细胞自噬对脂多糖介导血管通透性增加的作用  

Effects of autophagy on lipopolysaccharide-induced vascular hyper-permeability

作　　者：王盛标[1] 殷爽[2,3] 李云峰[1] 李翠玲[3] 李涛[1] 刘友坦[3] 

机构地区：[1]郴州市第一人民医院重症医学科,湖南郴州423000 [2]广东医科大学研究生学院,广东湛江524000 [3]南方医科大学深圳医院麻醉科,广东深圳518000

出　　处：《中华危重病急救医学》2016年第8期673-677,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81500066）

摘　　要：目的：探讨细胞自噬对脂多糖（LPS）介导血管通透性增加的影响。方法①体外实验：将人脐静脉内皮细胞（HUVEC）分为空白组、LPS组（LPS5mg/L刺激）、自噬抑制剂6-氨基-3-甲基嘌呤（3-MA）＋LPS组（5mmol/L3-MA预处理30min＋LPS5mg/L刺激）及自噬诱导剂雷帕霉素（RAP）＋LPS组（10nmol/LRAP预处理30min＋LPS5mg/L刺激）。4组于LPS刺激60min后检测跨内皮细胞电阻（TER）以反映内皮通透性；采用蛋白质免疫印迹试验（WesternBlot）检测自噬标志性蛋白微管相关蛋白轻链3（LC3Ⅱ/Ⅰ）和自噬相关基因Beclin-1的蛋白表达；采用流式细胞仪检测内皮细胞凋亡情况；采用荧光法检测天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）活性。②体内实验：将24只SD大鼠按随机数字表法分为4组，每组6只。对照组不予任何处理；模型组经尾静脉注射LPS10mg/kg；3-MA预处理组及RAP预处理组经尾静脉注射3-MA10mg/kg或RAP2mg/kg预处理30min后再注射LPS10mg/kg。荧光显微镜下观察肠系膜微静脉内荧光白蛋白的渗出情况，并计算血管内外荧光强度变化值（ΔI）以反映血管通透性。结果①在细胞水平，LPS组LC3Ⅱ/Ⅰ、Beclin-1、caspase-3活性及细胞凋亡率均较空白组明显增加，TER值明显降低。与LPS组比较，3-MA＋LPS组LC3Ⅱ/Ⅰ、Beclin-1、caspase-3活性及细胞凋亡率均明显降低，TER值明显升高〔LC3Ⅱ/Ⅰ蛋白：（288.2±33.3）%比（420.5±39.4）%，Beclin-1蛋白：（185.3±26.4）%比（293.3±36.1）%，caspase-3活性：（196.6±28.5）%比（339.5±25.4）%，细胞凋亡率：（9.50±0.99）%比（15.40±1.55）%，TER值：0.88±0.09比0.63±0.05，均P＜0.05〕；RAP＋LPS组LC3Ⅱ/Ⅰ、Beclin-1、caspase-3活性及细胞凋亡率进一步增加， TER值进一步降低〔LC3Ⅱ/Ⅰ蛋白：（519.6±45.2）%比（420.5±39.4）%，Beclin-1蛋白：（359.0±38.3）%比（293.3±36.1）%，caspase-3活性：（449.1±31.0）%比（Objective To investigate the effects of autophagy on lipopolysaccharide （LPS）-induced vascular hyper-permeability. Methods ① In vitro： Human umbilical vein endothelial cells （HUVECs） were randomly divided into blank group, LPS group （5 mg/L LPS stimulation）, autophagy inhibitor 6-amino-3-methyl purine （3-MA） ＋ LPS group （5 mmol/L 3-MA pretreatment for 30 minutes ＋ 5 mg/L LPS stimulation） and autophagy revulsive Rapamycin （RAP） ＋ LPS group （10 nmol/L RAP pretreatment for 30 minutes ＋ 5 mg/L LPS stimulation）. After LPS simulation for 60 minutes in four groups, endothelial permeability was detected by trans-endothelial electrical resistance （TER） determination. The protein expressions of autophagy marker protein microtubule-associated protein 1 light chain 3 （LC3 Ⅱ/Ⅰ） and autophagy related gene Beclin-1 were detected by Western Blot. Cell apoptosis was evaluated by using flow cytometry. Caspase-3 activity was detected by fluorometric assay kit. ② In vivo： 24 Sprague-Dawley （SD） rats were randomly assigned to four groups according to random number table, with 6 rats in each group. The rats in control group received no treatment; rats in model group were tail intravenous injected 10 mg/kg of LPS. The rats in 3-MA pretreatment and RAP pretreatment groups were tail intravenous injected 10 mg/kg of 3-MA or 2 mg/kg of RAP pretreatment for 30 minutes before 10 mg/kg LPS injection. The extravasation of FITC-albumin in mesenteric post-capillary venules was observed by fluorescence microscope. Then the change in fluorescence intensity of FITC-albumin between the intravascular and extravascular space （ΔI） were measured to reflect vascular permeability. Results ① In vitro, compared with blank group, the LC3 Ⅱ/Ⅰ protein, Beclin-1 protein, caspase-3 activity and rate of cell apoptosis in LPS group were increased, and the TER was decreased. Compared with LPS group, the LC3 Ⅱ/Ⅰ, Beclin-1, caspase-3 activity and rate of cell apoptosis in 3-MA＋LPS group were

关 键 词：细胞自噬 细胞凋亡 血管通透性 内皮细胞 脂多糖 

分 类 号：R459.7[医药卫生—急诊医学]