| Objective:Intestinal ischemia-reperfusion(I/R)injury often occurs in the process of hemorrhagic shock,small bowel transplantation and severe trauma.It can cause the decrease of small bowel contractility,the increase of microvascular permeability and the decrease of intestinal mucosal barrier function,which can lead to multiple organ failure.Orexins is a brain-gut peptide mainly distributed in the hypothalamus,but its receptors are widely distributed in central and peripheral tissues,including various parts of the gastrointestinal system,such as intermuscular plexus,submucosal plexus,gastrointestinal mucosa and smooth muscle.Orexins are mainly involved in the regulation of feeding,energy balance,cardiovascular function and stress.However,the role of orexins in intestinal ischemia-reperfusion injury,especially in the protection of intestinal motor function and intestinal nervous system after intestinal ischemia has not been reported.The aim of this study was to investigate the protective effect and the potential mechanism of orexin-A intravenous infusion on intestinal contractile activity,feeding and intestinal neurons before intestinal ischemia-reperfusion through the establishment of intestinal ischemia-reperfusion model.Methods:The jejunal smooth muscle strips of male Wistar rats were prepared and treated with10~-88 M,10~-77 M and 10~-66 M orexin-A respectively to observe their effects on contraction of jejunal muscle strips;Jejunal strips were incubated with SB-334867,nitric oxide synthase neuron type(nNOS),selective inhibitor N'-nitro-L-arginine(L-NNA),M receptor blocker atropine,or Na~+channel blocker tetrodotoxin(TTX),respectively,to observe the effect of orexin-A on the contractile effect of jejunal strips,and analyze the potential mechanism of this effect.The rat model of intestinal ischemia-reperfusion was established by clamping the superior mesenteric artery for 45 minutes and reperfusion for 90 minutes;Orexin-A(500pmol/Kg/min)was injected into the tail vein of rats for 30 minutes before intestinal ischemia-reperfusion to observe the effect of orexin-A on contractile activity of isolated jejunal smooth muscle strips;Orexin-A was perfused into tail vein for 30 minutes before intestinal ischemia-reperfusion to observe the intestinal propulsion rate and feeding changes after I/R in rats;The effects of orexin-A pretreatment on morphology and apoptosis of intestinal mucosal epithelial cells and neuronal expression of intestinal myenteric plexus in I/R rats were observed by fluorescence immunohistochemistry or TUNEL;Western blot and Elisa were used to detect the changes of apoptosis-related proteins(Bcl-2,Bax and Caspase-3)and peroxidation or inflammatory response-related factors(malondialdehyde,glutathione and myeloperoxidase).Results:Orexin-A could promote contraction of jejunal strips in a dose-dependent manner in normal rats(P<0.05-0.01).Preincubation of SB-334867 could completely block the contraction-promoting effect of orexin-A(P<0.05).If L-NNA(P<0.05),atropine(P<0.05)or TTX(P<0.05)were incubated in advance,orexin-A-induced contraction of muscle strips could be partially inhibited.In intestinal I/R model rats,the contraction frequency of jejunal smooth muscle strips was significantly decreased(P<0.05),and the area under the contraction curve was significantly reduced(P<0.05).The maximal contraction tension of jejunal smooth muscle strips was significantly decreased(P<0.05)when stimulated by acetylcholine(Ach).If orexin-A was infused into caudal vein for 30 minutes before intestinal ischemia-reperfusion,the contraction weakening of jejunal smooth muscle strips was significantly improved(P<0.05-0.01).Compared with sham-operated group,the 24 h cumulative intake of intestinal I/R rats decreased significantly from 1 to 7 days after operation(P<0.05-0.01),on the 5th day after operation,the propulsive rate of small intestinal carbon powder was also significantly decreased(P<0.01);If orexin-A infused into tail vein was used before intestinal ischemia-reperfusion,the food intake of I/R rats began to increase significantly on the 3rd day after operation compared with NS control group(P<0.05),and the rate of small intestinal carbon propulsion was also significantly increased(P<0.05).HE staining showed that intestinal villus was exfoliated,a large number of inflammatory cells infiltrated and capillaries dilated and congested in intestinal I/R rats.If rats were pretreated with orexin-A,the damage of jejunal mucosal epithelial cells was alleviated,and inflammatory cells infiltrated less.Neuron-specific enolase(NSE)fluorescence immunohistochemical staining showed that the number of NSE immunoreactive neurons in ischemia-reperfusion group(NS+I/R)was significantly decreased compared with NS+sham operation group(P<0.05).The number of NSE immunoreactive neurons in rats was significantly increased after orexin-A pretreatment(orexin-A+I/R)(P<0.05).Fluorescence immunohistochemical study of acetylcholinesterase(ChAT)showed that ChAT immunoreactive neurons interlaced with their nerve fibers under microscope.Compared with NS+sham operation group,the area of ChAT immunoreactive nerve and IOD of myenteric plexus in NS+I/R group were significantly decreased(P<0.05);Compared with NS+I/R group,the area of ChAT immunoreactive nerve(P<0.05)and IOD in orexin-A intervention group(orexin-A+I/R)increased significantly(P<0.05).Western blot results showed that compared with sham-operated rats,the expression of cholinergic receptor 3(M3)and Bcl-2 in jejunum of intestinal I/R rats decreased significantly(P<0.05),while the expression of Bax and Caspase-3 increased significantly(P<0.05);The expression of M3 and Bcl-2 in jejunum of rats increased significantly(P<0.05),while the expression of Bax and Caspase-3 decreased significantly(P<0.05)if the rats caudal vein were perfused with orexin-A before intestinal ischemia and reperfusion(P<0.05).The results of ELISA showed that compared with NS+sham operation group,the contents of MDA(P<0.05)and MPO(P<0.05)in jejunum tissue of rats in NS+I/R group increased significantly,while the contents of GSH decreased significantly(P<0.05);Compared with NS+I/R group,the contents of MDA and MPO in jejunum of rats in Orexin-A+I/R group decreased significantly(P<0.05),and the contents of GSH increased significantly(P<0.05).Conclusion:Orexin-A can promote the contraction of small intestinal muscle strips in a dose-dependent manner,which may be mediated by orexin-1 receptor(OX-1R)pathway,and NOS,Na~+channel and M receptor signaling pathway may also participate in the regulation of this process.Peripheral administration of orexin-A can improve the increase of intestinal oxygen free radicals,activation of neutrophils,apoptosis of intestinal epithelial cells and decrease of excitatory motor neurons in myenteric plexus induced by intestinal ischemia-reperfusion injury.Furthermore,it can promote small intestinal peristalsis and increase the intake of food in rats,which is conducive to promoting the rehabilitation of rats. |
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