Effects On Bezafibrate On Th17Differentiation From Peripheral Blood Of Primary Biliary Cirrhosis

ObjectivesPrimary biliary cirrhosis (PBC) is an autoimmune disease which is characterized as chronic, progressive, nonsuppurative inflammation of small bile ducts. How to treat PBC effectively, improve the prognosis and survival quality of patients are the current research hot spot. Foreign clinical practice found bezafibrate (BF) which is PPAR pan-agonists had a certain curative effect on PBC patients, but its mechanism was unknown. Our study found that the levels of Th17from peripheral blood in primary biliary cirrhosis patients were significantly higher than control, which suggested BF might interfere in Th17cell differentiation as its target function. So we will focus on studies addressing the mechanism of BF target Thl7in PBC from clinical samples analysis, providing the laboratory support for further revealing PBC pathogenesis and seeking the intervention measures that enhance the immune adjustment function.Methods1. Screening the best concentration of bezafibrate that influence Th17cells differentiationCD4+T cells were isolated from the peripheral blood of normal control by Ficoll-Hypaque density gradient centrifugation and MACS, then were cultured in four groups at1×109/L cells, four groups were added different bezafibrate concentrations.(1) medium;(2)10μM BF group;(3)50μM BF group;(4)100μM BF group;Using flow cytometry to evaluate the optimal concentration of BF that inhibited Th17cell differentiation.2. BF interfere in Th17cell differentiation from PBC patients in vitro(1) CD4+T cells were isolated from the peripheral blood of normal control and PBC patients by Ficoll-Hypaque density gradient centrifugation and MACS, two groups were added different BF concentrations.①medium group(2)50μM BF groupUsing flow cytometry to evaluate the the influence of bezafibrate on Th17differentiation.(2) CD4+T cells were isolated from the peripheral blood of normal control and PBC patients and were cultured nine days. The mRNA levers of the IL-17and IFN-γ were detected by RT-PCR.(3) CD4+T cells were isolated from the peripheral blood of normal control and PBC patients and were cultured for nine days. The protein levers of IL-17and IFN-γ were detected in the cellular supernatant by Enzyme-linked immuno-sorbent assay (ELISA).Results1.BF inhibited Thl7differentiation from the peripheral blood of normal control in a dose-dependence at a certain extent.50μM BF inhibited IL-17+cells differentiation were the lowest (0.1%～0.5%), average0.3%, which showed50μM concentration was the optimal dose.2.BF treated the CD4+T cells from the peripheral blood of normal control and PBC patients, the levels of IL-17+IFN-γ-and IL-17+IFN-γ+in CD4+T cells were all significantly lower than those in controls(P<0.05), while there were no significant differences of the levels of IL-17-IFN-Y+in CD4+T cells (P>0.05).3.BF treated the CD4+T cells from the peripheral blood of normal control and PBC patients, the levels of IL-17in the culture surpernatant were all significantly lower than those in control s (P<0.05), while there were no significant differences of the levels of IFN-γ (P>0.05).4. BF treated the CD4+T cells from the peripheral blood of normal control and PBC patients, the mRNA levels of IL-17were all significantly lower then those in controls (P<0.05), while the mRNA levels of IFN-γ were no significant differences (P>0.05).ConelusionPreliminary screening50μM bezafibrate was the best concentration that influence Thl7cells differentiation;In the Th17polarization condition, BF could significantly inhibit the levels of IL-17in CD4+T cells from the peripheral blood of PBC patients, but did not affect IFN-γ expression.