| Toxoplasmosis is a serious parasitic disease that endangers human and animal health.The prevention and treatment of toxoplasmosis has always been a hot topic in medical and veterinary research.Polysaccharide from Inonotus obliquus(IOP)as the main active component of traditional Chinese medicine Inonotus obliquus,has a wide range of pharmacological effects.Previous studies in our laboratory showed that IOP had significant anti-Toxoplasma gondii activity in mice and macrophages in vitro infected with Toxoplasma gondii.In order to further improve the anti-Toxoplasma gondii effect in vitro of IOP and its mechanism,the mouse spleen lymphocyte was selected as the experimental object to establish an model in vitro of Toxoplasma gondii infection.The immune regulatory mechanism of IOP on mouse spleen lymphocytes infected with Toxoplasma gondii was studied by observing the changes of inflammatory factors and chemokines of mouse spleen lymphocyte,and the phosphorylation levels of NF-κB and MAPKs signaling pathway key proteins.This will provide theoretical basis for the development and utilization of anti-Toxoplasma gondii drugs.In this experiment,the spleen lymphocytes of mice were isolated from healthy Kunming mice by using the mouse spleen lymphocyte separation kit.The toxicity of IOP on mouse spleen lymphocytes was detected by CCK-8 method and the safe dosage of IOP was determined.After the establishment of mouse spleen lymphocyte model infected with Toxoplasma gondii in vitro,ELISA method was used to detect inflammatory factors IL-1β,IL-4,IL-6,TNF-α and IFN-γ in the cell supernatant.RT-PCR method was used to detect the expression of IL-1β,IL-4,IL-6,TNF-α,IFN-γ,MIP-1 and MCP-1.Western blot method was used to measure the expression of TLR2,TLR4,and the phosphorylation levels of IκBα,p65,p38,JNK and ERK1/2 in NF-κB and MAPKs signaling pathways.In order to further clarify the target and molecular mechanism of IOP as an anti-Toxoplasma gondii drug,NF-κB inhibitor BAY 11-7082,p38 inhibitor SB203580 and JNK inhibitor SP600125 were used to pretreat the spleen lymphocytes of mice infected with Toxoplasma gondii before the administration of IOP,the content of TNF-α in the supernatant of cells was measured again by ELISA methodThe results of CCK-8 showed that IOP had no significant cytotoxic effect on mouse spleen lymphocytes in the concentration range of 0-100 μg/mL.and the concentrations of low,medium and high dose groups of IOP were 12.5 μg/mL,25μg/mL and 50 μg/mL.The results of ELISA showed that IOP could decreased the contents of IL-1β,IL-4,IL-6,IFN-γ and TNF-α in the supernatant of mouse spleen lymphocytes infected with Toxoplasma gondii.The results of RT-PCR showed that IOP could inhibit the over expression of IL-1β,IL-4,IL-6,TNF-α,IFN-γ,MIP-1 and MCP-1 mRNA in the mouse spleen lymphocytes infected with Toxoplasma gondii in a dose-dependent manner.Western blot results showed that IOP could down-regulate TLR2 and TLR4 expression in the mouse spleen lymphocytes infected with Toxoplasma gondii,and IOP could inhibit the over phosphorylation of p65 and IκBαin NF-κB signaling pathway and p38,JNK in MAPKs signaling pathway.By observing the effect of IOP on TNF-α secretion after pretreatment with specific inhibitors,the results showed that the inhibition of IOP on inflammatory cytokine was weakened in varying degrees by BAY11-7082,SB203580 and SP600125.In conclusion,IOP can inhibit the excessive inflammatory response caused by Toxoplasma gondii infection by regulating NF-κB,p38 and JNK signaling pathways in mouse spleen lymphocytes,and thus play an anti-Toxoplasma gondii role in the mouse spleen lymphocytes infected by Toxoplasma gondii through the mechanism of immunoregulation. |
