Effects Of Trichinella Spiralis And Its Serine Protease Inhibitors On Autophagy Of Host Small Intestinal Cells

In eukaryotes,autophagy is an innate defense mechanism,which degrades pathogens through lysosomal system and maintains the homeostasis of body.Trichinella spiralis(T.spiralis),as a pathogen,endangers all tissues and organs of host,and causes damage of tissues and cells and physiological dysfunction after invasion.In recent years,the study of the interaction between parasites and host cells autophagy has become a hot spot.More and more studies indicate that parasites can not only cause autophagy of host cells,but also evade autophagy defense system of host cells through various ways.However,there are few studies on the correlation between T.spiralis and host cells autophagy.Therefore,this study mainly explored the effects of T.spiralis and its serine protease inhibitors(TsSPIs)on the autophagy of host small intestinal cells,which laid a foundation for further exploring the pathogenesis of host intestinal dysfunction post T.spiralis infection,and also provided some experimental and theoretical basis for the prevention and treatment of trichinellosis.In order to explore the effect of T.spiralis infection on the autophagy of host small intestinal cells,a model of T.spiralis infected mice was established,the small intestines of uninfected mice and mice infected with T.spiralis on the 1,3,7,15 and 21 days were collected,and the expression levels of autophagy important proteins in small intestinal cells and the injury of small intestine were detected using Western blot and H&E staining.The results showed that the expression of autophagy important proteins in cells of duodenum,jejunum and ileum was changed significantly,and jejunum villi appeared edema and epithelial compactness decreased.The expression trend of proteins was consistent with the trend of small intestine injury,which revealed an upward trend from 1 to 7 days post infection,peaked on day 7,and a downward trend from 7 to 21 days.The autophagy of jejunal cells was the most serious in comparison to duodenal cells and ileal cells.Thus,the jejunum of uninfected and 7 days post infection(dpi 7)was selected as the object of subsequent research,and the autophagy-related indexes were detected by transmission electron microscopy(TEM),immunohistochemistry(IHC),and fluorescence quantitative PCR(qPCR)to further explore the influence of T.spiralis infection on the autophagy of the host small intestinal cells.The results showed that autophagosomes and autolysosomes were abundantly formed in the jejunal cells of mice,and autophagy marker protein microtubule-associated protein light chain 3B(LC3B)was substantially expressed in stromal cells of jejunum lamina propria after T.spiralis infection.In addition,the expression of autophagy-related genes was changed significantly.The results indicated that T.spiralis infection could cause autophagy of host small intestinal cells.To explore the effect of T.spiralis infection on autophagy pathway,including adenosine5’-monophosphate-activated protein kinase(AMPK)-mammalian rapamycin target protein(mTOR)-autophagy-related gene 1/2(ULK1/2),Atg12-Atg5-Atg16L1,phosphatidylinositol 3-kinase(PI3K)-serine/threonine-protein kinase(Akt)-mTOR,mitogen-activated protein kinase(MAPK)-mTOR and protein 53(p53)-mTOR,and Western blot was used to detect the expression levels of autophagy pathway-related proteins in jejunum of uninfected and dpi 7.The results showed that the expression of p-ULK1/ULK1,p-AMPK/AMPK,p-p53/p53 and Atg16L1 was up-regulated,while the expression of p-mTOR/mTOR,p-Akt/Akt and p-p38 kinase(p38)/p38 was downregulated post T.spiralis infection,indicating that T.spiralis might induce autophagy of host small intestinal cells by regulating these five signaling pathways.To explore the effect of TsSPIs on autophagy and autophagy pathway in the small intestinal cells of host,the models of TsSPIs stimulated Porcine’s small intestinal epithelial cells line-J2(IPEC-J2)in vitro and mice jejunum in vivo were established,and their autophagy-related indexes were detected using Western blot,TEM,H&E staining,immunofluorescence,IHC and qPCR.The results showed that TsSPIs could induce autophagy in the small intestinal cells of host and activate autophagy-related pathways.T.spiralis Serpin-type serine protease inhibitor(TsAdSPI)has more advantages in inducing autophagy of host cells in contrast with the T.spiralis Kazal-type serine protease inhibitor(TsKaSPI).In conclusion,T.spiralis infection could cause autophagy of host small intestinal cells,and its secreted TsSPIs plays an important role in the process of host small intestinal cells autophagy induced by T.spiralis.They might induce autophagy of host small intestinal cells by regulating signaling pathways,such as AMPK-mTOR-ULK1/2,Atg12-Atg5-Atg16L1,PI3K-Akt-mTOR,MAPK-mTOR and p53-mTOR.