HDAC6抑制剂通过保护肾小球内皮细胞线粒体稳态和EMT改善糖尿病肾病  

Histone deacetylases 6 inhibitor improves diabetic nephropathy by protecting mitochondrial homeostasis and inhibiting epithelial⁃mesenchymal transition of glomerular endothelial cells

作　　者：陈小丽 罗富里 童梦瑶 CHEN Xiaoli;LUO Fuli;TONG Mengyao(Department of Rheumatology,Affiliated Hospital of Jinggangshan University,Ji’an 343000,China;Department of Nephrology,Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine,Nanchang 330006;Respiratory Department,the Second Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine,Nanchang 330006)

机构地区：[1]井冈山大学附属医院肾病风湿免疫科,江西吉安343000 [2]江西中医药大学附属医院肾病科,南昌330006 [3]江西中医药大学第二附属医院呼吸科,南昌330006

出　　处：《中国比较医学杂志》2023年第10期71-80,共10页Chinese Journal of Comparative Medicine

基　　金：江西省青年科学基金资助项目(20192BAB215051)。

摘　　要：目的 探讨组蛋白去乙酰化酶6(histone deacetylases 6,HDAC6)特异性小分子抑制剂Tubastatin A对糖尿病肾病(diabetic nephropathy, DN)小鼠肾损伤的保护作用和机制。方法 C57BL/6J小鼠随机分为3组:对照组、DN组和Tubastatin A组。DN组和Tubastatin A组行包膜下肾切除术以切除右肾,并通过腹膜内注射STZ诱导DN。Tubastatin A组接受Tubastatin A治疗,每3 d 1次,连续治疗8周。RNA测序分析DN组和Tubastatin A组肾组织中差异表达基因。通过透射电子显微镜评估线粒体受损情况,以及DHE染色估计肾组织中的活性氧(reactive oxygen species, ROS)水平。将小鼠肾小球内皮细胞(mice glomerular endothelial cell, mGEC)暴露于高葡萄糖(high glucose, HG)培养基或40 mmol/L甘露醇(对照),加入或不加入Tubastatin A处理。采用蛋白质印迹分析HDAC6、肾损伤标志物KIM1和上皮细胞-间充质转化(epithelial-mesenchymal transition, EMT)标志物的表达情况,以及流式细胞仪检测细胞中线粒体ROS和细胞凋亡情况。结果 DN小鼠肾组织和暴露于HG的mGEC细胞中HDAC6表达上调,并与KIM1水平升高一致。组织学分析显示DN小鼠的显著形态学变化,包括肾小球肥大、肾小球系膜基质积聚、肾小球基底膜增厚、肾小管基底膜增厚和出现肾小球、肾小管间质纤维化;Tubastatin A治疗缓解了这些不良改变。与对照DMSO相比,Tubastatin A在HG处理下显著降低了mGEC细胞中肾损伤分子1(kidney injury molecular1,KIM1)、HDAC6、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、N-钙粘蛋白、波形蛋白表达(P<0.05),并上调E-钙粘蛋白表达(P<0.05)。透射电子显微镜显示Tubastatin A组小鼠的肾小球内皮细胞受损线粒体的比例较DN组显著降低(P<0.01)。Tubastatin A组小鼠肾组织中ROS水平较DN组降低(P<0.01)。RNA测序结果表明,与DN组小鼠相比,Tubastatin A组小鼠肾组织中与ECM-受体相互作用和与三羧酸(TCA)循环相关的基因富集。在mGEC细Objective To investigate the protective effect and mechanism of histone deacetylase 6(HDAC6)-specific small molecule inhibitor tubastatin A on renal injury in diabetic nephropathy(DN)mice.Methods C57BL/6J mice were randomly divided into control,DN and tubastatin A groups.Mice in DN and tubastatin A groups were intraperitoneally injected with 80 mg/kg STZ daily for 3 days after removal of one kidney.The tubastatin A group received tubastatin A treatments every 3 days for 8 weeks.RNA-sequencing of differentially expressed genes was performed in kidney tissue of DN and tubastatin A groups.Mitochondrial damage was assessed by transmission electron microscopy.ROS levels in kidney tissue were estimated by DHE staining.Mouse glomerular endothelial cells(mGECs)were exposed to high glucose(HG)medium or 40 mmol/L mannitol(control)with or without tubastatin A treatment.Western blot was used to analyze expression of HDAC6,kidney injury marker KIM1,and Epithelial-Mesenchymal Transition(EMT)markers.Flow cytometry was used to detect mitochondrial ROS and apoptosis in cells.Results HDAC6 expression was upregulated in DN mouse kidney tissue and mGECs exposed to HG,which was consistent with the increased level of KIM1.Histological analysis showed significant morphological changes in DN mice,including glomerular hypertrophy,mesangial matrix accumulation,glomerular basement membrane thickening,tubular basement membrane thickening,and the presence of glomerular intertubular fibrosis.Tubastatin A treatment alleviated these changes.Compared with DMSO as the control,tubastatin A significantly decreased expression of KIM1,HDAC6,α-SMA,N-cadherin and vimentin(P<005)and up-regulated E-cadherin expression(P<005)in mGECs under HG treatment.RNA-sequencing revealed enrichment of genes related to ECM-receptor interactions and the tricarboxylic acid cycle in kidney tissue of the Tubastatin A group compared with the findings in the DN group.Transmission electron microscopy showed that the proportion of damaged mitochondria in glomerular endotheli

关 键 词：组蛋白去乙酰化酶6 Tubastatin A 糖尿病肾病 线粒体 肾小球内皮细胞 

分 类 号：R-33[医药卫生]