Effect Of Paeoniflorin On The Apoptosis Of Retinal Ganglion Cells In Rats With Chronic Ocular Hypertension

Objective: The rat model of chronic ocular hypertension was established by standard cauterization of the superior scleral vein.The effect of Paeoniflorin?PF?on apoptosis of retinal ganglion cells was clarified.To observe the changes of expression of upstream nuclear transcription factor E2 related factor 2?Nrf2?and downstream heme oxygenase-1?HO-1?proteins,preliminarily reveal the mechanism of PF inhibiting RGCs apoptosis in chronic glaucoma,and provide theoretical basis for the clinical application prospect of the drug.Methods:1.Establishment of chronic ocular hypertension model in SD rats: Thirty male SD rats were divided into blank control group,high intraocular pressure model group and paeoniflorin treatment group with right eye as experimental eye by random number table method.A rat model of chronic ocular hypertension?standard cauterization of superior scleral vein method?was established.The eyelid was opened with traction suture at the midpoint of the upper and lower eyelids.The conjunctival sac was flushed with 0.9% normal saline.The conjunctival sac was fixed at 1.5mm behind the corneal limbus and continuously cut at 6 13 clockwise.The subconjunctival fascia and muscles were gently and carefully separated.A total of 3 superficial scleral vein total branches?12:00,10:00 and 8:00?were exposed on both sides of the superior rectus muscle and below the outer rectus muscle.The vein total branches were cauterized with a table electrocoagulation hemostat.The cauterization is successful when the venous blood flow disappears at the cauterization site,the proximal vein is hyperemia and engorgement,and the distal vein blood flow disappears into a white line.In the blank control group,only the bulbar conjunctiva was cut and the superficial scleral vein was not treated.TONO-Pen AVIA tonometer was used to measure intraocular pressure.the intraocular pressure after operation was 1.7 times higher than that before operation,which was regarded as successful modeling.Paeoniflorin group received intraperitoneal injection of paeoniflorin?20mg/Kg?on time in rats,while the rest received intraperitoneal injection of 0.5ml of normal saline once a day for two weeks.Intraocular pressure was measured and recorded before,30 min after,7 days and 14 days after modeling.2.Tissue examination: On the second day after the last injection,the rats were killed under excessive anesthesia,the right eyeball was quickly removed,part of retrobulbar optic nerve tissue was retained,and 4% paraformaldehyde was fixed for 24 hours.Paraffin embedded retina pathological sections were stained with hematoxylin eosin staining to observe pathological changes of retina in each group of rats.Immunohistochemical method was used to detect the expression changes of Nrf2 and HO-1 in retina tissues of rats in each group.TUNEL apoptosis was used to detect the apoptosis of RGCs in each group of rats.3.Statistical analysis:All data were analyzed by SPSS 25.0 statistical software.The measurement data were described by mean standard deviation?`x± s?and compared between groups by analysis of variance of single factor repeated measurement.Multiple comparisons were made between the two groups and LSD-t test was used.Paired T test was used for comparison of self-control before and after.The counting data is expressed as [cases?%?],and the correlation analysis of the constituent ratios of two or more samples uses X² test,with p<0.05 being statistically significant.Results:1.Intraocular pressure: The intraocular pressure difference at different time points was statistically significant?spherical analysis: X²=6.66,P=0.247;F time?3,81?=560.672,P=0.000?,Before modeling,there was no significant difference among the three groups?F=1.191,P = 0.319?;The intraocular pressure of the model group and paeoniflorin group increased 30 min after modeling?tm=-25.086,tPF=-24.912,both P = 0.000?;7 days and 14 days after the model was established,the intraocular pressure of the model group and paeoniflorin group were higher than that of the blank control group?F1 w =388.43,F2 w =281.504,all P=0.000?.The intraocular pressure of the model group and the treatment group had no significant difference at each time point?F=1.223,P=0.283?.2.HE: Compared with the model group and paeoniflorin group,the blank control group has more regular retina structure,clearer layers,more regular arrangement of inner and outer nuclear layers,and more RGCs layer cells(t_{model group} =7.965,t PF group =3.833,both P=0.000);At the same time,the number of RGCs layer cells in paeoniflorin treatment group was higher than that in model group,and the difference was statistically significant?t=-5.014,P=0.000?.3.Tunel detection: No apoptotic cells were found in the blank control group.TUNEL-positive RGCs cells were found in the single visual field of the model group and paeoniflorin group,and the model group?12.1±1.52 /visual field?was more than paeoniflorin group?5.3±1.7 /visual field?,with statistical significance?t=9.410,P=0.000?.4.Immunohistochemical staining: NRF2/HO-1 was scattered in tan granules in the cytoplasm of RGCs of chronic ocular hypertension rats.In the model group,2 cases?20%?showed high expression of Nrf2 and HO-1,while 8 cases?80%?showed high expression of Nrf2 and HO-1 in the paeoniflorin treatment group.Through X² test,the difference between the two groups was statistically significant?X²=5,P=0.023?;The score difference between the model group and paeoniflorin group was analyzed by t test?Nrf2:t=-5.304,P=0.000;HO-1:t=-4.506,P=0.000?,Nrf2 and HO-1 proteins were significantly expressed in RGCs of paeoniflorin group?P<0.05?.Conclusions:1.Paeoniflorin can inhibit RGCs apoptosis in rats with chronic ocular hypertension.2.Paeoniflorin inhibits RGCs apoptosis in chronic ocular hypertension rats by up-regulating NRF2/HO-1 expression.