摘要
目的探讨硫化氢(H2S)是否通过调控c-Jun氨基端激酶/活化蛋白-1(JNK/AP-1)信号通路对大鼠小肠缺血/再灌注(I/R)损伤起保护作用。方法按随机数字表法将30只雄性Wistar大鼠分为假手术组(Sham组)、I/R组、H2S供体硫氢化钠干预组(I/R+NaHS组),每组10只。用无损伤血管夹阻断肠系膜上动脉法制备I/R损伤模型(缺血60 min、再灌注120 min)。I/R+NaHS组再灌注前10 min经大鼠尾静脉注射100μmol/kg的NaHS,随后按1.07 mmol·kg-1·h-1输注直到再灌注120 min。采用敏感硫电极法测定血浆H2S浓度。采用分光光度法检测小肠组织丙二醛(MDA)、超氧化物歧化酶(SOD)水平。对小肠病理组织切片行苏木素-伊红(HE)染色并进行Chiu评分以评价肠黏膜损伤程度;采用蛋白质免疫印迹试验(Western blotting)检测小肠组织磷酸化JNK(p-JNK)、JNK、AP-1、BCL-2蛋白表达。结果与Sham组比较,I/R组固有层破坏,出血、溃疡,Chiu评分明显升高(分:4.80±0.63比0.70±0.09,P<0.01);血浆H2S浓度和回肠组织SOD活性明显降低〔H2S(μmol/L):17.29±1.40比34.62±1.48,SOD(kU/g):5.38±0.93比20.56±1.85,均P<0.01〕,回肠组织MDA含量明显升高(μmol/g:16.06±1.71比4.80±0.92,P<0.01),回肠组织BCL-2蛋白表达明显降低(BCL-2/β-actin:0.32±0.06比0.79±0.05,P<0.01),p-JNK、AP-1蛋白表达明显升高(p-JNK/β-actin:0.69±0.03比0.10±0.03,AP-1/β-actin:0.82±0.02比0.22±0.02,均P<0.01)。与I/R组比较,I/R+NaHS组上皮层与固有层中度分离,部分绒毛顶端破损,Chiu评分明显下降(分:2.90±0.56比4.80±0.63,P<0.01);血浆H2S浓度和回肠组织SOD活性明显升高〔H2S(μmol/L):24.48±1.84比17.29±1.40,SOD(kU/g):10.29±1.26比5.38±0.93,均P<0.01〕,回肠组织MDA含量明显降低(μmol/g:7.88±1.01比16.06±1.71,P<0.01),回肠组织BCL-2蛋白表达明显升高(BCL-2/β-actin:0.44±0.06比0.32±0.06,P<0.01),p-JNK、AP-1蛋白表达明显降低(p-JNK/β-actin:0.54±0.05比0.69±0.03,AP-1/β-actin:0.66±0.04比0.82±0.02,均P<0.01)。Sham组、I/R组、I/R+NaHS组间回肠组织JNK表达差异无统计学意义(JNK/β-actin:0.63±0.02、0.66±0.02、0.64±0.02,P>0.05)。结论H2S通过下调JNK/AP-1信号通路表达减轻氧化应激水平,对大鼠小肠I/R损伤起保护作用。
Objective To investigate whether hydrogen sulfide(H 2S)protects against intestinal ischemia/reperfusion(I/R)injury in rats by regulating c-Jun N-terminal kinase/activator protein-1(JNK/AP-1)signaling pathway.Methods Thirty male Wistar rats were divided into sham operated group(Sham group),I/R group,and H 2S donor sodium hydrosulfide(NaHS)intervention group(I/R+NaHS group),with 10 rats in each group.The I/R injury model was established by blocking the superior mesenteric artery with a non-traumatic vascular clip,with 60 minutes of ischemia followed by 120 minutes of reperfusion.In the I/R+NaHS group,100μmol/kg of NaHS was injected through the tail vein 10 minutes before reperfusion,followed by continuous infusion of 1.07 mmol·kg-1·h-1 until the end of the 120-minute reperfusion period.Plasma H 2S concentration was measured using a sensitive sulfur electrode.Malondialdehyde(MDA)and superoxide dismutase(SOD)levels in the small intestine tissue were assayed spectrophotometrically.Histological sections of the small intestine were stained with hematoxylin-eosin(HE)staining and scored using the Chiu scoring system to assess the degree of intestinal mucosal injury.Western blotting was used to detect the protein expressions of phosphated-JNK(p-JNK),JNK,AP-1,and BCL-2 in the small intestine tissue.Results Compared with the Sham group,the I/R group exhibited damage to the lamina propria,hemorrhage,and ulceration,with a significantly higher Chiu score(4.80±0.63 vs.0.70±0.09,P<0.01);plasma H 2S concentration and SOD activity in the ileum tissue were significantly reduced[H 2S(μmol/L):17.29±1.40 vs.34.62±1.48,SOD(kU/g):5.38±0.93 vs.20.56±1.85,both P<0.01],while MDA level was significantly elevated(μmol/g:16.06±1.71 vs.4.80±0.92,P<0.01);expression of BCL-2 protein in the ileum tissue was significantly down-regulated(BCL-2/β-actin:0.32±0.06 vs.0.79±0.05,P<0.01),while expressions of p-JNK and AP-1 proteins were significantly up-regulated(p-JNK/β-actin:0.69±0.03 vs.0.10±0.03,AP-1/β-actin:0.82±0.02 vs.0.22±0.02,both P<0.01).Compared with the I/R group,the I/R+NaHS group showed moderate separation between the epithelial and lamina propria layers,with partial damage to the tips of the villi;the Chiu score was significantly lower(2.90±0.56 vs.4.80±0.63,P<0.01);plasma H 2S concentration and SOD activity in the ileum tissue were significantly increased[H 2S(μmol/L):24.48±1.84 vs.17.29±1.40,SOD(kU/g):10.29±1.26 vs.5.38±0.93,both P<0.01],while MDA level was significantly reduced(μmol/g:7.88±1.01 vs.16.06±1.71,P<0.01);expression of BCL-2 protein in the ileum tissue was significantly up-regulated(BCL-2/β-actin:0.44±0.06 vs.0.32±0.06,P<0.01),while expressions of p-JNK and AP-1 proteins were significantly down-regulated(p-JNK/β-actin:0.54±0.05 vs.0.69±0.03,AP-1/β-actin:0.66±0.04 vs.0.82±0.02,both P<0.01).There was no statistically significant difference in the expression of JNK in the ileum tissue among the Sham group,I/R group,and I/R+NaHS group(JNK/β-actin:0.63±0.02,0.66±0.02,0.64±0.02,respectively,P>0.05).Conclusion H 2S exerts a protective effect on intestinal I/R injury in rats by down-regulate the expression of the JNK/AP-1 signaling pathway,as well as reducing oxidative stress levels.
作者
童斐
卢根林
吴爱兵
姜仁鸦
Tong Fei;Lu Genlin;Wu Aibing;Jiang Renya(Department of General Surgery,Longyou County People's Hospital,Quzhou 324400,Zhejiang,China;Department of Oncology,Zhanjiang Central People's Hospital,Zhanjiang 523808,Guangdong,China;Department of Hepatobiliary Pancreatic Surgery,Quzhou City People's Hospital,Quzhou 324002,Zhejiang,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2024年第11期1179-1182,共4页
Chinese Critical Care Medicine
基金
国家自然科学基金青年科学基金(81201672)
浙江省衢州市医疗卫生"258"重点学科人才工程培养专项基金(2021-19)
浙江省衢州市医学重点学科建设专项基金(2023-7)。
