来氟米特对糖尿病大鼠肾组织甘露糖结合凝集素表达的影响

Effect of leflunomide on renal expression of mannose-binding lectin in diabetic rats

目的探讨糖尿病大鼠血清及肾组织甘露糖结合凝集素(mannose-binding lectin,MBL1)表达变化及来氟米特治疗后对其表达的影响。方法将56只8周龄清洁级雄性SD大鼠,按随机数字表法分为对照组(n=16)与模型组(n=40);模型组给予链脲佐菌素制作糖尿病大鼠模型,造模成功后再随机分为糖尿病组和来氟米特组,来氟米特组给予来氟米特[5 mg/(kg·d)]治疗性灌胃,对照组及糖尿病组给予等体积生理盐水灌胃,连续8周。分别于成模后4周及8周处死大鼠,检测各组大鼠24 h尿蛋白定量、血糖、血清肌酐、尿素氮,肾脏标本测肾脏质量,PAS染色观察肾脏病理改变。ELISA法检测血清MBL1水平,采用免疫组化、半定量RT-PCR及Western blot检测肾组织MBL1表达。结果与对照组大鼠比较,4、8周糖尿病组及来氟米特组大鼠肾质量指数、血清肌酐、尿素氮、血清MBL1及24 h尿蛋白定量差异均有统计学意义(P<0.01);来氟米特组大鼠肾质量指数、血清肌酐、尿素氮、血清MBL1[4周(1.117±0.067)μg/L vs(1.249±0.054)μg/L,8周(1.222±0.074)μg/L vs(1.470±0.132)μg/L]及24 h尿蛋白定量较糖尿病组大鼠明显降低(尿素氮P<0.05,余指标P<0.01);对照组肾组织MBL1有少量表达,糖尿病组肾组织MBL1表达增加,来氟米特组各时间点与糖尿病组比较均显著降低(P<0.01),相关分析显示血清及肾组织MBL1表达与24 h尿蛋白定量、血清肌酐、尿素氮、肾质量指数、平均肾小球横截面积、平均肾小球体积呈显著正相关。结论 MBL1参与糖尿病肾病发生、发展,来氟米特可以通过减低血清及肾组织MBL1的水平,降低炎症反应,减轻糖尿病大鼠肾脏损害。

Objective To investigate the expression of mannose-binding lectin （MBL1） in the serum and renal tissue and preventive effects of leflunomide （LEF） in diabetic nephropathy rats. Methods Fifty-six SD male rats （8 weeks old, weighing 180 to 220 g） were randomly divided into control group （group N, n=16） and diabetes model group （n=40）. Diabetic rat model was induced by intra-peritoneal injection of streptozotocin. The diabetic rats were randomly divided into group of diabetic mellitus （group DM） and leflunomide treatment group （group LEF）. Group LEF were treated with leflunomide （5 mg/kg every day） through intra-gastric infusing, and group DM and group N were treated with equal volume of normal saline. The rats were sacrificed in 4 and 8 weeks after establishment of diabetic nephropathy model, and then 24 hours urine protein, blood glucose, serum creatinine, and blood urea nitrogen were detected. PSA staining was used to observe the pathological changes in the renal tissues. Serum level of MBL1 was detected by ELISA, and its expression in the renal tissue was detected by immunohistochemical assay, RT-PCR and Western blotting. Results Compared with control group, the blood glucose, renal mass index, serum creatinine, blood urea nitrogen and 24 hours urine protein were significantly increased in DM group and LEF group （P〈0.01）. The renal mass index, serum creatinine, blood urea nitrogen, serum MBL1 （4 weeks： 1.117±0.067 vs 1.249±0.054 μg/L; 8 weeks： 1.222±0.074 vs 1.470±0.132 μg/L） and 24 hours urine protein was significantly lower in the group LEF than group DM （P〈0.05 for blood urea nitrogen, P〈0.01 for other indexes）. MBL1 was mildly expressed in normal control group renal tissue, significantly enhanced in group DM, and was significantly decreased in group LEF than in group DM at the 2 time points （P〈0.01）. Correlation analysis showed that the change of serum and renal MBL1 expression was positively correlated with renal mass index, serum creatinine, blood urea nitrogen, 24 hours urine protein, mean glomerular area and mean glomerular volume. Conclusion MBL1 participates in the incidence and development of diabetic nephropathy. Leflunomide attenuates inflammatory response and ameliorates renal injury through reducing expression of MBL in serum and renal tissue.