Regulation Of Fisetin On Immunological System Of Mice And Its Protective Effects On Autoimmune Hepatitis Model Mice

Objectives1. To investigate the effects of fisetin on the macrophages and T lymphocytes behaviors.2. To study the protective effects of fisetin to autoimmune hepatitis model mice.Materials and Methods1. In vitro:BALB/c mice were sacrified, macrophages and T lymphocytes were prepared respectively. The effect of fisetin on NO secretion of macrophages stimulated by LPS and IFN-y was detected by Greiss Kit, and the effect of fisetin on phagocytosis of macrophages was measured by flow cytometry plus fluorescent microbeads. T lymphocytes toxic effect of fisetin was estimated by MTT assay, the expression levels of CD69 and CD25 of T lymphocytes stimulated by Con A were measured after staining with fluorescent monoclonal antibody using flow cytometry assay. CFDA-SE staining plus flow cytometry assay was employed to analyze the proliferation of lymphocytes. DIOC6(3) staining was employed to analyze the apoptosis of T cell induced by DEX. H2DCF-DA staining was used to analyze the ROS production of lymphocytes.2. In vivo:Con A (20 mg/Kg) was injected into caudal vein of the mice to establish the AIH model. The expression level of ALT, histological section, hepatic index and splenic index were used to eveluate the protective effects of fisetin on AIH model mice. To study the protective mechanism of fisetin, two-color fluorescent antibody staining were used to detect the activation of T cells of AIH model mice, MTT assay was used to detect the proliferation of T cells of AIH model mice. Greiss Kit was uesed to evaluate the NO levels of macrophages of AIH model mice.3. All the data of flow Cytometry were obtained by CELLQuest and were analyzed by ModFitLT 3.2 software. The data were analyzed by Excel and were presented as mean ±standard deviation (x±s). Independent sample t-test were used to compare the two groups. When P<0.05 it is determined the results had significant differences, P<0.01 isdetermined the results had extremely significant differences.ResultsOur results showed that fisetin inhibited NO production of macrophages stimulated by LPS and IFN-y. And phagocytosis of macrophages was significantly inhabited by fisetin. The expression of CD69 and CD25 of T lymphocytes were inhabited by fisetin significantly.The proliferation of T lymphocytes was inhabited by fisetin significantly, and the PI of 48 h reduced from 1.27±0.13 to 1.13±0.16, 1.07±0.12 and 1.02±0.06 respectively, the PI of 72 h reduced from 1.59±0.03 to 1.37±0.03,1.31±0.01 and 1.16±0.02 respectively. DIOC6(3) staining implied that fisetin promoted the apoptosis of T lymphocytes induced by Dex. Fisetin inhabited the ROS production of T cells.The injection of Con A through caudal vein could establish the AIH mode stablely, hepatic index reached peak value at 10 h, splenic index reached peak value at 6 h. the expression level of ALT reached peak value at 8 h. Intraperitoneal injection of fisetin (5 mg/Kg) could significantly reduced the level of ALT, hepatic index and splenic index of the AIH model mice. Histological section showed that inflammatory cells infiltration was relieved by fisetin treatment. Fisetin inhibited the expression of CD69, proliferation of T lymphocytes, reduced the NO expression of serum of AIH model mice.ConclusionsThe immunoregulation properties of FIS on murine T lymphocytes mainly display on the inhibition of CD69, CD25, proliferation and ROS production of T lymphocytes in vitro.Combining with the inhibition of FIS on NO secretion and phagocytosis of murine macrophages, we found that FIS had potential effect on immunosuppressive. Fisetin can protect the AIH model mice, these data provide useful information in which FIS could develop as a new immunointervention reagent.