消胀贴膏外敷对小鼠肝硬化腹水的消退作用及对腹腔血管通透性的影响

目的评价消胀贴膏外敷对实验性肝硬化腹水的药效作用,探讨其影响腹腔血管通透性的作用机制。方法以二甲基亚硝胺(DMN)腹腔注射,药效评价实验模型动物随机分为模型对照组、安慰剂对照组、消胀贴膏低、中、高剂量组共5组,机制实验分为模型对照组、消胀贴膏中剂量组共2组,均设正常对照组。消胀贴膏组脐部外敷消胀贴膏,安慰剂对照组脐部外敷空白贴膏,每日1贴,共7d,模型对照组不敷贴任何药物。称量或测量模型动物的腹水量、腹围、体重,HE与天狼星红染色观察肝组织炎症与纤维化,生化检测血清ALT活性和Alb、TBil与一氧化氮(NO)含量,蛋白质印迹法分析肝组织血管内皮生长因子(VEGF)表达水平,通过测定小鼠腹腔积液及肝组织中渗漏伊文氏蓝(EB)含量的方法检测动物腹腔血管通透性。结果敷药前,各组模型动物的腹围、体重等基线资料一致。给药结束,与正常组比较,模型对照组小鼠的体重、腹围、腹水、腹水体重比等明显增加,腹腔积液及肝组织中渗漏EB含量明显增多,血清NO浓度及肝组织中VEGF蛋白表达明显增加。与模型对照组比较,安慰剂对照组的腹水量、腹围与体重等无明显改变。与安慰剂对照组比较,消胀贴膏中、高剂量组的腹水量、腹水体重比值明显下降,高剂量组的腹围、体重也明显下降;高剂量组与低剂量组比较,腹围、体重明显下降。各剂量消胀贴膏对肝功能及肝组织病理均无明显影响。与模型对照组比较,中剂量消胀贴膏组小鼠腹腔积液中渗漏EB含量降低,血清NO浓度及肝组织中VEGF蛋白表达明显减少。结论消胀贴膏明显减少肝硬化腹水模型动物的腹水量,中剂量(0.25 cm2消胀贴膏,652.9 mg生药/kg小鼠)开始发挥药效,其部分机制在于下调肝硬化腹水模型动物的肝组织VEGF表达、降低血清NO含量,从而抑制其异常增强的腹腔血管通透性。

Effect of Xiaozhang Tie on cirrhotic ascites and peritoneal vascular permeability in mice

Objectives To evaluate the effects of external application of Xiaozhang Tie on ascites and the mechanism related to vascular permeability of peritoneum and liver in cirrhotic mice.Methods Dimethylnitrosamine was injected intraperitoneally for four weeks to induce liver cirrhosis in mice.After ascites were identified by ultrasound,the animals were randomly divided into five groups(n=10 each): model(untreated control);placebo(plaster matrix only control),low-dose Xiaozhang Tie(0.125 cm2 plaster);mid-dose Xiaozhang Tie(0.25 cm2 plaster);and high-dose Xiaozhang Tie(0.5 cm2 plaster).Uninduced mice(n=10) were used as normal controls.Xiaozhang Tie(652.9 mg crude drug/kg weight) was applied by external application to the umbilical once daily for one week.The amount of ascites,abdominal circumference,and body weight were recorded.Inflammation and fibrosis of liver tissue were observed by hemotoxylin-eosin and Sirius rose staining.Serum alanine aminotransferase(ALT),albumin,total bilirubin(TBil),and nitric oxide(NO) were tested.Liver expression of vascular endothelial growth factor(VEGF) was assessed by Western blotting.Evans blue(EB) staining in ascites and liver tissue was used to evaluate vascular permeability of peritoneum and liver.Results Before external application,the abdominal circumference and body weight of cirrhotic mice were comparable.After external application,amount of ascites,abdominal circumference,body weight,ascites index,EB in ascites and liver tissue,serum NO,and VEGF protein expression in liver tissue were all higher than in the normal group.The amount of ascites in the placebo group and model group was similar.Compared to the placebo group,the amount of ascites and ascites index were lower in the Xiaozhang Tie mid-dose and high-dose groups.The abdominal circumference and body weight were lower in the high-dose group than in either the placebo or low-dose groups.Three dosages of Xiaozhang Tie did not produce any observable effects on liver function or pathology.Compared to the model group,EB in ascites,serum NO,and VEGF protein expression in liver tissue were obviously decreased in the Xiaozhang Tie mid-dose group.Conclusion Xiaozhang Tie administered as 0.25 cm2 plaster external application may reduce ascites in cirrhotic mice.Furthermore,the mechanism of this effect may involve attenuation of VEGF expression in liver tissue,reduction of the NO level in serum,and resolving the abnormally higher vascular permeability in peritoneum.