Effects Of Nourishing-yin And Clearing-heat Method On Treg/Th17Cells And Renal Pathology Of MRL/Lpr Mice

ObjectiveTo investigate the effects of nourishing-yin and clearing heat method on the regulatory T cells/Th17cells and renal pathology of MRL/lpr mice with spontaneous systemic lupus erythematosus; To explain the mechanism of nourishing-yin and clearing heat method on SLE by the modulation of Treg/Th17.MethodsThirty-two Female MRL/lpr mice were divided equally into four groups: model group、herbal medicine group、prednisone group and integrative group, eight female C57BL/6mice were included in the control group were. Mice in the model group and the control group were perfused intragasticly with normal saline (17.5mL·kg-1·d-1); Mice in the herbal medicine group were perfused intragasticly with concentrated solution of modified Qinghaobiejia decoction(Ingredients and the proportion of their dose:Artemisia apiacea10g [decocted later]、Turtle shell30g [decocted first]、Cornu bubali20g [decocted f irst]、Cortex Moutan10g、Radix Rehmanniae20g、Gentiana10g and Radix Scrophulariae10g; density:1.1g dired herbs/ml; dosage:19.25g·kg-1·d-1); Mice in the prednisone group were perfused intragasticly with solution of Prednisone acetate (8.75mg·kg-1·d-1); Mice in the integrative group were given both the concentrated solution of modified Qinghaobiejia decoction and the solution of Prednisone acetate (Dosages were in accord with the herbal medicine group and the prednisone group). The course of treatment was7weeks. The body weight of mice were scaled weekly; The urine protein were detected before and after the treatment; The level of anti-dsDNA in the serum were detected after treatment by ELISA. The proportions of CD4+CD25+/CD4+、Foxp3+/CD4+CD25+、 Foxp3+/CD4+and IL-17+/CD4+cells in the splenic mononuclear cell suspension in the mice were detected by flow cytometry. Tissues of kidneys were stained by H&E、PAS、Masson and PASM methods and observed by micorscope. Activity indexes (AI) and chronic indexes (CI) of the glomerular lesions were scored. Statistical software Statall.0and Concise Statiatics14.0were used for analysis of data.ResultsProportions of CD4+Foxp3+cells、CD4+CD25+Foxp3+cells、CD4+CD25-Foxp3+cells and Th17cells in all the group had significent differences(P<0.05respectively).Mice in the herbal medicine group and the prednisone group had significantly higher proportions of CD4+Foxp3+cells (P<0.05)、higher proportions of CD4+CD25-Foxp3+cells (P<0.05), and lower proportions of Th17cells (P<0.05)、lower proportions of CD4+CD25+Foxp3+cells (P<0.05) than the model group. The renal lesions of mice in the model group included:Thickening of the capillary wall, proliferation of mesangial cells and endothelial cells, leukocyte infiltration were observed in some glomeruli, crescents、adhesion of cyst wall、hyaline thrombus and wire-loop lesion were detected occasionally; chronic lesions, for example the glomerular hyalinization or glomerular sclerosis、leukocyte infiltration of the renal intertitium and angiectasis were detected occasionally. AI scores of all the groups had significent difference(P<0.05),The herbal medicine group、prednisone group and integrative group had significantly lower AI scores than the model group(P <0.05). AI scores of all the MRL/lpr mice correlated inversely with the proportions of CD4+Foxp3+cells (Spearman’s rho=-0.5934, P<0.05) and the proportions of CD4+CD25-Foxp3+cells (Spearman’s rho=-0.5914, P<0.05); AI scores of all the MRL/lpr mice correlated positively with the proportions of Th17cells (Spearman’s rho=0.4958, P<0.05). Serum level of anti-dsDNA in all the groups had significent difference (P<0.05). In contrast to the model group, the herbal medicine group had significantly lower level of anti-dsDNA(P<0.05), but the levels of anti-dsDNA in the prednisone group and integrative group were not significently lower than that of the model group (P>0.05respectively). The decrement of urine protein (before and after treatment) in all the groups had significent differences(P<0.05). The decrement of urine protein in the herbal medicine group was significantly higher than that of the prednisone group(P<0.05). The increments of body weight before and after treatment (AWeight) of all the groups had significent difference (P<0.05). The increments of body weight in the model group and the prednisone group were significantly lower than that of the control group (P<0.05), The increments of body weight in the herbal medicine group and the integrative were not lower than that of the control group (P>0.05).Conelusion(1) Both nourishing-yin and clearing heat method and prednisone can ameliorate the renal lesions of MRL/lpr mice; In point of controlling the level of anti-dsDNA, nourishing-yin and clearing heat method maybe superior to prednisone; nourishing-yin and clearing heat method may attenuate the disadvantage of prednisone on the increment of body weight.(2)Nourishing-yin and clearing heat method and prednisone may not up-regulate the proportion of CD4+CD25+Foxp3+cells (the conventional Treg cell) of MRL/lpr mice, but they can up-regulate the expression of Foxp3molecule on the CD4+T cells, and up-regulate the proportion of CD4+CD25-Foxp3+cells. The CD4+CD25-Foxp3+cells may perform the immunosuppression effects of Treg cells to a certain degree. Meanwhile, both nourishing-yin and clearing heat method and prednisone can reduce the proportion of Th17cells, which induce the autoimmune response in SLE.(3)Nourishing-yin and clearing heat method and prednisone may ameliorate the renal lesions of MRL/lpr through modulating the proportions of regulatory T cells and Th17cells. The modulating effect of nourishing-yin and clearing heat method may be one of its pharmacological action on SLE.