A Preliminary Study Of The Early Cell Damage Mitochondria Lysis Time Curve And Cyclosporin A On Mitochondrial Membrane Stabilizing Effect

Objective Trauma causes the body’s systemic inflammatory response syndrome, oreven multiple organ failure. SIRS pathogenesis and prevention have not yet fullyunderstand. Now many experimental studies confirm when the body damage, ruptureof the tissue cells, the mitochondria released into the circulatory system, stimulate theinflammatory response upon mitochondrial cleavage, large-scale, then the occurrenceof SIRS, and lead to MODS. Is a stable mitochondrial membrane organelles, bodyinjury, how long it will be released by the cells, and how long it will be cracking, areunclear.This experiment will attempt to reveal the early cell damage mitochondria intothe blood after lysis time curve, while observing the membrane protective effect of thedrug cyclosporin A on mitochondrial membrane stabilizing effect.Methods The research at the cellular level, the molecular level, to explore thestructural stability of the mitochondrial membrane, and inhibition of mitochondrialmembrane rupture, the mechanism of inhibition as a source of damage-associatedmolecular patterns start uncontrolled SIRS, carry out research from the molecular,cellular, stating mitochondrial release into the blood cell damage, its membranerupture time curve and its suppression factors. And mitochondrial damage controlsurgery, discover new target for prevention and treatment of the body after severetrauma SIRS as the introduction of an early warning mechanism.Part1: Take6g rat liver tissue were extracted use the sucrose method and kit methodfrom the mitochondria purity suitable for mitochondrial research experiments to evaluate the the mitochondrial extraction quality, discover rat liver cells mitochondriaextraction methods, as well as mitochondrial extract the evaluation of the quality toprovide experimental basis.Part II: Fresh rat mitochondria from rat liver extract, use the concentration of MAOindicates concentration of mitochondria, the concentration of MAO the200U/mlmitochondrial suspension in the20serum culture, within12hours at different timepoints (half an hour) the determination of the content of the medium MAO, Cox andMDH. Observation on the mitochondrial membrane and matrix respective specificenzyme concentration time curve, the peak time analysis of the mitochondrialmembrane lysis.Part III: Fresh rat mitochondria from rat liver will be again the MAO concentration of200U/ml of mitochondrial suspension5ml placed in20ml of serum in the culture,Addition of1ml of100mg/ml cyclosporin A drug on mitochondrial membraneprotection, is still12hours at different time points (every half hour) Determination ofmedium in the inner mitochondrial membrane and matrix in each specific enzyme COXthe content of the MDH. Set the control group, Add1ml of physiological saline. Thecontent of the two groups was measured time curve, statistical analysis, to observewhether there is a clear inhibitory effect of the drug on mitochondrial membranerupture.Results1. Using the kit method and the method of cane sugar were extracted mitochondrialsuccinate dehydrogenase and acid phosphatase purification factor was notstatistically significant (P>0.05), But, use kit method Purification factor ofgreater than sucrose extraction of mitochondria on alkaline phosphatase, the difference was statistically significant (P <0.05). That the use of mitochondrialisolation kit extracted mitochondrial membrane pollution less than sucrose lawextraction, more suitable for the extraction mitochondrial step for futureexperiments.2. The content of mitochondria in vitro culture Cox and MDH in3hours,3.5hours toreach the peak. The experimental results show that the culture solution of MAOcontent did not change significantly over time, but the content of the COX andMDH with time gradually increased, reaching the crest, and then gradually falling.Thus the complete mitochondrial released by cells after about3.5hours to reach thecleavage peak.3. control group in COX MDH peak appeared in3hours,3.5hours, peak1.384U/l34.568U/l; of COX MDH peak in the experimental group appeared in6hours,6.5hours, peak1.376U/l,31.994U/l. The two sets of data at each time point wereused for statistical analysis,3hours of COX control group was significantly higherthan the experimental group (P <0.05);3.5hours, the MDH concentration in thecontrol group was also significantly higher than the experimental group (P <0.05).Therefore, the addition of cyclosporin A drug action mitochondria, completemitochondrial cleavage in serum was significantly delayed (P <0.05) and a slightdecrease in peak.Conclusion Body injury after cell rupture the complete mitochondrial release intothe blood, after3.5hours to reach the peak of its cleavage, the addition of cyclosporinA drug can significantly delay the lysis of mitochondria, and reduce its peak level.