Giardia Duodenalis Regulates Tight Junction Protein Expression And NO Production Via ROS-AMPK/mTOR Autophagy Pathway In Intestinal Epithelial Cells

Giardia duodenalis(Giardia),as a non-invasive protozoan parasite,infects the small intestine of humans and many mammals and causes symptoms such as abdominal pain,diarrhea and malabsorption in the host.Autophagy is a catabolic process in cells responsible for digesting damaged organelles,proteins,and lipids in the cytoplasm and then removing them via lysosomes.This process is critical for cell survival and pathogen infection.It has been proved that autophagy is involved in regulating the destruction of tight junction protein(TJP)in host cells by parasites.And autophagy is involved in regulating the production of nitric oxide(NO)in cells,which is closely associated with parasitic infection.It is unknown whether Giardia can induce autophagy in host cells.In this study,the interaction model between Giardia and intestinal epithelial cells(IECs)was established to explore whether Giardia can induce autophagy and the effects of autophagy on giardiasis-related TJP expression and NO production,which provides a new theoretical basis for studying the pathogenesis of Giardia.After Giardia interacted with IECs,the expression levels of some autophagy-related genes were detected by real-time fluorescence quantitative PCR(q PCR)and Western blot.With the increase of Giardia stimulation time,the transcription and protein expression levels of autophagy-related genes such as LC3,Beclin1 and ULK1 gradually increased.Immunofluorescence assay also showed a significant increase in intracellular LC3 protein.Western blot results showed that p62 protein was also significantly down-regulated.These results indicated that the formation of autophagosomes increased after Giardia stimulation in IECs.Western blot and immunofluorescence assay showed that the autophagy inhibitor Chloroquine(CQ)significantly affected the expression of Giardia-induced LC3 and p62,suggesting that Giardia could induce autophagy in IECs.Next,the pathway of inducing autophagy was explored.Western blot assay was used to detect the expression of several proteins in the autophagy pathway after Giardia stimulation,and the results showed that Giardia induced the autophagy through AMPK/m TOR pathway in IECs.The production of reactive oxygen species(ROS)in the cells was detected by fluorescence probe DCFH-DA labeling,and the results showed a large accumulation of ROS in IECs after Giardia infection.The effects of ROS inhibitor N-acetylcysteine(NAC)on autophagy and AMPK/m TOR pathway were detected by q PCR,immunofluorescence and Western blot.The results indicated that Giardia could induce autophagy through ROS-AMPK/m TOR pathway in IECs.It has been shown that pathogens-induced autophagy is involved in down-regulation of TJP expression,and one of the pathogenic mechanisms of Giardia is the disruption of the intestinal barrier through the disruption of TJP.In this experiment,the expression of TJP: Claudin-1,Claudin-4,Occludin and ZO-1 were detected.Western blot results showed that the expression of these four TJP gradually decreased with the increase of Giardia stimulation time.To determine the effect of Giardia-induced autophagy by ROS-AMPK/m TOR on TJP,cells were pretreated with autophagy inhibitor 3-methyladenine(3-MA),CQ,m TOR inhibitor Rapamycin(Rapa)and NAC,respectively.Both q PCR and Western blot experiments indicated that Giardia autophagy induced by ROS-AMPK/m TOR pathway was involved in the destruction of TJP in IECs.A large number of literatures have studied the relationship between autophagy and NO release,and Giardia can reduce NO release to escape the host immune response in IECs.Therefore,this study explored whether the Giardia-induced autophagy was related to the reduction of NO production in IECs.3-MA can effectively alleviate the reduction of NO release induced by Giardia.Rapa and NAC significantly reduced and up-regulated NO production.These results suggested that Giardia-induced autophagy by ROS-AMPK/m TOR pathway was involved in the reduction of NO production.In addition,a large number of literatures have proved the regulation of autophagy on ROS.So in this study,3-MA and CQ were used to test the relationship between Giardia-induced autophagy and intracellular ROS,and the results showed that both 3-MA and CQ could lead to the further accumulation of ROS,indicating that Giardia-induced autophagy can in turn regulate the generation of ROS.In conclusion,this study proved that Giardia can induce autophagy in IECs through the ROSAMPK/m TOR pathway for the first time,and the autophagy is involved in the destruction of TJP and reduction of NO production caused by Giardia infection in IECs,which may provide favorable conditions for Giardia colonization and sustained infection.Therefore,our study can further reveal the potential pathogenesis of giardiasis from the perspective that giardiasis induces autophagy of IECs to participate in the pathogenesis of giardiasis,and provide a new reference for future clinical research on giardiasis.