Effect And Mechanisms Study Of αlpha Crystallin On Retinal Ganglion Cells Of Rat

BackgroundsHow to protect the retinal ganglion cells(RGCs) and promote axonal regeneration after optic nerve injury has become a focus all over the world.Recently,the effects of injuried lens on the axon/neurite re-growth in retinal ganglion cell were investigated in adult mice,which showed,in vivo,injuried lens promoted successful regeneration of retinal ganglion cell axons passing the optic nerve lesion site.In vitro,retinal ganglion cells from lens-lesioned adult mice grew significantly longer neurites than those from mice with intact lens,however,the role of lentogenic factors and/or cytokines in promoting RGCs survival after injuried lens is still unknown.The most possible involved factors are the water-soluble proteins released from lens after injury,which have been observed mainly consisted by three proteins:α-crystallin,β-crystallin andγ-crystallin.α-crystallin,with an average molecular weight of approximately 800 kDa,takes 35%of the whole lens weight and possess chaperone-like functions,α-crystallin is composed of two 20 kDa soluble polypeptides designated asαA andαB at a 3:1 ratio.Moreover,αlpha-A andαlpha-B crystallins can inhibit the activation of caspase-3.In our recent work we have isolated and identified theα-crystallin,β-crystallin andγ-crystallin from the Long Evans rats' lens,and then added them respectively to the culture medium of retinal ganglion cells in according to the group in order to find out which crystallin has the effect on neurite outgrowth and survival of rats retinal ganglion cells.The ends suggest thatα-crystallin plays an important role in regulating RGCs survival and axon growth.However,some specialists show anxieties about the purities ofα-crystallin that was isolated from the Long Evans rats' lens by us.Secondly,the functions ofα-crystallin in optic nerve injury fields have not been studied in detail,and the more important thing is no one report the mechanisms ofα-crystallin on RGCs.Thirdly,the pure bovineα-crystallin can be bought from Sigma Company.Fourthly,the production ofα-crystallin has been limited in rat resource;some big animals such as bovine are superiority to rat economically.So a further study needs to be done about the effects and mechanisms of bovineα-crystallin on RGCs of Long Evans rat in vitro.PurposeThe aim of this study is to investigate the effect of bovine and ratα-crystallin on RGCs in vitro firstly,and to observe the effect and possible mechanisms of bovineα-crystallin on RGCs exposed to H₂O₂.Thirdly,to detect the target location ofα-crystallin on RGCs with immunofluorescence staining and immunohistochemistry staining under confocal laser scanning microscope,light microscope and immunoelectron microscopic.At last,to detect the target substance ofα-crystallin on RGCs with WesternBlot and to get its information by peptide mass fingerprinting analysis.Interaction phenomen ofα-crystallin and other cells were observed too.MethodsRGCs were cultured in DMEM/F12 medium.They were identificated by thy1.1 antibody.The changes of RGCs in vitro were observed after the culture medium were added with bovineα-crystallin at different concentrations(10^-4g/L,10^-5g/L,10^-6g/L,10^-7g/L) or ratα-crystallin at 10^-4g/L concentration for different time.The RGCs were cultured in DMEM/F12(control group) and DMEM/F12 containingα-crystallin at different concentrations(α-crystallin groups).The number of axons,the length of the longest axons of RGCs were calculated and compared with the control group in the 3^rd hour,12^th hour,1^st day,2^nd day and 3^rd day.The effect ofα-crystallin on RGCs was detected under phase-contrast microscope.To observe the changes of RGCs exposed to H₂O₂ in vitro during the period of intervention ofα-crystallin(10^-4g/L),the survival rate of RGCs were calculated under microscope and compared with the control group in the 3^rd hour,12^thhour,24^thhour and 2^ndd.The activity of SOD,LDH and the content of MDA in medium were detected by biochemistry technology.Laser scanning confocal fluorescence microimaging system detected the calcium ions level by fluo3/AM method. Immunohistochemistry and WesternBlot assayed the level of AKT in RGCs.At last,the target substance ofα-crystallin on RGCs was detected by immunofluorescence method under confocal laser scanning microscope and by immunohistochemistry method under light microscope and immunoelectron microscopic method.The target substance ofα-crystallin on RGCs was verificated by WesternBlot and analyzed by peptide mass fingerprinting.Interaction phenomen ofα-crystallin and other cells including 293 cells,ECV cells,retinal pigment epithilial cells,myocardial cell and macrophage was studied by immunofluorescence methods.ResultsOur results showed that the length of the longest axons and number of axons of RGCs were higher inα-crystallin group than those in the control group,and the effect ofα-crystallin on RGCs was dose-response,10^-4g/L was shown to be the bestα-crystallin concentration,suggesting thatα-crystallin improves the survival of RGCs and the regeneration of axons of RGCs in vitro.The survival rate of RGCs and activity of SOD decreased with time after H₂O₂ added.In the same time,the activity of LDH and content of MDA increased.The level of AKT decreased too significantly but the calcium ions level increased.While inα-crystallin group,the activity of SOD,the level of AKT increased significantly,in the same time,the activity of LDH,the content of MDA and calcium ions decreased significantly.The interaction betweenα-crystallin and RGCs was detected positive by immunofluorescence with confocal laser scanning microscope.The target substance ofα-crystallin on RGCs is on the membrane of RGCs,which was also verified by immunofluorescence staining,immunohistochemistry staining and immunoelectron microscopic observing and WesternBlot.The target matter was analysized by peptide mass fingerprinting method.The results about interaction phenomen ofα-crystallin and 293 cells,ECV cells were as same as that and RGCs,while interaction ofα-crystallin and retinal pigment epithilial cell,myocardial cell and macrophage were different from that ofα-crystallin and RGCs,They were detected positive with immunofluorescence staining withα-crystallin antibody even not interacting withα-crystallin.Conclusionsα-crystallin improve the survival of RGCs and the regeneration of axons.A-crystallin reduce the free radical damage effect on RGCs by H₂O₂.The mechanisms have relationship with inhibitting calcium ions flowing into RGCs and increasing the activity of antioxidant enzyme and AKT.The target substance ofα-crystallin on RGCs is on the membrane of RGCs,which may be involved in the mechanisms of the effect ofα-crystallin on RGCs. A-crystallin has not only action on RGCs but also on other cells.We can add ectogenesisα-crystallin to protect RGCs when they were injuried.