Abstract:

Abstract:Objective
To study the influence  of the specific inhibitor of autophagy 3-methyladenine（3-MA）in apoptosis of retinal ganglion cells (RGCs),and to clarify the protective effect of 3-MA on RGCs under  hypoxic micro-environment.Methods The RGCs at logarithmic growth phase were collected and divided into control group (PBS),3-MA group (6 mmol•L^-1 3-MA),CoCl2 group(300 μmol•L^-1 CoCl2) and 3-MA combined with CoCl2 group(6 mmol?L-13-MA+300 μmol•L^-1 CoCl2).MTT method was conducted to evaluate the optimal concentration of CoCl2 and the  inhibitory rate of proliferation of RGCs in various groups; Acridine orange/ Ethidium bromide (AO/EB) staining was used to detect the morphology of RGCs;  Monodansylcadaverine (MDC) staining was used to detect the autophagic vacuoles of RGCs;Western blotting method was used to test the expression levels of  apoptotic proteins P53,Bax,caspase-9,caspase-3,iASPP and autophagic  protein LC3 in RGCs in various groups. Results The 30%inhibitory concentration( IC30 )of CoCl2 was 300 μmol?L-1.Compared with CoCl2 group,the  inhibitory rate of proliferation of RGCs in 3-MA+CoCl2 group was decreased(P<0.05).The apoptosis body and autophagic vacuole in RGCs in various groups were observed under fluorescence microscope.The Western blotting results showed that compared with control group,the expression levels of  P53,Bax,caspase-9,caspase-3,and  LC3 in RGCs in the other groups    were increased(P<0.05) and the expression level of  iASPP   was decreased(P<0.05);compared with CoCl2 group,the expression levels of P53,Bax,caspase-9,caspase-3,and LC3 in RGCs in 3-MA +CoCl2 group were decreased(P<0.05) and the expression level of  iASPP   was increased(P<0.05); compared with 3-MA group,the expression levels of P53,Bax,caspase-9,caspase-3,and LC3 in RGCs in 3-MA +CoCl2 group were increased(P<0.05) and the expression level of  iASPP was decreased(P<0.05).Conclusion  Under  hypoxic micro-environment,the inhibitor of autophagy 3-MA has protective effect on apoptosis of RGCs induced by CoCl2.

Key words: retinal ganglion cells, 3-methyladenine, autophagy, apoptosis, hypoxia