| Objective:To study the mechanism of retinal injury and apoptosis of retinal ganglion cells(RGC)caused by acute ocular contusion,and the protective mechanism of Chufeng Yisun Decoction on retina and optic nerve.Methods:Select age-appropriate healthy ordinary rabbits,weighing about 2kg,all male,no ocular fundus diseases.Using the ’ imitation Allen ’ method,30 rabbits were successfully modeled.They were randomly divided into 6 groups:blank group,model group,experimental high,medium and low dose groups,and control group.The experimental high,medium and low groups were given Chufeng Yisun Decoction,and the control group was given mecobalamin.Administration for 14 days.HE staining,histochemistry,TUNEL apoptosis detection and RT-qPCR detection were used respectively.Results:1.Apparent morphological changesThere was no abnormal change in the ocular surface of the normal group;in the model group,the rabbit eyeball conjunctiva hemorrhage,cornea and eyelid edema,conjunctival sac secretions,central cornea had been hit by heavy objects,there is no smooth eye surface morphology,blood in the anterior chamber,peeping was not clear,slow reflection of light;in the experimental group of Chufeng Yisun Decoction,the conjunctival hemorrhage of rabbit eyeball disappeared,the edema of cornea and eyelid disappeared,the conjunctival sac had no secretion,the corneal surface was smooth,the anterior chamber could be clearly detected,and the light reflection speed became faster.In the mecobalamin group,there were still bleeding points on the conjunctiva,corneal and eyelid edema improved,conjunctival sac still had a small amount of secretion,the corneal surface was smooth,and there was still a small amount of blood in the anterior chamber,but the ophthalmoscope could peep in and the light reflection was improved.2.HE staining of retinal morphological changesThe morphology of the retina of the normal group was arranged in an orderly manner,the structural level was clear,the boundary was obvious,the inner nuclear layer and the outer nuclear layer were closely arranged,the staining of each layer was uniform,and the cell morphology was complete and orderly.In the model group,edema appeared in each layer of the retina,the outer nuclear layer gap became larger and the edema was obvious.In addition to the wind Yisun Decoction low dose group,the outer nuclear layer gap was slightly large,the inner nuclear layer gap becomes larger and the cavity appears;in the middle dose group of Chufeng Yisun Decoction,the inner and outer nuclear layers were closely connected,the inner nuclear layer was empty,and the number of ganglion cells increased.The inner nuclear layer of the high-dose group of Chufeng Yisun Decoction was empty,and the layers were also arranged in order;in the mecobalamin group,each layer of edema,inner nuclear layer edema and cavity,and ganglion cells were less.3.Positive expression results of immunohistochemical protein(1)Immunohistochemical Apaf-1 protein results showed that compared with the blank group,the positive expression of Apaf-1 protein in the model group increased(P<0.01);compared with the model group,the positive expression of Apaf-1 protein in the high,medium and low dose groups and the mecobalamin group decreased(P<0.01).The positive expression of Chufeng Yisun Decoction high and medium dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.05).(2)The results of immunohistochemistry showed that the positive expression of Bax protein in the model group was higher than that in the blank group(P<0.01).Compared with the model group,the positive expression of Bax protein in the high,medium and low dose groups and the mecobalamin group decreased(P<0.01).The positive expression of Chufeng Yisun Decoction high and medium dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.01).(3)Immunohistochemical Bcl-2 protein results showed:Compared with the blank control group,the positive expression of Bcl-2 protein in the model group was decreased(P<0.01).Compared with the model group,the positive expression of Bcl-2 protein in the high,medium and low dose groups and the mecobalamin group increased(P<0.01).The positive expression of middle dose of Chufeng Yisun decoction was higher than that of mecobalamin group,the difference was statistically significant(P<0.05).(4)The results of immunohistochemistry showed that the positive expression of Caspase-3 protein in the model group was higher than that in the blank group(P<0.01).Compared with the model group,the positive expression of Caspase-3 protein in the high,medium and low dose groups of Chufeng Yisun Decoction decreased(P<0.01),and the positive expression of Caspase-3 protein in the mecobalamin group decreased(P<0.05).The positive expression of Chufeng Yisun Decoction high and medium dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.01)(5)The results of immunohistochemistry showed that the positive expression of Caspase-9 protein in the model group was higher than that in the blank control group(P<0.01).Compared with the model group,the positive expression of Caspase-9 protein in the high,medium and low dose groups and the mecobalamin group decreased(P<0.01).The positive expression of Chufeng Yisun Decoction high and medium dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.01).(6)The results of immunohistochemical Cytc protein showed that the positive expression of Cytc protein in the model group was higher than that in the blank group(P<0.01).Compared with the model group,the positive expression of Cytc protein in the high,medium and low dose groups of Chufeng Yisun Decoction decreased(P<0.01),and the positive expression of Cytc protein in the mecobalamin group decreased(P<0.05).The positive expression of Chufeng Yisun Decoction high and medium dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.01).4.TUNEL apoptosis resultsCompared with the blank control group,the number of positive cells in the model group increased(P<0.01);compared with the model group,the number of positive cells in the high,medium and low dose groups and the mecobalamin group decreased(P<0.01).The number of positive cells in the high and medium dose groups was lower than that in the mecobalamin group,and the difference was statistically significant(P<0.05).5.RT-qPCR mRNA expression results(1)The expression of Apaf-1 mRNA showed that the expression of Apaf-1 mRNA in the model group was higher than that in the blank control group(P<0.01).Compared with the model group,the expression of Apaf-1 mRNA in the high,medium and low dose groups of Chufeng Yisun Decoction decreased(P<0.01),and the expression of Apaf-1 mRNA in the mecobalamin group decreased(P<0.05).The expression of middle dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.05).(2)The results of Bax mRNA expression showed that compared with the blank control group,the expression of Bax mRNA in the model group was increased(P<0.01).Compared with the model group,the expression of Bax mRNA in the high,medium and low dose groups of Chufeng Yisun Decoction decreased(P<0.01),and the expression of Bax mRNA in the mecobalamin group decreased(P<0.05).The expression of high dose group was lower than that of mecobalamin group,the difference was statistically significant(P<0.05).(3)The results of Caspase-3 mRNA expression showed that compared with the blank control group,the expression of Caspase-3 mRNA in the model group was increased(P<0.01).Compared with the model group,the expression of Caspase-3 mRNA in the high,medium and low dose groups and the mecobalamin group decreased(P<0.01).The expression in the medium-dose group was lower than that in the mecobalamin group,and the difference was statistically significant(P<0.05).(4)The results of Caspase-9 mRNA expression showed that compared with the blank control group,the expression of Caspase-9 mRNA in the model group increased(P<0.01).Compared with the model group,the expression of Caspase-9 mRNA in the high,medium and low dose groups and the mecobalamin group decreased(P<0.01).The expression of high and medium dose groups of Chufeng Yisun Decoction was lower than that of mecobalamin group,and the difference was statistically significant(P<0.01).(5)The results of Cytc mRNA expression showed that compared with the blank control group,the expression of Cytc mRNA in the model group was increased(P<0.01).Compared with the model group,the expression of Cytc mRNA in the high,medium,low dose and mecobalamine groups was decreased(P<0.01).The expression in the medium-dose group was lower than that in the mecobalamin group,and the difference was statistically significant(P<0.05).Conclusion:1.The intervention method of Chufeng Yisun Decoction can improve the pathological changes of ocular surface of ocular contusion,improve the damage of retina to a certain extent,have a positive effect on the acute stage of ocular contusion,protect the retina and optic nerve,and the effect is better than that of mecobalamin.2.Chufengyisun decoction can down-regulate the positive expression of Apaf-1,Caspase-3,Caspase-9,Bax,Cytc protein and mRNA,up-regulate the positive expression of Bcl-2 protein,thus inhibiting the occurrence of apoptosis and protecting the retina and optic nerve. |
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