Investigate The Protective Effect Of TPHK To Stress-Induced Intestinal Mucosa Injury

Objective:1.To study the protective effect of recombinant attenuated Salmonella strain carryinghypoxia-inducible factor-1α gene (HIF1α) and keratinocyte growth factor gene (KGF)(TPHK) against intestinal mucosal injury induced by rat intestinalischemia-reperfusion stress.2.To study the protective effect of recombinant attenuated Salmonella TPHK againstintestinal mucosal injury induced by rat intestinal ischemia-reperfusion stress ein thehigh-altitude hypoxic conditions.Methods:Part I:All144male Wistar rats were randomly divided into six groups: normal controlgroup (NC), model control group (TC), onlye attenuated Salmonella group (Ty21a),recombinant attenuated Salmonella carrying ehypoxia-inducible factor-1α geneegroup (TPH), recombinant attenuated Salmonella carrying the keratinocyte growthfactor gene in group (TPK), recombinant attenuated Salmonella carryinghypoxia-inducible factor-1α gene and keratinocyte growth factor genee group (TPHK),n=24.There are6rats at2h,6h,12h,24h in every goup. Before setting up animalmodel7days, rats started to take drug (109pfu a rat, only once every other day, a totalof4times). After Rats were taken medication7days, we eestablish intestinalischemia and reperfusion (I/R), an animal model by strictly following the asepticconditions. The plasma and small intestine tissue of rats were ecollected andpreserved in two groups:e one group for expression eof detection HIF1α and KGF,eSOD activity and MDA content detectione, and the other group for histopathologicaldetection,Results:1. HIF1α expression level of small intestine tissue turned to increase at2h in the experimental group than the NC group, reached a peak at6h and declined after24h.HIF1α expression levels were still significantly higher in the TPH group,and TPHKGroup than other treated groups. The experimental group (P <0.01); and TPHK groupHIF1α expression levels after I/R6h,12h,24h are significantly higher than the othertreated group (P <0.01).2. KGF expression level of small intestine tissue turned to increase at2h in theexperimental group than the NC group, GF expression level in reached a peak at6h,KGF expression levels reached a peak in the TC group, the Ty21a group, the TPHgroup at2h and then begin to decline, while the when KGF expression in the TPKgroup and TPHK group reached a peak at12h, then begin to decline, KGF expressionlevel in theTPK group, group TPHK is still high at the24h;and HIF1α expressionlevels in TPHK group are significantly higher than the other experimental groups (P<0.01) after I/R2h,6h,12h,24h.3. The SOD activity in intestinal tissue and plasma turned to increase at2h in theexperimental groups compared with NC group, reached a peak at6h, and then beginto decline, of SOD activity in TPH group, TPK group, and TPHK group is stillmaintained at a higher vitality at24h,; SOD activity inTPHK group are significantlyhigher than the other experimental groups (P <0.01) after I/R6h,12h,24h, with betteranti-oxidation; and SOD activity in plasma increaseing is more obviously.4. Compared with NC group, MDA content in intestinal tissue and plasma turned toincrease at2h in other groups, reached a peak at6h, then begin to decline, MDAlevels in intestinal tissue of TPHK group were significantly lower than the othergroups at12h (P <0.01); MDA content in intestinal tissue of TPK group, TPHK groupwas significantly lower than other experimental groups (P <0.01)24h, plasma MDAcontent in the group of the TPH, TPK group and TPHK group are also significantlylow other experimental group (P <0.01). And MDA level in TPHK group aresignificantly lower after I/R6h,12h,24h (P <0.01),approximate the normal group,the peroxides role of TPHK group were significantly lower than the TPH group andTPK group.5. Histopathological observation: after I/R injury, mucosa and glands in TPHK group is almost complete compared with the TC group, Ty21a group, TPH group and TPKgroup after24h of reperfusion. The crypt depth was deeper in the recovery process.There no inflammatory cells TPH group, TPK group and TPHK group, and themucosal glands, mucosa and mucosa lamina propria structure becomed nearly normalafter24h of reperfusion.Part II:Male Wistar rats were divided into normal group (NC), model control group (TC),TPHK prevention group (TPHK)3group,8/group. At radical high altitudes (3848mabove sea level Maxianshan), the stress-induced intestinal mucosal injury model inthe high altitude under hypoxic conditions were established through the superiormesenteric artery I/R (60min/60min)e. Before building the model3days, we startedto take each group drug (109pfu/only),(every other day administratio,4times), andturned to increase the rats were killed to collect specimens after7days,. Total of200μL plasma was sent to do biochemical detection in order to analyze major organsfunctional status, and expression levels of HIF1α and KGF in intestinal tissue weremeasured to analyze the target protein expression by ELISA, and SOD and MDAexpression of plasma and small intestinal tissue were analyzed oxidative stress bycolorimetric assay, and intestinal tissue was taken to observe histopathologicalchanges by HE staining.Results:1.There were the content of CK-MB and the AST, ALT,UREA and and CRE inTPHK group significantly lower than those in TC group (P <0.01), and close or lowerthan those in the normal group, which indicated that TPHK does not damage majororgans of the body;2. the HIF1α expression of intestinal tissue was significantly higher in TPHK groupthan TC group (P <0.01);3, the KGF expression of intestinal tissue was significantly higher in TPHK groupthan the TC group (P <0.01);4. in the SOD activity of plasma and small intestine was significantly higher in TPHK group than the TC group (P <0.01);5. the MDA content of plasma and small intestine was significantly lower in TPHKgroup than the TC group (P <0.01);6. The outline of small intestine mucosal and submucosal glands were clear andrelatively complete, and recess was obvious, and mucosal layer and lamina propriacombined integrity in TPHK group comprared to TC group.Conclusion:1. TPH, TPK and TPHK play a protective role on intestinal mucosal injury of ratsafter the I/R of intestine. The protective effect of TPHK is better than that of theTPH and TPK. It suggests that KGF and HIF1may have a synergistic effect.2. TPHK has protective effect on intestinal mucosal injury caused by I/R stress,particularly under hypoxic conditions in high altitude.