The Effects Of Complanatoside A On Photoreceptor And Microglial Cells In Mice With Retinal Degeneration

Retinal degeneration（RD）,a class of neurodegenerative diseases,is one of the leading causes of visual impairment worldwide.Its main pathological feature is the progressive death of neurons in the retina,resulting in the loss and apoptosis of many neurons in the retina,especially photoreceptor cells.In addition,the gradual activation of microglial cells in the retina in the process of degeneration leads to the morphology change from amoeboid cell morphology to round cell bodies,and gradually migrate to the outer nuclear layer of the retina.It also secretes many pro-inflammatory cytokines/chemokines,which promote inflammatory response and accelerate the loss of photoreceptor cells.Therefore,the activation of microglia is considered to be one of the typical pathological changes in retinal degenerative diseases.At present,there are no effective drugs and surgical methods to completely treat retinal degeneration,some treatments have been taken to delay the development of the disease by using nutrients.Astragalus complanatus,a leguminous plant,is the dried mature seed of Astragalus complanatus R.Br.,which is widely used in clinical Chinese medicinal materials.It contains a variety of amino acids,flavonoids,and other organic substances,which has the function of anti-inflammatory,anti-apoptosis,antioxidant characteristics and can be used for the treatment of dim eyesight,kidney deficiency,lumbago,and other diseases.Flavonoids are important bioactive compounds in Astragalus complanatus.Complanatoside A occupy the main components in the flavonoids of Astragalus Complanatus,which plays an important role in its pharmacological effects.For example,it has the effect of protecting the liver against liver fibrosis and inhibiting the secretion of inflammatory factors.Objective 1.To investigate the effects of complanatoside A on visual function in mice with retinal degeneration.2.To determine the effects of complanatoside A on the loss of photoreceptor cells and oxidative reaction during retinal degeneration.3.To determine the effects of complanatoside A on inflammatory factors and quantity of microglia activated during retinal degeneration.Method 1.Effect of complanatoside A on visual function of dark-adapted flash electroretinogram（F-ERG）in RD1 mice.RD1 mice were gavaged with complanatoside A 50mg/kg each time,3 times a day at 7 day after birth.After 10 days,F-ERG was used to detect the amplitudes of a,b and Ops waves to evaluate the effect of complanatoside A on the visual function of RD1 mice.2.Effect of complanatoside A on photoreceptor cells.RD1 mice were gavaged with complanatoside A 50mg/kg each time,3 times a day at 7 day after birth.After 10days,the effect of complanatoside A on the apoptosis of RD1 photoreceptor cells was evaluated by Tunel.661W cells,a cell line of mice retinal photoreceptor cell,were treated with H₂O₂ for 30 min and cultured with complanatoside A for 24 h.The viability and apoptosis of 661W cells were evaluated by CCK-8 and fluorescence immunohistochemical staining.3.Effect of complanatoside A on microglia.RD1 mice were gavaged with complanatoside A 50mg/kg each time,3 times a day at 7 day after birth.Ten days later,the number and morphology of retinal microglia were evaluated.The expression of IL-1β,IL-6,i NOS,CCL2 inflammatory factors in the whole retina were detected by RT-PCR.BV2 cells,a mice microglia cell line,were cultured in vitro and treated with LPS for 4 hours,followed by complanatoside A for 24 hours.RT-PCR and fluorescence staining were used to evaluate the effect of complanatoside A on the apoptosis of BV2cells and the expression of inflammatory factors such as IL-1β,IL-6,i NOS,CCL2.Result 1.Complanatoside A can improve a,b,Ops oscillation amplitude of RD1 mice,protect the visual function of retina,and delay the degeneration process of RD1 mice.2.Complanatoside A can effectively protect the structure of photoreceptor cell layer in the retina of RD1 mice,reduce the apoptosis of photoreceptor cells in the degeneration process of RD1 mice,and maintain the thickness of photoreceptor cells in the outer nuclear layer.After treating 661W cells with H₂O₂,complanatoside A can increase the viability of 661W cells and reduce the number of apoptotic cells in 661W cells.3.Complanatoside A can reduce the number of microglia activated during retinal degeneration of RD1,and reduce the expression of IL-1β,IL-6,i NOS,CCL2 in retinal tissue.After treating BV2 with LPS,the number of apoptotic cells and the expression of IL-1β,IL-6,i NOS,CCL2 inflammatory factors were increased,while down-regulating after the intervention of complanatoside A.Conclusion Complanatoside A can effectively protect the visual function of RD1 mice under dark adaptation,protect the photoreceptor cell layer in the retinal tissue structure,reduce the apoptosis number of photoreceptor cells,inhibit the number of activated microglia,and reduce the gene level expression of inflammatory factors in the retinal tissue.In addition,complanatoside A can enhance the cell activity,reduce the apoptosis,and inhibit inflammatory factor expression of activated BV2 cells.