参附注射液对再灌注期间肠黏膜上皮细胞凋亡的抑制

背景肠上皮细胞异常凋亡是缺血再灌注期间肠黏膜损伤的主要原因,参附注射液对小肠黏膜损伤有良好的防治作用.目的建立大鼠肠缺血再灌注模型,观察缺血再灌注时给予参附注射液对肠上皮细胞凋亡数目、凋亡相关基因半胱氨酸天冬氨酸蛋白酶-3、Bcl-2的表达及肿瘤坏死因子水平的影响.设计随机对照实验.单位武汉大学人民医院麻醉科.材料实验于2002-12/2003-06在武汉大学人民医院麻醉科实验室完成.选取健康清洁级SD大鼠24只,随机分为空白对照组、肠缺血再灌注组、参附注射液组,8只/组.方法各组大鼠给予氨基甲酸乙酯1 mg/kg腹腔注射麻醉,肠缺血再灌注组和参附注射液组用血管钳夹闭肠系膜上动脉1 h然后再灌注2 h,空白对照组不用血管钳夹闭肠系膜上动脉.参附注射液组于阻断前30 min静注参附注射液0.02 mL/g,空白对照组和肠缺血再灌注组静脉输注等量生理盐水.制备切片进行半胱氨酸天冬氨酸蛋白酶-3、Bcl-2蛋白的表达及细胞凋亡检测.主要观察指标①各组大鼠细胞凋亡指数的比较.②各组大鼠肠上皮细胞半胱氨酸天冬氨酸蛋白酶-3及Bcl-2基因的表达.③各组肠粘膜匀浆中肿瘤坏死因子含量的比较.结果实验选用24只大鼠,全部进入结果分析.①各组大鼠细胞凋亡指数的比较参附注射液组的凋亡指数明显低于肠缺血再灌注组,但比空白对照组高(7.75±1.89)%,(28.25±8.50)%,(4.25±2.63)%,P均＜0.01].②各组大鼠肠上皮细胞半胱氨酸天冬氨酸蛋白酶-3及Bcl-2基因的表达参附注射液组组半胱氨酸天冬氨酸蛋白酶-3的表达低于肠缺血再灌注组,但高于空白对照组(0.211 6±0.087 5),(0.354 7±0.077 8),(0.194 1±0.057 4),P＜0.01,P＞0.05];Bcl-2的表达在肠缺血再灌注组比空白对照组显著增高(P＜0.05),参附注射液组的表达比肠缺血再灌注组明显降低(P＜0.01).③各组肠黏膜匀浆中肿瘤坏死因子含量的比较砀缺血再灌注组肿瘤坏死因子的表达显著高于空白对照组和参附注射液组(189.7±56.3),(38.6±10.4),(47.5±18.7)mg/L,P均＜0.01],参附注射液组和空白对照组基本相似(P＞0.05).结论参附注射液通过抑制肿瘤坏死因子的含量、降低半胱氨酸天冬氨酸蛋白酶-3的表达、上调Bcl-2基因而抑制缺血再灌注期间肠黏膜上皮细胞的凋亡,从而减轻肠黏膜缺血再灌注损伤.

Suppression of intestinal epithelium apoptosis by shenfu solution during reperfusional period

BACKGROUND: The abnormal apoptosis of intestinal epithelial cells is the main cause of intestinal mucous membrane injury during ischemia reperfusion. Shenfu injection has good therapeutic effect on intestinal mucous membrane injury. OBJECTIVE: To investigate the influence of shenfu injection medication on intestinal apoptotic epithelial number, apoptosis-related caspase-3 and Bcl-2 gene expression, as well as the level of tumor necrosis factor (TNF)in intestinal ischemia reperfusion rat model.DESIGN: Randomized controlled experiment.SETTING:Department of Anesthesia, Renmin Hospital of Wuhan University.MATERIALS:This experiment was carried out in the laboratory of the Department of Anesthesia, Renmin Hospital of Wuhan University, between December 2002 and June 2003. Totally 24 healthy SD rats of clean grade were randomized into blank control group, intestinal ischemia reperfusion (IR) group and shenfu injection group with 8 rats in each group.METHODS: Rats in each group were anesthetized with intraperitoneal injection of ethyl carbamate at dosage of 1mg/kg; then in intestinal IR group and shenfu injection group rats' superior mesenteric artery was occluded with vascular clamp for 1 hour before 2-hour reperfusion,which was not conducted in blank control group. Rats in shenfu injection group were intravenously injected with shenfu injection at 30 minutes before occlusion at dose of 0.02 mL/g, which was replaced by the same volume of physical saline in blank control group and intestinal IR group. The expression of caspase-3 and Bcl-2 protein and cell apoptosis wee detected.MAIN OUTCOME MEASURES: ① Comparison of apoptotic index between groups. ② The expression of caspase-3 and Bcl-2 gene in rat intestinal epithelium. ③ Comparison of TNF content in intestinal mucous membrane homogenate between groups.RESULTS: Totally 24 rats were included in this experiment and all ehtered the result analysis. ①Comparison of apoptotic index between groups:The apoptotic index was obviously lower in shenfu injection group than in intestinal IR group, but higher than in blank control group (7.75-±1.89)%,(28.25±8.50)%, (4.25-±2.63)%, P ＜ 0.01]. ② The expression of caspase-3 and Bcl-2 gene in rat intestinal epithelium: caspase-3 expression was lower in shenfu injection group than in intestinal IR group, but higher than in blank control group (0.211 6±0.087 5), (0.354 7±0.077 8), (0.194 1±0.057 4) A,P ＜ 0.01, P ＞ 0.05]; Bcl-2 expression was remarkably higher in intestinal IR group than in blank control group (P ＜ 0.05), but obviously reduced in shenfu injection group compared to intestinal IR group (P ＜ 0.01). ③ TNF content of intestinal mucous membranehomogenate in each group: TNF content was remarkably higher in intestinal IR group than in blank control group and shenfu injection group (189.7±56.3), (38.6±10.4), (47.5±l8.7)mg/L,P ＜ 0.01], and basically the same in shenfu inj~tion group and blank control group (P ＞ 0.05).CONCLUSION: Shenfu injection can suppress intestinal epithelial apoptosis by reducing TNF content and caspase-3 e.pression as well as upregulating the expression of Bcl-2 gene during ischemia reperfusion, thereby attenuating ischemia reperfusion injury of intestinal mucous membrane.