The Effects Of GRh2 Against Toxoplasma Gondii Infection And Mechanism Of Inhibiting Microglia Activation

Objective:The purpose of the study was to establish an infection model of activation of microglia with BV2 cells and an acute Toxoplasma gondii(T.gondii)infection model infected with T.gondii virulent strain RH in vitro and in vivo,to study the anti-toxoplasma effect of ginsenoside Rh2(GRh2)and inhibit the neuroinflammation and mechanism of activation of microglia induced by T.gondii infection.Providing scientific basis for the treatment of Toxoplasma encephalitis(toxoplasmosis).Methods:(1)In vitro experiment,MTT assay was used to determine the safe concentration of GRh2 on BV2 cells.The anti-T.gondii effect of GRh2 was examined by morphological observations,immunofluorescence staining,trypan blue exclusion assay,RT-PCR and Western blot analyses.(2)In vivo experiment,T.gondii virulent strain RH infected female BALB/c mice intraperitoneally injected with 1 × 105 tachyzoites to establish an acute T.gondii infection model,4 hours after infection,the mice were orally administered with GRh2(25,50,100mg/kg/day)and sulfadiazine sodium(SD-Na)(100mg/kg/day)for 6 consecutive days,respectively,observe the clinical signs and symptoms of mice on day 2,4,and 6 and their survival rate was calculated for 10 days.At the sixth day,brain tissues were collected from all mice to quantify the parasite burden in the brain by QC-PCR assay.(3)The protein expression of Iba-1,IFN-γ,TNF-α,iNOS,TLR4,p-NF-κB,NF-κB p65,p-IκB-α and IκB-α in BV2 cells and mouse brain tissues and the expression of TRIF and MyD88 of BV2 cells were measured by western blot analysis.The culture supernatants of BV2 cells and serum of mice of NO production was determined using Mouse nitric oxide ELISA Kit.Immunofluorescence assay was used to detect Iba-1 and NF-κB p65 nuclear expression in BV2 cells.To explore the neuroinflammation of GRh2 on the activation of microglia induced by T.gondii infection and its mechanism.Results:(1)GRh2 improved the morphology of T.gondii-infected BV2 cells and increased cell viability and T.gondii inhibition rate in a dose-dependent manner.At the same time,GRh2 significantly decreased the SAG1 and T.g.HSP70 mRNA levels and the expression of T.gondii protein.GRh2 not only protected the host cell but also significantly directly and indirectly inhibited T.gondii proliferation in vitro.(2)GRh2 significantly improved the clinical signs and symptoms and prolonged the survival time of lethal dose of acute T.gondii-infected mice.SAG1 copy numbers of T.gondii by treated with GRh2 were much more decreased compared with untreated group.GRh2 has a protective effect on T.gondii-infected mice.(3)The enhanced Iba-1,IFN-γ,TNF-α and iNOS in T.gondii-infected BV2 cells and mouse brain tissues were significantly inhibited by GRh2 treatment.GRh2 treatment significantly down regulated the expression of TLR4 and TRIF proteins,while inhibiting T.gondii infection induced IκB-α degradation and NF-κB p65 nuclear translocation.However,the abundance of MyD88 was unaffected by the T.gondii infection or drug treatment.Conclusion:GRh2 has a good anti-T gondii effect in vitro and in vivo and the inhibition mechanism on microglia cell hyperactivation induced by inflammation mediators(IFN-γ,TNF-α and iNOS(NO))through TLR4/NF-κB signaling pathway provide a basic pharmacological evidence for the development of prevention and treatment of toxoplasmic encepharitis(toxoplasmosis)drugs.