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氢吗啡酮通过调节雷帕霉素靶蛋白介导的自噬改善脓毒症性脑病的研究 被引量:2

Hydromorphone ameliorates sepsis-associated encephalopathy by regulating mam-malian target of rapamycin-mediated autophagy
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摘要 目的探讨氢吗啡酮对脓毒症性脑病的影响,以及氢吗啡酮对脓毒症性脑病的神经保护与雷帕霉素靶蛋白(mTOR)信号通路介导的自噬的关系。方法无特定病原体(SPF)级健康雄性C57BL/6小鼠160只,8~10周龄,体重20~25 g,采用随机数字表法分为4组(n=40):假手术组(Sham)、脓毒症性脑病组(SAE)、脓毒症性脑病+氢吗啡酮组(SAE+HP)、脓毒症性脑病+氢吗啡酮+mTOR激动剂MHY1485组(SAE+HP+MHY)。采用盲肠结扎穿孔法(CLP)建立脓毒症模型,SAE+HP组于模型建立30 min后给予氢吗啡酮0.1 mg/kg,SAE+HP+MHY组于模型建立前1 d腹腔注射MHY148510 mg/kg,持续2 d,模型建立30 min后给予氢吗啡酮0.1 mg/kg。记录术后8 d生存情况。术后24 h处死小鼠,采用苏木精-伊红(HE)染色观察海马组织病理学结果,采用酶联免疫吸附试验(ELISA法)检测海马组织白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)的含量,采用蛋白质印迹法(Western blot)检测海马组织自噬相关蛋白p-mTOR、mTOR、微管相关蛋白1轻链3(LC3)-Ⅱ/LC3-Ⅰ、与B淋巴细胞瘤-基因2相互作用的肌球蛋白样螺旋蛋白(Beclin-1)的表达,免疫荧光染色观察海马组织LC3阳性细胞。CLP术后4~8 d行Morris水迷宫实验,记录逃避潜伏期和目标象限停留时间。组间比较采用单因素方差分析。结果SAE组海马组织IL-1β、IL-6、TNF-α水平高于Sham组[(231±20)ng/L比(111±15)ng/L、(191±20)ng/L比(55±3)ng/L、(351±20)ng/L比(80±6)ng/L,F=139.942、274.485、1055.362,P<0.05];SAE组p-mTOR/mTOR比值高于Sham组(1.412±0.057比0.313±0.046,F=1341.453,P<0.05),SAE组Beclin-1蛋白表达水平低于Sham组(0.332±0.021比0.976±0.053,F=755.084,P<0.05),SAE组LC3-Ⅱ/LC3-Ⅰ表达高于Sham组(0.942±0.03比0.344±0.022,F=226.552,P<0.05),差异均有统计学意义;SAE组海马神经元退行性改变,细胞核固缩、深染,免疫荧光染色SAE组海马CA1区神经元LC3荧光强度低于Sham组(6.15±2.12比17.5±3.07,F=56.020,P<0.05);SAE组小鼠逃避潜伏期延长,目标平台停留时间缩短[逃逸潜伏期:5 d(F=32.947),6 d(F=33.322),7 d(F=41.888),8 d(F=44.685);目标平台停留时间(F=16.210,P<0.05)],差异有统计学意义。SAE+HP组IL-1β、IL-6、TNF-α水平低于SAE组[(161±15)ng/L比(231±20)ng/L、(95±12)ng/L比(191±20)ng/L、(200±12)ng/L比(351±20)ng/L,F=48.244、104.252、257.444,P<0.05];SAE+HP组p-mTOR/mTOR比值低于SAE组(0.869±0.064比1.412±0.057,F=238.676,P<0.05),SAE+HP组Beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值高于SAE组(0.788±0.039比0.332±0.021、1.513±0.055比0.942±0.039,F=625.670、163.195,P<0.05);SAE+HP组海马神经元退行性病变改善;SAE+HP组LC3荧光强度高于SAE组(22.5±4.5比6.2±2.1,F=66.306,P<0.05),差异均有统计学意义,SAE+HP组小鼠认知功能改善。MHY1485逆转了SAE+HP组氢吗啡酮的神经保护作用。结论氢吗啡酮通过抑制mTOR信号通路,促进自噬,从而减轻脓毒症性脑病。 Objective To investigate the effect of hydromorphone(HP)on sepsis-associated encephalopathy(SAE),and the relationship between the neuroprotection of hydromorphone on sepsis-associated encephalopathy and the mam-malian target of rapamycin(mTOR)-autophagy signaling pathway.Methods Totally,160 SPF male C57BL/6 mice(8-10 weeks,20-25 g)were divided into four groups(n=40 each)by random number table:sham operation group(Sham),SAE group,SAE+HP group,SAE+HP+MHY1485 group.The sepsis model was established by cecal ligation and perforation(CLP).The SAE+HP group was given HP(0.1 mg/kg)via the tail vein 30 min after the establishment of model.The SAE+HP+MHY group was intraperitoneally injected with MHY148510 mg/kg one day before the establishment of sepsis model,for continuous 2 days.After model establishment,HP(0.1 mg/kg)was injected via the tail vein 30 min later.The survival was recorded 8 days after operation.The mice were sacrificed 24 h after surgery,and the hippocampus tissues were obtained for determination of contents of interleukin(IL)-1β,IL-6,tumor necrosis factor-alpha(TNF-α)by enzyme-linked immunosorbent assay.The ratio of p-mTOR/mTOR and microtubule-associated protein 1 light chain 3-Ⅱ/Ⅰ(LC3-Ⅱ/LC3-Ⅰ)and the expression of Beclin-1 were detected by Western blotting,and histopathological results were observed by hematoxylineosin staining.Immunohistochemical staining was used to observe the fluorescence intensity of LC3.Morris water maze test was performed 4-8 d after CLP,and the escape latency period and target quadrant residence time were recorded.Comparison between groups was performed by one-way ANOVA.Results The level of IL-1β,IL-6,TNF-αin SAE group was higher than that in Sham group[(231±20)ng/L vs.(111±15)ng/L,(191±20)ng/L vs.(55±3)ng/L,and(351±20)ng/L vs.(80±6)ng/L,F=139.942,274.485,1055.362,P<0.05].The ratio of p-mTOR/mTOR in SAE group was higher than that in Sham group(1.412±0.057 vs.0.313±0.046,F=1341.453,P<0.05).The expression level of beclin-1 in SAE group was lower than that in Sham group(0.332±0.021 vs.0.976±0.053,F=755.084,P<0.05).The ratio of LC3-Ⅱ/LC3-Ⅰin SAE group was higher than that in Sham group(0.942±0.03 vs.0.344±0.022,F=226.552,P<0.05).In SAE group,we observed the degenerative changes of hippocampal neurons and condensed and hyperchromatic nucleus.The fluorescence intensity of LC3 in hippocampal CA1 region in SAE group was lower than that in Sham group(6.15±2.12 vs.17.5±3.07,F=56.020,P<0.05).In SAE group,escape latency was prolonged and residence time of target platform was shortened(Escape latency:5 d F=32.947,6 d F=33.322,7 d F=41.888,8 d F=44.685;Residence time of target platform:F=16.210,P<0.05).The level of IL-1β,IL-6 and TNF-αin SAE+HP group was lower than that in SAE group[(161±15)ng/L vs.(231±20)ng/L,(95±12)ng/L vs.(191±20)ng/L,and(200±12)ng/L vs.(351±20)ng/L,F=48.244,104.252,257.444,P<0.05].The ratio of P-MTOR/mTOR in SAE+HP group was lower than that in SAE group(0.869±0.064 vs.1.412±0.057,F=238.676,P<0.05),and beclin-1 expression and LC3-Ⅱ/LC3-Ⅰin SAE+HP group were higher than those in SAE+HP group(0.788±0.039 vs.0.332±0.021,1.513±0.055 vs.0.942±0.039,F=625.670,163.195,P<0.05).The degeneration of hippocampal neurons was improved in SAE+HP group.The LC3 fluorescence intensity in SAE+HP group was higher than that in SAE group(22.5±4.5 vs.6.2±2.1,F=66.306,P<0.05),and MHY1485 could abolish the neuroprotective effect of HP.Conclusion HP could mitigate sepsis-associated encephalopathy by promoting autophagy via inhibiting mTOR signaling pathway.
作者 张静 王庆元 李心怡 咸淑悦 金奇彦 王焱林 Zhang Jing;Wang Qingyuan;Li Xinyi;Xian Shuyue;Jin Qiyan;Wang Yanlin(Department of Anesthesiology,Zhongnan Hospital of Wuhan University,Wuhan 430071,China;Department of Anesthesiology,the People’s Hospital of Tuanfeng,Huanggang 438800,China)
出处 《中华实验外科杂志》 CAS 北大核心 2022年第1期76-80,共5页 Chinese Journal of Experimental Surgery
基金 国家自然科学基金(81871553、82002033)。
关键词 氢吗啡酮 脓毒症 脓毒症性脑病 雷帕霉素靶蛋白 自噬 Hydromorphone Sepsis Sepsis-associated encephalopathy Mammalian target of rapamycin Autophagy
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