The Study Of Bezafibrate On Monocytes From Primary Biliary Cirrhosis Patients

Primary biliary cirrhosis (PBC) is an autoimmune disease and characterized by chronic non-suppurative destructive cholangitis and liver cirrhosis, it is mainly caused by the breakdown of self-tolerance and hyper-responsive autoimmunity. Ursodeoxycholic acid (UDCA) is the first-line drugs of PBC, yet a part of patients resistance to it. However, for its good therapeutic effect and less side effects, an increasing number of clinical reports showed that bezafibrate (BF) has entered the clinical treatment of patient with PBC, but there is still lack of systematic study about BF on PBC disease mechanism.Innate immunity is the basis of the adaptive immune response, and monocyte, which is one of the main effector cells of innate immunity, plays an important role on the onset of infection and some autoimmune diseases by its unique phenotypic characteristics and functional features. The experiment we have been completed showed the imbalances of monocytes in PBC, they were closely related to the inflammation and damage index of liver as well as Th1 cells. However, There is no relevant evidence of whether BF has an active and effective role in monocytes to inhibit the development of disese on early stage.ObjectivesTo observe the effects of BF on the monocytes phenotype and function of patients with PBC, as well as whether the monocytes, through regulating by BF, can affect CD4+ T cells differentiation, and further explor its potential molecular mechanisms,in order to provide a novel angle and laboratory basis for BF antagonistic to PBC.Methods1.20ml peripheral blood were collected from PBC patients, peripheral blood mononuclear cells were isolated from it and further isolated CD14+ cells. Then the CD14+ cells were cultured with different concentrations of BF(0μM、50 ii M、100μM、200μM) for 48h. Gene expression level of CD16. TNF-α、IL-1β and IL-6 in CD14+ cells were detected by RT-PCR; f low cytometry assay was carried out to detect percentages of CD16、CD80/CD86/HLA-DR molecules on CD14+ cells and the cell apoptosis.2. Peripheral blood mononuclear cells were isolated from the peripheral blood of PBC patients and further isolated CD14+ cells and CD4+ T cells. The CD14+ cells were pre-cultured with BF or not, and then co-cultured with CD4+T cells, IFN-γ+ CD4+T (Th1)、IL-17+ CD4+T (Th17) and Ki-67+CD4+T frequencies were mesured by flow cytometry. Repeated the above experiment using the transwell culture system, then flow cytometry was used to detect the frequencies of Thl cells. Gene expression level of IL-12p35、IL-12p40 and IL-18 in CD14+ cells under BF were mesured by RT-PCR.Results1. Different concentrations of BF in the study had no cytotoxicity on monocytes (P>0.05);2. BF inhibited the mRNA and membrane surface antigen level of CD16 on monocytes by concentration dependent (P<0.05)’, especially in 200μMBF, the inhibition ratio could reach 44% and 58% respectively;3. BF inhibited the gene level of proinflammatory cytokines, such as TNF-a, IL-1β and IL-6 (P<0.01), the inhibition degree could reach 49%、53% and 42% in 200μM BF;4. Different concentrations of BF had no effect on the costimulatory molecules of monocytes (P>0.05), such as the expression of CD80、CD86 and HLA-DR;5. Different concentrations of BF could not induce apoptosis of the monocytes (P>0.05);6. The monocytes could not indirect inhibit the proliferation of Thl cells by BF regulation (P>0.05);7. The monocytes could indirect inhibit the differentiation of Thl cells by BF regulation (P<0.05), but not Th17 cells(P>0.05);8. Whether the monocytes treated with BF or not, there was no obvious difference between direct cell contact and indirect cell contact of the co-culture system on Thl cells proliferation (P>0.05), the monocytes pretreated by BF on both co-culture system could inhibit Thl cells differentiation (P<0.05);9. BF inhibited the gene level of IL-12p40 and IL-18 (P<0.05) except for IL-12p35(P>0.05).ConclusionsBF could inhibite the expression level of CD16 and proinflammatory factors (TNF-α、IL-1β and IL-6) in a concentration dependent manner. And the monocytes could indirect inhibit the Thl cells differentiation by BF, of which the possible mechanism was related to the inhibition of soluble molecules IL-12p40 and IL-18 on monocyres.