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中国临床药理学与治疗学 ›› 2023, Vol. 28 ›› Issue (12): 1331-1338.doi: 10.12092/j.issn.1009-2501.2023.12.002

• 基础研究 • 上一篇    下一篇

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

施珊红,林威远,张杰群,郑燕玲   

  1. 福建医科大学附属第二医院肾内科,泉州  362000,福建
  • 收稿日期:2023-04-23 修回日期:2023-09-15 出版日期:2023-12-26 发布日期:2023-12-21
  • 通讯作者: 林威远,男,硕士,主任医师,研究方向:肾病。 E-mail: qzlinweiyuan@163.com
  • 作者简介:施珊红,女,硕士,住院医师,研究方向:肾脏疾病的基础与临床研究。 E-mail: qzshish@163.com
  • 基金资助:
    福建省卫生健康科技计划项目(2022G0184)

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

SHI Shanhong, LIN Weiyuan, ZHANG Jiequn, ZHENG Yanling   

  1. Department of Nephrology, Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China 
  • Received:2023-04-23 Revised:2023-09-15 Online:2023-12-26 Published:2023-12-21

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(10 μmol/L大黄素处理MCs)、M-EMO组(25 μmol/L大黄素处理MCs)、H-EMO组(50 μmol/L大黄素处理MCs)、H-EMO+miR-96-5p-NC组(50 μmol/L大黄素处理MCs后转染miR-96-5p-NC)、H-EMO+miR-96-5p-minic组(50 μmol/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、M-EMO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。

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

Abstract:

AIM: To investigate the injury of emodin (EMO) in reduce of glomerular mesangial cells (MCs) in lupus nephritis by targeting forkhead protein K2 (FOXK2) through miR-96-5p. METHODS: The contents of 24 h urine protein, serum urea nitrogen (BUN) and serum creatinine (Scr) in MRL/faslpr mice (lupus nephritis group) and MRL/MPJ mice (control group) were detected. MCs were separated, purified and divided into: MCs group (MCs without any treatment), L-EMO group (MCs treated with 10 μmol/L Emodin), M-EMO group (MCs treated with 25 μmol/L Emodin), H-EMO group (MCs treated with 50 μmol/L Emodin), H-EMO+miR-96-5p-NC group (MCs treated with 50 μmol/L Emodin and transfected with miR-96-5p-NC), and H-EMO+miR-96-5p-minic group (MCs treated with 50 μmol/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 fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-96-5p. Western 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 protein 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), aspartate specific cysteine proteinase-3 (cleaved Caspase-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-96-5p-minic group obviously increased the miR-96-5p expression, inflammatory factor levels, A450 value and Bcl-2 protein level (P<0.05), and obviously decreased FOXK2 level and cell apoptosis rate (P<0.05). CONCLUSION: EMO can reduce the injury of lupus nephritis MCs by down-regulating miR-96-5p and then up-regulating FOXK2.

Key words: emodin, miR-96-5p, forkhead box protein K2, lupus nephritis, glomerular mesangial cells

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