Effect Of Omentin-1 On H₂O₂ Induced PC12 Cell Injury And Its Mechanism

Background: Ischemic brain injury is a disease in which the blood supply to the local brain tissue is blocked,resulting in irreversible neurological impairment and even death.Worldwide,it is the leading cause of adult disability and the second leading cause of death.Every year,scholars at home and abroad invest a lot of manpower and material resources to study its prevention and treatment and methods of improving prognosis,but the methods of timely and effective prevention and treatment and prognosis are still limited.Cerebral ischemia and hypoxia can cause oxidative stress?OS?,which can destroy cell membranes,proteins and DNA,and Caspase-3 is activated to promote neuronal cell apoptosis and participate in the pathogenesis of ischemic brain injury.Therefore,the prevention of oxidative stress-induced neuronal apoptosis is an important strategy for the treatment of ischemic brain injury.Omentin-1 is a newly discovered adipogenic factor.Many studies have found that circulating omentin-1 plays an important regulatory role in the pathophysiological process of various diseases and has anti-inflammatory,antioxidant and anti-apoptosis effects.Omentin-1 May be a biological indicator to determine the risk and severity of acute ischemic stroke.Recent studies in vivo have shown that omentin-1 can inhibit not only the apoptosis of myocardial cells after ischemia,but also the apoptosis of neurons after ischemia.However,the effect of omentin-1 on oxidative stress-induced apoptosis in ischemic brain injury is still unclear.Therefore,this study investigated the effect of omentin-1 on H₂O₂ induced oxidative stress injury in PC12 cells.It will lay a foundation for the next research of this subject.Objective: To study the effect of Omentin-1 on oxidative stress induced PC12 cell damage and its mechanism,and to preliminarily explore its effect and mechanism on the prevention and treatment of ischemic brain injury.Methods:?1?PC12 cells were cultured in vitro and their cell morphology was observed.?2?CCK-8 method was used to detect the effect of different concentrations of H₂O₂ on PC12 cell proliferation for 2h and to select the optimal damage concentration,so as to establish the hydrogen peroxide damage cell model for the experiment.?3?The effect of omentin-1 on cytotoxicity of PC12 cells was detected by CCK-8 method.?4?We randomly divide cells into three parts,such as normal control group,H₂O₂ group and H₂O₂+Omentin-1 treatment group,and we employed CCK-8 method to test the effect of omentin-1 on the survival rate of PC12 cells which were induced by hydrogen peroxide.?5?TUNEL method was used to detect apoptosis in each group.?6?Changes in the content of reactive oxygen species?ROS?in cells were detected by the reactive oxygen species detection kit;?7?At the same time,we employed Western Blot to test the effects of omentin-1 on the expression levels of Bcl-2,Bax and Caspase-3 in every group.Results:?1?The result that CCK-8 was used to test indicated that the growth of PC12 cells has no obvious variation under different concentrations of omentin-1 treatment at different time,and the difference makes no sense in statistics?P > 0.05?.?2?We usually employed CCK-8 method to test the effect of omentin-1 on cell viability after H₂O₂ induced PC12 cell injury.The results made clear that contrast to the normal control group,cells which was in the H₂O₂ model group were reduced in size,cell viability was obviously shrunk,and the difference makes no sense in statistics?P < 0.05?.Contrast to the model group,the cellular morphology of the omentin-1 pretreatment group was slowly getting better,the cell viability was distinctly increased,and the difference makes sense in statistics?P < 0.05?.?3?The TUNEL method showed that the apoptosis rate in the H₂O₂ model group increased significantly compared with the normal control group?P < 0.05?.Compared with the H₂O₂ model group,the apoptosis of cells in the omentin-1 pretreatment group showed a concentration dependence.With the increase of the concentration of omentin-1,the apoptosis rate gradually decreased,and the apoptosis rate was the most significant when the concentration of omentin-1 was 500 ng/ml,and the difference makes sense in statistics?P < 0.05?.?4?ROS test results indicated that compared with the normal control group,ROS that was in the H₂O₂ model group was distinctly increased,and the difference makes sense in statistics?P < 0.05?.Contrast to the model group,ROS which was in the omentin-1 treatment group was clearly slided,and the difference makes sense in statistics?P < 0.05?.?5?Western blot detection results stated clearly that compared with the normal control group,the expression level of Bcl-2 that was in the H₂O₂ model group was clearly went down,and the difference makes sense in statistics?P < 0.05?,however,the expression levels of both Caspase-3 and Bax were evidently raised,and the difference makes sense in statistics?P < 0.05?.Compared with the model group,the expression level of Bcl-2 which was in the omentin-1 treatment group were clearly raised,nevertheless,the expression levels of both Caspase-3 and Bax were significantly decreased.After PC12 cells were pretreated with different concentrations of omentin-1,the results stated clearly that the expression level of Bcl-2 protein did not amend obviously while omentin-1 concentration was increased?P > 0.05?,contrast to the normal control group.With the increase of omentin-1 concentration,Caspase-3 protein expression changed insignificantly,and the difference makes no sense in statistics?P > 0.05?.Nevertheless,Bax protein expression decreased with the increase of omentin-1 concentration,the difference makes sense in statistics?P < 0.05?.Conclusions: 1.Omentin-1 pretreatment has a protective effect on H₂O₂induced PC12 cell damage.2.Omentin-1 pretreatment can protect PC12 cells from oxidative stress-induced apoptosis by reducing ROS production.3.Omentin-1 preconditioning inhibited H₂O₂ induced PC12 cell apoptosis by down-regulating Bax pro-apoptotic protein.