Effects Of Oxymatrine On Respiratory Oxygen Consumption And Energy Synthesis Of Brain Mitochondria From Rat Exposed To Simulated High Altitude Hypoxia And Its Anti-hypoxic Effectiveness

The damnification and malfunction of the tissue and organ are main threat againstorganism which primarily exposed to high altitude hypoxia. The most important purpose ofthe high altitude medicine is to reduce the incidence of a high altitude disease and improvethe survival and labour ability of human body. The malfunction of mitochondrial, which isthe main location of oxygen consumption, is the key pathophysiologic process in hypoxicdisease. The lower efficiency of oxygen consumption and energy production caused byuncoupling between oxidation and phosphorylation is the characteristics of mitochondrialmalfunction. Uncoupling proteins (UCPs), which located in inner membrane ofmitochondria and resulted in proton leak, is the primary factor leaded to uncouple ofoxidative phosphorylation. There are increase in uncoupling oxygen consumption anddecreases in mitochondria membrane potential (MMP) and ATP synthesis in rat’s brainmitochondria after exposure to simulated high altitude hypoxia. The Chinese medicine isextensive and profound. Chinese traditional medicine has been widely used in clinic. Andthe Chinese herebal monomers, which are characterized by clear constructure, unitaryelement and corresponding effect, is an ideal object in laboratory study. Oxymatrane(OMT), an alkaloid extracted from sophora Flavescens, possesses many pharmacologicaleffects. In this experiment, Chinese herebal monomers which could effectively inhibit theuncoupling and improve oxygen consumption of mitochondria were screened by testing theinfluence of12monomers on the oxidative phosphorylation of mitochondria in vitro.Furthermore, we observed the effects of OMT on the energy production of mitochondriaand the role of the UCPs in the process. At last, the anti-hypoxic effectiveness of OMT invivo was investigated in mice. This experiment could provide laboratory demonstrates forprevention and treatment of high altitude disease.Methods1. Adult male SD rats were randomly divided into sea level control group and high altitude hypoxic group. The latter was exposed to a hypobaric chamber simulated5000mhigh altitude for23h/d for3d. Rat was sacrificed by decapitation in sea level andsimulativing high altitude, respectively. Brain mitochondria were isolated by centrifugationmethod. After treated with different concentrations of12Chinese herbal monomers,mitochondria oxidative respiratory function was measured by Clark oxygen electrode.Mitochondrial membrane potential was detected by Rhodamine123method. The monomerswhich could elevate mitochondrial respirative activity of hypoxia-exposed rat brain werethen screened.2. Adult male SD rats were randomly divided into normal control, hypoxic control,normal intervention and hypoxic intervention groups. The drug intervation models of braintissue and isolation mitochondria in vitro were set up. The effects of2mmol/L of OMT (1hand5min) on oxidative phosphorylation of mitochondria and the role of UCPs in theprocess were then studied. Mitochondria oxidative respiratory function was measured byClark oxygen electrode. Mitochondrial membrane potential was detected by Rhodamine123method. The UCPs’ activity and content was detected by [~3H]-GTP binding method. Thesize of adenine nucleotide pool (ATP、ADP、AMP) in mitochondria was measured by highperformance liquid chromatography(HPLC). The mRNA and protein expression of UCP4and UCP5in rat brain were determined by Real Time RT-PCR and Western blot analysisrespectively.3. Healthy Kunming mice were randomly set into normal group and drug interventiongroups. Mice were intraperitoneally injected different doses of OMT or Ginsenoside Rg1before hypoxic administration in different times. The standard toleration time of mice tohermetic hypoxia was used to evaluate the anti-hypoxic ability of drugs.Results1. OMT and Ginsenoside Rg1could promote the mitochondrial oxidativephosphorylation efficiency and elevate the mitochondrial membrane potential ofhypoxia-exposed rat by inhibiting the oxygen consumption in state4respiration ofmitochondria. Treatment with1.5mmol/L of OMT and Ginsenoside Rg1could increasemitochondrial RCR of hypoxia-exposed rats’ brain by33.04%and34.25%respectively.Treatment with2mmol/L of OMT and Ginsenoside Rg1could increase MMP by18.13%and16.15%respectively.2. Compared with normal control group, the activity and content of mitochondrial UCPs in hypoxic control group significantly increased during hypoxia exposure, which K_ddecreased40.39%and Bmaxincreased105.26%respectively. Also UCP4and UCP5mRNA expression increased2.87-fold and2.54-fold respectively and their proteinexpression significantly increased in hypoxia control group. OMT could significantlyinhibit in UCPs activities and expressions in vitro. The K_dincreased by44.08%and theBmaxdecreased by20.90%in hypoxic intervention group copared with hypoxic controlgroup; Meanwhile, mRNA and protein expression of UCP4and UCP5in hypoxicintervention group remarkably decreased compared with hypoxic control group. In fact,the mRNA of UCP4and UCP5in former group decreased to the0.46fold and0.48fold ofthe latter group.3. There were significant decreases in ST3, RCR, and MMP of mitochondria fromhypoxia-exposed rat brain, which were lower18.44%,42.06%, and18.78%in hypoxiccontrol group than in normal control group respectively. But ST4in hypoxic control groupwas significant higher40.38%than in normal control group. Treatment with2mmol/L ofOMT resulted in significant inhibition in ST4oxygen consumption (26.03%), whileincrease in RCR and MMP (47.10%and18.13%) in vitro compared with normal group.4. Compared to normal control group, the ATP content, ATP/ADP ratios, adenineenergy charge (AEC) and ATP/(ATP+ADP+AMP) in cerebral mitochondria weredecreased (37.80%,24.59%,48.34%,29.79%and39.02%respectively) while the AMP andATP content were increased (29.51%and18.51%) in hypoxic control group. OMTtreamentment resulted in significant increases of ATP content, ATP/ADP ratios, AEC andATP/(ATP+ADP+AMP) and decrease of ADP content in vitro in hypoxic interventiongroup compared with hypoxic control group.5. Intraperitoneally injection of OMT before hypoxia could extend the survival time ofmice exposed to hermetic hypoxia. The optimal intervention dose was0.3g/kg and optimalintervention time is1h asministration.Conclusion1. OMT, Ginsenoside Rg1and Saffron could elevate the oxidative phosphorylationefficiency of brain mitochondria from hypoxia-exposed rat in vitro. Meanwhile, OMT andGinsenoside Rg1could also increase the cerebral MMP of hypoxia-exposed rat in vitro.2. OMT could markedly inhaibit the uncoupling of mitochondria induced by hypoxiaexposure and increase the energy production. The mechanism may be related with the inhabitated activity of UCPs and expressions of the UCP4and UCP5inhibition.3. OMT administration could markedly increase the survival time of mice exposed tohermetic hypoxia (about2.8times). This indicated that OMT has anti-hypoxic effectivenessto mice.