The Protective Effect Of Carbachol On Gastrointestinal Dysfunction

Objective:By vitro cell models, animal experiments and preliminary clinical observations,we study the protective effect of injury after intestinal I/R injury and bowel dysfunction; exploring effective dose and the possible mechanisms. The objective is for providing experimental evidence and technical methods and feasibility analysis on Carbachol of the prevention and treatment of invasive burn shock resuscitation after intestinal I/R injury and MODS clinical studiesMethods:The first part, Carbachol on intestinal epithelial cells and intestinal macrophages proinflammatory genes and oxygen radical production after stimulation of oxidative stress:Experiments were randomly divided into those①Experimental group:rats IEC-6+carbachol+H2O2,observing the expression of cytokines and macrophages and smooth muscle cells releasing reactive oxygen species after carbachol about H2O2stimulation of intestinal epithelial cells;②Control group I:rat IEC-6+saline,observing intestinal epithelial cells and smooth muscle macrophages to release reactive oxygen species and expression levels of cytokines without H2O2stimulation of rat;③Control group II:rats IEC-6+H2O2,observing macrophages and smooth muscle cells release reactive oxygen species and expression of cytokines level after H2O2after stimulation of rat intestinal epithelial cells;④Control group III: rat IEC-6cells+carbachol observing smooth muscle macrophage cells release reactive oxygen species and the expression of cytokines without H2O2stimulation of carbachol on rat intestinal epithelial cells Then testing the content of TNF-a,IL-6,MDA and NO of the IEC each group;detecting expression changes of TNF-amRNA and IL-6mRNA each group by RT-QPCR.The second part,Carbachol on the effects of intestinal function,intestinal blood flow,intestinal local and systemic inflammatory in intestinal I/R injury animal.The animals were randomly divided into experimental group Group l:the carbachol (IM)+intestinal part of the I/R injury;experimental group2groups:of carbachol (oral)+intestinal part of the I/R injury;experimental group3:the carbachol (injection of intestinal bag)+intestinal part of the I/R injury;control group1:the carbachol (oral or intramuscular injection or intestinal bag)+sham operation;control group2:saline (oral or intramuscular injection or intestinal bag)+intestinal part of the I/R injury.Respectively,we detected DAO activity in the intestinal mucosa,TNF-a and the content of MDA and TNF-a mRNA expression at different time points;measuring intestinal mucosal blood flow change by doppler blood flow meter;And intestinal absorption and emptying ability,histopathological examination of the intestinal mucosa.The third part,Carbachol on the intestinal function intestinal blood flow,intestinal local and systemic inflammatory effects of intestinal I/R injury in animal.Selecting extensive burns (>30%) TBSA Ⅲ°rea>10%) of100patients.Inclusion criteria:not taking fluid resuscitation and/or despite fluid resuscitation,within4hours after injury,but urine output<40ml/h and/or PHI<7.1.After the consent of the patient’s family,They were divided into those:①Carbachol treatment group (n=50):In addition to burns after delayed resuscitation delayed resuscitation conventional treatment given immediately Kabbah choline1.0mg/day,administered through the tube or gastric mucosal pH pipe in for5days;②Conventional treatment group (n=50):Do not use the carbachol,By delaying resuscitation burned conventional treatment methods (including conventional rehydration,dopamine and oxygen free radical scavenger). After entering the program,The mediators of inflammation,intestinal analysis,organ function parameters and peripheral blood CD14+monocyte HLA-DR expression rate, peripheral blood lymphocyte apoptosis were measured after4hours,24hours and3days,5days.Results:1、Carbachol studies have shown that oxidative stress stimulation of intestinal epithelial cells and intestinal the macrophage proinflammatory genes and oxygen radical production:TNF-a, IL-6, MDA and NO content; and TNF-A mRNAand IL-6mRNA expression level of control group III were significantly higher than the other groups (P<0.01);experimental group was compared to control group III,TNF-a, IL-6, MDA and NO content; and the TNF-amRNA and IL-6mRNA expression levels were significantly decreased (P<0.01).2、Carbachol on intestinal I/R injury in animal intestinal function, intestinal blood flow, intestinal local and systemic inflammatory impact study:The levels of inflammatory factors, permeability, organ dysfunction of control group2were significantly higher than the other group (P<0.01); mucosal blood flow, absorption and discharge function were significantly lower than those of the other groups (P<0.01); experimental group was compared to the control group, the indicators significantly improved (P <0.01). In addition, the comparison among the three experimental groups,The indicators of experimental group3was better than that of the other two groups (P <0.05).3、Carbachol treatment of burns with delayed fluid resuscitation after intestinal I/R injury and organ dysfunction preliminary clinical studies:the carbachol treatment group was compared to the conventional treatment group, inflammatory factor levels were significantly lower (P<0.01),intestinal and organfunction parameters were significantly improved (P＜0.01), the immune indicators also were improved significantly (P<0.01).Conclusion:1、Carbachol can reduce oxidative stress injury of the IEC in vitro; The mechanism of carbachol reducing the IEC oxidative stress injury may be:Reducing the production of IL-6、TNF-a、MDA and NO when oxidative stress in intestinal epithelial cells.2、Carbachol can reduce intestinal ischemia-reperfusion injury; the mechanism of carbachol alleviating intestinal ischemia and reperfusion injury may be:to reduce the production of inflammatory cytokines in the intestinal ischemia and reperfusion, increased intestinal ischemia-reperfusion mucosal blood flow;enhanced intestinal absorption and discharge capacity.3、Carbachol can improve mucosal blood flow, improve the immune system, so as to reduce delayed resuscitation burned patients after intestinal I/R injury, protect their organ by reducing inflammation.