| Objective Through the observation of tight junction protein Claudin-3,ZO-1 and Occludin in hemorrhagic shock(hemorrhagic shock HS)regulation changes and expression of CORM-2 in rat ileum in the study of CORM-2 on hemorrhagic shock in rat ileum of tight junction proteins Claudin-3,ZO-1 and Occludin and the role of internal mechanism for CORM-2 used in clinic treatment of intestinal mucosal barrier to provide shock treatment strategies and new theoretical basis for injury.Method(1)60 male Sprague-Dawley rats were randomly divided into normal control group(group C,n=15),group HS(group B,n=15),CORM-2[A group(n=15,5mg/kg)],drug group,iCORM-2[D group(n=15,5mg/kg)] inactivation group,45 minutes before operation,intraperitoneal injection was given drug intervention.(2)The model of hemorrhagic shock was prepared and divided into groups: after the carotid artery puncture was completed,the average arterial pressure MAP(mean arterial pressure,MAP)stabilized 10 min began to prepare the model,and the shock state between the experimental rat MAP35-40 mmHg and 120 min was maintained from the carotid blood release or reinjection(MAP>40mmHg blood release,<35mmHg reinjection).Group B(group HS)was injected with the same dose of DMSO(5mg/kg)in the same dose of saline solution before the operation of 45 min to prepare the hemorrhagic shock model;group A(group CORM-2)before the operation of 45 min intraperitoneal injection intervention drug CORM-2(5mg/kg)to prepare the hemorrhagic shock model;D group(iCORM-2 group)before the operation of intraperitoneal injection intervention drugs,preparation In the model of hemorrhagic shock,iCORM-2 was the inactivation of CORM-2;group C(normal control group)was injected with the same dose ofDMSO(5ml.Kg-1)in the same dose of normal saline before operation,only the carotid artery was placed and the shock model was not prepared.(3)After 24 h,the rats in each group were killed.After collecting blood from inferior vena cava,the plasma and serum were centrifugally separated.The levels of plasma D lactic acid and serum TNF-TNF-were detected by ELISA.The operation was carried out according to the instructions of the kit.(4)Paraffin embedded tissue from Appendix 10 cm ileum,using HE staining to observe the pathological changes of the intestinal mucosa under light microscope,including histological examination,mucosal thickness and villus height measurement and intestinal mucosal injury score(Chiu s);intestinal epithelial cell ultrastructure and the changes of tight junction were observed by transmission electron microscopy.(5)The distribution and expression of close connexin Claudin-3,ZO-1 and Occludin in intestinal tissue were detected by immunohistochemistry.(6)The expression of close connexin Claudin-3,ZO-1 and Occludin in intestinal tissue was detected by Western Blot.Result1.Pathological changes of ileum in four groups of ratsNaked eye observation: there was no obvious abnormality in ileum tissue in group C;in group B and group D,the ileum tissue was swelling and the color was dark red;the ileum tissue in group A was slightly swollen and the color was basically restored to normal,which was significantly less than that in group B and D.Light microscopic observation: in group C,the structure of intestinal mucosa villi under complete and clear,neatly arranged and close the light microscope,the glands are also very complete;B group and D group significantly change the structure of ileal villi of intestinal mucosa edema,thickening and necrosis,glands cell necrosis and dissolution;comparison of ileal tissue injury compared with B group,A group and D group was significantly reduced.B group and D group compared with C group,the intestinal mucosa thickness and villus height significantly reduced,calculated Chiu,s scores increased significantly(P<0.01);A group andB group and D group results,intestinal mucosa thickness and villus height increased,the calculated Chiu,s score was significantly lower(P<0.01).observation under electron microscope:The microvilli in the ileum mucosa of group C were rich in microvilli and the activity of goblet cells was strong and the intercellular connection was close.The microvilli structure of group B and D was destroyed and partially disappeared,the goblet cells were vacuolated,the ileum epithelial cells were loose,disorderly and closed,and the part of the ileum tissue in group A had been restored to normal structure,The secretion activity of goblet cells was enhanced,and the tight junctions between cells were basically intact.Compared with group B and group D,the injury was significantly reduced.2.Changes of plasma D lactic acid and serum TNF-α in four groups of ratsCompared with group C,plasma D lactate increased significantly in group B and group D(P<0.01),and serum TNF-α level increased significantly(P<0.01).Compared with B group and D group,plasma level of D lactate decreased significantly in group A and sserum TNF-αalso showed a downward trend.3.Changes in the expression of tightly connected structural proteins in the ileum of four groups of ratsThe intestinal barrier injury induced by hemorrhagic shock can reduce the expression of Claudin-3,ZO-1 and Occludin protein in intestinal epithelial cells(P<0.01).Pretreatment with CORM-2 can increase the expression of Claudin-3,ZO-1 and Occludin protein(P<0.01).Conclusion CORM-2 can protect the intestinal barrier of rats with hemorrhagic shock by increasing the expression of tight connexin Claudin-3,ZO-1 and Occludin in the ileum epithelial cells of rats with hemorrhagic shock. |
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