Research About The Protective Effect Of Rhodiola On Hypoxic Brain Injury And Its Mitochondrial MPTP Mechanism

ObjectiveTo study the protective effect of rhodiola to hypoxia injury and its mechanism of mitochondria,this study through copying of oxygen model,function and appearance observation,biochemical and pathological examination,flow cytometry,etc,a variety of molecular and cell biology,in vivo and in vitro these two levels to observe rhodiola rosea protection to hypoxia,at the same time,by testing changes of mitochondrial membrane potential,respiratory function,Ca²⁺release,Caspase-3 activity,hope from the perspective of mitochondrial function to clarify mechanisms of rhodiola rosea.MethodsPart One The effect of nerve protective and mitochondrial membrane MPTP of damage in mice brain by hypoxia making by rhodiola pretreatment1.by building,simulated hypoxia in mice model,through the pathological examination anoxia in mice brain neurons damage,on the different hypoxia height and time conditions;biochemical testing groups of mice brain LDH,SOD,LD,MDA expression found that,different hypoxia height and different hypoxia time being different to the change of these metrics,to confirm the hypoxic brain injury programme.2.Through the rhodiola intervening beforehand,detection of oxygen survival time,and through MWM to observe the protective effect of salidroside on hypoxia tolerance and learning and memory ability of mice after hypoxia.3.Isolated brain tissue immediately after hypoxic brain injury,at the thalamus in the middle of the 1/3 thick slices,using TUNEL staining to observe the apoptosis of hippocampal CA1 neurons in mice,isolation hippocampus from brain tissue,making0.5 x 0.5 x 1mm³ tissue block in the uplift of the central of hippocampus,ultrathin section,morphological changes of neurons in hippocampal CA1 area of mice were observed by transmission electron microscope.4.Extracting brain mitochondria after hypoxic brain injury,testing mitochondrial respiratory function（ST3、ST4、RCR）,MPTP permeability,MMP,to observe the protective effect of salidroside pretreatment on brain mitochondrial function in mice with hypoxic brain injury,to analysis of the mechanism of Rhodiola sachalinensis against hypoxia and its mechanism of mitochondrial MPTP.Part Two The effect of hypoxia PC12 cells making by rhodiola glucoside pretreatment1.Choosing PC12 cells with good growth in logarithmic phase,adding cell suspension was made by adding proper amount of growth liquid,The concentration of cell culture medium for salidroside:5μM,10μM,15μM,20μM,25μM,30μM,40μM,60μM,80μM,adding in cell culture plate respectively,to determine the cell vitality by MTT,calculateing Rhodiola glucoside ID50,to determine the dose of rhodiola glucoside pretreatment,and set low,medium and high three kinds of different pretreatment dose;2.PC12 cells being divided into control group,hypoxia group,HPC group,low,medium and high dose of rhodiola pretreatment group,a total of 6,combined with MPTP blocker CsA and agonist ATR intervention,detection rhodiola glucoside pretreatment effect on hypoxia induced PC12 cells by MTT,LDH release rate;3.PC12 cells being divided into control group,hypoxia group,HPC group,low,medium and high dose of rhodiola pretreatment group,a total of 6,combined with MPTP blocker CsA and agonist ATR intervention,using flow cytometry to detect cell apoptosis rate,using Western Blot to analysis the Bcl-2 expression,detection caspase3 activity of cells,to analysis rhodiola glucoside pretreatment on the effect of hypoxia induced PC12 cells apoptosis and relations with MPTP;4.Reference to the foregoing group,using TMRE method to detect the mitochondrial membrane potential changes,detection of Ca²⁺concentration,observation of rhodiola glucoside pretreatment effect on hypoxia PC12 cells mitochondrial function.ResultsPart One The effect of nerve protective and mitochondrial membrane MPTP of damage in mice brain by hypoxia making by rhodiola pretreatment1.By building,simulated hypoxia in mice model,through the pathological examination anoxia in mice brain neurons damage,on the different hypoxia height and time conditions,each group of mice state performance was not completely the same,the mortality of mice were different,10000m altitude hypoxia 1 h mortality of50%,as the extension of anoxia time,mortality rate increased to 100%,the 7000m and 8000m altitude hypoxia group each time the highest mortality rate is only 10%.Biochemical testing groups of mice brain LDH,SOD,LD,MDA expression and brain hippocampal HE staining results found that different hypoxia height and different hypoxia time being different to the change of these metrics.8000 meters 18h of oxygen groups of mice brain CA1 area morphology change is obviously,damage degree is relatively heavy,the scheme in the anoxic brain damage being intended to be 8000 m height of oxygen 18 h;2.Through the rhodiola intervening beforehand,detection of oxygen survival time,compared with the hypoxia group,rhodiola can significantly increasing anoxic survival in mice;Localization cruise and space exploration ability detected that,after hypoxia,cruise trajectory is disorder,frequency in platform removed quadrant where time and space to explore decreasing significantly,shows that lack of oxygen causing the mice brain injury,affect memory ability in mice;and different concentrations of rhodiola,rhodiola oral liquid can improve the brain hypoxia injury in mice.3.After rhodiola rosea pretreatment,TUNEL detection in mice brain tissue apoptosis CA1 area:control group mice brain neurons apoptosis ratio rarely,hardly see tan or brown color cell;hypoxia mice brain tissue in the CA1 area tan（or brown）significantly increased the number of cells;neurons apoptosis ratio of pre hypoxia group eased;brain neurons in CA1 area number of positive cells of different concentrations of rhodiola intervention group were significantly reducing compared with hypoxia group.Mice brain of each group was detected the mitochondrial morphology by TEM.Nuclei nuclear membrane of hippocampus CA1 area neurons of control group mice was clear,the density of chromatin in nuclear uniformed,the cytoplasm in mitochondria was rich,form was normal,distribution was uniform.The hippocampus CA1 area neurons in the nucleus of hypoxia mice appeared irregular pycnosis,heterochromatin edge set,mitochondria swelling,crest fractured or disappeared.low dose and high dose group of rhodiola rosea nucleus pycnosis,some mitochondria swelling,part of mitochondrial crest fractured or fuzzy.Proved that oral liquid,rhodiola rosea and rhodiola could protect from brain neurons apoptosis caused by hypoxia,and it could reduce the structural damage of mitochondrial cell mitochondria,which is caused by hypoxia in varying degrees,It was suggested that rhodiola can reduce the rate of the apoptosis of brain cells caused by hypoxia,and might be related to the protection of mitochondrial structure.4.Hypoxia could significantly reduce the ST3 neuron mitochondria content（P<0.01）;and different concentrations of rhodiola,rhodiola oral liquid could significantly improve the ST3 level（P<0.01）.ST4 expression in the hypoxia group significantly increased（P<0.01）,and different concentrations of rhodiola,rhodiola oral liquid can reduced the ST4 content（P<0.01）.Mitochondrial reaction system situation changes after adding Ca²⁺absorbance,could reflect the mitochondrial membrane permeability of Ca²⁺indirectly.The OD of each group after adding Ca²⁺is on the decline,the OD of hypoxia group decreasing degree significantly higher than that of control group（P<0.01）,Compared with hypoxia group,the OD of the rest intervention group were decreased significantly lower than that of hypoxia group（P<0.01）,High concentrations of Ca²⁺caused each group mouse mitochondrial membrane potential decline,results showing that hypoxia mice mitochondrial membrane potential change is most significant（P<0.01）,and significantly higher than the other groups,the mitochondrial MMP changed in each pretreatment group were significantly lower than the hypoxia group（P<0.01）.These above results suggested,lack of oxygen could damage mitochondrial respiration,increase the mitochondrial MPTP through permeability,cause the mitochondrial membrane potential to change,rhodiola pretreatment could reduce lesions caused by hypoxia,playing a role in protecting the mitochondrial function of neurons form hypoxic brain damage.Part Two The effect of hypoxia PC12 cells making by rhodiola glucoside pretreatment1.According to the survival rate of cells treated with different concentrations of rhodiola glucoside after MTT intervention,the ID50 value was estimated to be about32.17μM2.After using different concentrations of rhodiola glucoside intervention,significantly increasing the cell vitality;after adding MPTP inhibitor Cs A,the cell vitality rising significant;after adding MPTP agonist ATR,compared with the hypoxia group,the cell vitality further lowered.CsA could promote the protective effects of rhodiola glucoside,the ATR can inhibit its function.3.Apoptosis-related test results showed that,hypoxia could significantly increase the PC12 cell apoptosis rate（P<0.01）,different concentrations of rhodiola glucoside could significantly reduce the proportion of apoptosis cells（P<0.01）.MPTP activator ATR could further the marked increase in the proportion of apoptosis cells the;And CsA MPTP inhibitors could reduce the proportion of each group.Caspase 3 activity of hypoxia group increased significantly,significantly higher than that of control group（P<0.01）,HPC,different concentrations of rhodiola glucoside could significantly reduce the activity of Caspase 3,but being higher than the control group.And MPTP inhibitor CsA could further inhibit the activity of Caspase 3.MPTP agonist ATR can increase the activity of Caspase 3.Hypoxia group cells antiapoptotic proteins expression of Bcl-2 significantly increased,the expression of Bcl-2 quantity of HPC group increased the most obvious,the expression of Bcl–2 of different concentrations of rhodiola glucoside significantly increased.The results showed that,the Bcl-2 increasing in hypoxia,oxygen beforehand,the Bcl–2 raising more.These results suggested that rhodiola can reduce the apoptosis rate of hypoxia PC12 cells,and be related to Bcl-2 and Caspase 3 pathway.4.Mitochondrial MPTP functional test results showed that,hypoxia PC12 cells mitochondrial membrane potential decreased,Rhodiola glucoside and CsA could alleviate hypoxia PC12 cells reducing mitochondrial membrane potential;The ATR could increase mitochondrial membrane potential.The mitochondrial Ca2+concentration testing showed that,hypoxia group could significantly increase intracellular Ca2+concentration,MPTP inhibitor Cs A could reduce intracellular Ca2+concentration,MPTP agonist ATR could increase intracellular Ca2+concentration;Rhodiola glucoside of each dose group effectively inhibited cell Ca2+concentration increasing,and presented a dose dependent.It was suggested that Rhodiola was preempted to protect the mitochondrial MPTP of hypoxia PC12 cells.ConclusionHypoxia could cause brain damage in mice,affected the function of mitochondria;HPC,Rhodiola rosea could inhibit the injury by the oxygen.And Rhodiola rosea mainly through inhibition of the mitochondrial membrane pore MPTP open,to stop the mitochondrial membrane potential changing and mitochondrial membrane Ca²⁺concentration inside and outside difference,inhibitory neurons apoptosis occurred to against hypoxia injury.