论文部分内容阅读
目的:保护素D1(PD1)是一个潜在的抗炎症脂蛋白分子,本实验探讨其治疗早期糖尿病肾病(DN)肾纤维化的作用及机制。方法:用链脲佐菌素125 mg/kg 2次腹腔注射C57BL/6J雌性小鼠,建立早期DN小鼠模型。糖尿病模型成功后,用PD1(0.08 mg·kg-1·d-1)腹腔注射治疗,设正常鼠及DN鼠为对照。治疗8周后检测各组小鼠24 h尿蛋白及尿白蛋白定量、体重、肾重、肾重/体重比、血清及尿肌酐和肌酐清除率;用PAS染色法检测肾小球系膜区基质/肾小球面积比,用免疫荧光染色法检测肾皮质中巨噬细胞的数量,用Western blotting检测肾小球纤连蛋白(FN)和α-平滑肌肌动蛋白(α-SMA)的表达,以及肾小球足突细胞特异性上皮标志蛋白zonula occludens-1(ZO-1)和P-cadherin的表达;同时,体外用高糖刺激小鼠巨噬细胞株RAW264.7,检测PD1对其分泌肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的抑制作用。体外用转化生长因子β1(TGF-β1)刺激小鼠足突细胞株,用Western blotting检测PD1对其诱导足突细胞上皮-间充质转化(EMT)中上皮细胞标志蛋白P-cadherin和ZO-1减少的恢复作用,及间充质细胞标志蛋白成纤维细胞特异性蛋白1(FSP1)和α-SMA过表达的抑制作用。结果:PD1能减少DN小鼠肾小球系膜基质的积聚、24 h尿蛋白及尿白蛋白定量、体重、肾重和肾重/体重比,抑制异常增高的肌酐清除率。PD1能减少DN小鼠肾皮质中巨噬细胞的数量,抑制DN小鼠肾小球FN和α-SMA的表达,恢复足突细胞特异性上皮标志蛋白ZO-1和P-cadherin的表达。PD1能抑制高糖诱导RAW264.7分泌TNF-α和IL-1β,能抑制TGF-β1诱导足突细胞FSP1和α-SMA表达的增加以及ZO-1和P-cadherin表达的减少。结论:PD1能减轻早期DN小鼠肾纤维化,其部分机制可能通过抑制肾脏的炎症及足突细胞EMT。
PURPOSE: The role of PD1 in the treatment of early diabetic nephropathy (DN) and its mechanism is explored in this study. Methods: C57BL / 6J female mice were injected intraperitoneally with streptozotocin (125 mg / kg) twice a day to establish an early DN mouse model. After successful diabetic model, PD1 (0.08 mg · kg-1 · d-1) was injected intraperitoneally, and normal mice and DN rats were used as control. After 8 weeks of treatment, 24 h urinary proteinuria and urinary albumin, body weight, kidney weight, kidney weight / body weight ratio, serum creatinine and creatinine clearance rate of 24 hours were detected; PAS staining was used to detect the mesangial area Matrix / glomerular area ratio, the number of macrophages in the renal cortex was detected by immunofluorescence staining, and the expression of glomerular fibronectin (FN) and α-smooth muscle actin (α-SMA) was detected by Western blotting , And the expressions of zonula occludens-1 (ZO-1) and P-cadherin in glomerular foot process-specific epithelial markers. Meanwhile, the macrophage cell line RAW264.7 was stimulated with high glucose in vitro, Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) inhibition. The expression of P-cadherin and ZO-1 in epithelial cells induced by PD1 was detected by Western blotting. The expression of P-cadherin and ZO- 1 reduced recovery and the inhibition of mesenchymal cell marker protein Fibroblast-specific protein 1 (FSP1) and α-SMA over-expression. Results: PD1 could reduce the accumulation of glomerular mesangial matrix in DN mice, 24 h urinary protein and urinary albumin quantitative, body weight, kidney weight and kidney weight / body weight ratio, inhibited abnormally elevated creatinine clearance rate. PD1 can reduce the number of macrophages in renal cortex of DN mice, inhibit the expression of FN and α-SMA in glomeruli, and restore the expression of foot process-specific epithelial markers ZO-1 and P-cadherin. PD1 inhibited the secretion of TNF-α and IL-1β by RAW264.7 induced by high glucose, and inhibited the increase of FSP1 and α-SMA expression and the decrease of ZO-1 and P-cadherin expression in foot process induced by TGF-β1. Conclusion: PD1 can reduce renal fibrosis in early DN mice, and its mechanism may be through inhibition of renal inflammation and footpulmonary EMT.