摘要
目的探讨硫化氢(H2S)对小肠缺血/再灌注损伤(IRI)大鼠磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路表达的影响。方法将30只雄性Wistar大鼠按随机数字表法分为假手术组(Sham组)、IRI组、H2S供体硫氢化钠(NaHS)干预组(IRI+NaHS组),每组10只。采用无损伤血管夹夹闭肠系膜上动脉(SMA)60 min、再灌注120 min的方法建立大鼠肠IRI模型;Sham组仅分离SMA后关腹。恢复SMA血流前10 min,IRI+NaHS组经尾静脉注入100μmol/kg NaHS后以1.07 mmol·kg^-1·h^-1的速度维持输注至再灌注120 min;Sham组和IRI组则给予等体积生理盐水。实验结束后取下腔静脉血,采用敏感硫电极法测定血浆H2S浓度。取血后处死大鼠取回肠组织,采用苏木素-伊红(HE)染色观察组织病理学改变并进行Chiu评分;用蛋白质免疫印迹试验(Western Blot)检测磷酸化Akt(p-Akt)、Akt、PI3K、活化的天冬氨酸特异性半胱氨酸蛋白酶9(caspase-9)、哺乳动物雷帕霉素靶蛋白(mTOR)蛋白表达。结果与Sham组比较,IRI组肠黏膜组织结构紊乱、水肿,绒毛断裂、脱落,病理评分明显升高(分:4.21±0.15比0.15±0.03,P<0.01),血浆H2S水平明显降低(μmol/L:26.72±3.17比38.34±5.24,P<0.01),回肠组织p-Akt、PI3K、caspase-9、mTOR蛋白表达明显升高(p-Akt/GAPDH:2.67±0.12比0.24±0.05,PI3K/GAPDH:1.42±0.07比0.57±0.08,caspase-9/GAPDH:4.23±0.61比0.13±0.02,mTOR/GAPDH:2.17±0.23比0.23±0.02,均P<0.01)。与IRI组比较,IRI+NaHS组肠黏膜病理改变减轻,病理评分明显下降(分:1.56±0.02比4.21±0.15,P<0.01),血浆H2S水平明显升高(μmol/L:32.36±2.45比26.72±3.17,P<0.01),回肠组织p-Akt、PI3K蛋白表达进一步升高(p-Akt/GAPDH:5.12±0.08比2.67±0.12,PI3K/GAPDH:3.14±0.05比1.42±0.07,均P<0.01),而caspase-9、mTOR蛋白表达明显降低(caspase-9/GAPDH:2.12±0.24比4.23±0.61,mTOR/GAPDH:1.37±0.28比2.17±0.23,均P<0.01)。结论H2S通过上调PI3K/Akt信号通路表达,下调caspase-9、mTOR表达,从而减轻IRI大鼠肠损伤。
Objective To explore the effect of hydrogen sulfide(H2S)on expression of phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)signal pathway in rats with intestinal ischemia/reperfusion(IRI)injury.Methods Thirty male Wistar rats were divided into sham operation group(Sham group),IRI group,and H2S precursor sodium hydrosuphide(NaHS)intervention group(IRI+NaHS group)by random number table method,with 10 rats in each group.The animal model of IRI was established by 60 minutes superior mesenteric artery(SMA)blockage with non-invasive vascular clamp and 120 minutes reflow.SMA was dissociated and peritoneum cavity was closed in Sham group.The rats in IRI+NaHS group was received NaHS(100μmol/kg bolus+1.07 mmol·kg^-1·h^-1 infusion)10 minutes prior to the onset of reperfusion,while the rats in IRI group and Sham group were received equal volume of normal sodium.Blood in vena cava was collected.H2S was detected by sensitive sulfide electrode.Rats were sacrificed after blood collection.Histopathology change was observed by hematoxylin-eosin(HE)staining,ileal pathological score was studied by Chiu score.The protein expressions of phosphated Akt(p-Akt),Akt,PI3K,cleaved caspase-9,mammalian target of rapamycin(mTOR)were determined by Western Blot.Results Compared with the Sham group,there was intestinal mucosa structure disorder edema and shedding villous fracture in the IRI group.Ileal pathological score in IRI group was significantly increased(4.21±0.15 vs.0.15±0.03,P<0.01),while plasma H2S in IRI group was significantly decreased(μmol/L:26.72±3.17 vs.38.34±5.24,P<0.01).Ileal p-Akt,PI3K,caspase-9 and mTOR protein in IRI group were significantly increased(p-Akt/GAPDH:2.67±0.12 vs.0.24±0.05,PI3K/GAPDH:1.42±0.07 vs.0.57±0.08,caspase-9/GAPDH:4.23±0.61 vs.0.13±0.02,mTOR/GAPDH:2.17±0.23 vs.0.23±0.02,all P<0.01).Compared with the IRI group,pathological changes of intestinal mucosa in the IRI+NaHS group was improved,ileal pathological score was significantly decreased(1.56±0.02 vs.4.21±0.15,P<0.01),plasma H2S was significantly increased(μmol/L:32.36±2.45 vs.26.72±3.17,P<0.01)and ileal p-Akt,PI3K were significantly increased(p-Akt/GAPDH:5.12±0.08 vs.2.67±0.12,PI3K/GAPDH:3.14±0.05 vs.1.42±0.07,both P<0.01),while caspase-9,mTOR in IRI+NaHS group were significantly decreased(caspase-9/GAPDH:2.12±0.24 vs.4.23±0.61,mTOR/GAPDH:1.37±0.28 vs.2.17±0.23,both P<0.01).Conclusion H2S attenuates intestinal injury in IRI rats by up-regulating PI3K/Akt signal pathway and down-regulating caspase-9 and mTOR.
作者
卢根林
吴爱兵
王宏宾
Lu Genlin;Wu Aibing;Wang Hongbin(First Department of General Surgery,Longyou County People's Hospital,Longyou 324400,Zhejiang,China;Oncology Center,the Hospital Affiliated to Guangdong Medical University,Zhanjiang 523808,Guangdong,China;Department of Hepatopancreato-biliary Surgery,the Hospital Affiliated to Qinghai University,Xining 810001,Qinghai,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2020年第10期1247-1250,共4页
Chinese Critical Care Medicine
基金
国家自然科学基金青年科学基金项目(81201672)
浙江省衢州市科技指导性项目(2019136)
浙江省义乌市人才引进立项项目(2012-R-04)。
关键词
缺血/再灌注损伤
肠
硫化氢
磷脂酰肌醇3-激酶/蛋白激酶B信号通路
Intestinal ischemia/reperfusion injury
Hydrogen sulfide
Phosphatidylinositol 3 kinase/protein kinase B signal pathway
