| Objective:Intestinal ischemia-reperfusion(I/R)injury is a common clinical disease,which can not only cause local intestinal tissue damage,but also damage the distal organs.Fucoidan is a kind of water-soluble heteropolysaccharide from marine brown algae and invertebrates.The current research shows that fucoidan has protective effect on the ischemia-reperfusion injury of heart,liver and brain.However,the effect of fucoidan on intestinal or brain injury induced by intestinal ischemia-reperfusion has not been reported.The purpose of this study was to investigate the protective effect and potential mechanism of fucoidan pretreatment on ileum and hippocampus injury in rats with intestinal I/R model.Methods:1.Experimental group: 240 adult male Wistar rats were randomly divided into 3 groups(n= 80):(1)For the sham group,normal saline(NS)was administered(2 ml/kg/day)by gavage for 30 days before operation,and abdominal operation was performed on the 31 st day to separate the superior mesenteric artery without clamping the superior mesenteric artery;(2)For the I/R group,NS was administered by gavage for 30 days(2 ml/kg/day)before the operation.On the 31 st day,I/R operation was performed,i.e.the superior mesenteric artery was occluded with noninvasive vascular clamp for 1.5 h,and the reperfusion lasted for 6h;(3)For the Fucoidan+I/R group,fucoidan(200 mg/kg/day)was administered by gavage for 30 days before ischemia-reperfusion,and the superior mesenteric artery was clamped for 1.5 h and reperfusion for 6 h on the 31 st day.2.Hematoxylin eosin(HE)staining was used to observe the pathomorphological changes of ileum and hippocampus in intestinal I/R rats,and TUNEL staining was used to observe the effect of fucoidan on apoptosis of ileum epithelial cells and hippocampus neurons in I/R rats;3.To prepare ileal smooth muscle strips and observe the effect of fucoidan on contractile activity of isolated ileal smooth muscle strips in intestinal I/R rats;4.Single-cell discharge recording was used to observe the effects of fucoidan on the activity of inhibitory gamma aminobutyric acid(GABA)sensitive neurons and excitatoryglutamate(Glu)sensitive neurons in the hippocampal CA1 region of I/R rats.5.Enzyme-linked immunosorbent assay(ELISA)was used to observe the effect of fucoidan on inflammatory factors(tumor necrosis factor-?(TNF-?),interleukin-6(IL-6)and interleukin-1 ?(IL-1 ?)in ileum and hippocampus of intestinal I/R rats.6.Biochemical analysis used to observe the effect of fucoidan on oxidative stress index malondialdehyde(MDA),glutathione(GSH)or superoxide dismutase(SOD)in ileum and hippocampus of intestinal I/R rats7.Western blot was used to observe the effects of fucoidan on the expression of apoptosis related proteins(Cleaved Caspase-3,Bcl-2,Bax),mitogen activated protein kinase signal pathway related proteins(p38MAPK,JNK,ERK)and phosphorylated JNK(p-JNK),ERK(p-ERK)and p38 MAPK(p-p38MAPK)proteins in ileum and hippocampus of intestinal I/R rats.Results:1.The effect of fucoidan on the morphology in ileum,cortex or hippocampus of intestinal I/R rats showed that the villi of intestinal mucosa of rats in sham group were arranged orderly without obvious damage.In the I/R group,the intestinal mucosa was disorganized with a large number of villi falling off,accompanied by serious damage of glands and infiltration of a large number of inflammatory cells.If the rats were pretreated with fucoidan,the ileal mucosa was less damaged,the infiltration of villous inflammatory cells and the denudation of villi was reduced.In the center,the neurons in the cerebral cortex and hippocampal CA1 area of intestinal I/R rats were irregularly arranged,nuclear pyknosis and hyperchromatism,vacuolation of cytoplasm,unclear boundary between nuclear membrane and nucleolar were found under the microscope.However,the neurons in the cerebral cortex and hippocampal CA1 area of intestinal I/R rats pretreated by fucoidan were relatively complete,and there were fewer pyknosis and necrosis neurons(P < 0.01).2.The effect of fucoidan on the contractility in intestinal muscle strips of intestinal I/R rats showed that compared with sham group,the spontaneous contraction frequency of ileal smooth muscle strip(P < 0.01)and the area under the spontaneous contraction curve(P < 0.01)in I/R group significantly decreased,and the contraction response of intestinal smooth muscle to acetylcholine(Ach)(P < 0.01)significantly decreased.Compared with the I/R group,the spontaneous contraction frequency and area under the spontaneous contraction curve of rats in Fucoidan+I/R group were significantly increased(P < 0.01),and the response to ACh was significantly increased(P < 0.01).But still significantly lower than sham group(P < 0.01).3.The effect of fucoidan on the discharge activity of neurons in hippocampal CA1 area of intestinal I/R rats showed that the discharge frequency of GABA sensitive neurons or glutamate sensitive neurons in I/R group was significantly higher than that in sham group during intestinal ischemia(P < 0.01).Although there was a significant decrease during intestinal perfusion,it was still higher than that in sham group(P < 0.01).Compared with the I/R group,the GABA-N discharge frequency in Fucoidan+I/R was significantly increased(P < 0.01)and the Glu-N discharge frequency was significantly decreased(P < 0.01)during the whole intestinal ischemia or reperfusion period.However,the discharge frequency of in GABA-N or Glu-N in Fucoidan+I/R group was significantly higher than that in sham group(P < 0.01).4.The effect of fucoidan on inflammatory factors in ileum and hippocampus of intestinal I/R rats showed that compared with the sham group,the levels of TNF-?(P <0.01),IL-6(P < 0.01)and IL-1 ?(P < 0.01)in the ileum and hippocampus of rats in I/R group were significantly higher than those in the sham group.Compared with the I/R group,the levels of TNF-?(P < 0.05 or P < 0.01),IL-6(P < 0.01),IL-1 ?(P < 0.01)in the ileum and hippocampus of rats in the Fucoidan+ I/R group were significantly decreased,but still significantly higher than those in the sham operated group(P < 0.05 or P < 0.01)5.The effect of fucoidan on the oxidative stress index in ileum and hippocampus of intestinal I/R rats showed that compared with Sham group,the content of MDA in intestine(P < 0.01)and hippocampus(P < 0.05)of rats in I/R group significantly increased,but GSH content and SOD activity significantly decreased(P < 0.01).Pretreatment with fucoidan significantly inhibited MDA content(P < 0.05 or P < 0.01)in intestine and hippocampus,increased GSH(P < 0.05 or P < 0.01)content and SOD(P <0.01)activity.Compared with Sham group,the content MDA content in intestine and hippocampus of rats in Fucoidan+I/R group significantly increased(P < 0.05 or P < 0.01),but the level of GSH(P < 0.05 or P < 0.01)and SOD(P < 0.01)was significantly decreased.6.The effect of fucoidan on apoptosis of ileum and hippocampus cells of intestinal I/R rats was studied,The results of TUNEL staining showed that compared with the sham group,the TUNEL positive cell index in the ileum mucosa of rats in I/R group increased significantly(P < 0.01),If the rats were pretreated with fucoidan,the TUNEL positive cell index of ileal mucosa and hippocampus in rats was significantly decreased(P < 0.01),but still significantly higher than that in the sham group(P < 0.01).In the center,TUNEL positive cell index in hippocampus of rats in I/R group was significantly higher than that in sham group(P < 0.01),while fucoidan pretreatment could significantly reduce TUNEL positive cell index in hippocampus(P < 0.01),but still significantly higher than that insham group(P < 0.05).The results of western blot showed that compared with the sham group,the expression of Bax(P < 0.01)and cleaved caspase-3(P < 0.05)in ileums and hippocampus of rats in I/R group significantly increased,while the expression of Bcl-2(P< 0.01)protein decreased.Fucoidan treatment significantly decreased Bax(P < 0.05 or P< 0.01)and cleaved caspase-3 expression(P < 0.05 or P < 0.01),and increased the Bcl-2(P < 0.05 or P < 0.01)expression following intestinal I/R.However,the expression of Bax(P < 0.05)and cleaved caspase-3(P < 0.01)in ileum and hippocampus of rats in Fucoidan+I/R group were still higher than that in sham group,and the expression of Bcl-2(P < 0.01)was still lower than that in sham group(P < 0.01).7.The effect of fucoidan on the expression of MAPK signal pathway related proteins in the ileum and hippocampus of intestinal I/R rats showed that the levels of p-ERK(P <0.01),p-JNK(P < 0.01)and p-p38MAPK(P < 0.01)in the ileum and hippocampus of rats in I/R group were significantly higher than those in sham operation group.However,compared with I/R group,fucoidan pretreatment significantly reduced p-p38 MAPK,p-JNK,and p-ERK levels(P < 0.05 or P < 0.01)in the ileum and hippocampus,but still significantly higher than that in sham operation group(P < 0.05).Conclusion:Fucoidan has protective effect on intestine and brain tissue of intestinal ischemia-reperfusion rats by inhibiting oxidative stress inflammatory reaction and apoptosi,which may be related to MAPK signaling pathway. |
PDF Full Text Request