内质网应激介导的细胞凋亡在原花青素减轻小鼠肠缺血再灌注损伤的作用

Role of endoplasmic reticulum stress-mediated apoptosis in proanthocyanidins-induced attenuation of intestinal ischemia-reperfusion injury in mice

目的评价内质网应激介导的细胞凋亡在原花青素减轻小鼠肠缺血再灌注损伤的作用。方法SPF级健康成年雄性C57BL/6小鼠30只,8~10周龄,体质量20~25 g,采用随机数字表法分为5组(n=6):假手术组(S组)、假手术+原花青素组(S+PC组)、肠缺血再灌注组(I/R组)、肠缺血再灌注+原花青素组(I/R+PC组)和肠缺血再灌注+原花青素+衣霉素组(I/R+PC+TM组)。采用夹闭肠系膜上动脉60 min,再灌注120 min的方法建立小鼠肠缺血再灌注损伤模型。S+PC组、I/R+PC组、I/R+PC+TM组缺血前1周每天灌胃给予原花青素100 mg/kg,S组和I/R组连续7 d给予等量生理盐水;I/R+PC+TM组于缺血前24 h腹腔注射内质网应激激动剂衣霉素1 mg/kg。再灌注120 min时处死小鼠,取小肠组织,采用ELISA法检测二胺氧化酶(DAO)水平;光镜下观察小肠组织病理学结果并行Chiu评分;采用TUNEL法测定细胞凋亡情况,计算细胞凋亡指数(AI),Western blot法检测葡萄糖调节蛋白78(GRP78)、转录因子C/EBP同源蛋白(CHOP)以及cleaved caspase-3、Bax、Bcl-2的表达水平,计算Bcl-2/Bax比值。结果与S组相比,I/R组小肠组织Chui评分、DAO水平和AI升高,GRP78、CHOP、cleaved caspase-3和Bax表达上调,Bcl-2表达下调,Bcl-2/Bax比值降低(P<0.05),小肠组织发生病理学损伤,S+PC组上述指标差异无统计学意义(P>0.05);与I/R组相比,I/R+PC组小肠组织Chui评分、DAO水平和AI降低,GRP78、CHOP、cleaved caspase-3和Bax表达下调,Bcl-2表达上调,Bcl-2/Bax比值升高(P<0.05),小肠组织病理学损伤减轻;与I/R+PC组相比,I/R+PC+TM组小肠组织Chui评分、DAO水平和AI升高,GRP78、CHOP、cleaved caspase-3和Bax表达上调,Bcl-2表达下调,Bcl-2/Bax比值降低(P<0.05),小肠组织病理学损伤加重。结论原花青素可通过抑制内质网应激介导的细胞凋亡,减轻小鼠肠缺血再灌注损伤。

Objective To evaluate the role of endoplasmic reticulum stress-mediated apoptosis in proanthocyanidins-induced attenuation of intestinal ischemia-reperfusion(I/R)injury in mice.Methods Thirty SPF healthy adult male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 5 groups(n=6 each)by a random number table method:sham operation group(S group),sham operation+proanthocyanidins group(S+PC group),intestinal I/R group(I/R group),intestinal I/R+proanthocyanidins group(I/R+PC group)and intestinal I/R+proanthocyanidins+tunicamycin group(I/R+PC+TM group).The superior mesenteric artery was clamped for 60 min and reperfused for 120 min to establish a mouse intestinal I/R injury model.Proanthocyanidin 100 mg/kg was given by intragastric gavage every day 1 week before ischemia in S+PC group,I/R+PC group and I/R+PC+TM group,and the equal volume of normal saline was given for 7 consecutive days in S group and I/R group,and endoplasmic reticulum stress agonist tunicamycin 1 mg/kg was intraperitoneally injected at 24 h before ischemia in I/R+PC+TM group.The mice were sacrificed at 120 min of reperfusion,and the small intestinal tissues were taken for microscopic examination of the histopathological changes(using light microscopy)and for determination of the level of diamine oxidase(DAO)(by enzyme-linked immunosorbent assay),cell apoptosis(by TUNEL method),glucose regulatory protein 78(GRP78),C/EBP-homologous protein(CHOP)and cleaved caspase-3,Bax and Bcl-2(by Western blot).Intestinal damage was assessed and scored according to Chiu,and the apoptosis index(AI)and Bcl-2/Bax ratio were calculated.Results Compared with S group,the Chiu′s score,level of DAO and AI were significantly increased,the expression of GRP78,CHOP,cleaved caspase-3 and Bax was up-regulated,the expression of Bcl-2 was down-regulated,the ratio of Bcl-2/Bax was decreased(P<0.05),and the pathological damage occurred in the small intestinal tissue in I/R group,and no significant change was found in the aforementioned indexes in S+PC group(P>0.05).Compared with I/R group,the Chiu′s score,DAO level and AI were significantly decreased,the expression of GRP78,CHOP,cleaved caspase-3 and Bax was down-regulated,the expression of Bcl-2 was up-regulated,the ratio of Bcl-2/Bax was increased(P<0.05),and the pathological injury to the small intestinal tissue was significantly reduced in I/R+PC group.Compared with I/R+PC group,the Chiu′s score,level of DAO and AI were significantly increased,the expression of GRP78,CHOP,cleaved caspase-3 and Bax was up-regulated,the expression of Bcl-2 was down-regulated,and the ratio of Bcl-2/Bax was decreased(P<0.05),and the pathological damage to the small intestinal tissue was aggravated in I/R+PC+TM group.Conclusions Proanthocyanidins can alleviate intestinal I/R injury by inhibiting endoplasmic reticulum stress-mediated cell apoptosis in mice.