Effects And Mechanism Of Methane-saline Through Upregulation Of HO-1 Expression In Intestinal Injury Induced By CLP-induced Sepsis In Rats

Objective: sepsis is a systemic inflammatory response caused by severe infection,often accompanied by systemic organ dysfunction such as acute lung injury,coagulation dysfunction,circulatory failure,and intestinal barrier disruption.Sepsis release a large number of inflammatory mediators to cause damage to the intestinal barrier,a large number of bacteria translocation into the blood,causing damage to other organs,eventually causing multiple organ failure or even death.Methane is the simplest kind of alkane,and it may be the most abundant organic material on Earth.In the natural environment,methane is degraded by organisms in an oxygen-deficient environment and is widely distributed as a harmful gas in the atmosphere.One of the ways in which methane is produced in the human body is damage to mitochondria under hypoxic conditions.In recent years,biogas methane studies have shown that damaged mitochondria exert resistance against oxidative stress injury under the treatment of methane in a mammal model of ischemia,reperfusion injury such as nerves,mesenteries,liver,myocardium,and diabetic retinopathy models.Body protection.Nuclear factor E2 related factor 2(Nrf2)in the Nrf2/HO-1 signaling pathway and the heme oxygenase-1(HO-1)induced by it can counteract ROS-induced oxidation Injury damage.The role of Methane saline(MS)in reducing intestinal injury in sepsis rats is not fully understood.This study intends to investigate the effect of MS treatment on the expression of HO-1 and its regulatory targets in the intestinal tissue of sepsis rats,and to provide a reference for clarifying the therapeutic mechanism.Methods: 160 adult male Sprague-Dawley rats were housed 8 to 10 weeks old and weighed 220 to 280 g.The random number method was divided into 5 groups: sham group,sepsis group(sepsis),sepsis +MS group(sepsis+MS),sepsis+ZnPPIX group(sepsis+ZnPPIX),sepsis+MS+ZnPPIX group(sepsis+MS+ZnPPIX).The cecal ligation and puncture(CLP)method establishes a sepsis model.10% MS treatment 10ml/kg was given intraperitoneally to the MS group 0.5h,12 h and 24 h after the sham operation or CLP respectively.The other groups were given normal saline.30mg/kg ZnPPIX was given by intraperitoneal injection 1 hour before surgery to the rats of Sepsis+ZnPPIX group and sepsis+MS+ZnPPIX group.Part I: Randomly divide 100 rats into the above 5 groups(n=20)and record the 1-day,2-,3-,5-,and 7-day survival rates in each group.A further 60 rats were randomly divided into the above 5 groups(n=12).Rats were sacrificed at 6 h and 18 h after modeling.The levels of SOD and MPO in rat intestine tissues were measured at each time point.Tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)were detected in serum and intestinal tissues.Interleukin 10(IL-10)levels.The histopathological changes of ileum were observed at 6 and 18 h after operation in each group,and the intestinal permeability was measured(FITC fluorescence labeling method).The second part: Sixty rats in the upper group were sacrificed 6 and 18 h after surgery.a Western blot was used to observe the expression of Occludin in the intestinal mucosa of rats in each group at each time point,and the expression of HO-1 and Nrf2 in intestinal tissues.Expression;The expression of tight junction protein,HO-1 mRNA and Nrf2 mRNA in rat intestine was determined by real-time fluorescence quantitative polymerase chain reaction(qPCR).Results: part 1: The survival rate of the saline-treated group was 100%.In the sepsis group,the survival rate of rats decreased from 85% at 24 h to 40% at 48 h;MS treatment improved the 7-day survival rate of sepsis rats(P<0.05);ZnPPIX injection group The survival rate of rats decreased(P<0.05).The intestinal histopathological scores and intestinal permeability of septic rats were higher than those of shamoperated rats(P<0.05).MS treatment significantly relieved these changes(P<0.05).Intestinal tissue injury increased after ZnPPIX injection(P< 0.05);In addition,MS treatment increased sepsis serum and intestinal tissue IL-10 and SOD levels,decreased MPO levels and TNF-?,IL-1? expression levels(P <0.05),injection of ZnPPIX to TNF-?,The levels of IL-1? and MPO increased,and the levels of SOD and IL-10 decreased(P<0.05).Part 2: There was significant difference between the sham group and other group in expressions of HO-1 and Nrf2 protein and m RNA(P <0.05),HO-1 activity was increased(P <0.05)Saline treatment could further up-regulate HO-1 and Nrf2 expression(P <0.05)and HO-1 activity(P <0.05)in septic rats.Compared with the sham operation group,the expression of Occludin in small intestine mucosa of other groups decreased significantly(P <0.05)at 6 h and 18 h after operation,and the concentration of tight junction protein(P <0.05),ZnPPIX inhibition HO-1 expression decreased Occludin activity,but also reversed the treatment of intestinal injury in septic rats(P <0.05).Conclusion: Methane saline can increase the activity of SOD in the mucosa of rats,decrease the release of proinflammatory cytokines and infiltration of MPO,increase the activity of Occludin in small intestine mucosa,reduce the damage of intestinal mucosa and reduce the intestinal permeability Sexual and intestinal inflammation,which induces the expression of HO-1 through Nrf2,thereby reducing intestinal damage.