Protective Effect Of Adoptive Transfer Of R-SjCystatin Induced Macrophages On Mice With Sepsis

Background: Sepsis is one of the serious complications of clinically critical patients,with acute and dangerous onset and high mortality.Sepsis is known as severe tissue injury caused by the activation of immune cells by pro-inflammatory factors,especially in the systemic inflammatory response syndrome(SIRS)stage of sepsis.Mononuclear macrophages served as infection-resistant cells in the natural immune response of sepsis and antigen-presenting cells in specific immunity,have been widely concerned about the role of function alternations in the sepsis immuno-suppression.According to the different phenotypes and functions after activation,macrophages can be divided into classic activated macrophages(M1)and alternative activated macrophages(M2).The former secretes pro-inflammatory cytokines to aggravate inflammation,while the latter secretes interleukin-10(IL-10)and other anti-inflammatory factors to improve excessive inflammation and promote tissue repair.Studies have shown that the amount of M1 in tissues significantly increased during sepsis,which might be one of the mechanisms leading to sepsis immune paralysis.Epidemiological studies have shown that there was an inverse relationship between worm infections and sepsis.The mechanism involved is to reduce the polarization of macrophages to M1 by blocking toll-like receptor 4(TLR4)after worm infection,and promote the release of anti-inflammatory factors such as TGF-? and IL-10,thereby regulating excessive inflammation.Although worm therapy is considered to be safe and effective,the application of living parasites still carries the risk of adverse side effects.Therefore,the adoption of worm-derived proteins that replaces worm infections for the treatment of sepsis can avoid the shortcomings of infected worms and play a therapeutic role.In this experiment,Schistosoma japonicum cysteine protease inhibitor(r-SjCystatin)was used to observe its ability to regulate the polarization of macrophages and adoptive transfer of r-SjCystatin polarized macrophages to sepsis mice.To observe its therapeutic effect to explore its protective mechanism against sepsis.Objective: 1.Explore the regulatory effect of r-SjCystatin on macrophages in vitro 2.Observe the therapeutic effect of adoptively transferred macrophages induced by r-SjCystatin on sepsis mice and investigate the potential application value of worm proteinMethods: 1.Construct,express and purify r-SjCystatin protein: take the preserved strains and put it in culture medium to expand culture,and take another culture medium to induce the expression of r-SjCystatin;the culture supernatant was concentrated and dialyzed,then the protein was purified by low-pressure chromatography system;the purified protein was dialyzed for a night.After the dialysate was concentrated by ultrafiltration tube,the target protein was identified by gel electrophoresis and the protein concentration was determined by BCA.2.BMDMs were captured and cultured in conditioned medium for 5 days.The flow cytometry(F4/80,CD11b)was used to identify the maturity.Then,mature BMDMs were harvested and divided into four groups: negative control group(LPS group),positive control group(IL-4+IL-10 group),protein alone group(r-SjCystatin group),protein and LPS costimulation group(r-SjCystatin +LPS group).Cells and culture supernatant were collected 24 hour post-treatment,and the polarization indexes of CD11c(M1)and CD206(M2)cells were detected by flow cytometry and immunofluorescent staining.The levels of IFN-?,IL-6,IL-10 and TGF-? in the culture supernatant were measured by ELISA.3.Using cecal ligation and puncture(CLP)to prepare mouse sepsis model,BALB/c mice were randomly divided into 4 groups: CLP model group,CLP+BMDMs group,CLP+LPS-BMDMs group and CLP+r-SjCystatin-BMDMs group.CLP group: only for sepsis modeling;CLP+BMDMs group: adoptively transfer macrophages after sepsis modeling;CLP+LPS-BMDMs group: adoptively transfer LPS-treated macrophages after sepsis modeling;CLP+r-SjCystatin-BMDMs group: adoptively transfer r-SjCystatin-treated macrophages after sepsis modeling.30 minutes after CLP,cells prepared in advance were suspended in 100 ul PBS and injected into the tail vein of mice by 1×106 cells.The mice were sacrificed under anesthesia 12 hours after adaptive transfer,and serum,kidney,heart,lung and liver tissue were collected.The levels of ALT(alanine aminotransferase),AST(aspartate aminotransferase),BUN(blood urea nitrogen),and Cr(creatinine)in the sera of mice were detected by using an automatic biochemical analyzer.Histopathological examination and histological score were performed after HE staining.Result: 1.Gel electrophoresis identification results showed that the soluble recombinant protein was successfully expressed and the relative molecular mass(Mr)of the purified r-SjCystatin protein was 11.3 k Da,which was equivalent to the molecular mass of the deduced peptide gene product(11.3 k Da).2.Flow cytometry results showed that the maturity of bone marrow-derived macrophages cultured in M-CSF was significantly higher than that in the blank group(all P < 0.001).Compared with the LPS group,the CD206+ expressions in macrophages of r-SjCystatin and r-SjCystatin+LPS were significantly increased(both P < 0.01)and the levels of IL-10 and TGF-? in the cell supernatant were significantly increased(P < 0.01).Compared with LPS group,CD86+ expressions in macrophages of r-SjCystatin and r-SjCystatin+LPS groups were significantly decreased(both P < 0.001)and the levels of IFN-? and IL-6 in cell supernatants were significantly decreased,the difference was statistically significant(P < 0.05);3.Compared with CLP group and CLP+LPS-BMDMs group,the concentrations of ALT,AST,BUN and Cr in sera of mice with adoptive transfer of r-SjCystatin-treated macrophage were significantly reduced and the difference was statistically significant(P < 0.05).Meanwhile,glomerular damage,renal tubular cell edema and inflammatory cell infiltration were significantly alleviated;myocardial fiber rupture and myocardial cell degeneration and necrosis were significantly ameliorated;alveolar congestion,alveolar wall thickness and cell infiltration were significantly mitigated;hepatic cord disorder and hepatocytes swelling and inflammatory cell infiltration were significantly alleviated and the histological scores of kidney,heart,lung,and liver decreased significantly(P < 0.05).Conclusion: 1.r-SjCystatin protein could regulate the polarization of BMDMs to M2 with anti-inflammatory properties in vitro,and inhibit the transformation of M1.2.Adoptive transfer of macrophages regulated by r-SjCystatin could significantly improve the clinical symptoms and tissue and organ damage of CLP mice with sepsis.