Study On The Mechanism Of Regulating The Activity Of Calpain And Inhibiting The Apoptosis Of Photoreceptor Cells In MNU Rats By Bujing Yishi Tablet

Objective: The death of photoreceptor cells is mainly regulated by apoptosis.Autophagy,as a protective mechanism of organism,may be involved in it,and calpain activation can inhibit autophagy.The aim of this study was to explore the mechanism of regulating calpain activity and resuming autophagy in order to protect photoreceptor cells from apoptosis by Bu Jing Yi Shi tablet,which has protective effect on photoreceptor cells.In order to provide some scientific basis and therapeutic target for the research field of photoreceptor cells protection of traditional Chinese medicine.Methods:the rat retinal photoreceptor apoptosis model was established by intraperitoneal injection of MUN.The rats were randomly divided into 4 groups(normal group,model group,vitamin A group,Chinese medicine group)and each group will be treatment for 14 days.The morphology and apoptosis of retinal photoreceptor cells were observed by HE staining and TUNEL method.Immunohistochemical technique was used to detect the expression of cysteine protease-3(caspase-3)and calpain in retinal tissue and to observe the effect of Bu Jing Yi Shi tablet on the regulation of calpain activity on apoptosis of photoreceptor cells.The protein expression of microtubule-associated protein light chain 3 in retinal photosensitive cells was detected by immunoblotting,and the effect of Bu Jing Yi Shi tablet on autophagy of photoreceptor cells was observed.Results: 1.After modeling,HE staining showed that the total retinal thickness of retinal photoreceptor cells decreased,the structure of each layer arranged irregularly,the number of cell layers of each layer decreased and even the photoreceptor cell layer disappeared.Tunel showed that the apoptosis of retinal photoreceptor cells increased gradually,reached the maximum at 5 days,and decreased gradually into the later stage of apoptosis on the 7th day,indicating that the model was successful.2.(1)In the normal group,the cells of each layer were arranged neatly and without swelling,and in the model group,the total thickness of retina decreased,the structure of each layer was disordered and loose,the number of layers of cells in each layer decreased,and the structure was not clear.The thickness of retina in vitamin A group and traditional Chinese medicine group was significantly better than that in gmodel group,but it was lower than that in normal group,The cells in traditional Chinese medicine group were more neatly arranged and the structure was clear than in vitamin A group.(2).compared with the normal group,the average optical density of photoreceptor cells in each group increased significantly(p<0.05),suggesting that apoptosis occurred in each group.Compared with the model group,the average optical density of retinal photosensitive cells in vitamin A group and traditional Chinese medicine group decreased significantly(p<0.05),and the level of apoptosis was down-regulated.Compared with group A,the average optical density of retinal photoreceptor cells decreased and the number of apoptotic cells decreased significantly(p<0.05).The results of Tunel detection showed that: model group >vitamin A group > traditional Chinese medicine group > normal group.The results of Tunel detection were as follows: model group > vitamin A group > traditional Chinese medicine group > normal group.(3).calpain score of traditional Chinese medicine group was 1 ~ 2,caspase-3 score was 3 ~ 6;calpain score was 4 ~ 5,caspase-3 score was 6 ~ 8 in vitamin A group;calpain score was 4 ~ 6,caspase-3 score was 9 ~ 12 in model group;In normal group,calpain score was 0 ~ 1,caspase-3 score was 0 ~ 2;The decrease of the activity of calpain and the inhibition of caspase-3 in the traditional Chinese medicine group showed that the regulation of the activity of troponin could regulate the apoptosis of the photosensitive cells,and Bujing Yizhi tablet could inhibit the activity of calpain and down-regulate the apoptosis of the photosensitive cells,which was better than that of the vitamin A group.Capase-3 score: model group > vitamin A group >Chinese medicine group > normal group;calpain score: model group > Chinese medicine group > normal group.(4).the gray value of LC3 protein in TCM group was significantly higher than that in other groups(P < 0.05).Compared with the traditional Chinese medicine group,the gray value of LC3 protein expression in vitamin A group,normal group and model group decreased significantly(P < 0.05).The expression of LC3: Chinese medicine group > normal group > vitamin A group > model group;The results showed that Bujing Yizhi tablet could regulate autophagy and increase autophagy level and the autophagy level of vitamin A group was lower than that of traditional Chinese medicine group,suggesting that autophagy pathway might also participate in photosensitive cell apoptosis,and the increase of autophagy level could inhibit apoptosis.Conclusion: In the rat retinal photoreceptor apoptosis model induced by MUN,calpain was activated,and then the activity of caspase-3 was up-regulated,the expression of LC3 was down-regulated,and the autophagy of photoreceptor cells was inhibited,which resulted in apoptosis.Bujing Yizhi tablet may reduce the activity of calpain,down-regulate the expression of caspase-3 and increase the content of LC3,so as to restore autophagy and reduce the apoptosis of photoreceptor cells.