Inhibition Of Toxoplasma Gondii CPC1 Protein On Host NF-κB Signaling Pathway And Its Molecular Mechanism

Toxoplasma gondii is an obligate intracellular parasite that can cause toxoplasmosis in humans and animals.Toxoplasmosis brings enormous economic and social burdens to society and is an important foodborne disease.In China,the average infection rate of T.gondii is 7%-8% and it is increasing year by year.Objective: After the T.gondii enters the body,the body’s natural immune pathway is activated,producing a series of cytokines and inflammatory factors to clear T.gondii.T.gondii can achieve immune evasion by regulating the body’s natural immune pathway.The NF-κB signaling pathway is an important pathway in innate immunity.It has been found that T.gondii can inhibit the activation of NF-κB signaling pathway,but the specific molecules and mechanism of action are not clear.This study focused on the effect of T.gondii Cathepsin C1(Tg CPC1)protein on NF-κB signaling pathway and its molecular mechanism to provide new clues and ideas for understanding the relationship between T.gondii and innate immunity.Methods: 1.The effect of Tg CPC1 on the activity of NF-κB promoter was detected by the dual luciferase reporter gene method.2.Overexpress a set of components on the NF-κB signaling pathway to explore possible sites of Tg CPC1 protein action.3.The effect of Tg CPC1 protein on the expression of inflammatory factors downstream of NF-κB signaling pathway was analyzed by QPCR.4.Western blot analysis was used to detect the effect of Tg CPC1 protein on the expression of NF-κB signaling pathway protein.5.RNA sequencing technology to detect the effect of Tg CPC1 protein on RNA expression in TLR4-HEK293 T cells.Results: 1.The dual luciferase reporter gene results showed that Tg CPC1 can inhibit NF-κB promoter activity.2.Overexpression of TRAF6,IKKα,IKKβ and p65,Tg CPC1 can inhibit NF-κB promoter activity,indicating that Tg CPC1 may act downstream of p65 or p65.3.QPCR results showed that Tg CPC1 can inhibit the expression of IL-1β,IL-6,IL-8,IL-12 and TNF-α.4.Western blot results showed that Tg CPC1 can inhibit the phosphorylation of p65.5.RNA sequencing results showed that Tg CPC1 significantly up-regulated EPO.In HEK293 T cells,Tg CPC1 nolonger inhibits IL-6 and IL-12 expression after silencing EPO with sh RNA.At the same time,Tg CPC1 up-regulated HIF-1α,and after inhibiting HIF-1α with 2-Me OE2 inhibitor,Tg CPC1 no longer inhibited NF-κB promoter activity.Conclusion: Tg CPC1 can inhibit NF-κB signaling pathway by inhibiting phosphorylation of p65 and up-regulating HIF-1α/EPO response axis.