Effects Of Exogenous H₂S On Opa1 Of Retinal Cells In Rats With Acute Ocular Hypertension

Objective:To investigate the effect of regulation of the expression and distribution of OPA1 protein by exogenous H₂S on apoptosis of retinal cells in SD rats with acute ocular hypertension.Methods:The AOH model of SD male rats was established by increasing the intraocular pressure by anterior chamber perfusion.Sodium hydrosulfide（NaHS）was used as a donor to provide exogenous H₂S.52 healthy SD rats without healthy eye disease were randomly divided into control group,AOH group and acute high intraocular pressure（AOH）+NaHS according to random number table method.The latter two groups were divided again into six time periods-1h,6h,12h,24h,48h and 72h after the model establishment,respectively.The biochemical method was used to detect the level of retinal hydrogen sulfide.The apoptosis of RGCs was detected by terminal-dUTP nick end labeling（TUNEL）.The real-time quantitative polymerase chain reaction（qPCR）was used to detect retinal OPA1mRNA.Western blotting（WB）was used to detect the expression of OPA1 protein and CytC in cytoplasm and mitochondria.The expression of OPA1 in the retina was detected by immunohistochemistry（IHC）.The morphology of mitochondria was observed by transmission electron microscopy.Results:（1）The level of H₂S:compared with the control group,the level of H₂S in all AOH groups dropped to different extents（P=0.00）except the groups of 1h（P=0.411）and24h（P=0.411）;while compared with the control group,the level of H₂S in all AOH+NaSH groups increased to different extents（P=0.00）except the groups of 48h（the level of H₂S had no statistical significance,P=0.350）and 72h（the level of H₂S dropped,P=0.00）.The H₂S of AOH+NaSH group was higher than that of AOH group at each time point,and the difference was statistically significant（P<0.05）.（2）The apoptosis detected by TUNEL:compared with the control group,the AOH+NaSH group had no significant increase in the apoptosis of RGCs at 1h after the model establishment（P>0.05）;the apoptosis increased at all time periods of the AOH group and the AOH+NaSH group（except the group of 1h）（P<0.05）;and although the apoptosis trend of the AOH+NaSH group was the same with the AOH group,its apoptosis index at all time periods showed an apparent decline compared with the AOH group,and the differences were statistically significant（P<0.05）.（3）The mRNA expression of OPA1 in rat retina was detected by qPCR.The mRNA expression of OPA1 in AOH+NaSH and AOH groups was decreased compared with the control group（P<0.05）.The difference was statistically significant.The AOH+NaSH group and AOH group were compared at different time points.The expression of OPA1mRNA in the 1h group was lower than that in the AOH group.The mRNA expression of OPA1 was higher than that in the AOH group at the other time points.The difference was statistically significant（P<0.05）.（4）Detection of expression of OPA1 and Cyt C in mitochondria and cytoplasm by WB assay.The protein expressions of OPA1 and Cyt C in the mitochondria of AOH group were gradually decreased compared with the control group,and the difference was statistically significant（P<0.05）.AOH+NaSH group Compared with the control group（P<0.05）;the expression of OPA1 and Cyt C in the AOH+NaSH group was lower than that in the AOH group except for the AOH group,and the expression of OPA1 and Cyt C was higher than AOH at the remaining time points.There was no statistical significance in the other groups except for 12h（P=0.574,P=0.562）（P<0.05）.In the cytoplasm,the time points of the two groups were gradually increased compared with the control group,the difference was statistically significant（P<0.05）;the AOH+NaSH group was compared with the AOH group at each time point,OPA1 and Cyt C The expression was lower than that of the AOH group,and the difference was statistically significant（P<0.05）.（5）IHC detects the expression of retinal OPA1:Compared with the control group,the expression quantity of OPA1 protein in both the AOH group and the AOH+NaSH group decreased at all time periods except that the expression quantity of OPA1 protein in the AOH+NaSH group did not decrease significantly at 1h;and compared with the AOH group,the expression quantity of OPA1 protein in the AOH+NaSH group was higher at all time periods,and the differences were statistically significant at all time periods（P<0.05）except 6h（P=0.253）and 24h（P=0.078）.（6）The mitochondrial morphology observed by transmission electron microscope:compared with the control group,mitochondrial damage in the AOH group was deteriorated with the time passing,and mitochondrial swelling,cytoplasmic vacuolation and autophagic vacuole could be observed（they were the clearest at 24h）;in the AOH+NaHS group,mitochondrial damage increased with time,mitochondria swelled,cytoplasmic vacuoles and autophagic bodies were observed,and mitochondria morphology was not significantly different at 48h and 72h.AOH+NaHS group and mitochondria lesions were alleviated at different time points compared with AOH group.Conclusion:Hydrogen sulfide may reduce the damage of RGCs in acute high intraocular pressure by regulating the expression and distribution of OPA1,and its protective effect is 24 hours after intervention.