摘要
目的 探讨补体C3a受体在db/db小鼠糖尿病肾病发病中的作用,为糖尿病肾病的防治提供新靶点.方法 12只8周龄雄性2型糖尿病(db/db)小鼠及6只同窝野生型(db/m)小鼠饲养于无特定病原体级环境.实验分组及干预:db/m组、db/db组、C3a受体拮抗剂组,每组6只,其中C3a受体拮抗剂组为db/db小鼠腹腔注射C3a受体拮抗剂(SB290157,10 mg/kg)进行干预,2 d腹腔注射1次,连续注射8周.收集血、尿样本,检测小鼠体重、血糖、血肌酐、血尿素氮、尿微量白蛋白/尿肌酐、尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG)水平;收集肾组织,采用HE、PAS、Masson染色观察肾组织病理变化,免疫组化、免疫荧光及Western印迹检测肾组织C3及C3a受体表达,Western印迹检测肾组织肾损伤分1(Kim-1)、α平滑肌肌动蛋白(α-SMA)、紧密连接蛋白1(ZO-1)、波形蛋白、E钙黏蛋白(E-cadherin)的表达,免疫荧光分析α-SMA、ZO-1、Kim-1表达及分布,免疫组化分析白细胞介素1(IL-1)、肿瘤坏死因子α(TNF-α)表达,TUNEL法检测肾组织细胞凋亡情况.结果 与db/m组相比,db/db组小鼠体重、空腹血糖、尿微量白蛋白/尿肌酐及尿NAG均明显较高(均P＜0.01);与db/db组相比,C3a受体拮抗剂组小鼠体重、空腹血糖、尿微量白蛋白/尿肌酐及尿NAG均较低(均P＜0.01);三组血肌酐和血尿素氮的差异均无统计学意义(均P＞0.01).与db/m组相比,db/db组小鼠肾小球肥大,肾小管上皮细胞坏死、脱落,肾小管扩张,C3、C3a受体蛋白表达均明显较高(均P＜0.01);与db/db组相比,C3a受体拮抗剂组肾小球病变轻微,肾小管上皮细胞轻度坏死,肾小管扩张不明显.db/db组小鼠肾组织Kim-1、IL-1、TNF-α蛋白表达均高于db/m组,而C3a受体拮抗剂组Kim-1、IL-1、TNF-α蛋白表达均低于db/db组(均P＜0.01).与db/m组相比,db/db组小鼠肾小管上皮细胞α-SMA、波形蛋白蛋白表达均明显较高,ZO-1、E-cadherin蛋白表达均明显较低(均P＜0.01);与db/db组相比,C3a受体拮抗剂组α-SMA、波形蛋白蛋白表达均明显较低,ZO-1、E-cadherin蛋白表达均明显较高(均P＜0.01).与db/m组相比,db/db组小鼠肾组织凋亡细胞数量增加;与db/db组相比,C3a受体拮抗剂组凋亡细胞数量减少.结论 db/db小鼠肾组织C3和C3a受体表达明显升高.拮抗C3a受体可降低db/db小鼠体重、血糖、尿微量白蛋白/尿肌酐及尿NAG,减轻肾脏病理损伤,抑制肾组织炎症、细胞凋亡和肾小管上皮间充质转化.
Abstract
Objective To investigate the role of complement C3a receptor in the diabetic nephropathy pathogenesis of db/db mice,and to provide a new target for prevention and treatment of diabetic nephropathy.Methods Twelve 8-week-old male mice with type 2 diabetes mellitus(db/db mice)and 6 wild-type(db/m)mice were reared in the special pathogen free environment.The mice were grouped into db/m group,db/db group and C3a receptor antagonist group,with 6 mice in each group.db/db model mice were intraperitoneally injected with C3a receptor antagonist(SB290157,10 mg/kg)once every two days for 8 weeks in C3a receptor antagonist group.Blood and urine samples were collected,and body weight of mice,fasting blood glucose,serum creatinine,blood urea nitrogen,urinary microalbumin/urinary creatinine and urinary N-acetyl-β-D-glucosaminidase(NAG)were detected.Renal tissues were collected,and HE,PAS and Masson stainings were used to observe the pathological changes.Immunohistochemistry,immunofluorescence and Western blotting were used to detect the protein expression levels of C3 and C3a receptor.Western blotting was used to analyze the protein expression levels of kidney injury molecule-1(Kim-1),α-smooth muscle actin(α-SMA),zonula occluden-1(ZO-1),vimentin and E-cadherin in renal tissues.Immunofluorescence was used to analyze the protein expression levels and distribution of α-SMA,ZO-1 and Kim-1,and immunohistochemistry was used to analyze the protein expression levels of interleukin-1(IL-1)and tumor necrosis factor-α(TNF-α).TUNEL assay was used to detect apoptotic cells in renal tissues.Results Compared with db/m group,body weight,fasting blood glucose,urinary microalbumin/urinary creatinine and urinary NAG in db/db group were significantly higher,while these indicators in C3a receptor antagonist group were slightly lower than those in db/db group(all P＜0.01).There were no significant differences in serum creatinine and blood urea nitrogen among the three groups(all P＞0.01).Compared with db/m group,db/db group had glomerular hypertrophy,necrosis and exfoliation of renal tubular epithelial cells,and dilation of renal tubules,and C3 and C3a receptor protein expression levels were higher(both P＜0.01).Compared with db/db group,C3a receptor antagonist group had less glomerular lesions,mild necrosis of renal tubular epithelial cells and less tubular dilation.Compared with db/m group,the protein expression levels of Kim-1,IL-1 and TNF-αin kidney tissues of db/db group were significantly higher,while Kim-1,IL-1 and TNF-α in C3a receptor antagonist group were significantly lower than those in db/db group(all P＜0.01).Compared with db/m group,the protein expression levels of α-SMA and vimentin of renal tubular epithelial cells in db/db group were significantly higher,while the protein expression levels of ZO-1 and E-cadherin were significantly lower(all P＜0.01).Compared with db/db group,the protein expression levels of α-SMA and vimentin of renal tubular epithelial cells in C3a receptor antagonist group were significantly lower,and the protein expression levels of ZO-1 and E-cadherin were significantly higher(all P＜0.01).Compared with db/m group,the number of apoptotic cells of kidney tissues in db/db group was increased,while the number of apoptotic cells in C3a receptor antagonist group was reduced compared with db/db group.Conclusions The expression levels of C3 and C3a receptor of kidney tissues in db/db mice are significantly increased.Antagonistic C3a receptor can reduce the body weight,blood glucose,urinary microalbumin/urinary creatinine and urinary NAG,alleviate renal pathological injury,inhibit renal tissue inflammation,apoptosis and renal tubule epithelial-mesenchymal transition in db/db mice.
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