鞘氨醇-1-磷酸酯受体拮抗剂对抗肾小球基底膜肾炎的影响

Effects of sphingosine-1-phosphate receptor agonist FTY720 on anti-glomerular basement membrane glomerulonephritis in mice

目的观察鞘氨醇-1-磷酸酯受体拮抗剂720(sphingosine-1-phosphate receptor agonist 720,FTY720)对C57BL/6小鼠抗肾小球基底膜肾炎模型(Anti-GBM GN)的干预作用并对其作用机制进行探讨。方法选择健康雄性C57BL/6小鼠52只,给予兔IgG(500μg)加完全弗氏佐剂皮下注射进行预免疫,5d后从中随机抽取8只作为正常对照组,经尾静脉注射同剂量的非抗体性的正常兔血清,其余44只小鼠取灭活兔抗小鼠GBM抗血清0.3mL/20g经尾静脉注射。44只小鼠按随机化原则分组,FTY0.3mg/(kg·d)组(n=8)尾静脉注射6h后0.3mg/(kg·d)FTY720灌胃;FTY3mg/(kg·d)组(n=8)3mg/(kg·d)FTY720灌胃;FTY5mg/(kg·d)组(n=8)5mg/(kg·d)FTY720灌胃;FTY10mg/(kg·d)组(n=8)10mg/(kg·d)FTY720灌胃;Anti-GBM GN组(n=12)为模型组,同等剂量的无菌生理盐水灌胃。观察小鼠实验室指标及肾脏组织病理学改变情况。实时定量PCR检测小鼠脾脏鞘氨醇-1-磷酸脂(sphingosine-1-phosphate,S1P)受体S1P1～S1P5mRNA表达变化。流式细胞仪检测CD4+T细胞的凋亡情况。结果治疗组小鼠的尿蛋白、血肌酐、尿素氮和血胆固醇水平明显降低,血浆白蛋白明显增高;治疗组小鼠肾脏组织学病变明显减轻。与模型组相比,用药各组随着用药剂量增加S1P1、S1P2、S1P5的表达量逐渐减少,差异有显著性;S1P3、S1P4的表达量无明显变化,差异无显著性。流式细胞仪检测结果表明FTY720促进小鼠脾脏CD4+T细胞凋亡。结论 FTY720可能通过下调S1P1、S1P2、S1P5mRNA表达,促进CD4+T细胞凋亡,减轻抗肾小球基底膜肾炎模型的肾脏组织学改变,从而减少蛋白尿、稳定肾功能。

Objectives Mouse anti-glomerular basement membrane （GBM） glomerulonephritis is widely used as an animal model of nephritis because its biochemical and histological characteristics similar to human crescent nephritis.FTY720 is a novel immune modulator whose primary action is blood lymphocyte depletion through interaction with sphingosine-1-phosphate （S1P） receptor.In this study,we evaluated the potential therapeutic effects of FTY720 on a mouse model of anti-GBM nephritis.Methodology A total of 52 C57BL/6 mice were immunized with rabbit IgG in complete Freund＇s adjuvant.On 6th day after immunization,8 of the mice were randomly taken as the normal control group.Anti-GBM model of nephritis was induced in the other 44 mice by intravenous injection of rabbit anti-mouse GBM serum,and the mice were randomly divided into five groups：model group,FTY720 therapy group （0.3,1,3 or 10mg/kg/d）.FTY720 was orally administered daily for 14 days beginning from six hours after injection of rabbit anti-mouse GBM serum.Meanwhile,mice in the normal and model group received orally saline.Blood and urine samples were collected 14 days after induction of anti-GBM glomerulonephritis.Mice were then killed for histopathologic analyses.S1P receptor 1-5 mRNAs were measured by real-time PCR.Apoptosis of the CD4＋ T-lymphocytes were measured by flow cytometry.Results Anti-GBM glomerulonephritis was remarkably alleviated by FTY720 treatment in lower levels of proteinuria,serum creatinine and crescent formation,lower splenic S1P receptor 1,S1P receptors 2,and S1P receptors 5 mRNAs,and more apoptosis of CD4＋ T-cells in spleen.Conclusion FTY720 plays a protective role during the induction of glomerulonephritis by inhibiting expression of splenic S1P receptor 1,S1P receptors 2,and S1P receptor 5 mRNAs.