵州贵阳550003 [3]贵州医科大学贵州省常见病发病机制与药物研究重点实验室,贵州贵阳561113

出　　处：《贵州医科大学学报》2025年第2期157-166,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金资助项目(32160144);中国博士后科学基金(2023MD734162)。

摘　　要：目的探讨m6A甲基化修饰在肾纤维化中的作用及评价m6A甲基化抑制剂对肾纤维化的干预效果。方法使用C57BL/6小鼠建立单侧输尿管结扎(unilateral ureteral obstruction,UUO)诱导的小鼠肾纤维化模型,随机分为Sham组、UUO-3 d组、UUO-7 d组及UUO-14 d组,另设m6A抑制剂SAH干预组,SAH治疗第7天时处死小鼠;HE染色后进行肾损伤评分,运用Masson染色和Sirius red染色分析小鼠肾脏胶原累积情况;转化生长因子-β(transforming growth factor-β,TGF-β)以10μg/L的浓度处理HK-2细胞,分别在0 h、24 h、48 h和72 h时,采用m6A Dot blot检测总体m6A甲基化水平;通过RT-qPCR和IHC检测纤维化标志物(α-SMA、CollagenⅢ和FN1)、m6A相关基因(Mettl3、Mettl14、Alkbh5和FTO)的mRNA和蛋白表达。结果m6A甲基化修饰水平随着UUO术后时间的延长逐渐增加,而Mettl3、Mettl14、Alkbh5及FTO的mRNA或蛋白表达随着纤维化水平的增加而显著减少(P<0.01);TGF-β处理显著增加HK-2细胞中m6A的甲基化修饰水平,具有时间依赖效应(P<0.05);SAH显著抑制UUO诱导α-SMA、CollagenⅢ及FN1的蛋白表达(P<0.01),显著减少肾间质区域的胶原沉积,明显改善UUO诱导的肾损伤和肾间质纤维化(P<0.01)。结论m6A甲基化修饰在肾纤维化发展中起关键作用,m6A甲基化抑制剂SAH可通过调节其修饰水平显著改善肾纤维化。Objective To explore the function of m6A methylation in kidney fibrosis and evaluate the intervention effect of m6A inhibitors on kidney fibrosis.Methods Unilateral ureteral obstruction(UUO)induced kidney fibrosis model was established in C57BL/6 mice,which were randomly divided into Sham,UUO-3 d,UUO-7 d,and UUO-14 d groups.Additionally,m6A inhibitor SAH intervention group(SAH group)was set up,and the mice were sacrificed on 7 th day after SAH treatment.After HE staining,kidney injury was scored.Masson staining and Sirius red staining were used to analyze the accumulation of mouse renal collagen.HK-2 cells were treated with transforming growth factor-β(TGF-β)at a concentration of 10 ng/mL.At 0 h,24 h,48 h,and 72 h,m6A Dot blot was used to detect the overall m6A methylation levels.RT-qPCR and IHC were applied to detect the mRNA and protein expressions of fibrosis markers(α-SMA,CollagenⅢ,and FN1)and m6A-related genes(Mettl3,Mettl14,Alkbh5,and FTO).Results The level of m6A methylation was increased gradually with the extension of Unilateral ureteral obstruction(UUO)postoperative time,while the mRNA or protein expressions of Mettl3,Mettl14,Alkbh5 and FTO were decreased significantly with the increasing kidney fibrosis level(P<0.01).TGF-βtreatment markedly increased m6A methylation levels in HK-2 cells in a time-dependent manner(P<0.05).SAH effectively inhibited Unilateral ureteral obstruction(UUO)induced expressions ofα-SMA,CollagenⅢand FN1 proteins(P<0.01),reduced collagen deposition in renal interstitial regions and improved Unilateral ureteral obstruction(UUO)induced kidney injury and renal interstitial fibrosis(P<0.01).Conclusion m6A methylation plays a key role in the development of renal fibrosis.m6A methylation inhibitor SAH can significantly improve renal fibrosis by regulating its modification level.

关 键 词：肾纤维化 单侧输尿管结扎 N6-甲基腺苷修饰 脂肪质量和肥胖相关蛋白 

分 类 号：R737.9[医药卫生—肿瘤] R730.23[医药卫生—临床医学]