Sitagliptin In Improving Intestinal Mucosal Injury Associated With Severe Acute Pancreatitis By Nrf2

RESEARCH BACKGROUND AND PURPOSEIntestine is the most frequently involved organ in severe acute pancreatitis(SAP),related to systemic inflammatory response and oxidative stress induced by SAP.SAP and hyperglycemia during stress can also lead to tissue oxidative damage.Founded in the treatment of diabetes,sitagliptin(SIT)could improve organ function by reducing blood sugar,oxidative stress and tissue inflammatory through restraining the activity of DPP4 and/or non enzymatic effect.Whether the anti-inflammatory effect of sitagliptin can improve the intestinal mucosa barrier function of SAP,so as to reduce the incidence of SAP sepsis,and whether sitagliptin antagonize intestinal mucosal oxidative damage through Nrf2 pathway,protect SAP intestinal function,were not confirmed.The purpose of this research is to confirm the effect of sitagliptin in improving SAP related intestinal mucosal oxidation/reduction balance and reduce inflammatory response through Nrf2 pathway,and further to provide a theoretical basis for the rational use of sitagliptin in the treatment of SAP related intestinal injury.METHODS1.C57BL/6 mice were randomly divided into four groups(control,SAP,SAP+SIT100 and SAP+SIT200).The histopathology of pancreas and small intestine were compared.The concentration of serum IL-6,IL-1β,blood glucose and the activity of amylase,lipase,DPP4 were tested and compared.In small intestine,the concentration of MDA and the activity of SOD were compared,the mRNA and protein expression level of DPP4 and NF-κB were assayed.2.Rat small intestinal crypt epithelial cells(IEC-6)cultured with 10%serum high glucose(4.5 g/L)and 0.01 mg/ml insulin were divided into four groups:phosphate buffer solution(PBS),SIT,Lipopolysaccharide(LPS)and LPS+SIT group.Cell survival rate and intracellular ROS fluorescence intensity were compared.The mRNA and protein expression level of IL-6,IL-1β and TNF-α were assayed.3.Wild type(WT)mice and Nrf2-/-(KO)mice were randomly divided into three groups(control,SAP and SAP+SIT200).The histopathology of small intestine was compared,and the concentration of serum IL-6,IL-1β,blood glucose as well as the activity of DPP4 were compared.In small intestine,the concentration of MDA and the activity of SOD were compared,the mRNA and protein expression level of DPP4,NF-κB and Nrf2 were assayed.RESULTS1.Results of sitagliptin on inflammatory response and small intestinal mucosal oxidative damage in SAP model mice.Compared with the control group,inflammatory cell infiltration in pancreas and small intestinal mucosal injury were more serious,the concentration of serum IL-6,IL-1β increased and the activity of serum amylase,lipase,DPP4 increased.In small intestinal tissue,the concentration of MDA increased while the activity of SOD decreased,and the mRNA as well as the protein expression level of DPP4 and NF-κB increased in the SAP group.The results suggest that SAP model leads to systemic inflammatory response and oxidative damage of small intestinal mucosa in mice.Compared with the SAP group,the pathological changes of pancreas and small intestine were relieved in SAP+SIT100 and SAP+SIT200 group.The concentration of serum IL-6,IL-1β and the activity of serum amylase,lipase,DPP4 decreased.In small intestinal tissue,the concentration of MDA decreased while the activity of SOD increased,the mRNA and protein expression level of DPP4 and NF-κB decreased,in SAP+SIT100 and SAP+SIT200 group.Compared with SAP+SIT100 group,the protein expression level of NF-κB in small intestinal tissue decreased more obviously,while the activity of SOD increased significantly in SAP+SIT200 group.Moreover,there is no significant difference between SAP+SIT100 and SAP+SIT200 group in the concentration of serum IL-6 and IL-1β and the activity of serum DPP4 and the mRNA and protein expression level of DPP4 in small intestinal tissue.There was no difference in blood glucose among four groups.The results suggest that sitagliptin improves the inflammatory response and intestinal mucosal oxidative damage in SAP model mice.The result of increasing the dose of sitagliptin to further improvement the repair of small intestinal mucosal injury in mice was not closely related to the inhibition of DPP4 enzyme activity.2.Results of sitagliptin on oxidative and inflammatory factors of IEC-6 cells induced by LPS.After stimulated by LPS,the survival rate of IEC-6 cells reduced,and the level of intracellular ROS content increased.The mRNA and protein expression level of IL-6,IL-1β and TNF-α all increased.Compared with LPS group,the survival rate of IEC-6 cells improved in LPS+SIT group,intracellular ROS content decreased,the mRNA and protein expression level of IL-6,IL-1β and TNF-α decreased.The results suggest that sitagliptin reduces the ROS content and inflammatory factors level of IEC-6 cells induced by LPS.3.Results of sitagliptin on inflammatory response and small intestinal mucosal oxidative damage in SAP model Nrf2-/-mice.Compared with the WT-CTRL group,the mRNA and protein expression level of Nrf2 in intestinal tissue was lower in WT-SAP group.Compared with the WTSAP group,the mRNA and protein expression level of Nrf2 in intestinal tissue was higher in WT-SAP+SIT200 group.Compared with KO-CTRL group,the activity of serum DPP4 and the concentration of IL-6 and IL-1β increased significantly in KO-SAP group.Meanwhile,in small intestinal tissue,the concentration of MDA increased and the activity of SOD decreased,the mRNA and protein expression level of DPP4 and NFκB increased in KO-SAP group.Compared with KO-SAP group,the activity of serum DPP4 as well as the mRNA and protein expression level of DPP4 in small intestinal tissue decreased in KO-SAP+SIT200 group.However,there was no difference in small intestinal histopathology,the concentration of serum IL-6 and IL1β,the concentration of MDA,the activity of SOD and the mRNA and protein expression level of NF-κB in small intestinal tissue,between KO-SAP group and KO-SAP+SIT200 group.The results suggest that although sitagliptin can inhibit DPP4 enzyme activity in Nrf2-/-Amice,it does not improve the inflammatory response and small intestinal mucosal oxidative damage in SAP model.It shows that the antiinflammatory and antioxidant effect on small intestinal mucosa of sitagliptin depends on its activation of Nrf2,not closly related to the function of DPP4 enzyme inhibitor.CONCLUSIONSThis research founds that sitagliptin could reduce small intestinal mucosal oxidative stress,improve small intestinal mucosal oxidation/reduction balance and reduce inflammatory factors expression in small intestinal epithelial cells through activating Nrf2 pathway,and further relieve SAP related small intestinal mucosl injury.This effect is not closely related to the inhibition of DPP4 enzyme activity.