二甲双胍通过AMPK抑制mTORC1并减轻内毒素诱导的急性肺损伤
发布时间：2018-11-18 17:15
【摘要】：目的:急性肺损伤(acute lung injury,ALI)是临床上危及生命的急重症,急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是其严重形式。ALI发病机制复杂,病死率高,失控性炎症反应是ALI发生发展的关键机制。腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)是细胞内的重要代谢调节酶,但近年来研究表明AMPK可在多种体内外模型中发挥抗炎效应。临床上常用的降糖药二甲双胍(metformin,MET)可通过激活AMPK而发挥降糖及抗炎效应。本研究探讨了MET在ALI中的潜在药理学效应及相关机制。方法:选用雄性BALB/c小鼠,采用脂多糖(lipopolysaccharide,LPS)腹腔注射(15 mg/kg)模拟全身感染诱发的ALI小鼠模型,MET(400 mg/kg)在LPS注射前30 min经腹腔注入。为探讨MET的药理效应是否与AMPK有关,AMPK抑制剂Compound C(15 mg/kg)在MET注射前30 min经腹腔注入,或采用AMPK激活剂A-769662(30mg/kg)替代MET在LPS注射前30 min经腹腔注入。为探讨AMPK是否通过激活沉默信息调节因子1(sirtuin 1,SIRT1)或抑制哺乳动物雷帕霉素靶蛋白复合体1(mammalian target of rapamycin complex 1,mTORC1)发挥效应,SIRT1抑制剂EX527(10 mg/kg)或mTORC1激活剂3-BDO(100 mg/kg)在A-769662注射前30 min经腹腔注入。上述小鼠均在LPS注射后18 h断颈处死,收集血浆及肺组织标本,ELISA法检测血浆及肺组织中促炎细胞因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(Interleukin-6,IL-6)的水平,HE染色法观察肺组织形态学异常,免疫印迹法检测肺组织中AMPK的总蛋白及其磷酸化水平、mTORC1下游靶蛋白P70S6K1和4EBP1的总蛋白及其磷酸化水平。结果:(1)MET处理可抑制LPS诱导的ALI模型小鼠血浆和肺组织中促炎细胞因子TNF-α和IL-6的表达,这伴随有模型小鼠肺组织形态学异常的减轻。(2)MET处理也可使AMPK的磷酸化水平升高,而采用AMPK抑制剂Compound C抑制MET对AMPK的激活效应后,MET在模型小鼠中的抗炎及肺组织保护效应被阻断。(3)与此相一致,采用AMPK激活剂A-769662可模拟MET在ALI中的抗炎保护效应,表现为血浆中IL-6的水平明显降低、肺组织学异常显著减轻。(4)AMPK激活剂A-769662对IL-6产生的抑制效应不能被SIRT1抑制剂EX527所阻断,但mTORC1激活剂3-BDO可显著阻断A-769662对IL-6产生的抑制效应及其对肺组织损伤的保护作用。(5)LPS暴露可诱导肺组织中mTORC1下游靶蛋白P70S6K1和4EBP1的磷酸化水平升高,而MET处理可抑制P70S6K1和4EBP1的磷酸化;MET对P70S6K1和4EBP1的抑制效应也可被AMPK抑制剂Compound C阻断。结论:本研究的实验数据提示MET可在LPS诱导的ALI模型中发挥抗炎保护作用,其机制可能与MET激活AMPK从而下调mTORC1活性有关。本研究揭示了MET通过AMPK发挥抗炎效应的一个新机制,也提示MET可能在ALI中具有潜在的应用价值。
[Abstract]:Objective: acute lung injury (acute lung injury,ALI) is a critical clinical life-threatening disease, and acute respiratory distress syndrome (acute respiratory distress syndrome,ARDS) is a serious form. ALI has complex pathogenesis and high mortality. Uncontrolled inflammation is the key mechanism for the development of ALI. Adenylate activated protein kinase (AMP-activated protein kinase,AMPK) is an important metabolic regulator in cells, but recent studies have shown that AMPK can play an anti-inflammatory effect in various models in vivo and in vitro. Metformin (metformin,MET), a common hypoglycemic drug, can play an antidiabetic and anti-inflammatory effect by activating AMPK. This study investigated the potential pharmacological effects of MET in ALI and its related mechanisms. Methods: male BALB/c mice were injected intraperitoneally with lipopolysaccharide (lipopolysaccharide,LPS) intraperitoneal injection (15 mg/kg) to mimic the ALI mouse model, MET (400 mg/kg induced by systemic infection 30 min before LPS injection. To investigate whether the pharmacological effect of MET is related to AMPK, AMPK inhibitor Compound C (15 mg/kg was injected intraperitoneally 30 min before MET injection, or AMPK activator A-769662 (30mg/kg) was used to replace MET 30 min before LPS injection. To investigate whether AMPK plays an effect by activating silencing information regulatory factor 1 (sirtuin 1 + SIRT1) or inhibiting mammalian rapamycin target protein complex 1 (mammalian target of rapamycin complex 1 mTORC1. SIRT1 inhibitor EX527 (10 mg/kg) or mTORC1 activator 3-BDO (100 mg/kg) were injected intraperitoneally 30 min before A-769662 injection. All the above mice were killed at 18 h after LPS injection. Plasma and lung tissue samples were collected. The levels of proinflammatory cytokine tumor necrosis factor 伪 (tumor necrosis factor- 伪 (TNF- 伪) and interleukin-6 (Interleukin-6,) in plasma and lung tissue were detected by ELISA method. The total protein and phosphorylation of AMPK in lung tissue were detected by Western blot, and the total protein and phosphorylation level of P70S6K1 and 4EBP1 in the downstream of mTORC1 were detected by Western blot. Results: (1) MET could inhibit the expression of TNF- 伪 and IL-6 in plasma and lung tissue of ALI model mice induced by LPS. (2) MET treatment also increased the phosphorylation level of AMPK, while Compound C, a AMPK inhibitor, inhibited the activation of AMPK by MET. The anti-inflammatory and lung tissue protective effects of MET were blocked in model mice. (3) in accordance with this, AMPK activator A-769662 could mimic the anti-inflammatory effect of MET in ALI, which showed that the level of IL-6 in plasma was significantly decreased. (4) the inhibitory effect of AMPK activator A-769662 on IL-6 was not blocked by SIRT1 inhibitor EX527. However, mTORC1 activator 3-BDO significantly blocked the inhibitory effect of A-769662 on IL-6 and its protective effect on lung tissue injury. (5) LPS exposure induced an increase in the phosphorylation levels of P70S6K1 and 4EBP1, the downstream target proteins of mTORC1, in lung tissue. MET treatment inhibited the phosphorylation of P70S6K1 and 4EBP1. The inhibitory effect of MET on P70S6K1 and 4EBP1 could also be blocked by AMPK inhibitor Compound C. Conclusion: the experimental data of this study suggest that MET may play an anti-inflammatory role in LPS induced ALI model, and its mechanism may be related to the activation of AMPK by MET and down-regulation of mTORC1 activity. This study revealed a new mechanism of anti-inflammatory effect of MET through AMPK and suggested that MET may have potential application value in ALI.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965
本文编号：2340631
[Abstract]:Objective: acute lung injury (acute lung injury,ALI) is a critical clinical life-threatening disease, and acute respiratory distress syndrome (acute respiratory distress syndrome,ARDS) is a serious form. ALI has complex pathogenesis and high mortality. Uncontrolled inflammation is the key mechanism for the development of ALI. Adenylate activated protein kinase (AMP-activated protein kinase,AMPK) is an important metabolic regulator in cells, but recent studies have shown that AMPK can play an anti-inflammatory effect in various models in vivo and in vitro. Metformin (metformin,MET), a common hypoglycemic drug, can play an antidiabetic and anti-inflammatory effect by activating AMPK. This study investigated the potential pharmacological effects of MET in ALI and its related mechanisms. Methods: male BALB/c mice were injected intraperitoneally with lipopolysaccharide (lipopolysaccharide,LPS) intraperitoneal injection (15 mg/kg) to mimic the ALI mouse model, MET (400 mg/kg induced by systemic infection 30 min before LPS injection. To investigate whether the pharmacological effect of MET is related to AMPK, AMPK inhibitor Compound C (15 mg/kg was injected intraperitoneally 30 min before MET injection, or AMPK activator A-769662 (30mg/kg) was used to replace MET 30 min before LPS injection. To investigate whether AMPK plays an effect by activating silencing information regulatory factor 1 (sirtuin 1 + SIRT1) or inhibiting mammalian rapamycin target protein complex 1 (mammalian target of rapamycin complex 1 mTORC1. SIRT1 inhibitor EX527 (10 mg/kg) or mTORC1 activator 3-BDO (100 mg/kg) were injected intraperitoneally 30 min before A-769662 injection. All the above mice were killed at 18 h after LPS injection. Plasma and lung tissue samples were collected. The levels of proinflammatory cytokine tumor necrosis factor 伪 (tumor necrosis factor- 伪 (TNF- 伪) and interleukin-6 (Interleukin-6,) in plasma and lung tissue were detected by ELISA method. The total protein and phosphorylation of AMPK in lung tissue were detected by Western blot, and the total protein and phosphorylation level of P70S6K1 and 4EBP1 in the downstream of mTORC1 were detected by Western blot. Results: (1) MET could inhibit the expression of TNF- 伪 and IL-6 in plasma and lung tissue of ALI model mice induced by LPS. (2) MET treatment also increased the phosphorylation level of AMPK, while Compound C, a AMPK inhibitor, inhibited the activation of AMPK by MET. The anti-inflammatory and lung tissue protective effects of MET were blocked in model mice. (3) in accordance with this, AMPK activator A-769662 could mimic the anti-inflammatory effect of MET in ALI, which showed that the level of IL-6 in plasma was significantly decreased. (4) the inhibitory effect of AMPK activator A-769662 on IL-6 was not blocked by SIRT1 inhibitor EX527. However, mTORC1 activator 3-BDO significantly blocked the inhibitory effect of A-769662 on IL-6 and its protective effect on lung tissue injury. (5) LPS exposure induced an increase in the phosphorylation levels of P70S6K1 and 4EBP1, the downstream target proteins of mTORC1, in lung tissue. MET treatment inhibited the phosphorylation of P70S6K1 and 4EBP1. The inhibitory effect of MET on P70S6K1 and 4EBP1 could also be blocked by AMPK inhibitor Compound C. Conclusion: the experimental data of this study suggest that MET may play an anti-inflammatory role in LPS induced ALI model, and its mechanism may be related to the activation of AMPK by MET and down-regulation of mTORC1 activity. This study revealed a new mechanism of anti-inflammatory effect of MET through AMPK and suggested that MET may have potential application value in ALI.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965
【参考文献】
相关期刊论文 前3条
1 玄玲玲;侯琦;;AMPK与肺部炎症研究进展[J];药学学报;2014年08期
2 林志健;张冰;刘小青;;AMPK-ACC信号通路与相关代谢疾病的研究进展[J];中国糖尿病杂志;2013年05期
3 贾雪梅;杨光福;;急性肺损伤/急性呼吸窘迫综合征发病机制的研究进展[J];中国实用医药;2011年32期
，本文编号：2340631
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2340631.html
最近更新
教材专著
