Study On The Effects Of Carbachol On The Postoperative Intra-abdominal Adhesions And Its Mechanism In Rat

Object:1.To observe the effect of carbachol on a rat model of postoperative intra-abdominal adhesions;2.To study the effects of carbachol on angiogenesis of abdominal adhesion tissues and expression of VEGF of microvascular endothelial cell,and explored their mechanism related to the cholinergic anti-inflammatory pathway.Methods:1.Animal experiment:male Wistar rats were randomly assigned into eight groups:sham operated group(S group,n=8),abdominal adhesions group (A group,n=14),carbachol group(C group,n=14,60μg/kg.ml),nicotine group(N group,n=14,lmg/kg.ml),normal sodium group(NS group,n=8,lml/kg),α-Bgt group(αgroup,n=12,lμg/kg),carbachol+α-Bgt group(C/αgroup,n=12), nicotine+a-Bgt group(N/αgroup,n=12).Animal model of abdominal adhesion was produced by scraping on appendix of cecum,followed by a silicone path fixed on the lateral abdominal wall.Ten days after surgery,rats were sacrificed respectively,and the adhesion degree was evaluated by Chiang scale.Adhesion tissues were taken to observe histopathological changes and quantitate vessel density.The changes of VEGF were detected by immunohistochemistry method.2.Cell experiment:Rat pulmonary microvascular endothelial cells(PMVECs) cultured by tissue-sticking method were divided into seven groups:TNF-a group (T group,10ng/ml);carbachol+TNF-αgroup(five subset:Cl group,2mmol/l; C2 group,0.2mmol/l;C3 group,0.02mmol/l;C4 group,2μmol/l;C5 group, 0.2μmol/l;TNF-α,10ng/ml);nicotine+TNF-αgroup,(N group,nicotine, 0.2mmol/l,TNF-α,lOng/ml);α-Bgt+TNF-a group,(αgroup,α-Bgt,6μg/ml; TNF-α,10ng/ml);a-Bgt(6μg/ml)+carbachol(0.2mmol/l)+TNF-α(l0ng/ml) group,(C2/αgroup);α-Bgt(6μg/ml)+nicotine(0.2mmol/l)+TNF-α(10ng/ml) group,(N/αgroup);cell control group(B group,add culture medium).Cells were identified with CD31 and factorⅧby immunocytochemistry and cell morphology observed by inverted phase contrast microscope.Cell proliferations of rat PMVECs were determined by MTT method and VEGF contents detected by ELISA.Results 1.All fourteen rats in abdominal adhesion group developed obvious intra-abdominal adhesions with adhesion scores 6 to 12,indicating that an animal model of abdominal adhesion was successfully established.2.Cultured cells were found fusiform,slabstone arranged,monolayer growing and stained positive with factorⅧand CD31 antibody,indicating that the collected cells were rat PMVECs.3.Scores of adhesion,angiogenesis and expression of VEGF of adhesion tissues were significantly decreased in carbachol and nicotine treated group compared with those of adhesion group.a-Bgt treatment markedly reduced the inhibitive effect of carbachol and nicotine on abdominal adhesion.4. The rates of rat PMVECs proliferation and contents of VEGF stimulated by TNF-a were significantly decreased after treatments of carbachol and nicotine, and these inhibitive effects of carbachol and nicotine were markedly inverted through inhibition of rat PMVECsα7nAChR.Conclusion Carbachol had significant inhibitive effect on postoperative abdominal adhesion with its mechanism related to the decrease of TNF and VEGF, and the inhibition of angiogenesis by activation of cholinergicα7nAChR pathway.