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

Abstract

Objective To explore the therapeutic effects of sirolimus on nephritic mice with chronic rejection,and the possible mechanisms. Methods B6D2F1mice 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 randomly divided into two groups. Mice in the sirolimus-treated group were given sirolimus orally in a dose of 1 mg /( kg·d) for 12 consecutive weeks,while mice in the control group were administered with equal amounts of olive oil. Nephritis in mice was confirmed by urine protein determination and histopathological analysis. Enzyme immunoassay was used to detect the serum levels of anti-DNA antibodies including anti-ds DNA IgG,anti-ds DNA IgG1,anti-ds DNA IgG2a,anti-ss DNA IgG,anti-ss DNA IgG1,and anti-ss DNA IgG 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 collgen 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. Furthermore,the levels of anti-DNA antibodies and the transcriptional expression of lupus IL-6,TNF-α,IFN-γ,IL-1β,MCP-1,RANTES,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 surface 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 cells in spleen was significantly upregulated in sirolimus-treated group as compared with control group. Conclusion Sirolimus may inhibit the proliferation and activation of effector cells 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.