| Objective: To explore the antioxidant regulation of acupuncture for protection of dopaminergic neurons in the retinas of guinea pigs with Form Deprivation Myopia(FDM),and relevant molecular mechanism as well,thus providing a new method and theoretical basis for the prevention and control of myopia.Methods: This study was divided into three parts.The first part: 3-week-old tricolor guinea pigs were used to establish a myopia model by means of form deprivation.They were randomly divided into the blank group,the model group and the acupuncture group.On the day of modeling,the temple,Hoku point,Ganshu point,Shenting point,Baihui point,and Dazhui point of the acupuncture group received electroacupuncture intervention.After 2w and 4w,the changes in diopter and axial length were measured respectively.After 4w,the samples were taken,and the pathological changes in retinal morphology,structure and thickness were evaluated by HE staining.The content of Dopamine(DA)and dihydroxy-phenyl acetic acid(DOPAC)in retinal and vitreous body of guinea pigs was determined by enzyme linked immunosorbent assay(ELISA),and the protein expression of retinal tyrosine hydroxylase(TH)and the average positive cell count were detected by Western blot and immunofluorescence,respectively.The second part: The experimental animal grouping and intervention were the same as the first part.The retinal superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)were detected by chemical colorimetry.Retinal reactive oxygen species(ROS)was detected by flow cytometry.The expression of extracellular signal-regulated kinase 1/2(ERK1/2),P-ERK1/2 and nuclear factor erythroid 2 related factor 2(Nrf2)was detected by Western blot.The third part: 3-week-old tricolor guinea pigs were used to establish a myopia model by means of form deprivation.They were randomly divided into the blank group,the model group,the acupuncture group,the inhibitor NC group and the U0126-Et OH group.Electroacupuncture intervention was given on the acupuncture group,the inhibitor NC group and the U0126-Et OH group starting on the day of modeling.10μl of ERK1/2 specific inhibitor U0126-Et OH was injected into the vitreous cavity of the right eye in the U0126-Et OH group,and the same amount of NC was injected into the vitreous cavity of the right eye in the inhibitor NC group.The changes in diopter and axial length,ERK1/2 pathway transduction protein and downstream oxidative stress index,dopamine-related index and mean positive cell count in FDM guinea pigs were observed.Results: 1.Study on the effect of acupuncture on retinal dopaminergic neurons in guinea pigs with FDM(1)Effects of acupuncture on diopter,axial length,retinal shape and thickness of FDM guinea pigs1)Diopter:Left and right eyes: There were no significant differences between the left and right eyes in the blank group at 2w and 4w as well as at 0week in each group(P > 0.05),and the values of the right eyes in model group and acupuncture group at 2w and 4w were significantly smaller than those of the left eye in the tow groups(P < 0.05).Different groups: There were no significant differences in each group at 0w,2w and4 w in the left eye and 0w in the right eye(P > 0.05),and all groups at 2w and 4w in the right eye showed blank group > acupuncture group > model group(P < 0.05).Different time points: Each group of the left and right eyes showed 0w > 2w > 4w at different time points(P < 0.05),that is,the myopic shift gradually formed with time.2)Axial length:Left and right eyes: There were no significant differences between the left and right eyes in the blank group at 2w and 4w as well as at 0week in each group(P > 0.05),and the values of the right eyes in model group and acupuncture group at 2w and 4w were significantly greater than those of the left eye in the two groups(P < 0.05).Different groups: there were no significant differences in each group at 0w,2w and4 w in the left eye and 0w in the right eye(P > 0.05),and all groups at 2w and 4w in the right eye showed blank group < acupuncture group < model group(P < 0.05).Different time points: Each group of the left and right eyes showed 0w < 2w < 4w at different time points(P < 0.05),that is,the myopic shift gradually formed with time.3)Retinal shape and thickness:Model group showed significant pathological changes with thinning of the whole retinal layer,inner nucleus layer(INL),outer plexiform layer(OPL)and outer nuclear layer(ONL),whose values were significantly lower than those of blank group(P < 0.05);acupuncture group showed mild thinning of the whole retinal layer,INL,OPL and ONL,whose values were significantly lower than those of blank group except OPL(P < 0.05),but greater than those of model group(P < 0.05).(2)Effect of acupuncture on dopamine synthesis,metabolism and neuronal expression in the retina of FDM guinea pigsCompared with the blank group,DA and DOPAC concentrations in the vitreous and retina,the mean number of TH positive cells in the retina,and TH protein expression were significantly decreased in model group(P < 0.05);compared with the model group,the expression of the above parameters in the vitreous and retina was significantly increased in acupuncture group(P < 0.05).2.Study on the mechanism of acupuncture on ERK1/2 pathway and anti-oxidation in retina of FDM guinea pigsCompared with the blank group,the activities of SOD and GSH-Px in the retina of the model group and acupuncture group were significantly decreased(P < 0.05),and the MDA content and i NOS activity were significantly increased(P < 0.05);compared with the model group,the activities of SOD and GSH-Px in the retina of the acupuncture group were significantly increased(P < 0.05),and the MDA content and i NOS activity were significantly decreased(P < 0.05).ERK1/2,p-ERK1/2,and Nrf2 protein expression was significantly increased in the retina of model group and acupuncture group compared with blank group(P < 0.05);the above parameters were significantly decreased in acupuncture group compared with model group(P < 0.05).The FITC mean fluorescence intensity of ROS in model group and acupuncture group was significantly increased compared with blank group(P < 0.05);this index was significantly decreased in acupuncture group compared with model group(P < 0.05).3.Mechanism of acupuncture regulating antioxidant and pathway-related expression in retinal dopaminergic neurons of FDM guinea pigs under ERK1/2 inhibition(1)Effect of acupuncture on FDM guinea pigs diopter and axial length under ERK1/2 inhibition1)Diopter:Left and right eyes: There were no significant differences between the left and right eyes of each group at 0w,and blank group at 2w and 4w(P > 0.05),and the values of the right eyes of all groups except blank group at 2w and 4w were significantly smaller than those of the left eye(P < 0.05).Different groups: There were no significant differences in each group at 0w,2w and4 w in the left eye and 0w in the right eye(P > 0.05),there was no statistical difference between the model group at 2w and 4w in the right eye and the U0126-Et OH group,acupuncture group and inhibitor NC group(P > 0.05),and there were statistical differences in diopter between the other groups(P < 0.05),as shown by the blank group > acupuncture group ≈ inhibitor NC group > model group ≈ U0126-Et OH group.Different time points: Each group of the left and right eyes showed 0w > 2w > 4w at different time points(P < 0.05),that is,myopic shift gradually formed with time.2)Axial length:Left and right eyes: There was no significant difference between the left and right eyes of each group at 0w,2w,and 4w blank group(P > 0.05),and the values of the right eyes of the model group,acupuncture group,and inhibitor NC group and U0126-Et OH group at 2w and 4w were significantly greater than those of the left eyes in the four groups(P < 0.05).Different groups: there were no significant differences in each group at 0w,2w and4 w in the left eye and 0w in the right eye(P > 0.05),there was no statistical difference between the model group at 2w and 4w in the right eye and the U0126-Et OH group,acupuncture group and inhibitor NC group(P > 0.05),and there were statistical differences in axial length between the other groups(P < 0.05),as shown by the blank group < acupuncture group ≈ inhibitor NC group < model group ≈ U0126-Et OH group.Different time points: Each group of the left and right eyes showed 0w < 2w < 4w at different time points(P < 0.05),that is,myopic shift gradually formed with time.(2)Acupuncture under ERK1/2 inhibition regulates antioxidant and ERK1/2pathway and dopamine-related expression in the retina of FDM guinea pigsCompared with the blank group,the activities of SOD and GSH-Px in the retina of the other four groups were significantly decreased(P < 0.05),and the MDA content and i NOS activity were significantly increased(P < 0.05);compared with the model group,the activities of SOD and GSH-Px in the retina of the acupuncture group and inhibitor NC group and U0126-Et OH group were significantly increased(P < 0.05),and the MDA content and i NOS activity were significantly decreased(P < 0.05);compared with the acupuncture group and inhibitor NC group,the activities of SOD and GSH-Px in the retina of the U0126-Et OH group were significantly decreased(P < 0.05),and the MDA content and i NOS activity were significantly increased(P < 0.05).Compared with the blank group,the expression of ERK1/2,p-ERK1/2,and Nrf2 protein in the retina of guinea pigs was significantly increased in the model group,acupuncture group,and inhibitor NC group(P < 0.05);compared with the model group,the above parameters were significantly decreased in the acupuncture group and inhibitor NC group and U0126-Et OH group(P < 0.05);compared with the acupuncture group and inhibitor NC group,the above parameters were significantly decreased in the U0126-Et OH group(P < 0.05).Compared with blank group,the concentrations of DA and DOPAC in vitreous and retina and the relative expression of TH protein in retina were significantly decreased in the other four groups(P < 0.05);compared with model group,the above indicators were significantly increased in acupuncture group and inhibitor NC group and U0126-Et OH group(P < 0.05);compared with acupuncture group and inhibitor NC group,the above indicators were significantly decreased in U0126-Et OH group(P < 0.05).Compared with the blank group,the mean number of retinal TH positive cells was significantly decreased in the other four groups(P < 0.05);this index was significantly increased in the acupuncture group and inhibitor NC group compared with the model group(P < 0.05);and this index was significantly decreased in the U0126-Et OH group compared with the acupuncture group and inhibitor NC group(P < 0.05).Conclusion: In FDM guinea pigs,acupuncture can slow down the progression of diopter,delay the growth of axial length,and inhibit the progression of myopia.2.Acupuncture can improve the synthesis and metabolism of dopamine in the retina of FDM guinea pigs,reduce the loss of retinal dopaminergic neurons,reduce retinal thickness thinning,and protect retinal dopaminergic neurons.3.With the deepening of myopia,the expression of ERK1/2,p-ERK1/2 and downstream Nrf2 and oxidative factors increased,and the expression of antioxidant factors decreased in the retina of FDM guinea pigs,indicating that form deprivation may cause retinal dopaminergic neurons injury by activating ERK1/2 pathway.4.Acupuncture may exert its antioxidant effect on the retina of FDM guinea pigs by inhibiting the ERK1/2 pathway,reduce retinal dopaminergic neurons injury,and delay myopic progression.5.Acupuncture can inhibit the expression of ROS and improve the oxidative stress induced by form deprivation in the retina of FDM guinea pigs,providing a theoretical and experimental basis for future antioxidant prevention and treatment of myopia. |
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