摘要：
Objective To explore the protective effect of resveratrol (RSV) on intestine barrier injury induced by hemorrhagic shock and its mechanism in rats.Methods According to random number table method,sixty-four SPF grade male Sprague-Dawley (SD) rats were divided into four groups:Sham operation group (only the catheters were indwelled in arterial and venous passages after anesthesia),hemorrhagic shock model group (model group,the catheters were indwelled in arterial and venous passages after anesthesia,and 0.3 mL solvent was administrated after hemorrhagic shock),RSV group (the catheters were indwelled in arterial and venous passages after anesthesia,15 mg/kg RSV was administered after hemorrhagic shock),superoxide dismutase 2 (SOD2) specific inhibitor,2-Methoxyoestradiol (2-ME) group (on the basic treatment of RSV group,0.1 mmol/L 2-ME was administered).The hemorrhagic shock model was reproduced by femoral artery bleeding.After drug administration,all rats were divided into two parts.One part was used for observations on 24-hour survival rate and survival time,while in the other part,2 hours after the hemorrhagic shock,the blood was collected for determination of the content of serum D-lactic acid,and afterward the rats were executed to obtain small intestine tissues for the examination of histopathological changes and Chiu's score.Moreover,differences of expression levels of tight junction proteins (Occludin,Claudin,ZO-1) of small intestine tissue and the oxidative stress related indexes SOD2 activity and reduced glutathione (GSH),oxidized glutathione (GSSH),malonaldehyde (MDA) contents were compared among the groups.Results Compared with the sham group,the model group demonstrated decreased survival rate,SOD2 activity,GSH content,GSH/GSSH ratio,reduced survival time,significantly increased serum D-lactic acid activity,Chiu's score and MDA content,and decreased expressions of tight junction proteins in small intestine tissue.Compared with model group,the RSV group showed significant increased survival rate [75.0％ (6/8) vs.37.5％ (3/8)] and prolonged survival time (hours:21.0±4.3 vs.10.4±5.8,P ＜ 0.05),significantly decreased serum D-lactic acid (μg/L:380.18 ± 70.59 vs.500.88 ± 97.53) and Chiu's score (1.75 ± 0.71 vs.4.00± 0.53) in small intestine (both P ＜ 0.05),obviously increased expressions of tight junction proteins,SOD2 activity,GSH and GSH/GSSG [Occludin (gray value):0.89 ± 0.10 vs.0.43 ± 0.77,Claudin (gray value):0.78±0.06 vs.0.33 ± 0.05,ZO-1 (gray value):0.83 ± 0.06 vs.0.34 ± 0.07,all P ＜ 0.05],and the elevated SOD2 activity (kU/L:0.85 ± 0.12 vs.0.51 ± 0.11,P ＜ 0.05],as well as increased GSH content and GSH/GSSG ratio [GSH (μmol/L):7.25±1.01 vs.3.86±0.54,GSH/GSSG:6.39± 1.14 vs.1.56±0.25,both P ＜ 0.05] in the small intestine,and markedly reduced MDA content (ng/g:5.00± 1.31 vs.8.63±0.92,P ＜ 0.05).Compared with RSV group,the 2-ME group demonstrated significantly decreased survival rate [50.0％ (4/8) vs.75.0％ (6/8)] and further shorter survival time (hours:12.2 ± 5.7 vs.21.0±4.3),increased serum D-lactic acid (μg/L:463.88 ± 60.16 vs.380.18 ± 70.59),obviously elevated Chiu's score (3.13 ± 0.99 vs.1.75±0.71,P ＜ 0.05),decreased expressions of tight junction proteins [Occludin (gray value):0.55±0.04 vs.0.89±0.10,Claudin (gray value):0.38±0.05 vs.0.78±0.06,ZO-1 (gray value):0.41±0.04 vs.0.83±0.06,all P ＜ 0.05];moreover,the activity of SOD2,GSH content,GSH/GSSG ratio were greatly reduced [SOD2 activity (kU/L):0.58 ± 0.13 vs.0.85 ± 0.12,GSH (μmol/L):4.49 ± 0.52 vs.7.25 ± 1.01,GSH/GSSG:1.57 ± 0.39 vs.6.39 ± 1.14,all P ＜ 0.05],and increased MDA content (ng/g:6.25 ± 1.04 vs.5.00 ± 1.31,P ＜ 0.05).The small intestine tissue was basically normal in Sham group,and no significant pathological changes were seen;in the model group,the small intestine epithelial mierovilli were collapsed and the mucosal barrier was destroyed obviously;in the RSV group the damages of small intestine microvilli and barrier were markedly alleviated;in 2-ME group the pathological changes were more evident compared with those in the RSV group.Conclusion RSV can improve intestinal barrier injury following hemorrhagic shock in rats;its mechanism may be related to SOD2 activation.%目的 探讨白藜芦醇改善失血性休克肠损伤的作用机制.方法 SPF级雄性SD大鼠64只,按随机数字表法分为假手术组(Sham组,麻醉后仅行动静脉置管术)、失血性休克模型组(模型组,大鼠麻醉后行动静脉置管术,休克后给予0.3 mL溶剂)、白藜芦醇组(大鼠麻醉后行动静脉置管术,休克后给予白藜芦醇15 mg/kg)、超氧化物歧化酶2(SOD2)特异性抑制剂(2-ME)组(在白藜芦醇组基础上加用2-ME,浓度为0.1 mmol/L).采用股动脉放血的方法复制大鼠失血性休克模型.大鼠给药后分成两批,一批用以观察各组24 h存活率和存活时间;另一批在休克2h后取血测定血清D-乳酸含量,取小肠组织观察病理学改变和Chiu评分,并比较小肠组织紧密连接蛋白Occludin、Claudin、ZO-1的蛋白表达水平和氧化应激相关指标SOD2活性及还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)、丙二醛(MDA)含量的差异.结果 与Sham组比较,模型组大鼠存活率、SOD2活性、GSH、GSH/GSSG均降低,生存时间缩短,D-乳酸、Chiu评分和MDA含量均明显升高,小肠组织紧密连接蛋白表达减少.与模型组比较,白藜芦醇组大鼠存活率明显升高[75.0％ (6/8)比37.5％(3/8)],生存时间显著延长(h:21.0±4.3比10.4±5.8,P＜0.05),血D-乳酸含量、小肠组织病理评分明显降低[D-乳酸(μg/L):380.18±70.59比500.88±97.53,Chiu评分(分):1.75±0.71比4.00±0.53,均P＜0.05],紧密连接蛋白表达、SOD2活性、GSH和GSH/GSSG明显升高[Occludin蛋白(灰度值):0.89±0.10比0.43±0.07,Claudin蛋白(灰度值):0.78±0.06比0.33±0.05,ZO-1蛋白(灰度值):0.83±0.06比0.34±0.07,SOD2 (kU/L):0.85±0.12比0.51±0.11,GSH(μmol/L):7.25±1.01比3.86±0.54,GSH/GSSG:6.39±1.14比1.56±0.25,均P＜0.05],MDA显著降低(ng/g:5.00±1.31比8.63±0.92,P＜ 0.05).与白藜芦醇组比较,2-ME组大鼠存活率明显降低[50.0％ (4/8)比75.0％(6/8)],生存时间再度缩短(h:12.2±5.7比21.0±4.3,P＜ 0.05),D-乳酸含量增加(μg/L:463.88±60.16比380.18±70.59)和Chiu评分评分升高(分:3.13±0.99比1.75±0.71,P＜0.05),紧密蛋白表达降低[Occludin蛋白(灰度值):0.55±0.04比0.89±0.10,Claudin蛋白(灰度值):0.38±0.05比0.78±0.06,ZO-1蛋白(灰度值):0.41±0.04比0.83±0.06,均P＜0.05],SOD2活性、GSH、GSH/GSSG下降[SOD2活性(kU/L):0.58±0.13比0.85±0.12,GSH (pμmol/L):4.49±0.52比7.25±1.01,GSH/GSSG:1.57±0.39比6.39±1.14],MDA含量增加(ng/g:6.25±1.04比5.00±1.31,P＜ 0.05).Sham组小肠组织基本正常未见明显病理学改变;模型组小肠上皮绒毛倒塌,黏膜屏障破坏明显;白藜芦醇组小肠绒毛和小肠屏障的破坏明显减轻;2-ME组病理学改变较白藜芦醇组明显.结论 白藜芦醇能改善失血性休克大鼠的肠屏障损伤,其机制可能与激活SOD2有关.
