Research On MSCs Ameliorate Con A Induced Autoimmune Hepatitis Via Impacting Splenic CD4~+ T Cell Subsets

Objective: Autoimmune hepatitis(AIH) is a chronic inflammatory disease of the liver. Currently, corticosteroids, alone or in combination with azathioprine is the main treatment of the disease. However, not all the patients respond fully to the medication, and the long course of treatment is often associated with drug-related side effects. Thus, a novel effective method is required to control and cure this disease. Mesenchymal stem cells from bone marrow stroma are able to differentiate into multiple mesoderm-type cell lineages and play an important role in immune regulation. Studies have demonstrated that bone-marrow-derived mesenchymal stem cells(BMMSCs) have been successfully used in treatment of autoimmune diseases. The aim of this research is to evaluate the therapeutic effects of BMMSCs on Con Ainduced hepatitis and to elucidate the possible mechanism involved.Methods:Con A was injected intravenously into C57BL/6 mice to establish Con A-induced autoimmune hepatitis models. BMMSCs were isolated from bone marrow of the four limbs of three-week-old C57BL/6 mice and at passage three the specified surface markers and the osteogenic and adipogenic differentiation were detected. Six-to-seven-old female C57BL/6 mice were randomly divided into control group which was injected PBS only,MSC treatment group which was injected both Con A and MSCs,and PBS treatment group which was injected Con A and PBS. Each group contains five mice. The mice were sacrificed in 14 to 16 hours after injection.The level of alanine aminotransfersase(ALT),aspartate transaminase(AST)in peripheral blood were detected and the pathological changes in liver tissue was scored by knodell score system. Activation of splenic CD4+ T cells and the proportion of Th1,Th2,Th17 and Treg were detected by flow cytometry and the ratioof Th17/Treg was calculated. Finally the level of TNF-α,IFN-γand IL-4 in peripheral blood was analyzed by ELISA. Using q PCR to verify the changes of CD4+ T cell subsets between spleen and liver are consistent.Independent-sample t test was used for comparison between groups of measurement data.Results: 1. The BMMSCs we got from the bone marrow have the ability to differentiate into fat cells and osteogenesis cells,the surface makers we detected by Flow cytometry instrument are Complied. 2. Liver damage was reduced in MSC treatment group compared with PBS treatment group. 3. Splenic CD4+ T cell activation,the proportion of Th1,Th2 and the ratio of Th17/Treg,as well as the levels of TNF-α,IFN-γ and IL-4 in peripheral blood were increased in PBS treatment group,compared with control group,The above differences are statistically significant(P<0.01). 4. In contrast to PBS treatment group, splenic CD4+ T cell activation was not reduced(P=0.95)but the proportion of Th1,Th2 and the ratio of Th17/Treg,as well as the levels of TNF-α,IFN-γand IL-4 in peripheral blood were significantly decreased in MSC treatment group,The above differences are statistically significant(P<0.01). 5. the changes of CD4+ T cell subsets between spleen and liver are consistent. compared with control group, the proportion of Th1, Th2 and Th17 were significantly increased in PBS treatment group, the proportion of Treg was increased lightly. compared with PBS treatment group, the proportion of Th1, Th2 and Th17 were significantly decreased in MSC treatment group, the proportion of Treg was increased significantly.Conclusions:1. The BMMSCs we got from the bone marrows are the real BMMSCs. 2. BMMSCs can alleviate the Con A induced AIH. 3. BMMSCs can’t inhibiting CD4+ T cells’ activation. 4. MSCs ameliorated Con A induced acute hepatitis via impacting CD4+ T subsets. On the one hand, the frequencies of Th1 and Th2 cells in CD4+ T cells were reduced, which might lead to the decreased levels of pro-inflammatory cytokines such as TNF, IFN and IL-4 in peripheral blood. On the other hand, Treg cells were induced and Th17 cells were reduced, through which the balance of Th17/Treg were maintained. 5. the changes of CD4+ T cell subsets between spleen and liver are consistent.