The Effect Of The Apigenin On Microenvironment Of The Brain

Objective:In vitro studies of the effect of apigenin on the secretion of inflammatory cytokines,inflammation-related proteins,anti-inflammatory cytokines and neurotrophic factors in activated microglia.Methods:1.Dissect and separate the cerebral cortex of SD rat born in 24 hours,pancreatic enzyme digestion and mechanical blowing into single-cell suspension,inoculated in the medium for 14 days,vaccination in culture medium,Purified microglia cells and identified by CD1 lb/c immunocytochemistry.2.The purified microglia were treated with the same concentration of lipopolysaccharide(LPS)(100ng/ml)for different times(1h,2h,4h,6h,8h,10h).ELISA method was used to detect the changes in the secretion of inflammatory factors(IL-1),determine if the inflammatory factor secretes the most time for subsequent experiments.3.To investigated the effect of different concentrations(5uM/L?20 uM/L?40uM/L?60uM/L)of apigenin on microglia cell viability by MTT.The minimum concentration of microglia was selected for subsequent experiments.4.The experiment was randomly divided into 6 groups:blank control group,Lipopolysccharide(LPS)group,LPS+ apigenin5uM group,LPS+ apigenin20uM group,LPS+ apigenin40uM group and LPS+ apigenin60uM group.5.The expression of inflammatory factors(IL-1?TNF-a)?anti-inflammatory cytokines(IL-10)?and other nutritional factors(BDNF?PDNF?GDNF)were detected by ELISA.6.The transcription and expression levels of nitric oxide synthase(iNOS)were detected by real-time fluorescence quantitative RT-PCR and Western Blot.Results:1.After the mixed culture and purification of the microglia cells,immunocytochemistery for CD11b/c of positive,and the purity is 95%.2.The expression of IL-1 and TNF-a were up-regulated in LPS,the peak occured at 6h.3.In a certain concentration range(5-60uM),there was no significant effect on microglial cell viability.4.ELISA shows:The expression of inflammatory cytokines IL-1 and TNF-a were significantly increased in LPS group,while the expression of anti-inflammatory factor IL-10 decreased significantly.In the LPS+ API group,the inflammatory cytokines IL-1 and TNF-a were significantly decreased,and the expression of anti-inflammatory cytokines IL-10 was significantly increased,However,there was no significant difference between the levels of the different concentrations of apigenin.5.RT-PCR and Western blot shows:In the LPS group,the transcription and expression levels of iNOS genes were significantly higher than those of other groups.All LPS+API groups can significantly reduce the levels of iNOS.Conclusion:Apigenin inhibits the secretion of inflammatory cytokines and expression of inflammatory related proteins in activated microglia,and promote release of neurotrophic factor.It provides a theoretical basis for further experiments in animals.