Study On The Mechanism Of Sertraline On Microglia Activation And Neuronal Damage Induced By Toxoplasma Gondii Infection

Objective:By using Toxoplasma gondii(T.gondii)RH strain tachyzoites,we infect BALB/c mice in vivo and mouse microglial cell line(BV2 cells)in vitro to establish acute T.gondii infection model and microglia activation model or BV2 cell/Neuro-2a(N2a)neuroblastoma cell co-culture system to explore the intervention effect and mechanism of sertraline on microglia activation induced neuronal damage by T.gondii infection.The present study aimed to provides scientific basis for the treatment of T.gondii nervous system diseases.Methods:1)In vivo experiment:female BALB/c mice were selected as the research objects,and the mice were randomly divided into 4 groups:normal group(uninfected+untreated),model group(T.gondii-infected+ultrapure water),sertraline group(T.gondii-infected+10 mg/kg sertraline treatment)and sulfadiazine sodium group(positive control,T.gondii-infected+100 mg/kg sulfadiazine sodium treatment).Except for the normal group,the other groups were intraperitoneally injected with1×10~5 T.gondii to establish the mouse model of acute T.gondii infection.After 4 h,except for the normal group,the other groups were given 0.2 m L of ultrapure water or drugs by gavage,once a day,for 6 consecutive days.The neurological symptoms of mice were observed and scored every day.One hour after the last administration,the mice were dissected and the mesenteric lymph nodes(MLN)and brain tissue were separated.2)In vitro experiment:(1)Establishment of microglia activation model:BV2 cells were randomly divided into 6 groups:normal group(uninfected+untreated),model group(T.gondii-infected+0.1%dimethyl sulfoxide),sertraline low-dose group(T.gondii-infected+0.5μM sertraline treatment),sertraline high-dose group(T.gondii-infected+2μM sertraline treatment),sulfadiazine group(positive control,T.gondii-infected+100μg/m L sulfadiazine treatment)and C87 group[tumour necrosis factor-α(TNF-α)inhibitor,T.gondii-infected+2.5μM C87 treatment].Except for the normal group,the other groups were infected with the tachyzoites of the RH strain of T.gondii with a cell dose of 5 times.After 4 h of infection,each group were treated with corresponding drugs for 36 h,and the cell pellets were collected for subsequent experiments.(2)Establishment of a BV2 cells/N2a cells co-culture system:BV2 cells and N2a cells were seeded in the upper compartment of Transwell and 24-well plates respectively,and the BV2 cells were randomly divided into 6 groups:normal group,model group,sertraline high-dose group,sulfadiazine group,minocycline group(microglia inhibitor,T.gondii-infected+50μM minocycline treatment)and C87 group.After the BV2 cells were treated as described above,the Transwell was placed in a 24-well plate to establish a co-culture system with N2a cells and continued to culture for36 h.3)Quantitative competitive polymerase chain reaction(QC-PCR)was used to measure the T.gondii number in mouse MLN and brain tissue and BV2 cells;Nissl staining was used to detect the damage of neurons in the cerebral cortex of mice;Western blot was used to detect the expression levels of TNF receptor 1(TNFR1),TNF receptor-related factor 2(TRAF2)and TNF-αin mouse brain tissue and BV2 cells;Immunofluorescence staining was used to observe the the proliferation of T.gondii in BV2 cells,the nuclear translocation of nuclear transcription factor-κB p65(NF-κB p65)and the expression of apoptosis protein Annexin V in N2a cells in the co-culture system;Immunohistochemical staining was used to detect the expression of Ionized calcium-binding adapter molecule-1(Iba-1)(a specific microglial marker)and p-NF-κB p65 in mouse cerebral cortex.Results:1)Sertraline can effectively inhibit the proliferation of T.gondii in MLN and brain tissue of mice or BV2 cells.2)Sertraline can improve the neurological symptoms and neuronal damage in the cerebral cortex of T.gondii-infected mice.3)Sertraline can inhibit the overexpression of inflammatory factor TNF-αin the brain tissue and BV2cells infected with T.gondii.4)Sertraline can down-regulate the expression of TNFR1and TRAF2 in the brain tissue and BV2 cells infected with T.gondii,and has a significant inhibitory effect on the nuclear translocation of NF-κB p65 in T.gondii-infected BV2 cells.In addition,sertraline also significantly inhibiting the expression level of p-NF-κB p65 in Iba-1 positive cells in the brain tissue of T.gondii-infected mice.5)Sertraline has a good intervention effect on the apoptosis of N2a cells induced by BV2 cells activated by T.gondii infection.Conclusion:Sertraline has an anti-T.gondii effect and can inhibit the proliferation of intracellular T.gondii.Sertraline has a protective effect on neuronal damage caused by T.gondii infection,and its possible mechanism is inhibiting the activation of TNFR1/NF-κB signaling pathway in microglia and inhibit the production of large amount of inflammatory cytokines(TNF-α).