P115Mediated Golgi Stress In Oxidative Stress Model

Objective:To further elucidate the functional significance of p115in cerebral infarct, we use the H2O2intervened oxidative stress model here to mimic ischemia-like condition in vitro. Our study will focus on the role of p115in Golgi stress-sensing and its potential effects on apoptosis.Experimental Methods:1. We employed a stable oxidative stress model to mimic ischemia-like conditions in vitro.The neuronal injury was measured by the morphologic changes studied under light microscopes and detection of cell livability and apoptosis.2. Experimental grouping was designed as follows:control group; H202intervention group; H202intervention group with drug(sodium pyruvate). The latter two groups were divided into five different subsets according to the concentration of H2O2(20umol/l、40umol/l、60umol/l、80umol/l、100umol/l);3. The protein level of p115, p115-CTF, p53, p-p53(ser15) was determined by western blotting;4. The rates of apoptosis were measured by flow cytometric analysis;5. The rates of livability were measured by MTT;6. Confocal Immuno-fluorographs of p115and DAPI were taken to determine the sub-cellular location and fragment of p115in oxidative stress.Results:1. Compared to the control group, MTT number of the experiment group decreased and apoptosis rate increased in concentration-dependent manner(p<0.05), which indicated that different concentration of H2O2caused diverse severity of oxidative stress injury.2. The intervention of anti-oxidant drugs(sodium pyruvate) before H2O2resulted in significant increase in MTT number and decrease in apoptosis rate(p<0.05).3. After exposure upon80mmol/l H2O2,the expression of p115-CTF, p53, and p-p53(ser15) increased in a time manner.p115expression decreased in the mean time. After exposure upon H2O24hours, the expression of p-p53(serl5) increased to the top. p115-CTF and p-p53(ser15) expression increased and p115decreased remarkably in H2O280umol/l group compared with the40umol/l group(p<0.05). Expression of p53showed no significant difference between the two groups. In drug intervention group, all the four protein show no significant difference.4. Confocal Immuno-fluorographs showed that the p115labeled Golgi apparatus localized to a compact peri-nuclear ribbon, consisting of multiple vesicle-like structures arranged in close opposition to each other to form stacks. Along with the increased of H2O2concentration and time of H2O2intervention, the vesicle-like structures dispersed throughout the cytoplasm.Conclusion:1.p115is a Golgi-originated stress-related protein, and disperse in the process of oxidative stress along with the fragment of Golgi.2.The p115mediated Golgi fragmentation participates in the process of apoptosis, probably through the way of phosphorying p53.