The Role And Mechanism Of Epithelial-dendritic Cells Transition In Sepsis-associated Acute Intestinal Injury

Objects: To investigate the role and mechanism of intestinal epithelial-dendritic cell transition(EDT)in regulating sepsis-induced acute intestinal injury,systemic inflammatory response(SIRS),and multiple organ dysfunction syndrome(MODS).Methods: In vivo,Male C57BL/6(6-10 weeks)mice were exposed to cecal ligation and puncture(CLP)to induce sepsis.After CLP 6h,12 h,24h,the expression and location of DC-SIGN,the dendritic cell(DC)functional marker,were examined by RT-PCR,western blot,immunofluorescence,immunohistochemistry and flow cytometry.Intraperitoneal injection of DC-SIGN si RNA was used to inhibited DC-SIGN expression in mouse intestinal epithelial cells(IECs).To evaluate the role of EDT in acute inflammatory injury of intestine,lung,liver,and kidney through gross anatomy,hematoxylin-eosin straining,serum levels of ALT,AST,creatinine,and BUN,serum levels of cytokines(TNF-?,IL-1?,IL-6,IL-10,IFN-?,and MCP-1)assessed by ELISA,and 7-days survival rate of septic mouse.To study the potential mechanism of DC-SIGN regulates sepsis-associated acute intestinal injury-SIRS-MODS,the expression of total and phosphorylation of ERK1/2 and NF-??/p65 was detected by western blot.In vitro,a normal human IECs line(FHs74Int)was stimulated with lipopolysaccharide(LPS).The expression of DC-SIGN in FHs74 Int was detected by RT-PCR and western blot,to definite the optimal conditions of LPS stimulation.According to the results of mechanism in vivo,DC-SIGN si RNA transfection and specific inhibitor pretreatment were used to knockdown the expression of DC-SIGN and downstream mediators in vitro.The expression of phosphor-ERK1/2 and phosphor-NF-??/p65 was examined by western blot.The levels of cytokines in culture supernatant were measured by ELISA.Results: DC-SIGN expression and location mainly in septic mouse IECs,and was time-dependently upregulated by CLP.CLP significantly aggravated acute inflammatory injury of intestine,lung,liver,and kidney.CLP-induced phosphorylation of ERK1/2 and NF-??/p65 was significantly elevated,leading to the increase of systemic inflammatory cytokines.The above phenomena were effectively inhibited by DC-SIGN si RNA transfection in vivo,which alleviated multiple organ dysfunction and pathological injuries and increased the survival rate of septic mice.In vitro,DC-SIGN,phosphor-ERK1/2,and phosphor-NF-??/p65 expression in FHs74 Int was significantly upregulated by 200ng/m L LPS stimulation in 24 h.DC-SIGN si RNA transfection and ERK1/2 specific inhibitor SCH772984 abolished LPS-induced ERK1/2 and NF-??/p65 phosphorylation,resulting in the decrease of cytokines in culture supernatant.Conclusion: Sepsis induced EDT in the intestine.“DC-SIGN-ERK1/2-NF-??/p65” signaling pathway regulates cytokines release by IECs,and promotes acute intestinal injury and systemic inflammatory response.The inhibition of intestinal EDT exhibited protective effects on sepsis-associated SIRS and MODS.