Effects And Mechanisms Of Curcumin On Apoptosis Of Retinal Ganglion Cells In Chronic Ocular Hypertension Rats

Objective:By observing the effects of curcumin on retinal ganglion cells（RGCs）apoptosis and the expression of antioxidant proteins,such as glutamate-cysteine ligase modifier subunit（GCLM）and heme oxygenase-1（HO-1）in the rat model of chronic ocular hypertension,the mechanism by which curcumin protects against oxidative stress damage in the glaucoma model can be speculated and the theoretical basis for clinical studies can be provided.Methods:21 Spraque-Dawley（SD）rats were randomly divided into sham operation group,high intraocular pressure model group and curcumin treatment group,with 7 rats in each group.The rat models of chronic ocular hypertension were established by cauterization of the superior scleral veins in the high intraocular pressure model group and the curcumin treatment group,and the sham operation group only cut the conjunctiva without cauterization of the superior scleral veins;in addition,the rats in the curcumin treatment group were treated with curcumin at a dose of 4ml/kg once a day for 3 weeks,and the rats in the sham operation group and the high intraocular pressure model group were treated with pure water at a dose of 4ml/kg once a day for 3 weeks.The changes of intraocular pressure in the three groups were monitored before modeling,30min,1 week,2 weeks and3 weeks after modeling.After 3 weeks,all the rats were euthanized.HE staining was used to compare the morphological and pathological changes of retina,the number of RGCs and the thickness of ganglion cell layer（GCL）in the three groups;TUNEL was used to detect the apoptosis of RGCs and retinal cells in the three groups;the expression levels of GCLM m RNA and HO-1 m RNA in the retina of the three groups were compared by real-time fluorescence quantitative PCR;immunohistochemical staining and Western blot were used to detect the expression of GCLM and HO-1 protein in the retina of the three groups.Results:The intraocular pressure measurement results showed that there was no significant difference among the three groups before modeling（F=0.038,P>0.05）,which indicated that the groups were comparable;after controlling the grouping factors,the intraocular pressure data of the high intraocular pressure model group and the curcumin treatment group at each time point after modeling were significantly different from that before modeling,and were higher than that before modeling,indicating that modeling was successful.HE staining showed that compared with the sham operation group,the rats in the high intraocular pressure model group had disordered retinal morphology,unclear stratification,thinning of GCL（P<0.0001）,irregular arrangement of cells,reduced number of RGCs（P<0.0001）,and edema of inner and outer nuclear layers;under the same effect of high intraocular pressure,the retinal morphology of the curcumin treatment group was normal compared with the high intraocular pressure model group,with clear stratification,thickening of GCL（P<0.001）,regular arrangement of cells,increased number of RGCs（P<0.01）,and reduced edema of inner and outer nuclear layers.TUNEL assay showed that compared with the sham operation group,the apoptosis rate of RGCs in the high intraocular pressure model group and the curcumin treatment group increased,and the differences were statistically significant(P _{model group}<0.0001;P _{treatment group}<0.05);compared with the high intraocular pressure model group,the apoptosis rate of RGCs in the curcumin treatment group was significantly decreased（P<0.001）;the comparisons of the total apoptosis rate of retinal cells among the three groups were consistent with the apoptosis rate of RGCs.Real-time fluorescence quantitative PCR showed that compared with the sham operation group,the relative expression of GCLM m RNA in the retina of the high intraocular pressure model group and the curcumin treatment group increased,and the differences were statistically significant(P _{model group}<0.05;P _{treatment group}<0.001);compared with the high intraocular pressure model group,the curcumin treatment group had a significant increase in the relative expression of GCLM m RNA in the retina（P<0.05）;the comparisons of the relative expression of HO-1 m RNA among the three groups were consistent with that of GCLM m RNA.The results of immunohistochemistry staining and Western blot showed that the protein expression levels of GCLM and HO-1 were consistent with the results of real-time fluorescence quantitative PCR.Conclusions:1.Curcumin can inhibit the apoptosis of RGCs in the rat model of chronic ocular hypertension,as shown by the increase of RGCs number,the recovery of GCL thickness and the reduction of RGCs apoptosis rate;2.Curcumin inhibits the apoptosis of RGCs in the rat model of chronic ocular hypertension possibly by activating the antioxidant defense system and up-regulating the expression of antioxidant genes GCLM and HO-1 in Keap1-Nrf2-ARE signaling pathway.