大黄素通过miR-96-5p靶向叉头蛋白K2减轻狼疮性肾炎肾小球系膜细胞的损伤  

Emodin reduces the injury of glomerular mesangial cells in lupus nephritis by targeting forkhead protein K2 through miR-96-5p

作　　者：施珊红 林威远[1] 张杰群 郑燕玲 SHI Shanhong;LIN Weiyuan;ZHANG Jiequn;ZHENG Yanling(Department of Nephrology,Second Affiliated Hospital of Fujian Medical University,Quanzhou 362000,Fujian,China)

机构地区：[1]福建医科大学附属第二医院肾内科,福建泉州362000

出　　处：《中国临床药理学与治疗学》2023年第12期1331-1338,共8页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：福建省卫生健康科技计划项目(2022G0184)。

摘　　要：目的:探讨大黄素通过miR-96-5p靶向叉头蛋白K2(FOXK2)减轻狼疮性肾炎肾小球系膜细胞(MCs)损伤。方法:检测MRL/faslpr小鼠(狼疮性肾炎肾组)和MRL/MPJ小鼠(对照组)24 h尿蛋白以及血清尿素氮(BUN)和血肌酐(Scr)含量。MCs分离纯化后分为:MCs组(未做任何处理的MCs);L-EMO组(10μmol/L大黄素处理MCs)、M-EMO组(25μmol/L大黄素处理MCs)、H-EMO组(50μmol/L大黄素处理MCs)、H-EMO+miR-96-5p-NC组(50μmol/L大黄素处理MCs后转染miR-96-5p-NC)、HEMO+miR-96-5p-minic组(50μmol/L大黄素处理MCs后转染miR-96-5p-minic)。双荧光素酶报告基因实验验证miR-96-5p和FOXK2的靶向关系;实时荧光定量聚合酶链式反应(qRT-PCR)检测miR-96-5p表达;蛋白质印迹法(Western blot)检测FOXK2以及凋亡相关蛋白表达;酶联免疫吸附测定(ELISA)法检测MCs炎性因子水平;细胞计数试剂盒8(CCK-8)测定MCs活力;Annexin-V FITC/PI双染法检测MCs凋亡情况。结果:与对照组相比,狼疮性肾炎组24 h尿蛋白含量、血清BUN和Scr水平显著升高(P<0.05);与MCs组相比,L-EMO组、MEMO组、H-EMO组miR-96-5p的表达量、白介素1β(IL-1β)、白介素6(IL-6)、肿瘤坏死因子-α(TNF-α)水平、A450值、B淋巴细胞瘤-2(Bcl-2)蛋白水平依次显著下降(P<0.05),FOXK2水平、细胞凋亡率、Bcl-2相关的X基因(Bax)、天冬氨酸特异性半胱氨酸蛋白酶-3(cleaved-Caspase-3)蛋白水平依次显著升高(P<0.05),大黄素作用效果呈现剂量依赖性;与H-EMO组、H-EMO+miR-96-5p-NC组相比,H-EMO+miR-96-5p-minic组显著升高miR-96-5p的表达量、炎性因子水平、A450值以及Bcl-2蛋白水平(P<0.05),显著降低FOXK2水平以及细胞凋亡率(P<0.05)。结论:大黄素通过下调miR-96-5p进而上调FOXK2,从而减轻狼疮性肾炎MCs损伤。AIM:To investigate the injury of emo-din(EMO)in reduce of glomerular mesangial cells(MCs)in lupus nephritis by targeting forkhead pro-tein K2(FOXK2)through miR-96-5p.METHODS:The contents of 24 h urine protein,serum urea ni-trogen(BUN)and serum creatinine(Scr)in MRL/faslpr mice(lupus nephritis group)and MRL/MPJ mice(control group)were detected.MCs were sep-arated,purified and divided into:MCs group(MCs without any treatment),L-EMO group(MCs treated with 10μmol/L Emodin),M-EMO group(MCs treat-ed with 25μmol/L Emodin),H-EMO group(MCs treated with 50μmol/L Emodin),H-EMO+miR-96-5p-NC group(MCs treated with 50μmol/L Emodin and transfected with miR-96-5p-NC),and H-EMO+miR-96-5p-minic group(MCs treated with 50μmol/L Emodin and transfected with miR-96-5p-minic).Double luciferase report experiment was used to verify the targeting relationship between miR-96-5p and FOXK2.The real-time quantitative fluores-cent polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-96-5p.West-ern blot was used to detect the expression of FOXK2 and apoptosis related proteins.The enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors in MCs.cell count kit 8(CCK-8)was used to determine the activity of MCs.Annexin-V FITC/PI double staining was used to detect apoptosis of MCs.RESULTS:Compared with the control group,24 h urinary pro-tein content,serum BUN and Scr levels in the lupus nephritis group were significantly increased(P<0.05).Compared with the MCs group,the miR-96-5p expression,interleukin1β(IL-1β),interleukin6(IL-6),tumor necrosis factor-α(TNF-α),A450 value and B-lymphoblastoma-2(Bcl-2)protein in the L-EMO group,M-EMO group and H-EMO group were significantly decreased(P<0.05),the FOXK2 level,cell apoptosis rate,Bcl-2 related X gene(Bax),as-partate specific cysteine proteinase-3(cleaved Cas-pase-3)protein levels were significantly increased,respectively(P<0.05),the effect of Emodin was dose-dependent.Compared with the H-EMO group and H-EMO+miR-96-5p-NC group,H-EMO+miR-96-

关 键 词：大黄素 miR-96-5p 叉头蛋白K2 狼疮性肾炎 肾小球系膜细胞 

分 类 号：R593.242[医药卫生—内科学]