肾阳虚大鼠肝线粒体蛋白组变化及温补肾阳药作用机制的研究
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摘要
几十年来,国内外学者对肾阳虚证本质做了大量的工作,初步证实中医肾阳虚证具有现代病理生理学基础,并阐释了其中医理论的某些细节及其科学依据,对肾阳虚证进行了客观指标的研究,取得了不少的成果,但随着研究工作的深入,也带来了不少困惑与迷惘。中医“证”是疾病发生过程中不同阶段病因病机的高度概括,既然同一证有共同的临床表现和病理机制,那么肯定有共同的物质基础。蛋白质是生命活动的执行者,生物体生长、发育、疾病和中医证候等一切生命现象都是蛋白质表达调控及其效应的结果。国内许多专家认为蛋白质组学的研究方法及研究内容与中医学的整体观、辨证观有许多相似之处,蛋白质组学研究将是中医学现代化的重大突破口。因此,从蛋白质组学角度继续深入研究肾阳虚证的分子机制对于阐明肾阳虚证本质有着重要意义。
目的:
利用凝胶内差异显示电泳和质谱技术研究肾阳虚大鼠肝线粒体蛋白质组变化,并从肝线粒体蛋白质组角度阐明温补肾阳药治疗肾阳虚的分子机制。
方法:
应用4种不同方法制造大鼠肾阳虚模型(①反复多次注射氢化可的松,然后撤去激素造成激素依赖型肾阳虚模型;②服用丙基硫氧嘧碇造成药物性肾阳虚模型;③通过切除动物肾上腺,造成肾上腺功能缺陷的肾阳虚模型;④切除动物甲状腺,直接造成甲状腺功能缺陷的肾阳虚模型),分离正常大鼠、肾阳虚模型大鼠及温补肾阳药治疗后大鼠肝线粒体蛋白质,分别用荧光染料Cy3或Cy5标记。1个Cy3标记的样品,1个Cy5标记样品与等量Cy2标记的内标混合后在同一胶中进行电泳分离,双向电泳后经不同波长光激发扫描得到不同样品的蛋白质组图谱,经DeCyder Image Quant~(TM) V6.5软件分析,选择肾阳虚大鼠与正常大鼠中相差1.2倍以上的蛋白作为差异蛋白,并进行质谱鉴定。
结果:
1.多次反复注射氢化可的松,然后撤去激素造成的肾阳虚大鼠肝线粒体中与细胞保护作用相关的热休克蛋白60和70、与蛋白质代谢有关的肌氨酸脱氢酶和氨甲酰磷酸合成酶I、与核酸代谢有关的亚硫酸氧化酶、与氧化磷酸化相关的ATP合成酶及NADH脱氢酶表达量均增加。但是与糖代谢有关的二氢硫辛酰胺脱氢酶、与脂代谢相关的脂酰辅酶A脱氢酶、与精氨酸代谢相关的鸟氨酸氨基转移酶表达量均下降。应用温补肾阳药治疗后,二氢硫辛酰胺脱氢酶和ATP合成酶表达量增加,而脂酰辅酶A脱氢酶和NADH脱氢酶表达量降低。
2.服用丙基硫氧嘧碇造成肾阳虚大鼠肝线粒体中与机体防御系统相关的热休克蛋白60和谷胱甘肽过氧化物酶、与脂代谢相关的异戊酰辅酶A脱氢酶和酮脂酰辅酶乙酰辅酶A脱氢酶、与蛋白质分解代谢相关的氨甲酰磷酸合成酶I和鸟氨酸转氨甲酰酶表达均增加,但是与糖代谢相关的丙酮酸脱氢酶、与精氨酸代谢相关的鸟氨酸转氨酶表达量下调。应用温补肾阳药治疗后,热休克蛋白60、谷胱甘肽过氧化物酶、丙酮酸脱氢酶、鸟氨酸转氨酶表达量增加,而酮脂酰辅酶乙酰辅酶A脱氢酶α、氨甲酰磷酸合成酶I的表达量有所下调。
3.切除肾上腺造成的肾阳虚动物肝线粒体中与细胞防御系统相关的热休克蛋白60,过氧化氢酶以及谷胱甘肽过氧化物、与蛋白质分解代谢相关的氨甲酰磷酸合成酶I和3-巯基丙酮酸转硫酶、与核酸代谢有关的亚硫酸氧化酶、与氧化磷酸化相关的ATP合成酶和腺苷酸激酶同工酶5表达量均增高。但与糖代谢与柠檬酸循环相关的二氢硫辛酸脱氢酶,醛脱氢酶和丙酮酸脱氢酶表达量降低。经温补肾阳药治疗后,上述这些蛋白质表达量均有所增加。
4.切除甲状腺造成的肾阳虚大鼠肝线粒体中与细胞防御功能相关的热休克蛋白60,过氧化氢酶和谷胱甘肽过氧化物、与蛋白质分解代谢相关的氨甲酰磷酸合成酶I、与氧化磷酸化相关的ATP合成酶,乳铁传递蛋白,ATP酶质子通道蛋白和电子传递黄素蛋白表达量均增加。但与糖代谢相关的二氢硫辛酰胺支链酰基转移酶、与脂代谢相关的脂酰辅酶A脱氢酶、与精氨酸代谢相关鸟氨酸转氨酶表达量均降低。此外,还检测到与细胞内信号传导相关的类钙激活中性蛋白酶及鸟苷酸结合调节蛋白表达量也发生了显著的变化。经温补肾阳药治疗后,与细胞防御、糖类和脂类代谢、氧化磷酸化相关的蛋白质表达量上调,而氨甲酰磷酸合成酶I表达量减少。同时,与细胞内信号传导相关的蛋白质也得到相应的调节。
结论:
1.肾阳虚动物可能因糖类的分解代谢与柠檬酸循环受阻,线粒体膜的损伤导致能量缺乏而出现虚寒症状。
2.温补肾阳药通过调节糖代谢,消除自由基和过氧化物,维护线粒体膜的稳定性,保护线粒体正常生理功能,起到治疗肾阳虚证的作用。
For several decades,the researchers of all around the world had done great hard to reveal the essence of kidney-yang deficiency syndrome,and had tentatively confirmed the there are some modern pathophysiological basis.The details of some traditional Chinese medicine theory and its science nature had been partly explained,and some objective markes had been found.Though there many achievements on the study of kidney deficiency syndrome,some puzzle also remains.The Chinese medicine "syndrome"is the summary of pathology and physiology of the different phases of different diseases.Since thre are some common clinic manifestation and phathomechamism,there must be some common material basis.Protein is the executor of life activity.All life phenomenon such as the growth, develop,disease of organism,and Chinese medicine "syndrome" are the result of regulation and control of protein expression.Many domestic professors think that there are a lot of similarities between the methodology of proteomies and whole discrimination of Chinese medicine,the proteomics would be a great breakthrough of the research on modernization of Chinese medicine.Therefore,to research the molecular mechanisms of kidney-yang drficiency syndrome from the proteomics have great significance.
Objective:
Two-dimensional difference gel electrophoresis and mass spectrum were used to study the liver mitochondrial proteome of the Kidney-yang deficient rats and the molecular mechanism of Warm and Tonify Kidney-Yang Herbs was illuminated from the perspective of hepatic mitochondrial proteome.
Methods:
Four different methods were used to make the kedney-yang deficient rats(①the cortin were injected to the rats repeat many times,and withdraw the cortin.②take the PTU.③excise Animals's adrenal.④excise Animals's thyroid gland).Liver mitochondria proteins of normal rats,kidney-yang deficiency rats and the kidney-yang deficiency rats treated with warm and tonify kidney-yang herbs were separated and labeled with Cy3 or Cy5.Each Cy3-labeled sample and Cy5-labeled sample were mixed on the same 2-D gel along with a Cy2-labeled mixture of all samples as an internal standard.The gels were respectively imaged by Typhoon 9400 using different emission filter and spot-features were analyzed by DeCyder Image Quant~(TM) V6.5 in each paired normal/disease comparison,and using DIGE technology with internal standard,statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation.MALDI-TOF-MS was used to provide sensitive and accurate mass spectral data for the unique proteins that were changed in abundance more than 1.2 folder up or down between normal and kedney yang deficient rats.
Results:
1.The expression of heat shock protein60/70、sarcosine dehydrogenase、carbamoy-lphosphate synthetaseⅠ、ATP synthase、Sulfite oxidas、mitochondrial aldehyde dehydrogenase precursor and NADH dehydrogenase increased in the hormone deficient kidney-yang deficiency rats,while the expression of ornithine aminotransferase、2-oxoisovalerate dehydrogenase、Acyl-CoA dehydrogenase decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,the expression of 2-oxoisovalerate dehydrogenase,ATP synthase increased and the expression of Acyl-CoA dehydrogenase and NADH dehydrogenase decreased.
2.In the kidney-yang deficiency rats,the expression of heat shock protein 60, glutathione peroxidase,isovaleryl Coenzyme A dehydrogenase,Succinyl-CoA synthetase, hydroxyacyl-Coenzyme A dehydrogenase,carbamoyl-phosphate synthetaseⅠ,ornithine transcarbamylase increased,while the expression of pyruvate dehydrogenase and ornithine aminotransferase decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,the expression of heat shock protein 60,glutathione peroxidase,pyruvate dehydrogenase and ornithine aminotransferase increase,while carbamoyl-phosphate synthetaseⅠand hydroxyacyl-Coenzyme A dehydrogenase expressed decreasedly.
3.The expression of HSP60,Catalase,glutathione peroxidase,carbamoyl-phosphate synthetaseⅠ,3-mercaptopyruvate sulfurtransferase,sulfite oxidase,ATP synthase and Adenylate kinase isoenzyme 5 increased in the adrenalectomized Kidney-Yang deficiency Rats,while the expression of Dihydrolipoyl dehydrogenase,aldehyde dehydrogenase and pyruvate dehydrogenase decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,all these proteins expressed increasedly.
4.The expression of HSP60,Catalase,glutathione peroxidase,carbamoyl-phosphate synthetaseⅠ,ATP synthase,lactotransferrin,H+-transporting two-sector ATPase,alpha-ETF and calpain 12 increased in the thyroidectomized Kidney-Yang deficiency Rats,while the expression of oxoisovalerate dehydrogenase,Acyl-CoA dehydrogenase,Ornithine aminotransferase and GTP-binding regulatory protein decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,the expression of HSP60,Catalase,glutathione peroxidase,oxoisovalerate dehydrogenase,Acyl-CoA dehydrogenase,ATP synthase,lactotransferrin,H+-transporting two-sector ATPase, alpha-ETF and GTP-binding regulatory protein increased,and the expression of carbamoyl-phosphate synthetaseⅠand calpain 12 decreased.
Conclusion:
1.Because the carbohydrate catabolism and tricarboxylic acid cycle were restrained and mitochondrial membrane was damaged,the kidney-yang deficient rats was shot of energy and appeared the clinic deficiency-cold symptom.
2.Warm and Tonify Kidney-Yang Herbs could treated the kidney-yang deficiency by regulating the carbohydrate catabolism,eliminating the oxygen radicals and peroxides, maintaining stability of mitochondria and protecting normal function.
目的:
利用凝胶内差异显示电泳和质谱技术研究肾阳虚大鼠肝线粒体蛋白质组变化,并从肝线粒体蛋白质组角度阐明温补肾阳药治疗肾阳虚的分子机制。
方法:
应用4种不同方法制造大鼠肾阳虚模型(①反复多次注射氢化可的松,然后撤去激素造成激素依赖型肾阳虚模型;②服用丙基硫氧嘧碇造成药物性肾阳虚模型;③通过切除动物肾上腺,造成肾上腺功能缺陷的肾阳虚模型;④切除动物甲状腺,直接造成甲状腺功能缺陷的肾阳虚模型),分离正常大鼠、肾阳虚模型大鼠及温补肾阳药治疗后大鼠肝线粒体蛋白质,分别用荧光染料Cy3或Cy5标记。1个Cy3标记的样品,1个Cy5标记样品与等量Cy2标记的内标混合后在同一胶中进行电泳分离,双向电泳后经不同波长光激发扫描得到不同样品的蛋白质组图谱,经DeCyder Image Quant~(TM) V6.5软件分析,选择肾阳虚大鼠与正常大鼠中相差1.2倍以上的蛋白作为差异蛋白,并进行质谱鉴定。
结果:
1.多次反复注射氢化可的松,然后撤去激素造成的肾阳虚大鼠肝线粒体中与细胞保护作用相关的热休克蛋白60和70、与蛋白质代谢有关的肌氨酸脱氢酶和氨甲酰磷酸合成酶I、与核酸代谢有关的亚硫酸氧化酶、与氧化磷酸化相关的ATP合成酶及NADH脱氢酶表达量均增加。但是与糖代谢有关的二氢硫辛酰胺脱氢酶、与脂代谢相关的脂酰辅酶A脱氢酶、与精氨酸代谢相关的鸟氨酸氨基转移酶表达量均下降。应用温补肾阳药治疗后,二氢硫辛酰胺脱氢酶和ATP合成酶表达量增加,而脂酰辅酶A脱氢酶和NADH脱氢酶表达量降低。
2.服用丙基硫氧嘧碇造成肾阳虚大鼠肝线粒体中与机体防御系统相关的热休克蛋白60和谷胱甘肽过氧化物酶、与脂代谢相关的异戊酰辅酶A脱氢酶和酮脂酰辅酶乙酰辅酶A脱氢酶、与蛋白质分解代谢相关的氨甲酰磷酸合成酶I和鸟氨酸转氨甲酰酶表达均增加,但是与糖代谢相关的丙酮酸脱氢酶、与精氨酸代谢相关的鸟氨酸转氨酶表达量下调。应用温补肾阳药治疗后,热休克蛋白60、谷胱甘肽过氧化物酶、丙酮酸脱氢酶、鸟氨酸转氨酶表达量增加,而酮脂酰辅酶乙酰辅酶A脱氢酶α、氨甲酰磷酸合成酶I的表达量有所下调。
3.切除肾上腺造成的肾阳虚动物肝线粒体中与细胞防御系统相关的热休克蛋白60,过氧化氢酶以及谷胱甘肽过氧化物、与蛋白质分解代谢相关的氨甲酰磷酸合成酶I和3-巯基丙酮酸转硫酶、与核酸代谢有关的亚硫酸氧化酶、与氧化磷酸化相关的ATP合成酶和腺苷酸激酶同工酶5表达量均增高。但与糖代谢与柠檬酸循环相关的二氢硫辛酸脱氢酶,醛脱氢酶和丙酮酸脱氢酶表达量降低。经温补肾阳药治疗后,上述这些蛋白质表达量均有所增加。
4.切除甲状腺造成的肾阳虚大鼠肝线粒体中与细胞防御功能相关的热休克蛋白60,过氧化氢酶和谷胱甘肽过氧化物、与蛋白质分解代谢相关的氨甲酰磷酸合成酶I、与氧化磷酸化相关的ATP合成酶,乳铁传递蛋白,ATP酶质子通道蛋白和电子传递黄素蛋白表达量均增加。但与糖代谢相关的二氢硫辛酰胺支链酰基转移酶、与脂代谢相关的脂酰辅酶A脱氢酶、与精氨酸代谢相关鸟氨酸转氨酶表达量均降低。此外,还检测到与细胞内信号传导相关的类钙激活中性蛋白酶及鸟苷酸结合调节蛋白表达量也发生了显著的变化。经温补肾阳药治疗后,与细胞防御、糖类和脂类代谢、氧化磷酸化相关的蛋白质表达量上调,而氨甲酰磷酸合成酶I表达量减少。同时,与细胞内信号传导相关的蛋白质也得到相应的调节。
结论:
1.肾阳虚动物可能因糖类的分解代谢与柠檬酸循环受阻,线粒体膜的损伤导致能量缺乏而出现虚寒症状。
2.温补肾阳药通过调节糖代谢,消除自由基和过氧化物,维护线粒体膜的稳定性,保护线粒体正常生理功能,起到治疗肾阳虚证的作用。
For several decades,the researchers of all around the world had done great hard to reveal the essence of kidney-yang deficiency syndrome,and had tentatively confirmed the there are some modern pathophysiological basis.The details of some traditional Chinese medicine theory and its science nature had been partly explained,and some objective markes had been found.Though there many achievements on the study of kidney deficiency syndrome,some puzzle also remains.The Chinese medicine "syndrome"is the summary of pathology and physiology of the different phases of different diseases.Since thre are some common clinic manifestation and phathomechamism,there must be some common material basis.Protein is the executor of life activity.All life phenomenon such as the growth, develop,disease of organism,and Chinese medicine "syndrome" are the result of regulation and control of protein expression.Many domestic professors think that there are a lot of similarities between the methodology of proteomies and whole discrimination of Chinese medicine,the proteomics would be a great breakthrough of the research on modernization of Chinese medicine.Therefore,to research the molecular mechanisms of kidney-yang drficiency syndrome from the proteomics have great significance.
Objective:
Two-dimensional difference gel electrophoresis and mass spectrum were used to study the liver mitochondrial proteome of the Kidney-yang deficient rats and the molecular mechanism of Warm and Tonify Kidney-Yang Herbs was illuminated from the perspective of hepatic mitochondrial proteome.
Methods:
Four different methods were used to make the kedney-yang deficient rats(①the cortin were injected to the rats repeat many times,and withdraw the cortin.②take the PTU.③excise Animals's adrenal.④excise Animals's thyroid gland).Liver mitochondria proteins of normal rats,kidney-yang deficiency rats and the kidney-yang deficiency rats treated with warm and tonify kidney-yang herbs were separated and labeled with Cy3 or Cy5.Each Cy3-labeled sample and Cy5-labeled sample were mixed on the same 2-D gel along with a Cy2-labeled mixture of all samples as an internal standard.The gels were respectively imaged by Typhoon 9400 using different emission filter and spot-features were analyzed by DeCyder Image Quant~(TM) V6.5 in each paired normal/disease comparison,and using DIGE technology with internal standard,statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation.MALDI-TOF-MS was used to provide sensitive and accurate mass spectral data for the unique proteins that were changed in abundance more than 1.2 folder up or down between normal and kedney yang deficient rats.
Results:
1.The expression of heat shock protein60/70、sarcosine dehydrogenase、carbamoy-lphosphate synthetaseⅠ、ATP synthase、Sulfite oxidas、mitochondrial aldehyde dehydrogenase precursor and NADH dehydrogenase increased in the hormone deficient kidney-yang deficiency rats,while the expression of ornithine aminotransferase、2-oxoisovalerate dehydrogenase、Acyl-CoA dehydrogenase decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,the expression of 2-oxoisovalerate dehydrogenase,ATP synthase increased and the expression of Acyl-CoA dehydrogenase and NADH dehydrogenase decreased.
2.In the kidney-yang deficiency rats,the expression of heat shock protein 60, glutathione peroxidase,isovaleryl Coenzyme A dehydrogenase,Succinyl-CoA synthetase, hydroxyacyl-Coenzyme A dehydrogenase,carbamoyl-phosphate synthetaseⅠ,ornithine transcarbamylase increased,while the expression of pyruvate dehydrogenase and ornithine aminotransferase decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,the expression of heat shock protein 60,glutathione peroxidase,pyruvate dehydrogenase and ornithine aminotransferase increase,while carbamoyl-phosphate synthetaseⅠand hydroxyacyl-Coenzyme A dehydrogenase expressed decreasedly.
3.The expression of HSP60,Catalase,glutathione peroxidase,carbamoyl-phosphate synthetaseⅠ,3-mercaptopyruvate sulfurtransferase,sulfite oxidase,ATP synthase and Adenylate kinase isoenzyme 5 increased in the adrenalectomized Kidney-Yang deficiency Rats,while the expression of Dihydrolipoyl dehydrogenase,aldehyde dehydrogenase and pyruvate dehydrogenase decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,all these proteins expressed increasedly.
4.The expression of HSP60,Catalase,glutathione peroxidase,carbamoyl-phosphate synthetaseⅠ,ATP synthase,lactotransferrin,H+-transporting two-sector ATPase,alpha-ETF and calpain 12 increased in the thyroidectomized Kidney-Yang deficiency Rats,while the expression of oxoisovalerate dehydrogenase,Acyl-CoA dehydrogenase,Ornithine aminotransferase and GTP-binding regulatory protein decreased.After the kidney-yang deficiency rats were treated with warm and tonify kidney-yang herbs,the expression of HSP60,Catalase,glutathione peroxidase,oxoisovalerate dehydrogenase,Acyl-CoA dehydrogenase,ATP synthase,lactotransferrin,H+-transporting two-sector ATPase, alpha-ETF and GTP-binding regulatory protein increased,and the expression of carbamoyl-phosphate synthetaseⅠand calpain 12 decreased.
Conclusion:
1.Because the carbohydrate catabolism and tricarboxylic acid cycle were restrained and mitochondrial membrane was damaged,the kidney-yang deficient rats was shot of energy and appeared the clinic deficiency-cold symptom.
2.Warm and Tonify Kidney-Yang Herbs could treated the kidney-yang deficiency by regulating the carbohydrate catabolism,eliminating the oxygen radicals and peroxides, maintaining stability of mitochondria and protecting normal function.
引文
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