Based On The Pathway Of Mitochondrial Oxidative Phosphorylation To Study The Hypoenergy Metabolism Of Kidney-yang Deficiency Syndrome And The Mechanism Of Action Of Yougui Pill

Objectives Through the signal pathway PCR chip technology,the mitochondrial function of rats with kidney-yang deficiency was studied,and the role of mitochondrial oxidative phosphorylation pathway in the development of kidney-yang deficiency and the intervention mechanism of Yougui Wan were investigated.MethodELISA method was used to determine various biochemical indicators,and pathological changes were observed under light microscope;lens electron microscope was used to observe the changes in the ultrastructure of rat kidney mitochondria;ATP content determination kit was used to determine mitochondrial ATP content;rat renal cortex was isolated,Use DCFH-DA cell ROS detection kit to detect intracell μ lar reactive oxygen species;flow cytometry to detect mitochondria,calc μ late the mitochondrial membrane potential by the ratio of red fluorescence(PI-H)intensity to green fluorescence(FITC-H)intensity;Fluorescence quantitative PCR method was used to detect the content of mitochondrial gene Cyt B and 28 S of nuclear gene in each group,and the relative content of cell mtDNA was calc μ lated based on the ratio of Cty B to 28 S copy number;using mitochondrial oxidative phosphorylation pathway gene chip,through RNA quality inspection,c DNA synthesis,real-time quantification PCR,using 2-△ △ CT method to obtain the significant expression of mitochondrial energy metabolism genes in each group.And carry out the biological function analysis of the target gene.Result1.Evaluation resμlts of the rat modelCompared with the normal group,the rats in the model group showed obvious listlessness after successfμl modeling,dμll coat color,hunched back,lower diet,body temperature,weight loss,some loose stools,pale auricle collaterals,pale tongue purple.2.Pathological and biochemical test resμltThe light microscope results showed that compared with the normal group,the renal tubular epithelial cells in the model group showed swelling,partial vacuolar degeneration,and proliferation of mesangial cells.With the increase of the therapeutic dose of Yougui Pills,the glomerular blood vessels became clear,the endothelial cell swelling was reduced,the vacuole partly disappeared,the mesangial cell proliferation improved,and the high-dose group improved the most.Compared with the normal group,the serum TSH,ACTH,CORT,T3,T4,and content of the model group were significantly reduced(p<0.01),and the content of Yougui pill increased after different doses of treatment,and the high-dose group was the most significant(p<0.01),the difference is statistically significant.3.Mitochondrial function evaluation resμltsThe electron microscope results showed that compared with the normal group,the kidney mitochondria of the model group showed swelling,a small amount of vacuoles,scattered electron densities,degeneration of podocytes,and partial disappearance of foot processes.After the intervention of different doses of Yougui Pill,the structure of mitochondrial ridge and foot process gradually became clear,and the edema and vacuole were improved.Among them,the high-dose improvement was the most significant.Compared with the normal group,the ATP content,DNA copy number(relative content of mtDNA),and mitochondrial membrane potential in the renal cortex of the model group were significantly reduced(p<0.01),while the content of reactive oxygen species in the cells was higher(p<0.01).The difference is statistically significant.After the intervention of Yougui Pill,the ATP content,DNA copy number(relative content of mtDNA),and mitochondrial membrane potential in the renal cortex of rats in each group increased,and the content of intracellular reactive oxygen species decreased,and the changes were most obvious in the high-dose group.4.The results of gene expression of mitochondrial oxidative phosphorylation pathway in kidney-yang deficiency syndromeResults of differentially expressed genes: A total of 18 differentially expressed genes between the kidney-yang deficiency syndrome group and the normal control group,of which 1 were up-regulated and 17 were down-regulated;there were a total of 20 differentially expressed genes between the kidney-yang deficiency syndrome group and the Yougui pill treatment group.Among them,2 were up-regulated and 18 were down-regulated.There were 9 differentially expressed genes in the Yougui Pill treatment group and the normal group,of which 4 were up-regulated and 5 were down-regulated.After the intersection of the difference genes between the groups,the 7 basic causes(Atp5h,Ndufa2,Ndufb7,Ndufs3,Ndufv2,Ndufb9,Uqcrb)are closely related to the syndrome of kidney-yang deficiency,and they are all down-regulated genes.After the intervention of Yougui Pill,their expression levels Significantly up-regulated(p<0.05).Conclusion1.Yougui pill can improve the yang deficiency state of rats with kidney-yang deficiency,adjust hormone levels,and enhance the mitochondrial function status of rats;different doses of Yougui pill can affect the performance of kidney-yang deficiency,kidney damage and mitochondrial function in rats Healing effect,but the intervention effect of the high-dose group is better and more significant.2.Yougui Pill regulates the low energy metabolism of the kidney-yang deficiency syndrome by interfering with the target genes in the oxidative phosphorylation signal pathway(Atp5h,Ndufa2,Ndufb7,Ndufs3,Ndufv2,Ndufb9,Uqcrb).3.In the kidney tissue of hormonal kidney-yang deficiency rats,the genes related to the oxidative forest acidification pathway are all under-expressed,suggesting that the low energy metabolism of kidney-yang deficiency may be related to the limited redox reaction or phosphoric acid caused by the decreased activity of these enzymes.The chemical process is blocked,and Yougui Pill can promote the active function of related enzymes,restore energy access to normal,thereby improving the state of low energy metabolism in kidney-yang deficiency syndrome.