| Hypoxia is a frequent pathological process and is also a main factor of influencing human body in altitude environments. Mitochondrion (mt), the main place for organisms to use oxygen and an important structure for oxidative phosphorylation, is called energy factory. Liver is an important metabolism organ in which metabolism, synthesis, secretion, detoxication and immunization actively take place, so mitochondria in liver has a high rate of energy metabolism and its synthesis and use of energy play an important role in the normal function of liver. Previous researches have revealed that acute hypoxia could inhibit the transcription and translation of mitochondrial DNA (mtDNA) and cause damage to mitochondrial structure and function, but in chronic hypoxia, mitochondrial function could be restored to some extent. Mitochondrial function change of this kind is an important mechanism for organism to acclimatize hypoxia. The change of mitochondrial function is not only related to the change of its structure but also to the change of its related gene expression and the change of its genome sequence and number.Part 1st The initial research about the chang and significance of the copy number of mitochondrial genome in liver tissues of rats in the process of high altitude acclimatizationThere are few reports regarding the change law of the copy number of mtDNA in hypoxia. In this research, the correlation between the copy number of mitochondrial genome and altitude acclimatization and adaptation has been systematically studied by investigating the change laws of mtDNA copy number in the process of altitude acclimatization, the possible causes of the change in the copy number of mitochondrial genome, whether the change in the copy number of mitochondrial genome influences the function of mitochondrial oxidative phosphorylation via mRNA of mitochondrial cytochrome oxidase subunits, and the relationship between the change in the copy number of mitochondrial genome and the change in liver function.Methods Healthy male SD rats were divided into three groups and exposed to a simulated plateau 5000 meters above sea level in a low-pressure chamber for 5 days, 15 days, 30 days respectively, with 23 hours per day. The control group of rats stayed on plain. The blood of the rats in each group subjected to hypoxia was obtained through carotid artery cannula at simulated 5000 meters altitude, but the blood of the rats in the control group was got through carotid artery cannula on the plain. Examinations were conducted with regard to contents of malondialdehyde (MDA), homocysteic acid (HCY) in plasma, activity of superoxide dismutase (SOD), and the level of alanine aminotransferase (ALT) in serum. Then, the rats were decapitated to death and mitochondria in liver were isolated. The activity of complex enzymeâ…£(cytochrome oxidase) in liver mitochondria and the respiratory function of liver mitochondria were detected by means of Clark oxygen electrode. Hepatic tissue, which had been put in liquid nitrogen, was used to extract total DNA and the copy number of mitochondrial genome in liver was examined via quantitative PCR. Total RNA was extracted and quantitative RT-PCR was conducted to determine the expression levels of mRNA of mitochondrion transcription factor A (mtTFA), subunitâ… ,â…¡,â…¢(COXâ… ,COXâ…¡,COXâ…¢) of cytochrome oxidase.Results 1. Plasma MDA content of arterial blood of all hypoxia groups are obviously higher than that of the control group, among which the value of 15-day hypoxic group is obviously higher than the value of 5-day group and of 30-day group. HCY content and SOD activity of blood plasma of all hypoxia groups are obviously lower than the values of the control group, with the values of 15-day group is significantly lower than those of 5-day hypoxic group and of 30-day hypoxic group.2. The mtDNA copy number in rat liver tissue of the 15-day group and the 30-day group were obviously higher than those of the control group and of the 5-day group.3. The mtTFA mRNA level in rat liver tissue of the 15-day group and the 30-day group were obviously higher than those of the control group and of the 5-day group. 4. Between the mtTFA mRNA level or the copy number of mtDNA in liver and content of MDA in blood plasma,there existed a positive correlation relationship; Between the mtTFA mRNA level or the copy number of mtDNA in liver and content of HCY in blood plasma,there existed a negative correlation relationship;Between the mtTFA mRNA level or the copy number of mitochondrial genome in liver and activity of SOD in blood plasma,there existed a negative correlation relationship. Between the copy number of mtDNA and the mtTFA mRNA level, there existed a negative correlation relationship.5. The mRNA levels of liver mtDNA encoding COXI, COXII and COXIII the 15-day group and the 30-day group were obviously higher than those of the control group and of the 5-day group.6. The activity of complex enzymeâ…£(cytochrome oxidase) in liver mitochondria of all hypoxia groups are obviously lower than that of the control group, with the value of 30-day group higher than that of 15-day group. State III respiration (ST3), respiratory control ratio (RCR), and oxidative phosphorylation rate (OPR) in liver mitochondria of all hypoxia groups are obviously lower than the corresponding values of the control group, with RCR and OPR of 30-day group are higher than the corresponding values of 15-day group. State IV respiration in each hypoxic group is significantly higher than that of the control group, with the value of 30-day group higher than that of 15-day group.7. After consecutive 5-day and 15-day hypoxia at simulated 5000 meters above sea level, edema of part of liver mitochondria appeared, with vague membrane structure and disordered crista. After 30-day exposure to hypoxia at simulated 5000 meters above sea level, the shape and structure of liver mitochondria could be restored and became normal.8. Serum ALT level of arterial blood of the 15-day group and the 30-day group are obviously higher than that of the control group. The value of the 30-day group is obviously lower than the value of 15-day group. Between the values of the 30-day group and the 15-day group, there is no significant difference.Conclusion 1. In the process of acclimatization to hypoxia of the simulated high altitude,the mtDNA copy number in liver tissues of rats was significantly increased. There is positive correlation between the mtDNA copy number and the levels of reactive oxygen species in the bodies of rats, there is negative correlation between the mtDNA copy number and the contents of antioxidants and antioxidase in the bodies of rats, There is positive correlation between the mtDNA copy number and mRNA level of mitochondrial transcription factor A (mtTFA), that suggesting that the raise of the levels of reactive oxygen species in the bodies and the increase of the mRNA level of mtTFA is probably an important mechanism of the increase of the mtDNA copy number.2. In the process of acclimatization to hypoxia of the simulated high altitude,the mRNA levels of subunit I, II and III of cytochrome-oxydase of in rat liver mitochondria increased significantly, cytochrome oxidase activity restored gradually, oxidative phosphorylation function in liver mitochondria improve gradually. The mechanism of which is probably related to the increase of mtDNA copy number. The other words , In the process of hypoxia the increase of mtDNA copy numbe is probably an important mechanism of acclimatization of the bodies to high altitude hypoxia.3. Hypoxia could make structure and function of liver damaged. With the prolongation of time course of acclimatization, change of structure and function of liver restored gradually, the mechanism of which is probably related to partial recovery of oxidative phosphorylation function in liver mitochondria of rats in the process of acclimatization to hypoxia.In conclusion, In the process of acclimatization to hypoxia, the increase of the mtDNA copy number is probably an important reason of the improvement of function of liver mitochondria and liver function, which is probably an important mechanism of acclimatization of the bodies to high altitude.Part 2nd The research about mechanism of promotional effect of 1-Octacosanol on high altitude acclimatization1-Octacosanol is a kind of natural fatty alcohol with a long chain, many physiological functions, extensive sources but with no side effects. Our previous researches have demonstrated that 1-Octacosanol has obvious anti-hypoxic function, but its anti-hypoxic function does not lie in improvement of blood-oxygen transport efficiency in organisms. In order to identify mechanism of anti-hypoxic function of 1-Octacosanol, in this research, it has been investigated whether 1-Octacosanol educe its anti-hypoxic function via the copy number of mitochondrial genome. Methods Healthy male SD rats were randomly divided into two groups: administration group and control group, which were exposed to a simulated 5000 m high altitude in a low-pressure chamber for 30 days, with 23 hours per day. For 30 days, with one lavage per day, the administration group were administered 0.5%(m/v) 1-Octacosanol suspension according to 5 mg.kg-1 dosage while the control group were administered edible oil according to 1 ml.kg-1 dosage.The blood of the rats in both groups was obtained through carotid artery cannula at simulated 5000 m altitude. Then above-mentioned indexes were detected by the same methods.Results 1. MDA content of blood plasma of the group insulted by chronic hypoxia and simultaneously administered 1-Octacosanol of 5 mg per kg by lavage were obviously lower than that of the control group, but the plasma Hcy content and SOD activity of the former group were remarkably higher than those of the control group.2. The mtDNA copy number in liver tissue of chronically hypoxic rats simultaneously administered 5 mg per kg 1-Octacosanol by lavage were not significantly different from that of the control group.The mtTFA mRNA level in liver tissue of chronically hypoxic rats simultaneously administered 5 mg per kg 1-Octacosanol by lavage was not significantly different from that of the control group.There was no obvious difference in mRNA level of COXI, COXII and COXIII encoded by mtDNA in rat liver between the group insulted by chronic hypoxia and simultaneously administered 1-Octacosanol of 5 mg per kg by lavage and the control group.3. Cytochrome-oxydase activity of the chronically hypoxic group administered 5 mg per kg 1-Octacosanol by lavage was obviously higher than that of the control group. ST3 respiration of the administration group was not significantly difference from that of the control group, but ST4 respiration of the administration group was obviously lower than that of the control group. In addition, RCR and OPR of the administration group were obviously higher than the corresponding values of the control group.4. In liver cells of chronically hypoxic rats of the control group, hyperplasia, edema, lysis degranulation of endocytoplasmic reticulums appeared. Liver tissue structure of chronically hypoxic rats simultaneously administered 5 mg per kg 1-Octacosanol by lavage didn't take place obviously abnormal changes. 5. The index reflecting liver function (ALT) in administered group was significantly lower than that of the control group.Conclusion 1. 1-Octacosanol could significantly ameliorate impairments of structure and function of liver resulted from chronic hypoxia. Thus 1-Octacosanol can promote high altitude acclimatization very well.2. 1-Octacosanol could remarkably up-regulate cytochrome-oxydase activity, thus increasing oxidation respiration efficiency and ATP synthesis rate in liver mitochondria of chronically hypoxic rats. That is probably an important mechanism about promotional effect of 1-Octacosanol on high altitude acclimatization.3. 1-Octacosanol could not increase mRNA level of mtTFA and mtDNA copy number in liver tissue of chronically hypoxic rats. It could not increase mRNA levels of subunit I, II and III of cytochrome-oxydase also. But 1-Octacosanol could remarkably enhance antioxidation of bodies, relieve oxidative damage of bodies,which is probably an important mechanism about promotional effect of 1-Octacosanol on high altitude acclimatization.In brief, 1-Octacosanol could remarkably enhance antioxidation of bodies, decrease the levels of reactive oxygen species of the organism, relieve oxidative damage of bodies, thus elevating cytochrome-oxydase activity and mitochondrial function, further ameliorating liver function to promote high altitude acclimatization.Summary to the full textIn conclusion, in the process of acclimatization to hypoxia, the increase of the mtDNA copy number is probably an important reason of the improvement of function of liver mitochondria and liver function. 1-Octacosanol could remarkably enhance antioxidation of bodies, decrease the levels of reactive oxygen species of the organism, relieve oxidative damage of bodies, thus elevating cytochrome-oxydase activity and mitochondrial function, further ameliorating liver function to promote high altitude acclimatization.
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