小檗碱对糖尿病db／db小鼠骨骼肌蛋白质代谢的影响及机制研究

Effects of berberine on skeletal muscle protein metabolism in db/db mice and study of its mechanism

目的探讨小檗碱对糖尿病／胰岛素抵抗模型db／db小鼠骨骼肌蛋白质代谢及肌萎缩蛋白Fbox-1（Atrogin-1）表达的影响。方法db／db小鼠为研究对象，以野生型为对照组。予小檗碱（5mg·kg-1·d-1）腹腔注射3周，观察体重和食量；3周末处死，分离胫骨前肌和腓肠肌。胫骨前肌冰冻切片行层黏连蛋白免疫组化染色，荧光显微镜下观察并拍照，测量肌纤维横截面积并计算肌纤维面积分布图；同位素[14C]．苯丙氨酸掺入法检测肌肉蛋白合成，[3H]-酪氨酸释放率分析检测肌肉分解代谢；Northern印迹检测腓肠肌Atrogin-1和肌环指蛋白1（MurF-1）转录水平，Western印迹检测蛋白翻译水平。结果小檗碱能明显降低db／db小鼠血糖水平[（18．55±3．79对26．32±4．02）mmol／L，P〈O．01]，降低脂肪重量[（2．75±0．30对3．77±0．52）g，P〈O．05]，使胫骨前肌的肌重／胫骨长度比值及肌纤维横截面积下降，肌纤维面积分布图明显左移；小檗碱使野生型小鼠和db／db小鼠的蛋白质合成率下降18％～22％；蛋白质分解率升高24％～26％，使肌肉特异性的Atrogin-1、MurF-1转录和翻译水平增高，同时使真核转录因子3调节亚基（eIF3-f）蛋白水平降低。结论小檗碱具有降糖降脂作用，但可引起骨骼肌蛋白质合成下降，促进蛋白质分解代谢，加剧糖尿病的骨骼肌消耗，其机制与上调Atrogin-1和MurF-1表达，同时下调eIF3-f有关。

Objective To study the effect of berberine on diabetes or insulin resistance accompanied with reduction of skeletal muscle and wasting in db/db mice. Methods db/db mouse-a model of diabetes/insulin- resistance was studied, with the wild type mouse as control. After being treated with berberine （ 5 mg ·kg-1· d-1 ） for 3 weeks, the muscle size of tibia anterior （TA） of the animals was measured after staining with laminine/Hyosin Heavy chain-Slow using immunochemistry, then observed under fluorescent microscope and calculated with software. The rate of [ 14 C ] -Phenalanine incorporation into the muscle was measured to analyze the protein synthesis, and the [ 3 H ] - Tyrosine released into the medium was determined in order to analyze protein degradation. The mRNA expressions of muscle atrophy Fbox-1 （ Atrogin-1 ） and muscle ring finger-1 （ MuRF-1 ） were measured by Northern blot. Results With berberine treatment, blood glucose and fat levels were lowered [ （ 18.55 ± 3.79 vs 26.32 ± 4.02 ） mmol/L, P〈 O. O1 ; （ 2.75 ~± O. 30 vs 3.77± O. 52 ） g, P〈0.05 ], but tibia anterior muscle weight/length ratio and cross-section area were decreased, rates of protein synthesis in isolated muscles of db/db mice were decreased by 18% -22%, and the rates of degradation were significantly raised by 24%-26% after berberine treatment. There also was increased the transcription and translation of Atrogin-1 and MuRF-1, accompanied with decreased eukaryotic initiation factor 3 subunit （eIF3-f） protein level simultaneously. Conclusion Berberine improves hyperglycemia and insulin resistance by down-regulating blood sugar and body fat, but it causes reduced protein synthesis and minimally enhanced protein degradation. The mechanism might be related to berberine-induced up-regulating Atrogin-1, MurF-1 and down-regulating elF3-f.