Spermidine Protects Mouse Hippocampal Neurons HT22 Cells Against High Glucose-induced Cytotoxicity By Upregulation Of Cannabinoid Receptor 1

[Backgroud and Objective]High glucose（HG）has neurotoxic effects including cell senescence and apoptosis.Spermidine（Spd）,a natural polyamine,inhibits cellular senescence and apoptosis.Cannabinoid receptor 1（CB1）has neuroprotective effect.Therefore,in this study,we will use mouse hippocampal neurons HT22 cells to explore whether Spd inhibits HG-elicited neurotoxicity（including cell senescence and apoptosis）and whether CB1 receptor mediates this protection of Spd against HG-induced neurotoxicity.[Methods]1.Cell Counting Kit-8（CCK-8）was used to measure cell viability.2.Cell senescence was evaluated by senescence associated acidic-β-galactosidas（SA-β-Gal）staining.3.The growth of cells was determined by Trypan blue staining assays.4.The morphological of apoptotic cells were observed by Hoechst33258 staining.5.Flow cytometry（FCM）after Annexin/PI staining was used for evaluating the rate of cell apoptosis.6.Multi-function microplate detection system was used to determine the activity of Caspase-3 in HT22cells.Senescence-associated proteins(P16^INK4a and P21^CIPl)and the marker proteins of apoptosis（Bax and Bcl-2）were detected by Western Blotting.[Results]1.HG accelerates cellular senescence and apoptosis in HT22 cells.1.1 Treatment of HT22 cells with HG（13.5,27,40.5 mg/ml,48 h）significantly attenuated the viability of HT22 cells.1.2 Treatment of HT22 cells with HG（13.5,27,40.5 mg/ml,48 h）obviously increased the number of SA-β-gal positive cells,suppressed the growth of cells and increased the expression of senescence-associated proteins(P16^INK4a and P21^CIPl),suggesting that HG can induce cell senescence in HT22 cells.1.3 After treatment of HT22 cell with HG（13.5,27,40.5 mg/ml,48 h）,the apoptosis morphologic was significantly induced（such as nuclear condensation or nuclear fragmentation）,and the rate of cell apoptosis,the activity of Caspase-3 and the expression of pro-apoptotic protein（Bax）were increased,while the expression of anti-apoptotic protein（Bcl-2）was suppressed.These results showed that HG accelerates cellular apoptosis in HT22 cells.2.Spd inhibits HG-induced cellular senescence and apoptosis in HT22 cells.2.1 Pretreatment of HT22 cells with Spd（0.25,0.5,1μM,30 min）significantly reversed HG（27 mg/ml,48 h）-induced downregulation of cell viability,indicating the anti-cytotoxicity effect of Spd to HT22 cells.2.2 Preconditioning with Spd（0.25,1μM,30 min）in HT22 cells significantly reversed HG（27 mg/ml,48 h）-increased the number of SA-β-gal positive cells,-suppressed the growth of cells,and-upregulated the expressions of P16^INK4a and P21^CIP1.These results demonstrated that Spd prevents HG-induced cellular senescence in HT22cells.2.3 Spd markedly not only attenuated HG-increased the apoptosis morphologic changes（such as nuclear condensation or nuclear fragmentation）,the ratio of apoptosis,and the activity of caspase-3 in in HT22 cells,but also reversed HG-induced inclease in the expression of Bax and decrease in the expression of Bcl-2 in HT22 cells.These results showed that Spd inhibits HG-induced cell apoptosis in HT22 cells.3.CB1 receptor mediates the inhibitory effect of Spd on HG-caused cellular senescence and apoptosis in HT22 cells.3.1 HG（13.5,27,40.5 mg/ml,48 h）in HT22 cells inhibited the expression of CB1 receptor in a concentration-dependent manner,suggesting the possible relationship between HG-caused neurotoxicity and downregulation of CB1 receptor.3.2 Preconditioning with Spd（0.25,1μM,30 min）in HT22 cells significantly upregulated the expression of CB1 receptor in HG-exposed HT22 cells,indicating the involvement of the upregulated CB1 receptor in the inhibitory effect of Spd on HG-caused cellular senescence and apoptosis.3.3 AM251（10μM,30 min）,an inhibitor of CB1 receptor,reversed the protection of Spd against HG-caused the decrease in cell viability as well as the increases in cellular senescence and apoptosis.These results indicate that CB1 receptor mediates the protective effect of Spd on HG-caused cellular senescence and apoptosis in HT22 cells.[Conclusion]1.HG accelerates cellular senescence and apoptosis in HT22 cells;2.Spd inhibits HG-induced cellular senescence and apoptosis in HT22 cells.3.CB1 receptor mediates the inhibitory effect of Spd on HG-caused cellular senescence and apoptosis in HT22 cells.