The Morphological Alterations Of Golgi Apparatus And Intervention Effects After Oxygen-glucose Deprivation Followed By Reperfusion In HT22 Cells

Objective: To elucidate the morphological alterations of Golgi apparatus in HT22 cells upon Oxygen-glucose Deprivation Followed by Reperfusion,as well as potential effects via intervention of H89(the protein kinase A antagonists).Method: 1.We employed a stable OGD/R model to mimic ischemic-like conditions on HT22 cells, the HT22 cells were subjected to oxygen-glucose deprivation(OGD) for six hours, and were cultured respectively under normal culture conditions for 6 hours, 12 hours and 24 hours.2. The experiment were divided into control group, OGD/R group, OGD/R+H89 group. According to different reperfusion time, OGD/R group and OGD/R+H89 group were divided into three subgroups respectively at 6h, 12 h and 24h(OGD/R group includes three subgroups:OGD/R6h、OGD/R12h、OGD/R24h;OGD/R+H89 group includes three subgroups:OGD/R6h+H89、OGD/R12h+H89、OGD/R24h+H89).OGD/R+H89 group is as follows:normal culture medium containing 100μM H89 was added into culture the HT22 cells in the group upon oxygen-glucose deprivation(OGD) for six hours.3.Cellular viability and apoptosis were measured using the MTT assay and Hoechest 33258;the expressions of GM130 and GAAP were analyzed by Western blot; the morphological alterations of Golgi apparatus were determined by fluorescence microscope.Results: 1.The MTT results showed that cellular viability in HT22 cells of OGD/R group and OGD/R+H89 group were reduced significantly at 6-hour,12-hour,24-hour time points after OGD/R,compared to the normal control group( P < 0.05); the OD value of OGD/R12h+H89 group was significantly higher than OGD/R12 h group with statistically differences(P < 0.05);2. The results of the Hoechest 33258 test showed that the rate of apoptosis cells of OGD/R group and OGD/R+H89 group were significantly increased at 6-hour,12-hour,24-hour time points after OGD/R, compared to the control group(P < 0.05);However, there was no difference in the cell apoptosis rate of the corresponding subgroups between the OGD/R group and the OGD/R + H89 group(P > 0.05);3.Western blot test results showed: compared with normal group, the expressions of GM130 of OGD/R group and OGD/R+H89 group were lowered at 12-hour and 24-hour time points after OGD/R with statistically significant differences(P < 0.05),there was no difference between the corresponding subgroups of OGD/R group and OGD/R+H89 group(P > 0.05); the expressions of GAAP of OGD/R group were lowered significantly at 12-hour and 24-hour time points after OGD/R,compared to the normal control group(P < 0.05); compared with normal group, the expressions of GAAP of OGD/R+H89 group were lowered at 6-hour,12-hour and 24-hour time points after OGD/R with statistically significant differences(P < 0.05);the expressions of GAAP of OGD/R24 h + H89 group was significantly higher than OGD/R24 h group with statistically differences(P < 0.05); However, there was no significant difference in the expressions of GAAP at 6-hour and 12-hour time points after OGD/R between the OGD/R group and the OGD/R + H89 group(P > 0.05);4.The immunofluorescence results:Golgi apparatus showed swelling at 6-hour time point and obvious fragmentation at 12-hour,24-hour time points after OGD/R in HT22 cells of OGD/R group;however,the morphology of the Golgi apparatus in HT22 cells of OGD/R+H89 group was almost normal at 6-hour,12-hour time points,and showed obvious fragmentation at 24-hour time point after OGD/R; compared with the OGD/R group, the fragmentation of Golgi apparatus was reduced at 6-hour and 12-hour points after OGD/R to a certain extent.Conclusion: 1.The fragmentation of Golgi apparatus and GAS(Golgi apparatus stress) and apoptosis occured in HT22 cells upon Oxygen-glucose Deprivation Followed by Reperfusion,the potential mechanism may be related to the reduction of GM130 and GAAP expression levels.2.H89 couldn’t alleviate the fragmentation of Golgi apparatus and apoptosis in HT22 cells after OGD/R.