Research On Mitochondrial Metabonomics Methodology And Application In Cardiomyoblasts Hypoxia/Re-oxygenation Injury

Ischemia heart disease has become the top killer during the past decade.Various study focused on researching the pathogenetic mechanism and finding effective drugs.Cardiomyoblasts hypoxia/re-oxygenation model was used to simulate the procedure of ischemia injury.However,few report studied on the mitochondrial metabolomics during hypoxia/re-oxygenation injury among those researches.Recent researches proofed that nicotinamide-adenine dinucleotid and its reduced form acted an important role in tricarboxylic acid cycle thus was responsible for energy depletion and cell apoptosis.Therefore,nicotinamide,the precursor of nicotinamide adenine dinucleotide(NAD),was employeed to protect cardiomyoblasts from hypoxia/re-oxygenation injury.Due to the lack of the evaluation means towards mitochondrial separation,we first explored the evaluation means towards mitochondrial separation to do the characterization.Then mitochondria extraction method for H9c2 cells was optimized based on the evaluation tool.Thereafter,cellular and mitochondrial metabolites through injury/protection were detected and analyzed.Molecular verification towards the cardioprotective mechanism of nicotinamide was put forward and carried out.The main contents of the present dissertation are as follows:First,commercialized mitochondrial fluorescent probes were investigated in which MitoTracker Green FM was used as markers of mitochondrial.By measuring the probe fluorescence intensity in each component during the whole isolation procedure,a reliable evaluation method towards the mitochondrial separation was established.The isolation method towards H9c2 mitochondria was optimized,and final recovery of mitochondria was 24%.Then,the tricarboxylic acid cycle metabolites were selected as the target metabolites and detected using ion pair high performance liquid chromatography-triple-quadrupole mass spectrometry(HPLC-MS/MS)method.H9c2 cells and mitochondria isolated using the aforementioned method was used for metabolic detection.Finally,the protective concentration of nicotinamide towards cardiomyoblasts hypoxia/re-oxygenation injury was investigated.10 mM nicotinamide applied to cell shows a significant protective effect and increase the viability of hypoxia/re-oxygenation injured cells.Meanwhile,on the basis of the pre-work,metabolites of the model cells and their mitochondria was investigated.The results suggest that nicotinamide exerts a protective effect on cardiomyoblasts against H/R induced injury through both NADH/NAD regulation and reduction of reactive oxygen species(ROS)generation via SDH inhibition.