黄芪汤对慢性胆汁淤积继发肌少症大鼠骨骼肌细胞线粒体损伤的影响

Effects of Huangqi Decoction on mitochondrial damage of skeletal muscle cells in rats with sarcopenia secondary to chronic cholestasis

目的:基于骨骼肌细胞线粒体损伤探讨α-萘基异硫氰酸酯(ANIT)诱导慢性胆汁淤积继发肌少症大鼠的致病机制及黄芪汤的调控效应。方法:40只雄性Wistar大鼠随机分为正常组、模型组、低剂量黄芪汤组、高剂量黄芪汤组,每组10只。除正常组外,其余各组给予ANIT-橄榄油溶液灌胃,隔日1次,连续40周,诱导慢性胆汁淤积继发肌少症大鼠模型。于第33周首日分别给予低、高剂量(0.59、1.76 g/kg)黄芪汤灌胃,每日1次,连续8周。第40周末次给药后,收集外周血和肝、骨骼肌组织,记录肌肉质量、肌力,HE、天狼星红染色观察肝、骨骼肌组织病理学变化,透射电镜观察骨骼肌细胞线粒体超微形态结构,生化方法检测血清肝功能指标,JC-1法检测骨骼肌细胞线粒体膜电位,生物发光法检测骨骼肌组织三磷酸腺苷(ATP)含量,Western Blot检测骨骼肌MAFbx/Atrogin-1、MuRF1、LC3B、PINK1、Bnip3、UCP2蛋白表达。结果:模型组大鼠肌肉质量、肌力,肝、骨骼肌组织病理、骨骼肌细胞线粒体超微形态结构,血清肝功能指标,骨骼肌细胞线粒体膜电位、线粒体ATP含量,骨骼肌组织MAFbx/Atrogin-1、MuRF1、LC3B、PINK1、Bnip3、UCP2蛋白表达均发生显著改变(P<0.01)。两种不同剂量黄芪汤对大鼠肌力,肝、骨骼肌组织病理、骨骼肌细胞线粒体超微形态结构,血清肝功能指标,骨骼肌细胞线粒体膜电位、线粒体ATP含量,骨骼肌MAFbx/Atrogin-1、MuRF1、LC3B、PINK1、Bnip3、UCP2蛋白表达均有显著改善(P<0.05,P<0.01)。结论:骨骼肌细胞线粒体能量合成障碍,线粒体自噬过度,促进骨骼肌蛋白分解增加,可能是慢性胆汁淤积继发肌少症的重要病理机制之一;黄芪汤可通过改善骨骼肌细胞线粒体能量合成和线粒体自噬等损伤,减少肌蛋白分解,阻止骨骼肌萎缩,改善肌力,发挥防治慢性胆汁淤积继发肌少症的作用。

Objective:To explore the pathogenic mechanism of sarcopenia secondary to chronic cholestasis and the regulatory effect of Huangqi Decoction based on skeletal muscle cell mitochondrial damage.Methods:Forty male Wistar rats were randomly divided into normal group,model group,low-dose Huangqi Decoction group and high-dose Huangqi Decoction group,with 10 rats in each group.Except for the normal group,the other groups were givenα-naphthalene isothiocyanateolive oil solution once every other day for 40 weeks to induce the rat model of chronic cholestatic liver disease complicated with sarcopenia.The low and high-dose(0.59,1.76 g/kg)of Huangqi Decoction were given on the first day of the 33rd week,once a day for 8 consecutive weeks and ended at the end of the 40th week.The peripheral blood,liver and skeletal muscle tissue were collected.The muscle mass and strength were recorded.The histopathological changes of liver and skeletal muscle tissue were observed by HE and Sirius red staining.Transmission electron microscopy was used to observe the ultrastructural and morphological changes of mitochondria in skeletal muscle cells.The serum liver function indexes were detected by routine biochemical methods.JC-1 probe was used to detect mitochondrial membrane potential in skeletal muscle cells.Detection of ATP concentration in skeletal muscle tissue by bioluminescence.The expression of MAFbx/Atrogin-1,MuRF1,LC3B,PINK1,Bnip3 and UCP2 in skeletal muscle tissue were detected by Western Blot.Results:The muscle mass,muscle strength,histopathology of liver and skeletal muscle tissue,mitochondrial ultrastructure of skeletal muscle cells,serum liver function indexes,mitochondrial membrane potential and mitochondrial ATP content of skeletal muscle cell,and the expression of MAFbx/Atrogin-1,MuRF1,LC3B,PINK1,Bnip3 and UCP2 protein in skeletal muscle tissue were significantly changed in the model group(P<0.01).Both low-dose and high-dose Huangqi Decoction could significantly improve the muscle strength,histopathology of liver and skeletal muscle tissue,mitochondrial ultrastructure of skeletal muscle cells,serum liver function indexes,mitochondrial membrane potential and mitochondrial ATP content of skeletal muscle cell,and the expression of MAFbx/Atrogin-1,MuRF1,LC3B,PINK1,Bnip3 and UCP2 protein in skeletal muscle tissue(P<0.05,P<0.01).Conclusion:The disturbance of mitochondrial energy synthesis,excessive mitochondrial autophagy and the increase of skeletal muscle protein decomposition in skeletal muscle cells may be one of the main pathological mechanisms of sarcopenia secondary to chronic cholestasis.Huangqi Decoction can reduce the breakdown of skeletal muscle protein by improving mitochondrial energy synthesis and mitochondrial autophagy in skeletal muscle cells,thereby preventing skeletal muscle atrophy,improving muscle strength,and playing a role in the prevention and treatment of sarcopenia secondary to chronic cholestasis.