西罗莫司对慢性排斥反应诱发小鼠肾炎的治疗作用及可能机制

Therapeutic effects and possible mechanisms of sirolimus on nephritic mice with chronic rejection

目的探讨西罗莫司对慢性排斥反应诱发小鼠肾炎的治疗作用及可能的机制。方法建立DBA/2小鼠→B6D2F1(DBA/2×C57BL/6)小鼠的慢性排斥反应诱发的小鼠肾炎模型,随机分为两组西罗莫司治疗组和模型组。西罗莫司治疗组给予西罗莫司1 mg/(kg·d),连续治疗12周;模型组给予同样剂量的橄榄油。通过尿蛋白检测及组织病理学分析确定小鼠肾炎的发生。酶联免疫吸附(ELISA)检测血清中抗核酸抗体(抗-dsDNA IgG、抗-dsDNA IgG1、抗-dsDNA IgG2a、抗-ssDNA IgG、抗-ssDNA IgG1、抗-ssDNA IgG 2a)的水平;实时定量PCR检测肾脏中的炎性因子白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ、IL-1β、趋化因子单核细胞趋化蛋白(MCP)-1、调节正常T细胞表达和分泌因子(RANTES)、B细胞趋化因子(BLC)、转化生长因子(TGF)-β1和胶原蛋白(collgen)Ⅰ的水平;直接荧光标记检测外周血或脾中T、B淋巴细胞的活化水平及调节性T细胞(Treg)的比例变化。结果 12周观察期结束时,西罗莫司组小鼠肾炎的发病率明显低于模型组(P<0.05);病理结果显示,模型组的小鼠肾组织有明显的血管内膜增生及炎性细胞浸润,西罗莫司组的小鼠肾组织趋于正常;建模后5周,ELISA结果显示,西罗莫司组小鼠血清中自身抗体及抗核抗体水平较模型组显著降低(P<0.05);实时定量PCR结果显示,西罗莫司组小鼠肾IL-6、TNF-α、IFN-γ、IL-1β、MCP-1、RANTES、BLC、TGF-β1及collgen I的mRNA表达转录水平均明显低于模型组(P<0.05);流式测定结果显示,西罗莫司组小鼠外周血中活化/记忆性T细胞比例、脾中T、B细胞表面活化分子水平较模型组均显著降低(P<0.05);与模型组相比,西罗莫司组小鼠脾中CD4+FOXP3+Treg细胞的比例明显上调(P<0.05)。结论西罗莫司可能通过上调CD4+FOXP3+Treg细胞水平,抑制效应细胞的增殖活化,下调趋化因子和炎性因子的表达,从而有效延缓小鼠慢性排斥肾炎的发生。

Objective To explore the therapeutic effects of sirolimus on nephritic mice with chronic rejection, and the possi- ble mechanisms. Methods B6D2F1 mice transplanted with cell mixture of spleen, thymus, and lymph nodes from DBA/2 spleen, thymus, and lymph nodes cell mixture were used to construct murine model with chronic rejection, and the transplanted mice were ran- domly divided into two groups. Mice in the sirolimus-treated group were given sirolimus orally in a dose of 1 mg/（kg·d） for 12 consec- utive weeks, while mice in the control group were administered with equal amounts of olive oil. Nephritis in mice was confirmed by u- fine protein determination and histopathological analysis. Enzyme immunoassay was used to detect the serum levels of anti-DNA anti- bodies including anti-ds DNA IgG, anti-ds DNA IgG1, anti-ds DNA IgG2a, anti-ss DNA IgG, anti-ss DNA IgG1, and anti-ss DNAIgG 2a. Real-time PCR analysis was conducted to evaluate the gene expression of interleukin-6 （IL-6）, tumor necrosis factor-α （TNF-α） , interferon-γ （IFN-γ） , interleukin-1β （IL-1β） , monocyte chemoattractant protein 1 （MCP-1） , regulated upon activation normal T cell expressed and secreted （RANTES）, B lymphocyte chemoattractant （BLC）, transforming growth factor β1 （TGF-β1）, and coll- gen I. The proportion and activation of T and B lymphocytes in peripheral blood or spleen were determined by flow cytometric analysis. Results At the end of observation period, the incidence of nephritis in mice treated with sirolimus was significantly lower than that of mice treated with olive oil （ P 〈0.05 ）. Histopathological evaluation of kidney specimen showed evident vascular intimal hyperplasia and mononuclear cell infiltration in control group. In contrast, no obvious kidney lesions was observed in sirolimus-treated mice. Fur- thermore, the levels of anti-DNA antibodies and the transcriptional expression of lupus IL-6, TNF-α, IFN-α, IL-1β, MCP-1, RAN- TES, BLC, TGF-β1, and collgen I were significantly down-regulated in sirolimus-treated group as compared with control group. Flow cytometric analysis indicated that both of the proportion of activated/memory T cells in peripheral blood and the expression level of sur- face activation markers on T and B lymphocytes in spleen were significantly decreased in mice exposed to sirolimus as compared with those exposed to olive oil （P 〈0. 05）. On the other hand, the proportion of CD4＋ FOXP3＋ regulatory T ceils in spleen was signifi- candy upregulated in sirolimus-treated group as compared with control group. Conclusion Sirolimus may inhibit the proliferation and activation of effector ceils and downregulate the expression of chemokines and inflammatory factors through up-regulation of CD4＋ FOXP3 ＋ T cells, thus effectively ameliorating the progression of nephritis in mice.