Study On The Mechanism Of Arctigenin Inhibiting Toxoplasma Gondii-induced Microglia Activation And Neuronal Apoptosis

Objective: In this study,the mouse microglia cell line(BV2)and the mouse neuron cell line(N2a)were used as the research objects.The microglia cells activation model and the Transwell co-culture model were established by infecting the Toxoplasma gondii(T.gondii)RH strain tachyzoites in vitro experiment,and to explore the effect and mechanism of Arctigenin(AG)inhibit T.gondii infection-induced microglia cells activation induced neuronal apoptosis via Toll-like receptor 2(TLR2)/primary response gene 88(My D88)/ Nuclear factor kappa B(NF-κB)signaling pathway.Methods: Cell activation model: T.gondii infected BV2 cells with 5 times the amount of cells for 4 hours,and then treated the cells with different concentrations of liquid medicine for 36 hours.Samples were collected,and the effect of AG on the activation of microglia induced by T.gondii infection was detected by Western blot and immunofluorescence experiments.Western blot was used to investigate the effects of AG on the expression levels of adapter molecule-1(Iba-1),High mobility group box 1(HMGB1),synthase(i NOS),TLR2,My D88,Inhibitor of NF-κB p65(IκBα)and Nuclear factor kappa B p65(NF-κB p65)protein in BV2 cells.At the same time,immunofluorescence experiments were used to observe the effect of AG on the expression of Iba-1 in BV2 cells and the nuclear translocation of HMGB1 and NF-κB p65.Transwell co-culture model: N2 a and BV2 cells were respectively inoculated into the upper semipermeable membrane chamber of the 24-well plate Transwell co-culture model,and T.gondii tachyzoites were inoculated into the co-culture model at 5 times the amount of BV2 cells,And then transfer the chamber to a 24-well plate with N2 a cells to form a co-culture system.After 4 h,the culture medium of N2 a and BV2 cells were changed,and the expression of Annexin V of N2 a cell apoptosis protein was detected by immunofluorescence after 36 h treatment with different concentrations of drugs.Results: Western blot results show that AG can effectively inhibit the excessive activation of BV2 cells after T.gondii infection and reduce the expression of Iba-1.And AG can reduce the expression of i NOS,TLR2,My D88 protein in BV2 cells after T.gondii infection in a dose-dependent manner.The results of Western blot and immunofluorescence experiments showed that HMGB1 in the model group was transferred to the cytoplasm in large quantities after T.gondii infection,and the expression in the nucleus was significantly reduced.NF-κB p65 was transferred to the nucleus and the expression in the cytoplasm was reduced.After AG treatment,HMGB1 was significantly improved.Nuclear translocation with NF-κB p65.Immunofluorescence detection results showed that compared with the normal group,neuronal apoptosis in the model group was significantly increased,AG(10 μM,20 μM)or SD(100μg/m L)can effectively inhibit neuronal apoptosis.Conclusion:AG inhibited neuronal apoptosis induced by activation of microglia induced by T.gondii infection by regulating TLR2/My D88/NF-κB signaling pathway and downstream inflammatory mediator(i NOS and HMGB1).It will be provided a scientific basis for the research of prevention and treatment of T.gondii infection induced central nervous system(CNS)disease.