| It has become clear that the gut mucosa is highly susceptible to hypoxia,ischemia reperfusion injury and inflammation.Disruption of the intestinal epithelial lining is associated with the translocation of bacteria(BT)or bacteria-derived substances from the gut lumen into the blood stream.Gut dysfunction has been implicated as an initiating event in the development of septic complications and gastrointestinal tract has been defined as the "motor of system inflammatory response syndrome (SIRS)and multiple organ dysfunction syndrome(MODS)".So more and more studies are focusing on the protection of intestinal barrier during hemorrhagic shock and inflammation.Probiotics are live microbial food supplements or component of bacteria which have been shown to have beneficial effects on human health.Recent studies showed that probiotics could protect the intestinal barrier and decrease the endogenesis infection by stabilising the gut microbial environment.However,it is impossible to prevent the disruption of the intestinal barrier during severe infection,trauma and hemorrhagic shock by only using probiotics.Heme oxygenase(HO)is the rate-limiting enzyme in heme catabolism. So far,three distinct isoforms of riO(HO-1,HO-2 and HO-3)have been identified.Recently it was reported that HO-1,the inducible isoform of heme oxygenase,is involved in the fundamental protection of mucosal epithelialo cells of the rat intestine from oxidative damages that occur in sepsis.Therefore,in this study,we cloned the gene of rat HO-1 and engineered an Lactococcus lactis(a kind of probiotic)strain expressing biologically active rat HO-1.Then we evaluated the effects of recombinant Lactococcus lactis on the intestinal barrier in rats with hemorrhagic shock and endotoxemia.We aimed to finding a new way to protect the intestinal barrier during hemorrhagic shock and endotoxemia.Partâ… Expression of rat heme oxygenase-1 gene in Lactococcus LacKsObjective:To clone and express rat heine oxygenase-1(HO-1)gene in Lactococcus lactis.Methods:The rat HO-1 cDNA was amplified by reverse transcription-polymerase chain reaction(RT-PCR),and identified by DNA sequencing.The HO-1 gene digested with restriction endonuclease was ligated with plasmid pSEC.Then the recombinant plasmid was transformed into Lactococcus lactis NZ9000 by electroporation.The expression of heine oxygenase-1 gene induced by nisin was identified by SDS-PAGE and Western blot,and the activity of heine oxygenase-1 secreted by engineering Lactococcus lactis was measured by spectrophotography.Results:The relative molecular weight of expressed protein was about 32KD,and its expression level was about 7.0mg/L.The activity of heine oxygenase was 2.386 units/mg/h.Conclusions:The rat recombinant HO-1 with biological activity was expressed in the L.lactis NZ9000 strain.Partâ…¡The intestinal content of recombinant heme oxygenase-1 after intragatric administration the heme oxygenase-1-secreting Lactococcus lactis strainObjective:To invetigate the intestinal content of recombinant heme oxygenase-1 after intragatric administration the heme oxygenase-1-secreting Lactococcus lactis strain. Methods:Preparation of the heme oxygenase-1-secreting Lactococcus lactis strain and Lactococcus lactis suspension.SD rats were gavaged Lactococcus lactis strain by the different doses and the different garage times.ELISA and immunohistochemistry methods were employed to determine the content of HO-1 in ileal tissue.Results:The content of HO-1 in ileal tissue was more higher in low dose groups than high dose groups(P＜0.01);the content of HO-1 in ileal tissue was more higher in few garaging times groups than in more gavaging times(P＜0.01).Conclusions:The content of ileum HO-1 was not effectively increased simply dependently on increasing LL-HO-1 garage dose and garage times.Partâ…¢Protective effects ofLactococcus lactis producing heme oxygenase-1 on hemorrhagic shock-induced intestinal mucosal injury in ratsObjeetive:To investigate whether a genetically modified food-grade bacterium,Lactococcus lactis,could be used to deliver a biologically active heme oxygenase(HO)-1 into the rat intestine and whether this approach is successful to treat an intestinal mucosal injury induced by hemorrhagic shock.Methods:The therapeutic potential of this HO-1-secreting L.lactis was evaluated on hemorrhagic shock-induced intestinal mucosal injury in rats.After successful resuscitation,mean arterial blood pressure(MAP) was continued recorded at 5,10,20 and 30 minutes.After 1 h of resuscitation,the ileum was harvested for assessment of mucosal histological injury(Chiu's grade 0-5 by a blinded observer), myeloperoxidase(MPO)activity,bacterial translocation,tumor necrosis factor(TNF)-αand interleukin(IL)-10 concentrations.Results:Intragastric administration of LL-HO-1 strain in rats led to active delivery of HO-1 at intestinal mucosa(e.g.ileum).In LL-HO-1 groups,MAP and IL-10 were significantly increased in animals treated with LL-HO-1 as compared to LL and LL-HO-1+ZnPP(zinc protoporphyrin-â…¨,a HO-1 inhibitor)group.LL-HO-1 significantly decreased Chiu's grade,MPO activity,bacterial translocation,TNF-αin the ileum.The protective effect of LL-HO-1 could be abolished by ZnPP.Conclusions:These results suggest that intragastric administration of LL-HO-1 can reduce intestinal mucosal injury in rats with hemorrhagic shock.Partâ…£Intragastric administration with recombinant Lactococcus lactis producing Heme Oxygenase-1 prevents lipopolysaccharideinduced endotoxemia in ratsObjective:To evaluated the protective effects of intragastrically administered live Lactococcus lactis secreting bioactive HO-1 to treat intestinal mucosal injury induced by lipopolysaccharide(LPS)in rats.Methods:A model of rats with endotoxemia was reproduced in 32 healthy SD male rats.The rats were randomly divided into four groups (n=8):LL-HO-1 group,LL group,Gln group,LL-HO-1+ZnPP group. The rats received 1ml(5×1010CFU/ml).d-1LL-HO-1,1ml(5×1010CFU/ ml).d-1L.Lactis or 1g.kg-1.d-1glutamine for four days before LPS intraperitoneal injecion,and ZnPP was treatment intravenously once via tail vein before LPS treatment one hours.Twenty hours after LPS injection,the full-thickness distal ileum was harvested.The mortality, myeloperoxidase activitives,the pathologic changes,the contents of HO-1,tumor necrosis factor-αand interleukin-10 in the intestine were determined.Additionally,another rats were injected intraperitoneally with 20 mg.kg-1LPS for the study of survival and mortality.These animals were also divided into the LL-HO-1,LL,Glu and LL-HO-1+ZnPP groups(n=8 for each group),and their survivals were assessed every 12 hrs through 48 hrs.Results:Intragastric administration of recombinant L.lactis strain led to active delivery of HO-1 at the mucosa and significantly decreased morbidity and mortality of LPS-induced endotoxemia as confirmed by blinded macroscopic and microscopic inflammatory scores(Chiu's grade), myeloperoxidase(MPO)activity,mortality and TNF-αand IL-10 cytokines stimulation.This protective effect could be abolished by an HO-1 inhibitor,the zinc protoporphyrin-â…¨.Conclusions:a food-grade bacterium genetically modified to deliver bioactive HO-1 in situ exerts a protective effect against intestinal mucosal injury in rats with endotoxemia via modulation of the immune system. This novel approach may be beneficial for the maintenance of intestinal barrier and anti-inflammatory response of the lower intestine.Conclusions1.The rat recombinant HO-1 with biological activity was expressed in the L.lactis NZ9000 strain.2.The content of ileum HO-1 was not effectively increased simply dependently on increasing LL-HO-1 gavage dose and gavage times.3.Intragastric administration of LL-HO-1 can reduce intestinal mucosal injury in rats with hemorrhagic shock.4.Intragastric administration of LL-HO-1 exerts a protective effect against intestinal mucosal injury in rats with endotoxemia via modulation of the immune system.
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