Study On The Liver Mitochondrial Proteome Of Kidney-yang Deficient Rats And The Molecular Mechanism Of Warm And Tonify Kidney-Yang Herbs

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(â‘ the cortin were injected to the rats repeat many times,and withdraw the cortin.â‘¡take the PTU.â‘¢excise Animals's adrenal.â‘£excise 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 synthetaseâ… ,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 synthetaseâ… ,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 synthetaseâ… and hydroxyacyl-Coenzyme A dehydrogenase expressed decreasedly.3.The expression of HSP60,Catalase,glutathione peroxidase,carbamoyl-phosphate synthetaseâ… ,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 synthetaseâ… ,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 synthetaseâ… and 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.