The Mechanisms Of Coenzyme NADH In Cytoprotection Against I/R-induced Apoptotic Damage

BackgroundOLT is the most important progress in the treatment of end-stage liver disease. Though OLT was developed a lot in the past 30 years, such as the success rate of first procedure going up to 90%, the shortage of the donor liver significantly increased with the growing number of the patients waiting for OLT. In USA and other westen countries, the donor livers were got without I/R problem, but the donor livers were got from NHBD (non-heart-beating donor) in China which unavoidablly experienced I/R. Apart from the factors as surgical technics and expenses, the quality of the donor liver plays an important role in the restrict of efficacy of OLT in our country. Recently, research on the relationship of I/R with cell apoptosis shows cell apoptosis is an dynamic course related to the time, degree and reperfusion while going with I/R. Cell apoptosis goes before necrosis. And cell apoptosis after I/R may be the main cause of the primary graft non-function after OLT. So, it has great clinic significance to deeply go into the research on the mechanism of the cell apoptosis after I/R and the prevention way. In the course of cell apoptosis promoted by different factors, mitochondria are presented as the important executioner of apoptosis. Anti-apoptotic members probably function as mitochondia membrane stabilizing molecular. The reduced form of coenzyme NADH that helps many enzymes involved in energy production within mitochondria and many metabolic reactions in cells. NADHplays a vital role in the generation of adenosine triphosphate. NADH is crucial in process of repairing wounded cells. NADH is the most biologically potent antioxidant in nature and has the strongest positive effect in fighting the damage of free radicals. ObjectiveThe study was intended to clarify the molecular mechanisms of coenzyme NADH in cytoprotection against I/R-induced apoptotic damage. The apoptotic damage of I/R in vitro, the mitochondria regulation mechanism of coenzyme NADH in anti-apoptosis and the protection of NADH against I/R-induced liver damage. New I/R preventing strategy was provided to develop the latest cytoprotector in I/R preventing, establish new usage of coenzyme NADH. Methods1. MTT colorimetric assay was applied to detect cytotoxicity of I/R in human L02 hepatocytes, proliferation activity of cells influenced by NADH and cytotoxicity of I/R prevented and resisted by NADH. Apoptosis rate of cells was examined by flow cytometry. Apoptotic alteration was observed with TUNEL dyeing by fluorescence microscope, infrastructure of hepatocyte was observed by transmission electron microscope. The antagonism of coenzyme NADH in apoptosis induced by I/R was understood.2. Laser scanning confocal microscope was employed to detect mitochondria membrane potential A vm with fluorescent probe R123, free Ca2+ value with probe Flu-3-AM, pH value with probe SNARF-1-AM and ROS value with probe H2DCF in hepatocyte. The activity alternation of Caspase-3 and Caspase-8 was determined by colorimetric assay. The expression of Fas, P53, BC1-2 and P -actin gene were detected by RT-PCR. The expression of Cyto C and PARP protein were detected by Western blot. Cytoprotection ofNADH in apoptotic damage induced by I/R was explored to clarify the mechanism of mitochondria regulation pathway.3. Rat serum ALT, AST, LDH were deleted to observe the liver damage by I/R in vivo. The structural change was observed under TEM. Mitochondrial oxidation and phosphorylation were measured polarographically by determing oxygen consumption rate state 3 and state 4, respiration control rate and ADP/O ratio. PT-PCR was applied to detect the expression of Fas, P53, BC1-2 and ?-actin mRNA. DNA ladder of liver tissue was examined to illucidate the apoptotic effect caused by I/R. The expression of Fas, P53, BC1-2, FasL and Bax proteins were examined by westen blot procedure. Ultraviolet spectrophotometer was adopted to examine the concentration of intracellular NADH. The protections of NADH against I/R-induced liver damage were elucidated.