苦参碱干预狼疮样小鼠肾组织表达MCP-1、核因子-κB及肾损伤的作用

Effects of Matrine on Renal Injury Involved in Upregulation of Renal MCP-1 and NF-κB Expression in the Mice with Lupus Nephritis

目的探讨苦参碱对慢性移植物抗宿主病狼疮样小鼠肾组织单核细胞趋化蛋白1(MCP-1)、核因子B表达及肾小管-间质纤维化的影响。方法 6周龄Balb/c(B/C,H-2d)雌性小鼠与C57BL/6(B6,H-2b)雄性小鼠自行交配,获得杂交第一代雌性小鼠[(B/C×B6)F1(H-2d/b)]。无菌取出Balb/c雌性小鼠的胸腺、淋巴结和脾脏,分离淋巴细胞,按每只鼠5×107个活细胞,分别于实验的第1、4、7、10天免疫F1小鼠,末次免疫后,F1的小鼠被随机分为模型组、苦参碱组各23只,苦参碱组每天按30mg.kg-1.d-1腹腔注射苦参碱生理盐水,模型组、正常对照组予等量生理盐水注射,分别于实验的第8、16周末,随机取3组的小鼠各8只,收集24h尿量,采血取肾,检测24h尿蛋白定量和血肌酐,采用酶联免疫吸附法定量测定小鼠血清抗双链DNA(anti-ds-DNA)抗体、抗核抗体(ANA)、白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α;用PAS染色、免疫组化技术检测肾小球和肾小管间质中的MCP-1、核因子(NF)-κB蛋白的表达和分布,采用HPIAS-1000型全自动彩色图像分析系统,对免疫组化染色阳性信号进行扫描,半定量分析各组阳性表达的光密度值。结果治疗组小鼠的24h尿蛋白定量、血清肌酐与同期病理组比较,有明显的改善(P<0.05);anti-dsDNA、ANA、IL-6、TNF-α显著低于同期病理组(P<0.01);肾组织MCP-1、NF-κB表达较正常肾组织显著增高,但较同期病理组弱(P<0.05);肾小球硬化指数和肾小管间质损伤指数低于病理组。结论慢性移植物抗宿主病小鼠模型,具有狼疮性肾炎的典型特征,苦参碱可降低血清anti-dsDNA、ANA、IL-6、TNF-α的水平,对杂交F1代小鼠自身免疫反应有一定的抑制作用,并抑制NF-κB的活化,下调IL-6、TNF-α、MCP-1的表达,延缓肾小球硬化和肾小管间质纤维化的进展。

OBJECTIVE To investigate the effects of matrine on expression of renal MCP-1,NF-κB and tubulointerstial fibrosis in the mice with chronic graft versus host disease （cGVHD）lupus nephritis. METHODS Female （BALB/c×C57BL/6）F1 generation mice with lymphocytes were obtained from mother strain mice by intravenous injection at experiment day 1,2,4,6 and 8 respectively. They were randomly allocated into pathological model group （n=23） and matrine group （n=23）. The mice in matrine group were treated with matrine（30 mg·kg-1·d-1）by intraperitoneal injection. Eight and sixteen weeks later respectively,8 mice in each group were taken randomly for determining creatinine clearance （ccr）,24 h urinary protein quantitative measurement （24 h UPQ）. The levels of serum anti-dsDNA antibodies（anti-dsDNA）,anti-nuclear antibodies （ANA）,IL-6 and TNF-α were determined by ELISA method. The sections of kidneys were examined by PAS staining and immunohistochemical staining for semiquantitative morphological evaluation. RESULTS The levels of 24 h UPQ,Scr,anti-dsDNA,ANA,IL-6 and TNF-α in matrine group were lower than those in pathological model group. The magnitude of quantitative analysis in morphology showed that the expression of MCP-1 and NF-κB protein in matrine group decreased compared with pathological model group. In addition,glomerular sclerotic index and index of tubulointerstitial lesion was lower in matrine group. CONCLUSION The model of chronic graft-vs-host disease （cGVHD） has lupus-like humoral autoimmunity and renal disease,which can be decreased by matrine in the serum level of anti-dsDNA,ANA,IL-6,TNF-α and urine protein excretion rate. Furthermore,matrine may inhibit activation of NF-κB with down regulation of renal IL-6,TNF-α,MCP-1 expression. As a result,progress of glomerulosclerosis and renal tubulointerstial fibrosis were retarded.