Effects Of Two Kinds Of Serine Protease Inhibitors Of Trichinella Sprialis On The Immune Function Of Macrophages

Trichinelliasisis an important zoonotic parasitic disease,Trichinella spiralis can complete the parasitic process in vivo,which is inseparable in invasion of host and immune evasion.The research results show that the serine protease inhibitor(SPI)plays a key role in the process of parasite invasion of host and immune escape,etc.Some studies have found that Schistosoma haematobium can reduce the immunogenicity of its own and escape the host immune attack in SPI of body surface combined with human trypsin.Currently some SPIs have been isolated and identified.from Ascarid,Schistosomes,Brugia malayi,Haemonchus contortus,Trichinella spiralis and so on.In order to find out the role of SPI from Trichinella spiralis in host immune regulation,,this study researched the regulating effects of Trichinella spiralis adult serine protease inhibitor(TsAd SPI)and Trichinella Kazal-type serine protease inhibitor(TsKa SPI)on immune function of macrophages with the model of J774 A.1 macrophage in mice.Two gene expression vectors Ts Ad SPI and TsKa SPI with great activity,the laboratory have successfully constructed by prokaryotic expression,were induced by IPTG,and the expressed proteins were purified by electroelution,acquire recombinant proteins of TsAd SPI and TsKa SPI after dialysis renaturation.The effect of two kinds of SPIs with different concentrations on the proliferation of J774 A.1 was detected by CCK-8.J774 A.1 cells were stimulated with two SPI alone respectively or two SPIs respectively after LPS pre-stimulation,the level of mRNA expression and secretion of inflammatory cytokines TNF-alpha,IL-1beta,IL-6,IL-10,IL-12 and TGF-beta were detected respectively,and the effects of two kinds of SPIs on the JAK2/ STAT3 signal pathway were detected,used in Westen-blot.According to the TNF-alpha,IL-1beta,IL-6,IL-10,IL-12,TGF-beta and GAPDH housekeeping gene sequences published in Gen Bank,real-time PCR primers were designed and analysised to establish a fluorescence real-time PCR detection method.The consequence of CCK-8detection to TsAd SPI and TsKa SPI were basically consistent,compared with the PBS control group,effect of two kinds of SPIs on the proliferation of J774 A.1 showed the concentration dependent,two kinds of SPIs at low concentration on the proliferation of J774 A.1 were not affected;but when the concentration was reached 10 ?g/ m L,the proliferation activity of J774 A.1were obviously damaged by two kinds of SPIs,and with the increase of SPIs concentration,thecell proliferation of J774 A.1 significantly enhanced the degree of damage.Therefore,the working concentrations of TsAd SPI and TsKa SPI were both selected as 5 ?g/ mL.The detection results of SYBR Green I real-time fluorescence quantitative PCR showed that Ts Ka SPI and TsAd SPI can both inhibit the mRNA expression level of pro-inflammatory cytokines TNF-alpha,IL-1beta,IL-6 and IL-12 in different levels on J774 A.1 mice macrophage activated by LPS.And two kinds of SPIs can both promote the expression of anti-inflammatory cytokines IL-10 and TGF-beta on J774 A.1 macrophages of mice with different degrees.Regulation degrees of Ts Ad SPI and TsKa SPI to the expression and secretion of inflammatory cytokines in macrophages by are different from each other,but TsAd SPI and TsKa SPI can both regulate these inflammatory cytokines in inhibition or promotion,show that the two kinds of SPIs have the certain degrees of adjustment function on immune function of macrophages.To detect the conditions of secretion of pro-inflammatory cytokines and anti-inflammatory cytokines on macrophage in ELISA,results are consistent with the detection results of real-time fluorescence quantitative PCR,confirming the results that the two SPIs have the ability to regulate inflammatory cytokines on macrophage.At the same time,Westen-blot found that TsAd SPI and TsKa SPI could both induce the phosphorylation of JAK2 and STAT3 receptors,which indicated that two kinds of SPIs could affect the signal transduction of macrophage through regulating the JAK2/STAT3 signaling pathway.The results of this study showed that two kinds of serine protease inhibitors TsAd SPI and Ts Ka SPI of Trichinella spiralis can both regulate immune function of macrophages through the directly impacting to expression and secretion of inflammatory cytokines on macrophage and function of JAK2/STAT3 signaling pathway.Accordingly,we suggest that this two kinds of SPIs can affect the immune function of the host as effect molecules in regulating macrophages when the host was infected by Trichinella spiralis,to achieve immune escape,and to resist the host parasite elimination reaction and finish long-term survival in vivo.