Intervention Effect And Potential Mechanism Of Schistosoma Japonicum Polypeptide SJMHE1 In Mice With Sepsis

Objective:SJMHE1(Schistosoma japonicum peptide)is a derivative peptide of Schistosoma japonicum HSP-60(Heat shock protein).Previous studies have shown that SJMHE1 can prevent the occurrence of a variety of autoimmune and allergic diseases.Sepsis is a lifethreatening complication of infection,and excessive systemic inflammatory response is thought to be the cause of multiple organ damage in the early stages of sepsis.However,whether SJMHE1 can play a role in sepsis is not known.In this study,we established a mouse septicemia model using cecal ligation and perforation(CLP)technique to observe the effect of Schistosoma japonicum-derived polypeptide SJMHE1 on septicemic mice and elucidate the underlying mechanism.Methods:1.27 female CS7BL/6 mice aged 6-8 weeks were randomly divided into three groups by double-blind method: sham group,CLP group and SJMHE1 intervention group(CLP+SJMHE1group),with 9 mice in each group.50% CLP technique was used to construct a mouse model of moderate sepsis and SJMHE1 intervention was performed for survival study and analysis.2.Another 15 female C57BL/6 mice aged 6-8 weeks were randomly divided into three groups with 5 mice in each group as above.The mice mild septicemia model was constructed using 25% CLP technique and SJMHE1 intervention was performed.The mice were euthanized48 h later,and serum and alveolar lavage fluid(BALF)were collected.Meanwhile,lung,kidney and liver tissues and organs were collected in 4% paraformaldehyde fixative solution.3.ELISA was used to detecte the expressions of inflammatory factors IL-1β,TNF-α and IL-6 in serum of mice.At the same time,automatic biochemical analyzer was used to detecte the levels of creatine(Cr),urea nitrogen(BUN),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in mice serum.The cells in BALF were counted under microscope,and the wet-dry ratio(W/D)of lung tissue of mice was measured and recorded.Hematoxylin eosin staining(HE)was used to observe the damage and inflammatory infiltration of the collected mice tissues.4.Another 10 female C57BL/6 mice aged 6-8 weeks were randomly divided into experimental group and control group,with 5 mice in each group.After CLP induced sepsis,FITC-labeled SJMHE1(FITC-SJMHE1)was injected subcutaneously in the experimental group,while no CLP operation was performed in the control group.The mice were euthanized 24 h later,and the peritoneal macrophages of the mice were extracted.The combination of FITC-SJMHE1 and peritoneal macrophages was detected by flow cytometry.5.Mouse peritoneal macrophages were extracted and cultured in vitro after CLP treatment induced septicemia.After cell adhesion and stable growth,mouse peritoneal macrophages were incubated with FITC-SJMHE1 for different time(6,12,24,48 h),and the binding of FITClabeled SJMHE1 to macrophages in vitro was observed under laser confocal microscope.6.According to the pre-test results in the laboratory,mice peritoneal macrophages were treated with a gradient concentration of SJMHE1(0,0.1,0.2,0.5 μg /m L)for 24 h,and the effect of SJMHE1 treatment on cell proliferation activity was detected by CCK-8 method.The protein ratios of cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 were detected by western blotting,which were used as indicators to evaluate the levels of apoptosis.7.Mouse peritoneal macrophages were treated with 0.5 μg/m L SJMHE1 and/or 1 μg/m L LPS(Lipopolysaccharide).24 h later,the supernatant of cell culture was collected and the content of interleukin-1β(IL-1β)was detected by ELISA.Quantitative fluorescence PCR(q PCR)was used to detect IL-1β m RNA expression level in cells,and to evaluate the effect of SJMHE1 on the activation of peritoneal macrophages in mice.8.Mouse peritoneal macrophages were treated with 0.5 μg/m L SJMHE1 and/or LPS(1μg/m L),and cell proteins were extracted 24 h later.The amount of protein expression of IκB,p65 and p-IκB,p-p65 in NF-κB(nuclear factor kappa-B)signaling pathway were detected by western blots to evaluate the effect of SJMHE1 on NF-κB signaling pathway.Results:1.Compared with sham group,the mortality of mice in CLP-induced sepsis group was significantly increased.However,the application of SJMHE1 intervention one week before CLP surgery significantly reduced mortality due to sepsis.2.Compared with sham group,serum levels of IL-1β,TNF-α,IL-6,BUN,Cr,AST,ALT in CLP group and cell count in BALF were significantly increased,and SJMHE1 treatment decreased serum levels of IL-1β,TNF-α,IL-6,BUN and AST.And the cell count in BALF was significantly lower.3.SJMHE1 effectively reduced the inflammatory infiltration and damage of lung,kidney and liver tissue in CLP-induced septicemia mice.4.FITC-SJMHE1 was obviously bound to peritoneal macrophages in mice with septicemia,but no corresponding binding was observed in the PBS control group.5.The co-localization of FITC-SJMHE1 and peritoneal macrophages of septicemic mice was obvious in vitro.The binding was time-gradient dependent and obvious at 24 h and 48 h.6.Compared with non-SJMHE1-treated group,SJMHE1 gradient concentration(0.1,0.2,0.5 μg/m L)had no significant effect on cell viability.At the same time,SJMHE1 did not change the ratio of cleaved-Caspase-3/Caspase-3 and cleaved-Caspase-9/Caspase-9.The results showed that SJMHE1 had no cytotoxic effect on mouse peritoneal macrophages within the selected concentration range.7.LPS(1 μg/m L)treatment increased the concentration of IL-1β in mouse peritoneal macrophage culture supernatant,and the m RNA expression level of IL-1β was also significantly increased,while SJMHE1 treatment decreased the expression level of IL-1β and IL-1β m RNA.8.Compared with control group,stimulation of LPS made NF-κB signaling pathway activated.Meanwhile,the contents of phosphorylated IκB-α and p65 in NF-κB signaling pathway were decreased significantly in SJMHE1 intervention group.Conclusion:SJMHE1 plays a protective role in CLP-induced septicemia in mice,and the mechanism may be attributed to its binding to mouse peritoneal macrophages and its inhibition on NF-κB inflammation signaling pathway in the cells.Therefore,SJMHE1 provides a new idea for sepsis intervention.