二甲雙胍通過AMPK抑制mTORC1并減輕內(nèi)毒素誘導(dǎo)的急性肺損傷
發(fā)布時(shí)間：2018-11-18 17:15
【摘要】：目的:急性肺損傷(acute lung injury,ALI)是臨床上危及生命的急重癥,急性呼吸窘迫綜合征(acute respiratory distress syndrome,ARDS)是其嚴(yán)重形式。ALI發(fā)病機(jī)制復(fù)雜,病死率高,失控性炎癥反應(yīng)是ALI發(fā)生發(fā)展的關(guān)鍵機(jī)制。腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)是細(xì)胞內(nèi)的重要代謝調(diào)節(jié)酶,但近年來研究表明AMPK可在多種體內(nèi)外模型中發(fā)揮抗炎效應(yīng)。臨床上常用的降糖藥二甲雙胍(metformin,MET)可通過激活A(yù)MPK而發(fā)揮降糖及抗炎效應(yīng)。本研究探討了MET在ALI中的潛在藥理學(xué)效應(yīng)及相關(guān)機(jī)制。方法:選用雄性BALB/c小鼠,采用脂多糖(lipopolysaccharide,LPS)腹腔注射(15 mg/kg)模擬全身感染誘發(fā)的ALI小鼠模型,MET(400 mg/kg)在LPS注射前30 min經(jīng)腹腔注入。為探討MET的藥理效應(yīng)是否與AMPK有關(guān),AMPK抑制劑Compound C(15 mg/kg)在MET注射前30 min經(jīng)腹腔注入,或采用AMPK激活劑A-769662(30mg/kg)替代MET在LPS注射前30 min經(jīng)腹腔注入。為探討AMPK是否通過激活沉默信息調(diào)節(jié)因子1(sirtuin 1,SIRT1)或抑制哺乳動物雷帕霉素靶蛋白復(fù)合體1(mammalian target of rapamycin complex 1,mTORC1)發(fā)揮效應(yīng),SIRT1抑制劑EX527(10 mg/kg)或mTORC1激活劑3-BDO(100 mg/kg)在A-769662注射前30 min經(jīng)腹腔注入。上述小鼠均在LPS注射后18 h斷頸處死,收集血漿及肺組織標(biāo)本,ELISA法檢測血漿及肺組織中促炎細(xì)胞因子腫瘤壞死因子α(tumor necrosis factor-α,TNF-α)和白細(xì)胞介素-6(Interleukin-6,IL-6)的水平,HE染色法觀察肺組織形態(tài)學(xué)異常,免疫印跡法檢測肺組織中AMPK的總蛋白及其磷酸化水平、mTORC1下游靶蛋白P70S6K1和4EBP1的總蛋白及其磷酸化水平。結(jié)果:(1)MET處理可抑制LPS誘導(dǎo)的ALI模型小鼠血漿和肺組織中促炎細(xì)胞因子TNF-α和IL-6的表達(dá),這伴隨有模型小鼠肺組織形態(tài)學(xué)異常的減輕。(2)MET處理也可使AMPK的磷酸化水平升高,而采用AMPK抑制劑Compound C抑制MET對AMPK的激活效應(yīng)后,MET在模型小鼠中的抗炎及肺組織保護(hù)效應(yīng)被阻斷。(3)與此相一致,采用AMPK激活劑A-769662可模擬MET在ALI中的抗炎保護(hù)效應(yīng),表現(xiàn)為血漿中IL-6的水平明顯降低、肺組織學(xué)異常顯著減輕。(4)AMPK激活劑A-769662對IL-6產(chǎn)生的抑制效應(yīng)不能被SIRT1抑制劑EX527所阻斷,但mTORC1激活劑3-BDO可顯著阻斷A-769662對IL-6產(chǎn)生的抑制效應(yīng)及其對肺組織損傷的保護(hù)作用。(5)LPS暴露可誘導(dǎo)肺組織中mTORC1下游靶蛋白P70S6K1和4EBP1的磷酸化水平升高,而MET處理可抑制P70S6K1和4EBP1的磷酸化;MET對P70S6K1和4EBP1的抑制效應(yīng)也可被AMPK抑制劑Compound C阻斷。結(jié)論:本研究的實(shí)驗(yàn)數(shù)據(jù)提示MET可在LPS誘導(dǎo)的ALI模型中發(fā)揮抗炎保護(hù)作用,其機(jī)制可能與MET激活A(yù)MPK從而下調(diào)mTORC1活性有關(guān)。本研究揭示了MET通過AMPK發(fā)揮抗炎效應(yīng)的一個(gè)新機(jī)制,也提示MET可能在ALI中具有潛在的應(yīng)用價(jià)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 偽 (tumor necrosis factor- 偽 (TNF- 偽) 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- 偽 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R965
本文編號：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 偽 (tumor necrosis factor- 偽 (TNF- 偽) 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- 偽 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R965
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相關(guān)期刊論文 前3條
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