| OBJECTIVE:A rat model of Nonarteritic Anterior Ischemic Optic Neuropathy(NAION)was established by photodynamic method.To observe the retinal histopathological changes induced by hyperocular heat injection in rats and its effects on retinal ganglion cells(RGCs)apoptosis and the expressions of apoptosis factors Bcl-2 and Bax in NAION rats.To explore the therapeutic mechanism of "Guoyanre" acupuncture on NAION,verify the necessity of the selection of "Guoyanre" acupuncture,and provide theoretical basis for the clinical selection of the acupuncture on NAION.METHODS:Ten SD rats were randomly selected from 40 eligible rats and set as blank group.The remaining 30 rats were modeled by photodynamic method and evaluated comprehensively by Fundus Fluorescein Angiography.After successful modeling,the group was divided into three groups(n=10)by random number table method: the model group,the "Guoyanre" group,and he flat tonic and flat diarrhea group.Points selected Fengchi,Jingming,Taiyang.Fengchi point was treated with the flat tonic and flat diarrhea method and "Guoyanre" method in the flat tonic and flat diarrhea group and "Guoyanre" group.Jingming avoid eyeball and Taiyang oblique puncture 0.3cm,used the flat tonic and flat diarrhea method.Once a day for 14 consecutive days.Blank group and model group are not processed.After the intervention,the right eye was removed and treated,fixed and embedded to make paraffin sections.Morphological changes of RGCs were observed after HE staining.The expressions of RGCs apoptosis factors Bcl-2,Bax and Bax/Bcl-2 were detected by immunohistochemistry.The effect of TUNEL assay on the expression rate of positive apoptotic cells.The sections were photographed and the positive density value was counted.The final data were analyzed statistically using SPSS23.0 software.RESEULTS:1、HE staining of the retina and RGCs showed: in the blank group(FIG.A),the retinal structure of rats was complete,the tissue structure of each layer was complete,the RGCs cells were arranged neatly,the particles were clear,the cells were monolayer,closely arranged,the size was different,and there was no abnormal shape.In the model group(Figure B),the structure of choroid layer was damaged,the cells in the inner layer were obviously missing and the gap increased,the RGCs were obviously missing,some cells had abnormal morphology such as nuclear shrinkage,damaged or even lost,and the arrangement was relatively loose and disordered.The inner core layer cells in the flat tonic and flat diarrhea method group(FIG.C)and the "Guoyanre" group(FIG.D)were slightly damaged,and the rest of the cells were intact.Compared with the model group,the inner core layer damage in the two groups was significantly reduced,the loss degree of RGCs was less,the residual cell nuclei existed more,and the degree of nucleus abnormalities was less,The arrangement is dense and orderly.2、Immunohistochemical staining of Bcl-2 and Bax in rat retina:(1)In blank group,a small amount of Bax and a lot of Bcl-2 were expressed in RGCs.(2)The expression of RGCs Bax positive density was as follows:(1)Compared with model group(P<0.01),there was a statistical difference;(2)There were statistically significant differences between the model group and the flat tonic and flat diarrhea group(P<0.05)and the "Guoyanre" group(P<0.05);(3)There was no statistically significant difference between the flat tonic and flat diarrhea group and the "Guoyanre" group(P>0.05);(4)From the general trend,model group > flat tonic and flat diarrhea group > "Guoyanre" group > blank group.The results suggested that the expression of Bcl-2 was up-regulated by "Guoyanre" injection,and the up-regulation ability was stronger than that in the flat tonic and flat diarrhea group.(3)The expression of positive density of RGCs Bcl-2 showed:(1)Compared with the model group,the blank group had a statistically significant difference(P<0.05);(2)There were statistical differences between the model group and the flat tonic and flat diarrhea group(P<0.05)and the "Guoyanre" group(P<0.05),and the "Guoyanre" group had more significant differences;(3)There was a statistically significant difference between the flat tonic and flat diarrhea group and the "Guoyanre" group(P<0.05);(4)The density trend of Bcl-2positive RGCs in the four groups was as follows: blank group > "Guoyanre" group > the flat tonic and flat diarrhea group >model group.The results suggested that "Guoyanre" acupuncture could down-regulate the expression of Bcl-2,and the down-regulation ability was stronger than that of the flat tonic and flat diarrhea group.(4)The change of Bax/Bcl-2 ratio in each group:(1)Compared with the model group,the blank group had a statistically significant difference(P<0.01);(2)There were statistical differences between the model group and the flat tonic and flat diarrhea group(P<0.05)and the "Guoyanre" group(P<0.01),but the "Guoyanre" group had a more significant difference;(3)There was statistical difference between the flat tonic and flat diarrhea group and "Guoyanre" group(P<0.05);(4)From the trend of ratio of the four groups: model group >the flat tonic and flat diarrheagroup > "Guoyanre" group > blank group.The results indicated that the "Guoyanre" injection could lower the expression of Bax/Bcl-2 ratio compared with the flat tonic and flat diarrhea.3、The apoptotic rate of RGCs was detected by TUNEL:(1)There were a few apoptotic cells in the blank group,and the apoptotic rate of the model group was higher than that of the other three groups.The apoptotic rate of the "Guoyanre" group was lower than that of the flat tonic and flat diarrhea group.The mean trend of cell apoptosis rate was described as follows: blank group < "Guoyanre" group < the flat tonic and flat diarrhea group< model group.(2)The results of cell apoptosis rate were as follows:(1)Compared with model group(P<0.001),there was a statistically significant difference;(2)There were statistically significant differences between the model group and the two intervention groups(P<0.01).(3)There was no statistically significant difference between the flat tonic and flat diarrhea group and the "Guoyanre" group(P>0.05).CONCLUSION:The mechanism of "Guoyanre" acupuncture in the treatment of NAION may be to inhibit the damage of optic nerve apoptosis by enhancing the up-regulation of Bax,down-regulation of Bcl-2 and down-regulation of Bax/Bcl-2 ratio to reduce the apoptosis rate of RGCs cells,so as to play the role of RGCs anti-apoptosis and treatment of NAION. |
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