| Part Ⅰ:A mouse retinal ischemia reperfusion injury activates apoptosis-related pathways leading to retinal ganglion cells damagePurpose:Glaucoma is a kind of neurodegenerative diseases characterized by loss of retinal ganglion cells(RGCs),thinning of the optic nerve axon and visual field defects.However,the pathogenesis of glaucoma has not been fully elucidated.Therefore,this study aims to explore the underlying mechanisms of RGCs loss induced by retinal ischemia reperfusion(I/R)injury.Methods:A retinal I/R injury model was established in adult male C57BL/6J mice.RGC survival was detected by immunofluorescence staining.Retinal thickness was determined using hematoxylin and eosin(HE)staining.Retinal function was evaluated by Electroretinography(ERG).The protein levels of Brn3a,Bax,Bcl2,Cleaved Caspase3,HIF-1α,VEGF,p38,p53,PI3K and Akt were investigated using Western Blot analysis.Results:In comparison with the control group,RGCs survival was remarkably decreased in different experimental groups.As shown in HE staining,retinal thickness was significantly thinner in different I/R groups than in the controls.We found Brn3a expression was substantially reduced 3 and 7 days after retinal ischemic injury.We also revealed that the protein levels of Bax,Cleaved Caspase3,HIF-1α,VEGF and P-p38 were notably upregulated at 3 days following I/R injury than that of controls.Besides,p53 expression was significantly increased in the 3 and 7 days after I/R injury in comparison to the controls.Moreover,Western blot showed that P-PI3K and P-Akt protein levels were substantially decreased in the 3 and 7days ischemic groups compared the control group.Furthermore,as shown in the ERG,significant reductions in amplitudes of A-wave and B-wave were observed at different days following I/R injury in compared with the normal group.Conclusion:Our results suggest that retinal ischemia reperfusion injury elicited retinal ganglion cell loss and retinal dysfunction.Besides,we conclude that activation of the intrinsic apoptotic pathway mediated retinal ganglion cell death.Moreover,retinal ischemic injury can activate the HIF-1α/VEGF and p38/p53 pathways,while inhibiting PI3K/Akt pathway.Part Ⅱ:Resveratrol protects RGCs by inhibiting HIF-1a/VEGF and p38/p53 pathways while activating PI3K/Akt pathway in mouse retinal ischemia reperfusion injuryObjective:Resveratrol is a natural polyphenolic compound that has protective effects in retinal ischemia reperfusion(I/R)injury.However,the molecular mechanisms of resveratrol function on RGCs remains unclear.Therefore,the purpose of this part aimed to explore the potential mechanism of resveratrol in retinal ischemic injury.Methods:An acute glaucoma model was generated in C57BL/6J mice.Intraperitoneal injection of resveratrol was treated continuously for 5 days.Immunofluorescence staining was utilized to determine survived RGCs.HE staining was used to evaluate retinal thickness.Changes in Bax,Bcl2,Cleaved Caspase3,Brn3a,HIF-1α,VEGF,p53,p38,PI3K and Akt protein expression were identified by Western Blot.ERG was employed to assess retinal function.Results:Resveratrol administration significantly increased Brn3a-labelled retinal ganglion cells and retinal thickness elicited by retinal I/R injury at 7 days.Western blot showed that the expressions of Bax and Cleaved Caspase3 protein in the resveratrol treatment after3 day I/R injury were significantly lower in comparison with I/R injury at 3 days.Besides,Brn3a and Bcl2 protein levels were remarkably increased in the resveratrol treated I/R injury group at 3 days compared to the group with I/R injury alone at 3 days.Moreover,administration of resveratrol in I/R group at 3 days substantially attenuated the levels of HIF-1α,VEGF,P-p38 and p53 compared with the group following retinal I/R injury at 3 days.Furthermore,western blot revealed that the levels of P-PI3K and P-Akt were notably increased in the group treated with resveratrol 3 days post-injury compared with the group 3 days after injury without resveratrol treatment.Similarly,compared with the group at 3 days after I/R injury,both A-wave and B-wave amplitudes were also substantially elevated by resveratrol administration at 3 days after I/R injury.Conclusion:Our data demonstrated that resveratrol can mitigate retinal I/R injury elicited retinal ganglion cell loss and retinal dysfunction by inhibiting HIF-1α/VEGF and p38/p53 pathways while activating PI3K/Akt pathway.Part Ⅲ:Resveratrol protects retinal ganglion cells from mouse retinal ischemic injury by modulating the SIRT1/NF-κB axisPurpose:Glaucoma is a kind of blindness characterized by progressive degeneration of retinal ganglion cells,progressive atrophy of optic nerve axons and irreversible visual field defects caused by elevated intraocular pressure(IOP).So far,the treatment for glaucoma still mainly focus on lowering IOP.Thus,this section determined to investigate whether resveratrol can attenuate retinal ganglion cells death caused by retinal ischemia reperfusion injury via modulating SIRT1/NF-κB axis.Methods:This section utilizes an acute model of glaucoma.Resveratrol(an activator of SIRT1)was intraperitoneally injected for five consecutive days.Intravitreal injection of sirtinol(an inhibitor of SIRT1)was performed on the day of retinal I/R injury.Survival of RGCs was detected using immunostaining.TUNEL staining was utilized to assess retinal cells apoptosis.Retinal function was evaluated by ERG.Western blot was employed to examine the proteins of SIRT1,NF-κB,IL-6,Bax,Bcl2,Cleaved Caspase3 and Brn3a.The distribution and expression of SIRT1 and NF-κB in retinas were identified by immunofluorescence.Results:Resveratrol administration remarkably increased Brn3a-labelled retinal ganglion cells and mitigated retinal ganglion cell apoptosis elicited by retinal I/R injury.Moreover,resveratrol treatment also significantly decreased the protein levels of NF-κB,IL-6,Bax and Cleaved Caspase3,and increased the protein expressions of SIRT1 and Bcl2.Furthermore,resveratrol intervention substantially inhibited the reduction of ERG amplitudes induced by retinal I/R injury.More importantly,simultaneous administration of resveratrol and sirtinol abrogated the effects of resveratrol on down-regulating NF-κB protein expression and up-regulating SIRT1 protein level,as well as blocked the protective effect of resveratrol on RGCs reduction caused by retinal I/R injury.Conclusion:Our findings revealed that resveratrol can alleviate retinal ganglion cell loss and retinal dysfunction elicited by retinal I/R injury,and this neuroprotective action is partially modulated through the SIRT1/NF-κB axis.Taken together,our results further reinforced the potential function of resveratrol in treating glaucoma in the future. |
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