Experimental Research About Dysfunction Of Mitochondrial Autophagy Induced By Homocysteine In Cells

Objective The normal metabolism of homocysteine(Hcy)in the body is not only the physicalfoundation such as antioxidant,anti-aging and antidepressant,but also the important protective agent of the liver through embroiled in the reverse transportation of the high-density lipoprotein(HDL)and glutathione(GSH)synthesis.The abnormal metabolism of homocysteine which leads to hyperhomocysteinemia(HHcy)has been successfully confirmed its close connection with neurodegenerative diseases.However,its specific pathogenesis is not fully understood.A series of hypothesis including oxidative stress,excitatory amino acids toxicity,mitochondrial dysfunction,intracellular calcium overload,DNA damage and so on has been recognized to a certain extent.In recent years,lots of studies have shown that mitochondrial autophagy promotes the occurrence and development in neurodegenerative diseases.Based on previous research results,this subject is to investigate the relationship between elevated homocysteine levels and mitophagy.Methods PC 12 cells were treated with normal and homocysteine medium containing different concentrations for 24h to stimulate the damage of cell model by hyperhomocysteinemia,the proliferation of the cells was analysed using the CCK-8,and to decide the intervention concentration of Hcy.Mito-Tracker Green was used to detect mitochondria number of each group:Quantitative real time PCR and Western blot technologies were used to measure the expression levels of PGC-la、NRF1 and mtTFA mRNA and protein in Hcy-treated cells.laser cnfocal fluorescence microscopy was used to show the mitochondrial autophagy in living cells after employed fluorescence marker gene transfection technology;Finally,Western Blot analysis were used again to detect the protein expression levels in normal and Hcy-treated cells.ReSultSThe result showed that the viability of PC 12 cells was significantly inhibited exposured to Hcy for 24h,and the proliferation was detected in a dose-dependent manner.Mito-Tracker Green staining showed that the mass of mitochondria was increased in cells after treated with Hcy,indicating that Hcy can promote mitochondria biosynthesisunder the condition of stress.Results from Real-time PCR and western blot analysis showed that the expression level of PGC-1α、NRF-1 and mtTFA mRNA and Protein was increased in a dose-dependent manner after 24h by homocysteine treated.The results from fluorescence marker gene transfection showed that groups with homocystein treated shows decreasing mitochondrial autophagy with the increase of drug concentration while the normal grouppresents no or very little mitophagy;Besides,we also found that the increasing expression of protein Mfn2 which on behalf of the mitochondrial fusion and inhibiting mitophagy,also the decreasing expression of Drp1 that represents mitochondrial fission and promoting mitophagy.Conclusion Homocystein can inhibit the activity of PC 12 cells and reduce the cell viability;Homocystein can promote the mitochondria biosynthesis under the condition of stess so as to against the toxicity of Hcy;The increase quantity of mitochondria and lower survival rate indicate mitochondrial dysfunction;The mitochondrial dysfunction is associated with mitophagy;Mechanism of neurodegenerative diseases caused by Hhcy may be involved in mitophagy.