Study On Changs Of Mitochondrial DNA COX Gene, Expression And Function Of Small Intestine Epithelial Cells In Hemorrhagic Shock Rats And The Effectiveness Of Drug Intervention

Hemorragic shock can introduce the damages of tissue and Organs , especially,the small intestine. The small intestine served as an important target organ in hemorragic shock. Ischemia- hypoxia and the energy metabolism disorders played an important role in the pathogenesis of irreversible shock and multipe system organ failure. Mitochondria was organelle for energy transform. It can maintain the energy metabolism of cell and the structure and the function. Mitochondria contain their own DNA(mtDNA) and replication , transcription and translation system differented from nuclear DNA. Mitochondrial DNA(mtDNA) consist of 16596bp closed-loop doublestranded DNA which encodes 13 polypeptides of oxidative phosphorylation.The major function of mitochondria in human cells is to provide more than 90% ATP. The three largest subunits of cytochrome c oxidases (COX) are encoded by mitochondria DNA. COX is the terminal complex of the mitochondria respiratory chain. It is located in the mitochondrial inner membrane where it transfers electrons from ferrocytochrome c to molecular oxygen. It is the most important enzyme and the functions of mitochondria were impaired when COX was injuried. But what link cause the damages ? Is there the changes of gene expression or protein ? Is there any drug to treat ? The mechanism of the damages is unknow. It remains a matter for further investigation.Objective: (1) To study the damage and the change of expressions of mtDNA encoding COX genes(COX I , COX II and COX III subunits) of intestinal epithelial cells in hemorrhagic shock rats and to explore the relationship between the damages of structure and function induced by mutation of COX genes and to explain the mechanism of the energy metabolism disorder of mitochondria and the damages of cells induced by ischemia- hypoxia of intestinal epithelial cells.(2) To observe the protective effects of Panax notoginseng saponins Rg 1 on mitochondria and to study the effects of Panax notoginseng saponins Rgl on COX activity, and COX genes expressions, and mitochondrial morphology,and function, and survival rate of hemorrhagic shock rats.To explain the mechanism of effects of Panax notoginseng saponins Rg1.Methods: One hundred twenty Wistar rats were used and divided into three groups randomizely: hemorragic shock group, resuscitation group, resuscitation and Rgl treatment group. The present study has been carried using Wigger' s hemorrhagic shock model. Intestinal epithelial cells,mtDNA, mitochondria protein and RNA were isolated. The mutation of COX I, COX n and COX III genes were determined by polymerase chain reaction(PCR) with different primers of COX I, COX II and COX III and directly sequenced the products of PCR. Bioinformatics and computer homology modeling method were used to predict the trimetric space structure of the proteins coded by the normal genes and the mutant genes(COX I, COX II and COX III). Using experiment data and modeling resusts, the relationship between the structure and the function were analyzed.The changes of expressions of mtDNA encoding COX genes(COX I, COX II and COX III subunits) mRNA were determined by Reverse-transcriptional polymerase chain reaction(RT-PCR). The changes of expressions of COX I protein was examined by Western-blot. The mitochondrial morphologic changes were observed by an electron microscope and analysed quantitatively by an electron microscope image analysis system. Mitochondrial respiratory function was measured with Clark oxygen electrode. Cytochrome c oxidase activity was determined by ultraviolet spectrophotometer.Results: (l)The sequenced results of mtDNA COX I gene showed that there were thirteen diffuse point mutation from 5545 to 6245 and a insert mutation between 5692 and 5693 (5692 t 5693)and a lot of diffuse point mutation from 6260-6838 in hemorragic shock rats 5h group, and most of the mutation was G→ A. The sequenced results of mtDNA COX II gene showed that there were the point mutations at 7191(T→C), 7212(T→C), 7235(G →A), 7386(A→G), 7483(A→G)), 7542(C→G)in hemor...