Study On The Effect Of Trichinella Spiralis Sinfection And ES Antigen On NOD1 Receptor Pathway In Mice

Trichinellosis is a severe food-borne zoonotic nematodosis caused by Trichina.The interactions between various antigens produced by Trichina and host immune system during the infection were detrimental to the host health.Natural immune system is the first line of defense against pathogen invasion.NOD1 receptor is an important natural immune receptor with extensive existence in a variety of cells and tissues.It is generally recognized that NOD1 receptor initiates oligomerization after the activation by pathogens,and actives down-stream RIP2 via interactions between CARD-CARD structural domains of N terminus.General NOD1 receptor effector molecule RIP2 activates the downstream activation of pathogen,it can cause caspase-1,NF-?B,MAPKs signal pathway is activated,induced secretion of TNF-?,IL-1? and IL-6 and other inflammatory factors,which cause a variety of immune response.Research indicates the involvement of NOD1 receptor in multiple immune responses against bacteria,fungus,virus and parasites,however,studies on the infection of trichina is rarely reported.Therefore,this study emphasizes on the effect of trichina infection on NOD1 receptor pathway in host via the application of q PCR,Western blot and ELISA,in order to discuss the interactive mechanism between Trichina infection and host immune system.Based on the sequences of mice genes(NOD1,RIP2,NF-?B)and internal reference gene(GAPDH)registered at Gen Bank,primer was designed and synthesized,and the measurement method of q PCR was established.The expression levels of NOD1,RIP2 and NF-?B m RNA in mice celiac macrophages,small intestines,hearts,lungs and muscles were detected via q PCR at 0.5d,4d,7d,14 d,21d,28 d and 35 d after Trichina infection.The levels of NOD1,RIP2,NF-?B p65,NF-?B p-p65 in peritoneal macrophages were examined by Western blot.The levels of TNF-?,IL-1? and IL-6 in the supernatant of peritoneal macrophages and serum were measured by ELISA.Results of q PCR indicated two-peak tendency in expressions of all genes in mice celiac macrophages after Trichina infection,and NOD1,RIP2 and NF-?B m RNA displayed two peaks at 0.5d/21 d,4d /21 d and 0.5d /21 d respectively.In small intestines and hearts,target gene expressions were all elevated during different phases after Trichina infection,and reached peaks at 4d and 14 d after infection.For lungs,NOD1 m RNA expression was only slightly elevated at 7d after infection,while the m RNA expression of RIP2 was elevated in all infectious stages with peak value at 4d after infection.For muscles,the changes in gene expression were not significant at the early stage of infection;however,the m RNA expressions were significantly elevated from 21 d after infection with peak value at 28 d.Western blot results indicated the expressions of NOD1,RIP2 and NF-?B p-p65 in mice celiac macrophages were elevated to different extent after trichina infection,and the variation trend was similar to that of q PCR.ELISA results indicated the contents of TNF-? and IL-6 in supernate of mice celiac macrophage cultures and serums were significantly elevated,with no clear change of IL-1?.Moreover,this study applied the ES antigenic action of Trichina muscle lava on healthy mice celiac macrophages,and q PCR was utilized to detect the expressions of NOD1,RIP2 and NF-?B m RNA;Western blot was utilized to detect the expressions of NOD1,RIP2,NF-?B p65 and NF-?B p-p65;ELISA was utilized to detect the expressions of TNF-?,IL-1? and IL-6 in the supernate of cell cultures.Results of q PCR indicated dose and time dependent manner in gene expression within certain ES antigenic action concentration and periods,and the gene expression NF-?B m RNA displayed two peaks at 0.5d/21 d,4d/21 d and 0.5d/21 d respectively.In small intestines and hearts,target gene expressions were all elevated during different phases after Trichina infection,and reached peaks at 4d and 14 d after infection.For lungs,NOD1 m RNA expression was only slightly elevated at 7d after infection,while the m RNA expression of RIP2 was elevated in all infectious stages with peak value at 4d after infection.For muscles,the changes in gene expression were not significant at the early stage of infection;however,the m RNA expressions were significantly elevated from 21 d after infection with peak value at 28 d.Western blot results indicated the expressions of NOD1,RIP2 and NF-?B p-p65 in mice celiac macrophages were elevated to different extent after Trichina infection,and the variation trend was similar to that of q PCR.ELISA results indicated the contents of TNF-? and IL-6 in supernate of mice celiac macrophage cultures and serums were significantly elevated,with no clear change of IL-1?.Above results indicated Trichina infection activated mice NOD1 receptor,up-regulated the expressions of NOD1 pathway key molecule RIP2 and downstream molecule NF-?B,and promoted the secretions of cytokines of TNF-? and IL-6.ES antigen of Trichina muscle lava activated and adjusted the NOD1 pathway in healthy mice celiac macrophages.Additionally,Trichina infection induced immune tolerance of NOD1 receptor in host.However,due to the complexity of the interactions between Trichina and host immune system,the specific action mechanism needs to be further investigated.