基于CaMK4介导的细胞骨架重排探讨解毒祛瘀滋肾方对MRL/lpr狼疮小鼠足细胞损伤的影响  

Effects of Jiedu Quyu Zishen Prescription on podocyte damage via CaMK4-mediated cytoskeletal rearrangement in MRL/lpr mice

作　　者：宋子瑜 李莹 黄硕 范永升[2,3] 田丰源 SONG Ziyu;LI Ying;HUANG Shuo;FAN Yongsheng;TIAN Fengyuan(The First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310018,China;Zhejiang Chinese Medical University,Hangzhou 310053,China;The Second Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310005,China)

机构地区：[1]浙江中医药大学附属第一医院,杭州310018 [2]浙江中医药大学,杭州310053 [3]浙江中医药大学附属第二医院,杭州310005

出　　处：《中华中医药杂志》2024年第5期2357-2362,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：浙江省中医院学科高质量发展助推计划(No.2D02311)。

摘　　要：目的:从CaMK4介导的足细胞细胞骨架重排探讨解毒祛瘀滋肾方治疗狼疮性肾炎(LN)足细胞损伤的作用机制。方法:将8只C57BL/6小鼠设为对照组,24只MRL/lpr狼疮小鼠随机分为模型组、激素组、解毒祛瘀滋肾方组,每组8只。对照组和模型组灌胃蒸馏水,激素组灌胃强的松(5mg/kg)混悬液,解毒祛瘀滋肾方组灌胃解毒祛瘀滋肾方(36g/kg)水煎剂,连续干预8周。ELISA检测小鼠血清抗双链DNA抗体、肿瘤坏死因子α(TNF-α)和白细胞介素-6(IL-6)水平,全自动生化仪检测小鼠尿蛋白水平;HE染色观察肾脏病理改变;RT-PCR法检测肾脏Ssh1、Limk1、Cfl1 mRNA表达;免疫荧光染色法观察肾脏nephrin、synaptopodin、F-actin、G-actin、CaMK4表达。培养小鼠肾足细胞系(MPC-5),采用R848干预后,给予空白或解毒祛瘀滋肾方含药血清处理12 h,CCK-8法检测细胞活力;TUNEL法检测细胞凋亡,Fluo-4钙离子探针检测细胞内钙离子富集,免疫荧光染色法观察CaMK4表达。结果:体内实验中,与模型组比较,解毒祛瘀滋肾方组小鼠的血清抗双链DNA抗体、TNF-α和IL-6水平显著降低(P<0.01,P<0.05),尿蛋白水平显著降低(P<0.01),肾脏病理损伤改善,肾脏nephrin、synaptopodin蛋白表达水平上调;稳定细胞骨架相关转录因子Ssh1、Limk1、Cfl1 mRNA表达上升(P<0.05,P<0.01),肾脏F-actin表达水平上调,F/G-actin比率回升(P<0.05),同时肾脏CaMK4表达下降(P<0.01)。细胞实验中,与空白组比较,R848诱导细胞凋亡增多,胞内钙离子大量富集(P<0.01),CaMK4表达增加;解毒祛瘀滋肾方含药血清干预可减少细胞凋亡,减少内钙离子富集(P<0.01),下调细胞CaMK4表达。结论:解毒祛瘀滋肾方可能通过调控CaMK4的足细胞细胞骨架重排,改善LN中肾脏足细胞损伤,缓解SLE病情。Objective:To elucidate the mechanism of Jiedu Quyu Zishen Prescription(JP)in treating lupus nephritis(LN)podocyte injury via CaMK4-mediated podocyte cytoskeletal rearrangement.Methods:C57BL/6 mice served as controls,while MRL/lpr mice were randomized into model,PDN,and JP groups(n=8).Controls and models were gavaged with distilled water,PDN group received 5 mg/kg prednisone,and JP group received 36 g/kg JP decoction for 8 weeks.Serum anti-doublestranded DNA antibody,tumor necrosis factorα(TNF-α),and interleukin-6(IL-6)levels were assessed by ELISA;urinary proteins by automated biochemistry;renal pathology by HE staining;Ssh1,Limk1,and Cfl1 mRNA expressions by RT-PCR;nephrin,synaptopodin,F-actin,G-actin,and CaMK4 expressions by immunofluorescence staining.MPC-5,stimulated with R848to mimic lupus conditions,were treated with control-serum or JP-serum for 12 h.Cell viability,apoptosis,intracellular calcium,and CaMK4 were assessed.Results:JP group exhibited significantly lower serum anti-dsDNA antibody,TNF-α,IL-6 levels(P<0.01),reduced urinary protein levels(P<0.01),alleviates kidney pathology injury,upregulated nephrin and synaptopodin,and increased cytoskeletal stability transcription factors.Renal F-actin expression increased,F/G-actin ratio rebounded,while CaMK4expression decreased.R848 induced apoptosis in MPC-5,intracellular calcium increase(P<0.01),and elevated CaMK4;JP-serum reduced the apoptosis,intracellular calcium(P<0.01),and CaMK4 expression.Conclusion:JP may regulate CaMK4-mediated podocyte cytoskeletal rearrangement,ameliorating LN podocyte damage and SLE condition.

关 键 词：解毒祛瘀滋肾方 狼疮性肾炎 足细胞 细胞骨架 钙离子信号 

分 类 号：R285.5[医药卫生—中药学]