The Research On Alleviation Of LPS-induced Intestinal Inflammation By Treatment With Giardia Duodenalis In Intestinal Epithelial Cells

Giardia duodenalis is a zoonotic parasitic protozoa.Giardia is mainly parasite in the small intestine and can cause giardiasis along with the main symptoms including diarrhea,abdominal pain and weight loss,as well as a variety of complications such as arthritis,irritable bowel syndrome and chronic fatigue syndrome.Giardiasis has a global distribution with 280 million symptomatic cases reported annually,mainly children,immunocompromised people and the elderly.Although Giardia-caused intestinal mucosal inflammation symptoms are not obvious,Giardia co-infected with other pro-inflammatory gastrointestinal pathogens,has been reported to show a protective effect on the body,while the protective mechanism remains unclear.Cathepsin,as a lysosomal protease,exists in tissues and cells of most animals,which is closely related to the growth and development of parasites and their survival in the host.Recent studies have shown that Giardia duodenalis cathepsin B degrades CXCL8 secreted by intestinal epithelial cells treated by IL-1β and Clostridium typhimurium,and can directly reduce CXCL8-induced neutrophil chemotaxis.However,it is unclear which cathepsin B plays a role in this process.In this study,we attempted to reveal the role of Giardia and its recombinantion cathepsin B in LPS-induced inflammation in host intestinal epithelial cells.In this study,Caco-2 cells were stimulated by LPS to construct an in vitro enteritis model.Transcript and expression levels of TNF-α and IL-8 in Caco-2 cells were detected by q PCR and western blot analysis.The results showed that LPS stimulation promoted the secretion of TNF-αand IL-8 in Caco-2 cells in a time-dependent manner,which proved that the in vitro enteritis model was successfully constructed.In this study,Giardia was pre-incubated with cathepsin B inhibitors,then they were used to treated IECs.To verify its effect on the inflammatory response of Caco-2 cells themselves and on the LPS-induced enteritis model.The results of q PCR and western blot showed that Giardia stimulated Caco-2 cells to secret TNF-α and IL-8.When Giardia treated with cathepsin B inhibitor stimulated Caco-2 cells,the expression levels of TNF-α and IL-8 were significantly decreased compared with Giardia-stimulated cells without inhibitor pretreatment,which proved that cathepsin B could effectively induce inflammation.When Giardia treated with cathepsin B inhibitor stimulated LPS-induced enteritis model,and the expressions of TNF-α and IL-8 cytokines were significantly upregulated,indicating that cathepsin B could reduce inflammation.This study selected Giardia cathepsin B genes,which were processed by bioinformatics analysis to predict its hydrophilicity and hydrophobicity,signal peptide,secondary structure and tertiary structure,and the feasibility of prokaryotic expression of cathepsin B in vitro.Three kinds of cathepsin B(GL50803＿17516、GL50803＿15564 and GL50803＿16160)were expressed in vitro by prokaryotic expression system.Combined with the prokaryotic expression vectors of four cathepsin B(GL50803＿16486,GL50803＿14019,GL50803＿16779,GL50803＿29304)successfully constructed in the laboratory,a total of seven kinds of cathepsin B were purified.Their inflammatory and anti-inflammatory functions were investigated.Caco-2 cells and LPS-induced enteritis models were stimulated by recombinant cathepsin B,the expression levels of TNF-α and IL-8 were detected by q PCR and western blot to explore whether the recombinant cathepsin B caused inflammatory response and played a role in reducing inflammation.The results showed that compared with the control group,the recombinant cathepsin B could significantly increase the expression of cytokine TNF-α and IL-8,which proved that the seven recombinant cathepsin B could effectively induce inflammation.In a model of LPS-induced inflammation stimulated by the recombinant protein,the expression of cytokines TNF-α and IL-8 could be significantly down-regulated,which proved that the recombinant cathepsin B could effectively reduce LPS-induced inflammation.When different kinds of cathepsin B stimulates inflammation model induced by LPS,we found CTSB-1 and CTSB-8 can significantly decrease the secretion of inflammatory cytokines,CTSB-4 could not down-regulate the secretion of inflammatory factors.Although some cathepsin B significantly inhibited the expression of inflammatory cytokines.However,the degree of decrease is different,suggesting that different cathepsin B may play different functions and play a synergistic role in inducing anti-inflammation.Based on the mechanisms of LPS-induced inflammation mediated by TLR/NF-κB pathway,the effects of Giardia and recombinantion cathepsin B on NF-κB pathway were explored.Western blot and immunofluorescence were used to detect the phosphorylation level of p65 and its entry into the nucleus.The results showed that both Giardia and recombinant cathepsin B stimulation could both increase the phosphorylation level of p65 and inhibit the entry of p65 into the nucleus,which proved that Giardia and recombinant cathepsin could effectively inhibit activation of NF-κB pathway and play a role in reducing inflammation.To sum up,this study confirmed that Giardia stimulation could inhibit inflammation of the intestinal epithelial cells induced by LPS,and found the cathepsin B played a crucial regulatory role in this process.The present study demonstrated the mechanisms of Giardia-induced host protection,thus providing new references and basis for the study of giardiasis.