孔静,田平平,孟庆敏,卢雨微,张国忠,肖瑛,郭兵,石明隽
KONG Jing,TIAN Pingping,MENG Qingming,LU Yuwei,ZHANG Guozhong,XIAO Ying,GUO Bing,SHI Mingjun

• 1:贵州医科大学病理生理学教研室
• 2:贵州省常见慢性疾病发病机制及药物研究重点实验室
• 3:贵阳市第四人民医院

摘要(Abstract)：

目的:观察阿托伐他汀对糖尿病肾病(DN)小鼠肾组织SFRP1表达的影响,探讨阿托伐他汀延缓DN肾纤维化的可能机制。方法:30只雄性昆明小鼠均分为正常对照组(NC)组、糖尿病肾病(DN)组及阿托伐他汀治疗(Ato)组,DN和Ato组小鼠给予55 mg/kg链脲佐菌素(STZ)腹腔注射复制DN动物模型、NC组给予相同剂量无菌柠檬酸钠缓冲液;造模成功后第5周,Ato组小鼠给予阿托伐他汀20 mg/(kg·d)灌胃4周、NC组和DN组给予羧甲基纤维素灌胃;造模成功后第9周时处死小鼠,采用HE和Masson染色观察小鼠肾脏病理形态结构的变化,免疫组织化学染色观察小鼠肾组织β-catenin和SFRP1表达,Western blot检测小鼠肾组织SFRP1、Wnt4、β-catenin、E-cadherin、α-SMA和Col-Ⅳ蛋白表达,Real time-PCR检测小鼠肾组织SFRP1 mRNA表达。结果:HE和Masson染色见DN组肾小管管腔扩张,肾小管间质有蓝色胶原纤维沉积,Ato组上述病变减轻;免疫组织化学染色显示,β-catenin表达水平为DN组>Ato组>NC组,SFRP1表达水平为NC组>Ato组>DN组; Western blot结果显示,与NC组比较,DN组小鼠肾组织Wnt4、β-catenin、α-SMA及Col-Ⅳ表达上调(P<0.05)、SFRP1及Ecadherin表达下调(P<0.05),Ato治疗后Wnt4、β-catenin、α-SMA及Col-Ⅳ表达较DN组减少(P<0.05),SFRP1及E-cadherin表达增多(P<0.05); Real-time PCR结果显示,与NC组比较,DN组SFRP1 mRNA表达明显下调(P<0.05),经Ato治疗后SFRP1 mRNA表达较DN组增多(P<0.05)。结论:阿托伐他汀能抑制DN小鼠肾小管上皮细胞间质转化过程、减少肾间质细胞外基质沉积、延缓DN小鼠肾纤维化进程,可能与其上调SFRP1表达,进而增强对Wnt4/β-catenin信号通路的抑制作用有关。
Objective:To investigate the effect of atorvastatin on the expression of SFRP1 in renal tissue of diabetic nephropathy(DN) mice and explore the possible mechanism of atorvastatin in delaying the progression of DN renal fibrosis.Methods:Thirty male Kunming mice were randomly divided into normal control group(NC) group,diabetic nephropathy(DN) group and atorvastatin treatment(Ato) group.DN and Ato group mice were intraperitoneally injected with streptozotocin(STZ,55 mg/kg) to establish DN mouse model,and the same dose of sterile sodium citrate buffer was given to the NC group.At 5th week after establishing DN model,atorvastatin 20 mg/(kg·d) was given to the Ato group for four weeks.NC group and DN group were intragastrically administered with carboxymethylcellulose for 4 weeks.The mouse kidneys were collected at 9 th week.The pathological changes of kidneys were observed using HE staining and Masson staining.Immunohistochemical staining was performed to detect the expression levels of β-catenin and SFRP1.Western blot was used to examine the expression levels of SFRP1,Wnt4,β-catenin,E-cadherin,α-SMA and Col-IV.SFRP1 mRNA expression was determined by Real time-PCR.Results:HE and Masson staining showed that kidneys in DN group had renal tubular dilatation and blue collagen fiber deposition in the renal tubulointerstitium,and above lesions were alleviated in the Ato group.Immunohistochemical staining showed that the expression levels of β-catenin were ranked in the order of DN group>Ato group>NC group,and SFRP1 expression levels were ranked in the order of NC group>Ato group>DN group;Western blot analysis demonstrated that when compared with NC group,the expression levels of Wnt4,β-catenin,α-SMA and Col-IV in DN group was up-regulated(P<0.05),while SFRP1 and E-cadherin expression were down-regulated(P<0.05).When compared with DN group,Ato treatment decreased the expression levels of Wnt4,β-catenin,α-SMA and Col-IV(P<0.05),while the expression levels of SFRP1 and E-cadherin were upregulated(P<0.05).qPCR analysis showed that the expression levels of SFRP1 mRNA were significantly down-regulated in the DN group compared with the NC group(P<0.05),while Ato treatment significantly rescued its expression levels(P<0.05).Conclusion:Atorvastatin inhibits EMT in renal tubular epithelial cells of DN mice,reduces extracellular matrix deposition in renal interstitial cells and delays the progression of renal fibrosis in DN mice.Its effects might be associated with its upregulation of SFRP1 expression and its inhibition of Wnt4/β-catenin signaling.

关键词(KeyWords)： 糖尿病肾病;肾纤维化;阿托伐他汀;分泌型卷曲相关蛋白1;小鼠
diabetes nephropathy;kidney fibrosis;atovastatin;secreted frizzled-related protein 1;mouse

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81760131);; 贵州省重大常见疾病发病机制及药物防治研究科技创新人才团队[黔科合人才团队(2014)4007];; 贵州省教育厅招标项目[黔教合KY字(2015)370号];; 贵阳市科技支撑计划[黔科合支撑(2017)2837]

作者(Author): 孔静,田平平,孟庆敏,卢雨微,张国忠,肖瑛,郭兵,石明隽
KONG Jing,TIAN Pingping,MENG Qingming,LU Yuwei,ZHANG Guozhong,XIAO Ying,GUO Bing,SHI Mingjun

DOI: 10.19367/j.cnki.1000-2707.2019.01.003

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