HMB在小鼠急性呼吸窘迫综合征相关ICU获得性衰弱中的作用及其机制

Effect and mechanism of HMB on ICU acquired weakness associated with ARDS

目的探讨β-羟基-β-甲基丁酸(HMB)在小鼠急性呼吸窘迫综合征(ARDS)相关ICU获得性衰弱(ICU-AW)中的作用及其机制。方法将40只SPF级雄性C57BL/6小鼠随机分为对照组、假手术组、模型组及HMB组,每组10只。模型组及HMB组小鼠在气管内注入3μg/g脂多糖(LPS),制备ARDS相关ICU-AW模型,假手术组注入等量无菌水,对照组不予操作。造模第2天,HMB组给予340 mg/(kg·d)HMB灌胃,其余3组给予等体积生理盐水,连续灌胃2周。另取与HMB组相同处理的小鼠20只,随机分为ARQ-092组与Akt抑制剂对照组,每组10只。从造模第2天开始,每天在HMB灌胃10 h后口服Akt抑制剂(ARQ-092组)或等体积载体(Akt抑制剂对照组),持续给药12 d。测量各组小鼠前肢肌肉抓力、肌肉减少指数(SI);HE染色观察肺组织及肌肉组织病理变化;qRT-PCR检测小鼠腓肠肌中Akt、FoxO3a、Atrogin1及MuRF1的mRNA表达水平;Western blotting检测小鼠腓肠肌中Akt/FoxO3a通路相关蛋白表达水平。结果HMB组小鼠的前肢肌肉抓力及SI均明显高于模型组(P<0.05)。HE染色结果显示,对照组及假手术组肺组织结构正常;模型组肺泡间隔可见明显增厚、断裂,结构紊乱,炎性细胞浸润;HMB组肺组织损伤程度较模型组轻。对照组及假手术组小鼠的腓肠肌肌束结构正常;模型组出现肌纤维萎缩、数量减少,肌束结构破坏,横截面积减少;HMB组小鼠腓肠肌损伤程度较轻。与模型组比较,HMB组小鼠腓肠肌中Akt及FoxO3a mRNA表达水平均明显升高(P<0.05),Akt及FoxO3a蛋白磷酸化水平也明显升高(P<0.05),而Atrogin1及MuRF1的mRNA及蛋白表达水平均明显降低(P<0.05)。结论HMB可通过调控Akt-FoxO3a-MuRF1/Atrogin1信号通路在ICU-AW中发挥保护作用,可能对ICU-AW的防治具有重要价值,而Akt抑制剂ARQ-092能够逆转HMB的此种保护作用。

Objective To explore the role and mechanism ofβ-hydroxy-β-methyl butyric acid(HMB)in intensive care unit-acquired weakness(ICU-AW)associated with acute respiratory distress syndrome(ARDS).Methods Forty SPF grade male C57BL/6 mice were randomly divided into control group,sham operation group,model group,and HMB group,with 10 mice in each group.Model group and HMB group were treated with 3μg/g lipopolysaccharide(LPS)by intratracheal injection to prepare the ICU-AW model associated with ARDS.Sham operation group received the same amount of sterile water.No procedures for control group.On the second day of modeling,mice in HMB group were given 340 mg/(kg·d)HMB by intragastric administration,and mice in the other three groups were given an equal volume of normal saline for continuous intragastric administration for two weeks.Additional 20 mice were randomized into the ARQ-092 group and Akt inhibitor control group,with 10 mice in each group.On the second day of modeling,both groups were given Akt inhibitor(ARQ-092 group)or an equivalent volume of the carrier(Akt inhibitor control group)orally for 10 hours after HMB intragastric administration for 12 days.We evaluated the grasping force of forelimb muscles and measured the sarcopenia index(SI).HE staining was used to observe the pathological changes in lung and muscle tissues.The mRNA expressions of Akt,FoxO3a,Atrogin1,and MuRF1 in mouse gastrocnemius were tested by qRT-PCR,and the expression levels of Akt/FoxO3a pathway related proteins in mouse gastrocnemius was further detected by Western blotting.Results Compared with the model group,the grasping force and SI of forelimb muscle in HMB group were significantly higher(P<0.05).HE staining revealed regular lung tissue structure in control group and sham operation group.Alveolar septa in model group were thickened and fractured,with structural disorder and inflammatory cell infiltration.The injury degree of lung tissue in HMB group was lighter compared with the model group.We observed normal phenotypes of the muscle tracts of gastrocnemius in control group and sham operation group.In model group,we detected muscle fiber atrophy and decreased quantity,muscle bundle structure destruction,and decreased cross-sectional area.The injury degree of the gastrocnemius muscle in HMB group was mild.In addition,compared with model group,the mRNA expressions of Akt and FoxO3a in the gastrocnemius of HMB group were significantly increased(P<0.05),and the phosphorylation levels of Akt and FoxO3a protein were also increased(P<0.05),the levels of Atrogin1 and MuRF1 mRNA and protein expressions decreased(P<0.05).Conclusion HMB can play a protective role in ICU-AW by regulating the Akt-FoxO3A-MurF1/Atrogin1 signaling pathway,which may be valuable for ICU-AW prevention and treatment.The Akt inhibitor ARQ-092 reversed the protective effect of HMB.