Effect Of3-MA On Apoptosis Of Retinal Ganglion Cells Under Hypoxic Micro-environment

Background：Glaucoma is a group of neurodegenerative diseases, which ischaracterized by high intraocular pressure and retinal ganglion cellapoptosis[1]. In resent years, some studies have shown that retinalganglion cell autophagy also plays its greater impact in the process ofretinal ganglion cells apoptosis[2]. Autophagy is a process which the cellis self-decomposition and metabolic degradation, and the principle withthe participation of autophagic vesicles and lysosomal is the long-livedproteins in the cytoplasm or some damage organelles (such as theendoplasmic reticulum, Golgi apparatus, mitochondria) were degradated,then recycling and re-use of certain amino acids and protein. The growthof certain cells in the presence of basal levels of autophagy as a cleaningsystem cells to maintain cellular homeostasis. A variety of stressconditions (such as: hunger, oxygen stress and pathogen infection orcytotoxicity) can activate autophagy. In the process of tumor formation,inhibit tumor formation and promote tumor formation, this two-way role ofautophagy depends on the different cell backgrounds[4]. Bidirectional roleof autophagy phenomenon also exists in neurodegenerative diseases[5].3-methyladenine (3-MA) as a classical autophagy inhibitors has beenwidely used. At present, the research on autophagy inhibitor3-MA andapoptosis of retinal ganglion relations are fewer. We study the effect of3-methyladenineon and apoptosis of retinal ganglion cell by MTT,Immunofluorescence staining, RT-PCR, Western blotting and otherexperimental methods, and providing a theoretical basis for the method of treatment to neurodegenerative diseases including glaucoma.ObjectiveTo study the effect of the specific inhibitor of autophagy3-MA onapoptosis of retinal ganglion cell (RGCs), and to clarify the effect of3-MAon RGCs under the hypoxic micro-environment.MethodsThe RGCs at logarithmic growth phase were collected and divided intocontrol group (PBS),3-MA group, CoCl2group and3-MA combine withCoCl2group. MTT method was conducted to evaluate the optimalconcentration of CoCl2and the proliferation inhibitory rate of RGCs invarious groups; Acridine orange/Ethidium bromide (AO/EB) staining wasused to detect the morphology of RGCs; Monodansylcadaverine (MDC)staining was used to detect the autophagic vacuoles of RGCs; Westernblotting and RT-PCR methods were used to test the expression levels ofP53,Bax,caspase-9,caspase-3,iASPP and LC3.ResultsThe30%inhibitory concentration (IC30) of CoCl2was300μmol/L.Compared with CoCl2group, the proliferation inhibitory rate of RGCs in3-MA combine with CoCl2group was decreased(P＜0.05).The apoptosisbody and autophagic vacuole in RGCs were observed underfluorescence microscope. Western blotting and RT-PCR results showedthat compared with control group, the expression levels of apoptotic protein P53,Bax,caspase-9,caspase-3and the autophagic protein LC3of RGCs in the other groups were incresed (P＜0.05) and the expressionlevel of the apoptotic protein iASPP of RGCs in the other groups wasdecresed(P＜0.05);compared with CoCl2group,the expression levels ofP53,Bax,caspase-9,caspase-3,and LC3of RGCs in3-MA+CoCl2groupwere decresed(P＜0.05) and the expression level of iASPP of RGCs in3-MA+CoCl2group was incresed(P＜0.05).ConclusionUnder the hypoxic micro-environment, the inhibitor of autophagy3-MAhave protective effect on apoptosis of RGCs induced by CoCl2.