Effects Of CDK1on The Activity Of Caspase-2and Golgi Morphology In Oxidative Stress

Background and Objective:Oxidative Stress is considered to be an important factor of tissue and cell aging, and it occupies an important position in the pathogenesis of the diseases such as diabetes, hypertension and atherosclerosis.aIt also plays an important role in neurodegenerative disease.The study of oxidative stress can help us to understand the physiological and pathological processes of these diseases,and It is important for improving the treatment.In recent study,it has been found that oxidative stress is likely to be more than one organelle complicit complex biochemical processes.In these processes,Golgi apparatus plays an important role. Our research group proposed Golgi apparatus stress concept. This provides a new direction for the system research of oxidative stress mechanism.In cell mitosis and meiosis processes,there has been found the physiologic Golgi fragmentation.The Golgi fragmengtation ensures mitosis and meiosis processes.The Golgi fragmentation has also been found in oxidative stress and cell apoptosis caused by oxidative stress, and some proteins that regulate physiologic mitotic Golgi fragmentation also had the similar function in Golgi fragmentation caused by oxidative stress.Cyclin dependent kinases(CDKs) are important factors in the regulation of cell mitosis and meiosis processes. At the same time, CDK1has been found play a role in cell apoptosis. Caspase-2plays an important role in Golgi fragmentation in oxidative stress.Does CDK1play a role in apoptosis caused by oxidative stress? Does CDK1play a role in Golgi apparatus morphology change? Is these processes related to caspase-2? In this study,we will use mice neuroma N2a cells,by inhibiting CDK1activity, observing caspase-2activity,Golgi apparatus and cell apoptosis. We try to have a primary discussion about questions above.Methods:1Establish a H2O2oxidative stress model.Use flow cytometric analysis to measure Proliferation Index(PI). Select the appropriate concentration and working time of H2O2.2Experimental grouping was designed as follow:control group, H2O2injury group, roscovitine inhibited H2O2injury group.3Use flow cytometric analysis to measure the rate of cell apoptosis and use immunofluorescence to observe nuclear morphology change to study cell apoptosis.4Western blot to detect the expression of caspase-2.Use spectrophotometry to detect caspase-2activity. 5Use immunofluorescence to observe Golgi apparatus morphology change.Results:1Proliferation Index(PI):In the same working time, the concentration of H2O2is greater,the PI is smaller.On the same concentration, the working time of H2O2is longer, the PI is smaller. The best concentration and working time of H2O2is80uM and12h.2The rate of cell apoptosis:The rate of cell roscovitine inhibited H2O2injury group is higher than control group and is lower than H2O2injury group(P<0.05).3Nuclear morphology change:In control group,the nucleus size is uniform, and we can not see the nuclear shape obviously abnormal and can not see the nucleus concentration and fracture. In H2O2injury group, the nucleus size is not uniform, and the nucleus fragmentation and apoptotic body can be observed. In roscovitine inhibited H2O2injury group, the nucleus size is uniform, and the nucleus fragmentation can be seen occasionally but not obvious.4The expression of caspase-2:The expression of caspase-2from more to less order is H2O2group>roscovitine inhibited H2O2injury group>conrol group(P<0.05).5The activity of caspase-2:The activity of caspase-2from more to less order is H2O2group>roscovitine inhibited H2O2injury group>conrol group(P<0.05).6The Golgi apparatus morphology:In control group, Golgi apparatus is complete, around the nucleus and is not scattered.In H2O2injury group, Golgi apparatus is scattered, some nucei are covered by Golgi apparatus, but there is no typical Golgi apparatus cracking phenomenon.In roscovitine inhibited H2O2injury group, some of Golgi apparatus is scttered, but the nucei are covered less, and thers is no typical Golgi apparatus cracking phenomenon, too.Conclusion:1CDK1plays a role in the process of injury to N2a cell caused by oxidative stress.2In the process of injury to N2a in oxidative stress, CDK1is helpful for caspase-2activation and Golgi apparatus morphology change.