Effect Of Of Reg Proteins On Intestinal Mucosa Barrier In Rats With Severe Acute Pancreatitis And The Mechanism Of Reg Proteins

Objective: Severe acute pancreatitis (SAP) is a common clinical severeacute abdomen with an acute onset, rapid progression, complex complicationsand it’s mortality rate is about30%at present. In recent years, studies haveshown that intestinal barrier function damage caused by SAP, prone tointestinal barrier dysfunction (IBFD), increases the permeability of I ntestinalmucosa,which causes intestinal bacteria and endotoxin translocation,accelerates the process of sepsis, induces and aggravates the systemicinflammatory response syndrome (SIRS),Multiple organ dysfunctionsyndrome (MODS), and even causes death. Therefore, while treatment ing theprimary disease, the protection of intestinal mucosal barrier is also veryimportant. Recently, the role of Regenerating islet-derived protein（Reg）inthe progressions of SAP has attracted wide attention.Reg proteins are multifunctional molecules discovered in recent years,belonging to the c-type lection super family. Numerous studies have shownthat Reg proteins play important roles in many physiological and pathologicalevents. They found that Reg proteins can promote cell proliferation, regulateapoptosis, and inhibit inflammatory cytokines’ over expression and controlgrowth and proliferation of pathogenic microorganisms around the damagedarea. RegI, III regenerative gene ’s effect is getting more and more attention inacute pancreatitis (AP) especially. Now studies have shown that Reg cansuppress the NF-κB into the nucleus to regulate the TNF alpha, IL-6andother inflammatory factors’ over expression of to restrain inflammation.Other studies have shown that IL-22regulates the expression of Reg,tosuppress bacterial growth in the injury site.There are few studies about theRelationship between Reg proteins and damgge in intestinal mucosa barrier in patients with severe acute pancreatitis.In this study, we will measure RegI, III proteins and mRNA expression inSAP rats’ intestinal mucosa,and evaluate the relationship between the level ofRegI,III and intestinal mucosal barrier damage, We will analyze the change ofRegI and III in the small intestine after pyrrolidine dithiocarbamate (PDTC)pretreatment, which is a specific NF-κB pathway inhibitor，and further explorethe relationship between Reg family and NF-κB pathway.Methods:1Experimental animal group:120adult SD rats were randomlydivided into5groups: normal control (N) group, severe acute pancreatitis(S)group,1mg/kg PDTC pretreatment (P1) group,10mg/kg PDTC pretreatment(P10) group,100mg/kg PDTC pretreatment (P100) group. Each group wasdivided into two subgroups:12hours group and24hours group, there are12rats in each subgrroups.2Experimental models: The rats were given20%L-arginine (L-Arg)intraperitoneal injection at one-hour interval. In S groups, the rats were given20%L-Arg intraperitoneal injection by2.5g/kg,twice at one-hour interval（5.0g/kg rat’s weight）induce SAP. In N groups, the rats received the sameamount saline at the same time. In P1, P10, P100groups, the rat were givenPDTC1mg/kg,10mg/kg and100mg/kg intraperitoneal injection1h before thefirst injection of L-Arg. All rats were killed12h or24h after the injection ofL-Arg to collect blood, pancreas and intestinal tissue.3Experimental methods: The changes of pancreas, intestinal andsurrounding tissues were observed after laparotomy. The pathologic changesin pancreatic and intestinal tissues were observed and graded under opticalmicroscopy. ELISA was used to detect the expression of serum IL-22, TNF-αand I-FABP. The expression of intestine tissue’s RegI and III mRNA wereevaluated by RT-PCR. The levels of intestine tissue’s RegI, III and NF-κBwere detected by Western Blot.4Statistical Methods: Statistical analysis was performed by SPSS17.0forwindows. All data were presented as mean±SD. The significance was assumedat P<0.05. Results:1In SAP group, the pancreatic structure was disorganized andthe interstitial edema was obvious. Pancreatic acinar cell necrosis, bleeding,infiltration of inflammatory cells could be found, the situation got worse astime passes. HE staining of intestine showed epithelial cells’ necrosis, somevillous irregular or loss, the lamina propriety was disintegration, capillarieswere dilating or congestion and inflammatory cells was infiltrated in SAPgroup. We found changes in24h’s subgroup were more serious than the12hgroup. We applied Schmidt grading for the pathological score of pancreas use,Chiu grading for intestinal mucosa. Our data show that, in the SAP group，thescore of pancreas or intestinal mucosa was significantly higher (P <0.01) thanthe control group，in addition24h group was higher than12h group(P <0.05)，All these above date showed that the rats’ intestinal mucosa barrier damagewith SAP induced by L-Arg was successful.2After the pretreatment of different doses of PDTC, we found that thescore of pancreas in P1and P10group had a significant decrease compared withS group, especially P10group (P<0.05). However, the pancreas andsurrounding tissues of P100group were similar to the S group. The score ofintestine was not significantly different between P1012h and P112h group.There was significant decrease in P10024h group’s scores compared with P1group in either12h or24h (P<0.05). The score of S and P1group at24hincreased significantly than that of12h (P <0.05).3The expression of serum: S group was obviously higher than N group(P<0.05); the levels of IL-22in P1, P10and P100group significantly decreasedcompared with S group (P<0.05); Compared with P1group, P10groups werenot significantly different （P>0.05）In the P100group,we could foundincreased level of IL-22than P1group at12h or24h;IL-22in S group at24hincreased significantly than that at12h (P<0.05).4The expression of serum TNF-α: S group increased significantly thannormal group (P<0.01); P1,P10group significantly decreased compared with Sgroup (P<0.01), but P100group was not different from that in S group; Makinga Comparison with P1group, the TNF-α in P10group significantly decreased, but P100group overtopped P1group; S group and P100group at24h increasedsignificantly compared with that at12h (P<0.05).5The expression of serum I-FABP: S group significantly elevated thanthat in normal group (P<0.05); P1,P10group significantly decreased comparedwith S group (P<0.01), but P100group was not different from that in S group;Making a Comparison with P1group, the I-FABP in P10group significantlydecreased, but P100group overtopped P1group; S group at24h increasedsignificantly than that at12h (P<0.05).6The expression of intestinal NF-κBp65protein: S group wassignificantly higher than of N group (P<0.05); P1,P10group notable decreasedcompared with S group (P<0.05).P100group was not different from that of Sgroup; P10group significantly decreased than P1group, but P100group washigher than P1group; S group at24h increased significantly compared withthatof12h (P<0.05).7The expression of intestinal RegI、III protein: S group was obviouslyhigher than that of N group (P<0.01); P1, P10group significantly decreasedcompared with S group (P<0.01); P10group notable decreased than P1group,but P100group was higher (P<0.01); S and P100group at24h increasedcompared with that at12h (P<0.05).8The expression of intestinal RegI、III mRNA: S group was evidentlyhigher than N group (P<0.05); P1, P10group clearly decreased compared withS group (P<0.05); Compared with P1group, the expression of RegIII mRNAin P10group significantly decreased, but P100group overtopped than P1group;The expression of RegI mRNA in P10group decreased compared with P1group at12h,but no difference at24h was found; The expression of RegImRNA in P100group was higher than P1group; S group at24h increasedsignificant compared with that at12h (P<0.05).9RegI, III protein expression and intestinal mucosa pathological score,IL-22,I-FABP,TNF-α,NF-κBp65expression were positively correlated.Conclusion:1The expression of RegI, III proteins and mRNA werehigher in SAP rats’ intestinal mucosa, that suggests RegI, III many be adjustment factors in intestinal mucosa barrier and their expression are relatedto IL-22、TNF-αand other inflammatory cytokines.2The inhibition of PDTC to NF-κB pathway is dose related，and theresults suggest that1mg/kg,10mg/kg PDTC intervention,especially10mg/kgPDTC pretreatment, can reduce pancreas and intestinal mucosa damage byblocking the translocation of NF-κB.100mg/kg PDTC showed little protectionagainst SAP. Nevertheless, each indicator did not reach the level of the SAPgroup at12h group and24h group in the100mg/kg group.3The expression of RegI, III gene may be adjusted partly by the NF-κBpathway. The expression may be stimulated by intestinal mucosa injury orinflammation factors, to play a role to mucosa damage. The mechnismmay beinteraction between multiple pathways. We can not exclude the existence ofsynergy or antagonism effect between RegI, III genes. Whether or not overexpression of RegI, III will aggravate the intestinal mucosa damage of SAP,which needs to be further tested.