SMYD2介导高糖诱导小鼠肾成纤维细胞活化的机制研究  

Mechanism underlying the role of SMYD2 in mediating high glucose-induced activation of mouse renal fibroblasts

作　　者：左思洋 李霞 陈思羽 陈敏 彭睿 邹雪 龙合花 杨元 元辉雄 郭兵[3] 赵青青 刘丽荣 Zuo Si-yang;Li Xia;Chen Si-yu;Chen Min;Peng Rui;Zou Xue;Long He-hua;Yang yuan;Yuan Hui-xiong;Guo Bing;Zhao Qing-qing;Liu Li-rong(Center for Clinical Laboratories,The Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Guizhou Precision Medicine Institute,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Laboratory of Pathogenesis Research,Drug Prevention and Treatment of Major Diseases,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学附属医院临床检验中心,贵州贵阳550004 [2]贵州医科大学附属医院精准医学研究院,贵州贵阳550004 [3]贵州医科大学重大疾病发病机制研究与药物防治实验室,贵州贵阳550025

出　　处：《中国现代医学杂志》2024年第4期29-38,共10页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:32060202);贵州省科技计划项目[No:黔科合基础-ZK(2023)一般377]、[黔科合基础-ZK(2023)一般379]。

摘　　要：目的 探讨组蛋白赖氨酸甲基转移酶2(SMYD2)介导体外高糖环境下小鼠肾成纤维细胞(NRK-49F)活化的可能机制。方法 复制糖尿病(DM)小鼠模型,经全自动生化分析仪检测各组小鼠血糖(BG)、血肌酐(Scr)水平,经苏木精-伊红、Masson染色观察小鼠肾组织病理改变,经Western blotting检测各组小鼠肾组织纤维连接蛋白(Fibronectin)、Ⅰ型胶原蛋白(CollagenⅠ)、α-平滑肌肌动蛋白(α-SMA)、组蛋白H3赖氨酸4三甲基化(H3K4me3)、SMYD2、核转录因子-κB p65(NF-κB p65)及磷酸化NF-κB p65(NF-κB p-p65)蛋白表达,经免疫荧光检测各组小鼠肾组织α-SMA及SMYD2表达。复制体外DM细胞模型,使用正常糖(含5.5mmol/L葡萄糖)或高糖(含30.0 mmol/L葡萄糖)处理NRK-49F细胞,经Western blotting检测SMYD2、α-SMA及细胞外基质(ECM)成分蛋白表达;经CCK-8法检测SMYD2特异性抑制剂AZ505的细胞毒性;在AZ505处理/不处理的条件下,经Western blotting检测SMYD2、α-SMA、H3K4me3、Fibronectin、CollagenⅠ、NF-κB p65、NF-κB p-p65蛋白表达,经免疫荧光检测α-SMA、SMYD2及NF-κB p-p65在细胞中的表达及空间定位情况。结果 DM组血清BG、Scr水平高于NC组(P <0.05)。DM组肾组织α-SMA、Fibronectin、CollagenⅠ、SMYD2、H3K4me3、NF-κB p-p65蛋白相对表达量高于NC组(P <0.05),两组NF-κB p65蛋白相对表达量比较,差异无统计学意义(P>0.05)。HG刺激不同时间点NRK-49F细胞α-SMA、Collagen I、Fibronectin、SMYD2蛋白相对表达量比较,差异均有统计学意义(P <0.05)。40μmol/L组、60μmol/L组NRK-49F细胞存活率较0μmol/L组低(P <0.05)。HG组NRK-49F细胞Fibronectin、CollagenⅠ、SMYD2蛋白相对表达量较NG组高(P <0.05),HG+AZ505(20μmol/L)组较HG组低(P <0.05)。HG组NRK-49F细胞Fibronectin、CollagenⅠ、α-SMA、H3K4me3、SMYD2蛋白相对表达量较NG组高(P <0.05),HG+AZ505组较HG组低(P <0.05)。HG组NRK-49F细胞NF-κB p-p65蛋白相对表达量较NG组高(P <0.05),HG+AZ505组较HG组低(P <0Objective To investigate the possible mechanism underlying the role of the histone lysine methyltransferase SET and MYND domain-containing protein 2(SMYD2)in mediating the activation of mouse renal fibroblasts(NRK-49F)in a high glucose environment in vitro.Methods The mouse model of diabetes mellitus(DM)was established,and levels of blood glucose(BG)and serum creatinine(Scr)were measured via the automatic biochemical analyzer.The renal histopathological changes in mice were observed by hematoxylin-eosin and Masson staining.The protein expression levels of fibronectin,typeⅠcollagen(collagenⅠ),alpha-smooth muscle actin(α-SMA),histone H3 lysine 4 trimethylation(H3K4me3),SMYD2,nuclear factor kappa B p65(NF-κB p65)and phosphorylated NF-κB p65(NF-κB p-p65)in the kidney tissues of DM mice were detected via Western blotting,and the expressions ofα-SMA and SMYD2 in the kidney tissues of DM mice were also detected via immunofluorescence.The cell models of DM were replicated in vitro,where NRK-49F cells were treated with glucose of normal concentration(containing 5.5 mmol/L of glucose)or high concentration(containing 30 mmol/L of glucose).Then protein expressions of SMYD2,α-SMA and components of extracellular matrix(ECM)were measured by Western blotting,and cell counting kit-8 was used to detect the cytotoxicity of SMYD2-specific inhibitor AZ505.When treated with or without AZ505,protein expressions of SMYD2,α-SMA,H3K4me3,fibronectin,collagenⅠ,NF-κB p65 and NF-κB p-p65 were detected via Western blotting,and the expressions and distribution ofα-SMA,SMYD2 and NF-κB p-p65 in NRK-49F cells were detected via immunofluorescence.Results The levels of BG and Scr in the DM group were higher than those in the NC group(P<0.05).The relative protein expressions ofα-SMA,fibronectin,collagenⅠ,SMYD2,H3K4me3 and NF-κB p-p65 in the kidney tissues of the DM group were higher than those of the NC group(P<0.05).There was no difference in the relative protein expression of NF-κB p65 between the two groups(P>0.05).The relati

关 键 词：糖尿病 小鼠肾成纤维细胞 组蛋白赖氨酸甲基转移酶2 AZ505 

分 类 号：R587.1[医药卫生—内分泌]