Function Of Trichinella Spiralis Serine Protease Inhibitor In Invasion And Immune Regulation Of Trichinella Spiralis

Trichinellosis is a widespread food-borne parasitic zoonosis that infects through the ingestion of raw or undercooked meat containing infective muscle larvae(ML)of Trichinella.The different developmental phases of T.spiralis occur in a single host.During the parasitism of Trichinella spiralis,the host needs to expel pathogens,but the parasites need to reproduce and survive successfully in the host.Therefore,a complex host-parasite interaction relationship has gradually evolved in the long-term evolution process,and the serpin type serine protease inhibitor(TsSPI)of Trichinella spiralis may play an important role in this process.TsSPI)are expressed in adult worms(AW),newborn larvae(NBL)and muscle larvae(ML)of T.spiralis,with the ML stage demonstrating the highest expression level.This study aims to determine TsSPI functions in larval viability and invasion intestinal epithelial cells in vitro,as well as their development,survival,fecundity in vivo and the regulation of host immunity via RNAi,and the effect of TsSPI on macrophage polarization in vitro.In this study,RNA interference was used to explore the function of TsSPI in the life cycle of Trichinella spiralis.Three specific siRNAs(siRNA-153,siRNA-479,siRNA-986)were designed and synthesized.At the same time,the specific dsRNA TsSPI was synthesized in vitro.TsSPI-specific siRNAs and dsRNA were transfected into ML via soaking incubation.The results showed that RNAi could be effectively transferred to Trichinella spiralis muscle larvae.Silencing effect of TsSPI transcription and expression was determined using qPCR and Western blot,respectively.The results showed that the silencing effect of siRNA and dsRNA TsSPI was dose-dependent.With the increase of dose,the silencing effect was enhanced.However,2μM siRNA and 60ng/μl dsRNA-TsSPI were used to optimize the working concentration.2μM of siRNA-153,siRNA-479,or siRNA-986 or 60ng/μl dsRNA-TsSPI can effectively reduce the level of TsSPI gene transcription and protein expression.Si RNA–986 and dsRNA–TsSPI demonstrated the maximum silencing of TsSPI m RNA or protein expression and no significant difference exists between siRNA-986 and dsRNA-TsSPI.Therefore,60ng/μl dsRNA-TsSPI was used to optimize the working concentration for subsequent experiments.TsSPI gene silencing of Trichinella spiralis treated with dsRNA-TsSPI proved effectiv for 2 days,and the silencing effect was the best after 3days,and it still had significant silencing effect until 7 days.In the subsequent infection experiment,Trichinella spiralis had TsSPI gene silencing in the whole intestinal infection stage(3-7 days after infection).The results showed that RNAi induced by dsRNA–TsSPI had gene specificity.In this study,dsRNA mediated TsSPI gene silencing did not affect the molting and survival rate of larvae in vitro.Mice inoculated with Trichinella spiralis larvae transfected with dsRNA-TsSPI.The NBL production of female AW from mice inoculated with larvae soaked in dsRNA–TsSPI did not show a significant reduction compared with the control dsRNA group after culturing in vitro for 72 h.TsSPI gene silencing did not affect the fecundity of Trichinella spiralis.TsSPI may not directly affect the growth and development of Trichinella spiralis.In this study,after challenge with dsRNA–TsSPI-treated ML,mice exhibited a reduction of56.0% intestinal AW burden and 56.9 % ML burden.It showed that TsSPI gene silencing could affect the survival ability of AW and P 1 ML.The viability of AW was indirectly affected by the RNAi mediation of ML.The genetic analysis of dsRNA interference showed that the expression level of TsSPI of AW was still inhibited,but the expression level in P 1 ML had basically returned to normal level.Therefore,we speculated that the decrease in the number of P 1 muscle larvae in muscle might be caused by the decrease of AW.When mice were inoculated with larvae transfected with dsRNA-TsSPI,peritoneal macrophages were taken from mice after infection with 4 and 7 day.The expression of inflammatory cytokines in the culture supernatant of mouse peritoneal macrophages was detected by ELISA.Compared with uninfected mice,the contents of inflammatory cytokines TNF-α,IL-6,IL-1 β,IL-12 and anti-inflammatory cytokines TGF-β and IL-10 in the supernatant of peritoneal macrophages were increased in different degrees after infection with Trichinella spiralis.The mixed environment of pro-inflammatory cytokines and anti-inflammatory cytokines was conducive to the parasitization of Trichinella spiralis.Compared with 4 days after infection,the contents of pro-inflammatory cytokines TNF-α,IL-6 and IL-12 decreased in varying degrees after infection 7days except for IL-1β.Compared with the mice inoculated with untreated Trichinella spiralis,the contents of inflammatory cytokines in peritoneal macrophages of mice infected with TsSPI gene silencing were increased to varying degrees.The results showed that TsSPI gene silencing can significantly aggravate the host inflammatory response induced by Trichinella spiralis,which indicated that TsSPI may alleviate host inflammatory response by changing the balance of inflammatory factors.Western blot analysis of p65 phosphorylation in macrophages.The results showed that the level of p65 phosphorylation in peritoneal macrophages of mice infected with Trichinella spiralis was significantly higher than those in uninfected mice;The p65 phosphorylation in peritoneal macrophages of mice infected with Trichinella spiralis for 4 days was significantly lower than infection for 7 days,which indirectly proved that the host immunity caused by Trichinella spiralis changed from Th1 / Th2 mixed type to Th2 type with the passage of time.However,the level of p65 phosphorylation in peritoneal macrophages of mice infected with TsSPI gene silencing was significantly higher than that of mice infected with untreated Trichinella spiralis,indicating that TsSPI can alleviate the phosphorylation of NF-κB in macrophages induced by Trichinella spiralis infection,and then inhibit the host inflammatory response.qPCR was used to detect the m RNA expression of CAM marker i NOs and AAM marker Arg-1in peritoneal macrophages of mice infected with Trichinella spiralis.The results showed that the expression levels of i NOs and Arg-1 in peritoneal macrophages of mice infected with Trichinella spiralis were significantly higher than those in uninfected mice;Compared with 4 days after infection,the expression of i NOs in peritoneal macrophages was significantly decreased,while the expression of Arg-1 was significantly increased after 7 days.The results showed that Trichinella spiralis infection could regulate the polarization of host macrophages.Compared with the mice inoculated with untreated Trichinella spiralis,the relative expression of i NOs in peritoneal macrophages of mice infected with TsSPI gene silencing was significantly increased,while the relative expression of Arg-1 was significantly decreased,indicating that TsSPI plays an important role in regulating the polarization of host macrophages.The recombinant proteins TsSPI and mutspi were induced to express.Mutspi was a mutant TsSPI with decreased serine protease inhibitor activity.The recombinant proteins were using to research the effects of macrophage polarization.qPCR analysis showed that TsSPI could inhibit LPS mediated secretion of CAM related pro-inflammatory cytokines TNF-α,IL-6,IL-1 β,IL-12 and effective factor i NOs.Western blot analysis of p65 phosphorylation and protein expression of IRAK,IκB in macrophages showed that Tsspi could inhibit the degradation of IRAK,IκB and the phosphorylation of p65 mediated by LPS.The results showed that TsSPI could inhibit the degradation of IRAK,IκB and the phosphorylation of p65 mediated by LPS.Therefore,TsSPI can inhibit the activity of LPS mediated NF-κB pathway,further inhibit the release of proinflammatory cytokines TNF-α,IL-6,IL-1 β,IL-12 and CAM marker i NOs,and prevent macrophages polarizing to CAM.The recombinant proteins TsSPI and mu TsSPI were used to stimulate macrophages directly.qPCR analysis showed that TsSPI could effectively promote the transcription expression of AAM related anti-inflammatory cytokines TGF-β,IL-10 and effective factor Arg-1,and this regulatory function was weakened by using mutant stimulation.At the same time,Western blot was used to detect the phosphorylation of JAK2 and STAT3 in macrophages.The results showed that TsSPI could stimulate the phosphorylation of JAK2 and STAT3.Therefore,TsSPI can stimulate and activate JAK2 / STAT3 signaling pathway.Meanwhile,TsSPI can promote the secretion of anti-inflammatory cytokines TGF-β,IL-10 and AAM marker effector factor Arg-1,and promote macrophage polarization to AAM.In conclusion,dsRNA TsSPI can specifically and effectively inhibit the gene transcription and protein levels of TsSPI.TsSPI may not directly participate in the growth and reproduction of the parasite,but participate in the regulation of the interaction between Trichinella spiralis and its host by some extent.Tsspi gene silencing can regulate the polarization of macrophages induced by Trichinella spiralis,enhance the phosphorylation level of NF-κB,and promote the secretion of inflammatory cytokines in macrophages.The recombinant protein TsSPI can inhibit the activity of LPS mediated NF-κB pathway and regulate the expression of corresponding cytokines and effectors depending on serine protease inhibitory activity.In addition,TsSPI may activate JAK2 /STAT3 signal pathway.Therefore,TsSPI can directly regulate the polarization direction of macrophages.Tsspi may regulate the host immune response by regulating macrophage polarization,thereby affecting the secretion of inflammatory factors in the host,thus creating a favorable environment for the colonization of Trichinella spiralis in the host.