PI3K/Akt信号通路在盐酸戊乙奎醚抑制肢体缺血再灌注大鼠肠组织自噬中的作用  

Role of PI3K/Akt signaling pathway in penehyclidine hydrochloride-induced inhibition of autophagy in intestinal tissues in a rat model of limb ischemia-reperfusion

作　　者：康于庆 黄再青[2] 刘慧[2] 何志强[2] 朱俊峰[1] 古妙宁[3] 

机构地区：[1]上海市第六人民医院金山分院麻醉科,201599 [2]内蒙古医科大学第三附属医院麻醉科,包头市014010 [3]南方医科大学附属南方医院麻醉科,广州市510515

出　　处：《中华麻醉学杂志》2016年第8期934-937,共4页Chinese Journal of Anesthesiology

基　　金：内蒙古自然科学基金（2014MS0818）

摘　　要：目的 探讨磷脂酰肌醇3-激酶/丝氨酸-苏氨酸蛋白激酶（PI3K/Akt）信号通路在盐酸戊乙奎醚抑制肢体缺血再灌注大鼠肠组织自噬中的作用.方法 健康成年雄性SPF级SD大鼠64只,体重220～ 250 g,采用随机数字表法分为4组（n=16）：假手术组（S组）、肢体缺血再灌注组（LIR组）、盐酸戊乙奎醚处理组（PHC组）和盐酸戊乙奎醚＋PI3K抑制剂3-甲基腺嘌呤组（PM组）.采用夹闭股动脉3h,再灌注4h的方法制备大鼠肢体缺血再灌注损伤模型.于再灌注前15 min时,S组和LIR组静脉注射生理盐水1 ml,PHC组静脉注射盐酸戊乙奎醚0.15 mg/kg,PM组静脉注射盐酸戊乙奎醚0.15 mg/kg,同时腹腔注射3-甲基腺嘌呤10 mg/kg.于再灌注4h时,取静脉血样,采用ELISA法测定血清内毒素（LPS）和LDH水平;取肠组织,荧光显微镜下观察自噬小体;采用Western blot法检测肠组织自噬蛋白Beclin-1、微管相关蛋白1轻链3 Ⅰ（LC3 Ⅰ）和微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）的表达,计算LC3Ⅱ与LC3 Ⅰ表达水平的比值（LC3Ⅱ/LC3 Ⅰ）.结果 与S组比较,LIR组和PM组血清LPS和LDH水平升高,肠组织Beclin-1表达上调,LC3 Ⅱ/LC3 Ⅰ升高（P＜0.05或0.01）,自噬小体增多,PHC组上述指标差异无统计学意义（P＞0.05）;与LIR组比较,PHC组血清LPS和LDH水平降低,肠组织Beclin-1表达下调,LC3Ⅱ/LC3 Ⅰ降低（P＜0.05或0.01）,自噬小体减少,PM组上述指标差异无统计学意义（P＞0.05）;与PHC组比较,PM组血清LPS和LDH水平升高,肠组织Beclin-1表达上调,LC3Ⅱ/LC3 Ⅰ升高（P＜0.05或0.01）,自噬小体增多.结论 盐酸戊乙奎醚抑制肢体缺血再灌注大鼠肠组织自噬的机制与激活PI3K/Akt信号通路有关.Objective To investigate the role of phosphatidylinositol 3-kinase/serine-threonine kinase （PI3K/Akt） signaling pathway in penehyclidine hydrochloride-induced inhibition of autophagy in intestinal tissues in a rat model of limb ischemia-reperfusion.Methods Sixty-four healthy adult male SPF Sprague-Dawley rats,weighing 220-250 g,were randomly divided into 4 groups （n =16 each） using a random number table：sham operation group （group S）,limb ischemia-reperfusion group （group LIR）,penehyclidine hydrochloride group （group PHC）,and penehyclidine hydrochloride ＋ PI3K inhibitor 3-methyladenine group （group PM）.The animals were anesthetized with sevoflurane,and bilateral femoral arteries were clamped for 3 h followed by 4 h of reperfusion in LIR,PHC and PM groups.At 15 min before reperfusion,normal saline 1 ml was injected intravenously in S and LIR groups,penehyclidine hydrochloride 0.15 mg/kg was injected intravenously in group PHC,and penehyclidine hydrochloride 0.15 mg/kg was injected intravenously,and 3-methyladenine 10 mg/kg was injected intraperitoneally at the same time in group PM.At 4 h of reperfusion,venous blood samples were collected for determination of serum lipopolysaccharide （LPS） and lactic dehydrogenase （LDH） levels.The animals were sacrificed after blood sampling,and the small intestine was removed for determination of the expression of Beclin-1,microtubule-associated protein 1 light chain 3 Ⅰ （LC3 Ⅰ） and LC3 Ⅱ in intestinal tissues by Western blot.The ratio of LC3 Ⅱ expression to LC3 Ⅰ expression （LC3 Ⅱ/LC3 Ⅰ） was calculated.Results Compared with group S,the serum LPS and LDH levels were significantly increased,the expression of Beclin-1 in intestinal tissues was up-regulated,LC3 Ⅱ/LC3 Ⅰ was increased （P〈0.05 or 0.01）,and autophagosomes were increased in LIR and PM groups,and no significant change was found in the parameters mentioned above in group PHC （P〉0.05）.Compared with group LIR,the serum LPS and LDH levels were significa

关 键 词：1-磷脂酰肌醇3-激酶 蛋白质丝氨酸苏氨酸激酶 胆碱能激动剂 四肢 再灌注损伤 肠 自噬 

分 类 号：R614[医药卫生—麻醉学]