Influence Of Phloretin On Immunological System Of Mice And Its Protective Effects On Autoimmune Hepatitis Model Mice

Aim:To investigate the effect of phloretin on the T lymphocyte and macrophage behaviors and the protective effect of it to autoimmune hepatitis model mice.Methods:The expression levels of CD69 and CD25 on T lymphocytes stimulated with ConA were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Fluo-4/AM staining was used to detect intracellular Ca2+concentrations. CFDA-SE staining plus flow cytometry assay was employed to obtain the proliferation-related index (PI) of lymphocytes. Cell-cycle distribution of T lymphocytes was analyzed by propidium iodide staining. AnnexinⅤ-FITC/PI staining was employed to analyze the apoptosis of T cell induced by DEX.The proliferation effect of phloretinn on macrophage stimulated with LPS and IFN-y was analyzed using MTT assay, the effect of phloretin on NO secretion of macrophage in response to LPS and IFN-y was measured by Greiss Kit, and the effect of phloretin on phagocytosis of macrophage was detected by flow cytometry plus fluorescence microbeads.20 mg/Kg ConA was injected into mice through caudal vein to to establish the AIH model. ALT level, histological section, hepatic index and splenic index was used to eveluate the protective effect of phloretin on AIH model mice. To study the protective mechanism of phloretin. MTT assay, two-color fluorescent antibody staining and calcein/Co2+staining was used to detect the porliferation, activation and apoptosis of T cells of AIH model mice, and Greiss Kit was uesed to evaluate the NO levels. Results:Our results showed that phloretin significantly inhibited the expression of CD69 and CD25 of T cell. Phloretin inhibited Ca2+inflow and T cell proliferation, and the PI reduced from 1.41±0.13 to 1.34±0.16,1.19±0.12 and 1.07±0.06, respectively. The cell cycle distribution analysis showed that Ph could induce a cell cycle arrest in G0/G1 phase. Annexin-V/PI staining implied that phloretin promoted apoptosis of T cell induced by Dex.Phloretin inhibited proliferation and NO production of macrophage activated by LPS and IFN-y. And percent phagocytosis of macrophages was significantly reduced by phloretin.The injection of ConA through caudal vein could establish the stable AIH model, ALT level and hepatic index reached peak value at 10 h, splenic index reached peak value at 6 h. Intraperitoneal injection of 50 mg/Kg phloretin could significantly reduce ALT level, hepatic and splenic index of the AIH model mice. Histological section showed that inflammatory cells infiltration was relieved by phloretin treatment. Phloretin inhibited the proliferation and activation, induced apoptosis of T cell and reduced NO level of serum of AIH model mice.Conclusion:Phloretin can regulate the immunological system of mice, and it can protect the AIH model mice.