Genipin Adjust Mitochondrial Dynamics To Attenuate The Nerve Cell Apoptosis Induced By Oxidative Stress

OBJECTIVETo observe the effect of genipin on apoptosis of Neuro-2a cells induced by H₂O₂ and to explore the mechanism.METHODSFirst of all,Neuro-2a cells were cultured in vitro.Different concentrations of H₂O₂induced Neuro-2a cells’oxidative damage by the intervention of different period.Neuro-2a cells’viability was measured by MTT assay to screen the H₂O₂ treatment time.Flow cytometry was utilized to detect apoptosis rate by Annexin V-FITC/PI staining.Fluorescence microscope and flow cytometry was performed to detect the production of reactive oxygen species by DCFH-DA fluorescent staining.The suitable treatment concentration of H₂O₂ was screened through the above experiment.Then,Neuro-2a cells were pretreated by different concentrations of Genipin for an hour,and then they were given or did not give 400μmol/L H₂O₂ intervention for 6 hours.Neuro-2a cells’viability was measured by MTT assay to screen the H₂O₂ treatment time.Flow cytometry was utilized to detect apoptosis rate by Annexin V-FITC/PI staining.The suitable treatment concentration of Genipin was screened through the above experiment.Finally,Neuro-2a cells were randomly divided into normal control group,400μmol/L H₂O₂ group,40μmol/L Genipin group and 40μmol/L Genipin+400μmol/L H₂O₂group.Cell morphological alterations were observed by inverted phase contrast microscope.Flow cytometry was performed to detect the production of reactive oxygen species by DCFH-DA fluorescent staining.Messenger RNA expression of UCP2,UCP4,Mfn2 and DRP1 were measured by qRT-PCR.Western-Blot was used to detect protein expression of UCP2,UCP4,Mfn2 and DRP1.RESULTSDifferent concentrations of H₂O₂ induced Neuro-2a cells’oxidative damage by the intervention of different period.With the increase of concentration of H₂O₂ intervention and intervention in the extension of time,the vitality of Neuro-2a cell gradually lowers,and presents a certain concentration dependence and time dependence.Besides,the increase of H₂O₂ intervention concentration will cause gradual growth of cell apoptosis rate and the intracellular ROS level.By combining MTT cell vitality detection experiment,cell apoptosis rate determination and ROS qualitative quantitative detection results,400μmol/L is chosen as H₂O₂ concentration to induce the Neuro-2a cell oxidative damage.H₂O₂ intervention time is 6 h.Compared with the control group,the low concentration of Genipin（0.65⁴0μmol/L）had no significant effect on the activity of Neuro-2a cells,while the activity of the cells remarkably decrease when the intervention concentration is higher than 80（p<0.01）.In low concentration range（5⁴0μmol/L）,the concentration of cells apoptosis rate gradually declines as the pretreatment concentration of the given Genipin increases.And 40μmol/L is the strongest concentration to inhibit cell apoptosis（p<0.01）.By combining the validity of MTT cells and the cell apoptosis rate,40μmol/L is selected as the concentration to deal with the pretreatment of Genipin.Preliminary observed under a microscope,compared with normal control group,400μmol/L H₂O₂ intervention weakened after cell diopter,protuberant retraction,cells loose contact,part of the cell body,it become the cell survival rate decreased significantly,the apoptosis rate increased significantly,a significant rise in intracellular ROS generation,UCP2,DRP1 protein and mRNA expression in cells were significantly increased,Mfn2protein and mRNA expression were significantly lower（p<0.05 or p<0.01）,UCP4mRNA and protein expression has no obvious change;Give 40μmol/L Genipin to incubate cells in advance,compared with 400μmol/L H₂O₂ group,40μmol/L Genipin+400μmol/L H₂O₂ group was obviously improved,a significant rise in the cell survival rate,apoptosis rate dropped significantly,intracellular ROS generation significantly reduced,at the same time UCP2,DRP1 protein and mRNA expression were significantly decreased,Mfn2 protein and mRNA expression were significantly higher（p<0.05 or p<0.01）,but UCP4 protein and mRNA expression has no obvious change.CONCLUSIONSThe suitable concentration of Genipin has protective effect on Neuro-2a cells apoptosis induced by oxidative stress.The mechanism may be associated with inhibiting the excessive uncoupling of mitochondria,inhibiting the excessive generation of intracellular ROS,and promoting mitochondrial fusion to improve mitochondrial function.