温热中药对寒症模型大鼠肝线粒体蛋白质组的影响

Effects of warm and heat Chinese herbs on liver mitochondria proteome of model rats with cold syndrome

目的应用双向电泳和质谱技术研究温热中药对寒证模型大鼠肝线粒体蛋白质组的影响,阐述寒证与能量代谢的关系及温热中药的作用。方法提取正常大鼠、寒证模型大鼠和温热中药治疗后大鼠的肝线粒体蛋白质,经双向电泳分离后获得蛋白质组图谱,用Image Master 2D 6.0软件进行差异蛋白质分析,筛选出差异蛋白,进行质谱鉴定,用Western blotting法验证差异蛋白的表达。结果寒证大鼠肝线粒体中H+转运ATP合酶、电压依从性阴离子选择性通道蛋白1(VDAC)表达量增加,伴侣素groEL、丝氨酸蛋白酶抑制剂、Tu转运延伸因子、乙醛脱氢酶、二甲基甘氨酸脱氢酶、脂酰辅酶A脱氢酶、热休克蛋白60、酰基辅酶A脱氢酶、丝氨酸蛋白酶、电子传递黄素蛋白、腺苷酸激酶、DJ-1蛋白、羧酸酯水解酶、氨甲酰磷酸合成酶1表达量降低;应用温热中药治疗后F1-ATP合酶、H+转运ATP合酶表达量降低,丙酰辅酶A羧化酶、二氢硫辛酰胺支链酰基转移酶、Tu转运延伸因子、乙酰辅酶A酰基转移酶、脂酰辅酶A脱氢酶、丝氨酸蛋白酶、丙酮酸脱氢酶、3-巯基丙酮酸硫基转移酶、40 S核糖体蛋白、热休克蛋白1、烯酰辅酶A水解酶表达量增加。结论寒证模型大鼠线粒体内脂肪酸的β-氧化受阻,蛋白质的合成、折叠及分泌障碍,类固醇激素减少影响糖代谢,导致能量不足。应用温热中药治疗后,糖、脂代谢增强、蛋白质合成增加导致能量代谢活跃。

Objective The technologies of bidirectional electrophoresis and mass spectrum were used to study the effects of warm and heat Chinese herbs （WHCH） on the liver mitochondria proteome of rats with cold syndrome. Methods The mitochondrial proteins from the rmal rats, the rats with cold syndrome, and the rats treated with WHCH were separated with the bidirectional electrophoresis and the difference proteins were analyzed with Image Master 2D 6.0 software. MALDI-TOF MS and Western blotting were used to identify and verify the expression of difference proteins, respectively. Results In the rats with cold syndrome, the expression of ATP synthase and voltage-dependent anion-selective channel protein 1 increased, while the expression of chaperonin gro EL, serine protease inhibitor, translation elongation factor Tu, aldehyde dehydrogenase, dimethylglycine dehydrogenase, fatty acyl-coenzyme A （CoA） dehydrogenase, heat shock protein （HSP） 60, acyl-CoA dehydrogenase, serine protease, electron transfer flavoprotein, adenylate kinase, DJ-1 protein, carboxylic ester hydrolase, carbamoyl-phosphate synthetase 1 decreased. After the rats with cold syndrome were treated with WHCH, the expression of F 1-ATPase and H＋ transporting ATP synthase decreased, while the expression of propionyl-CoA carboxylase, dihydmlipoamide branched chain transacylase, translation elongation factor Tu, acetyl-CoA acylWansfemse, fatty acyl-CoA dehydrogenase, serine protease, pyruvate dehydrogenase, 3-mercaptopyruvate sulfurtransferase, 40S ribosomal protein, HSP 1, enoyl CoA hydratase increased. Conclusion Energy deficiency appeares in the rats with cold syndrome because of the B-oxidation block of fatty acid, protein synthesis, folding and secretion disorder, and steroid hormones decreasing in mitochondria of rats. After the rats with cold syndrome are treated with WHCH, the glucometabolism, lipid metabolism, and protein synthesis could be increased so as to make the energy metabolism active.