| Objective:To investigate the potential protection of baclofen on primary rat retinal ganglion cells against hypoxia-induced apoptosis.Methods:Primary rat retinal ganglion cells?RGCs?were cultured for the experiments.CoCl2 was applied to mimic hypoxia which occurs in retinal hypoxic disease.The apoptotic rates of hypoxia RGCs with or without baclofen treatment were assessed using Cell Viability Assay,TUNEL Assay,Hoechst stain,and flow cytometry.The GABAB2 gene was silenced by RNA interference,and GABAB2 mRNA was determined by quantitative RT–PCR.The expression of cleaved caspase-3,bcl-2,bax,Akt,phospho-Akt,PERK,phospho-PERK,eIF2?,phospho-eIF2?,ATF-4,CHOP were measured using western blotting.Results:After treated with 200?M CoCl2 for 24 hours,the percentage of apoptotic cells increased significantly?p<0.01 vs.basal level?.Cotreated with baclofen at the concentration of 100?M,the levels of the apoptosis related proteins were changed significantly,baclofen significantly decreased the percentage of apoptotic cells both in Hoechst staining and TUNEL staining?Hoechest:p<0.05 vs.basal level,TUNEL:p<0.05,vs.basal level?.Flow cytometry and Hoechst staining revealed that knockdown of GABAB2 decreased the baclofen protective effect on hypoxia-treated RGCs compare to siRNA-control group?p>0.05?.The expression of cleaved caspase-3,bax and bcl-2 in GABAB2-knockdown hypoxia RGCs treated with baclofen were similar to that without baclofen.In RGCs treated with MK2206 2Hcl,baclofen did not significantly reduce the percentage of apoptotic cells compared with the CoCl2 treated group?p>0.05?.While in the RGCs without treatment with MK2206 2Hcl,baclofen significantly reduced the percentage of apoptotic cells compared with the CoCl2 treated group?p<0.01?.Conclusions:Apoptosis of retinal ganglion cells induced by hypoxia was prevented by baclofen through increasing of Akt activity,suppression of PERK pathway and CHOP activation. |
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