| Purpose:1.To evaluate the effect of naringin on diabetic retinopathy?DR?by in vivo experiments.And to explore its effect on the expression of Glial fibrillary acidic protein?GFAP?,and to reveal the effect of Naringin on diabetic retinal Müller cells.2.The retinal Müller cells?rMC-1?were cultured in high glucose culture medium to simulate the diabetic environment.To evaluate the effect of Naringin on hyperglycemia-induced rMC-1 oxidative stress and inflammatory response,and to clarify the effect of Naringin on rMC-1.3.To evaluate the activation of NF-?B pathway in the retinal tissue of animal experiment and the rMC-1 in cell experiment,reveal the possible mechanism of Narigin to protect DR.Material and method:36 rats of 8-week-old healthy SPF grade SD?Sprague-Dawley?male?excluding estrogen?were selected and the body weight was 220±30g.The rats were fed with 1W.They were randomly divided into 6 groups:control group,high dose Naringin?80mg/kg?group,diabetes mellitus group,diabetes mellitus+low dose Naringin?20mg/kg?group,diabetes mellitus+middle dose Naringin?40mg/kg?Dose Naringin?80 mg/kg?group,each group of 6 rats.The rats in the diabetic model group were fasted at 12 hours before modeling and injected with 65 mg/kg streptozotocin?STZ?intraperitoneally.The rats were sacrificed on the 3rd and 5th day.Rats with blood glucose?16.7mmol/L were used for follow-up experiments.High dose Naringin?80 mg/kg?group,diabetes mellitus+low dose Naringin?20 mg/kg?group,diabetes+middle dose Naringin?40 mg/kg?group,diabetes+high dose Naringin?80 mg/kg?Intraperitoneal injection of the corresponding dose of Naringin,continuous injection for 12 weeks,the control group and diabetic rats were given equal volume of saline.The body weight and blood glucose were monitored every two weeks.At the end of the 12th week,the body weight and blood glucose were measured when the rats were fasted at night,to evaluate the effects of Naringin on body weight and blood glucose.Rats in each group were sacrificed by intraperitoneal injection of 3.5ml/kg?10%?of chloral hydrate in rats.The rats in the inferior vena cava were sacrificed and the retinal tissue was taken and fixed in 10%neutral formaldehyde solution.Paraffin embedded,histologically sliced.The effect of Naringin on the retinal ganglion cells was analyzed by HE staining and IPP?Image Pro Plus 6.0?.The number of retinal ganglion cells was measured and the number of cells was counted.Immunofluorescence assay was used to analyze the changes of GFAP expression in each group,and the effect of Naringin on Müller cells in DR was revealed.The rMC-1?cryopreserved?purchased from Qi's creatures,resuscitated in medium containing DEME,Ham's F-12.5%calf serum and 0.5 mg/ml gentamicin,37?,5%CO2,saturated humidity incubator to be cultured until the cell density of 70-80%,for low serum culture medium for growth,growth to logarithmic period.RMC-1 was cultured to a density of about 90%,and the cells were counted and seeded in three 96-well culture plates.The number of cells per well was 3×103,and 5 wells were designed in each group.3 times or more.The concentration of serum in the culture group was reduced to 0.2%after inoculation,and then placed in 37?,5%CO2 incubator in the following groups:control group,high glucose group,high glucose group+1?M Naringin group,5?M Naringin group,high glucose group+10?N Naringin group,high glucose group+25?M Naringin group,high glucose group+50?M Naringin group,high glucose group+100?M Naringin group,respectively,cultured 24h,48h,72h after MTT experiment,The optimal naringin was used for the concentration and cell culture time for subsequent experiments.The experimental settings were:blank control group,Naringin group,high glucose induction group?high glucose group?,high glucose induced+Naringin group?high glucose+ Naringin group?.The cells in the high glucose group and the high glucose+ Naringin group were induced with 25mM glucose.The cells of the high glucose+Naringin group were pretreated with the best concentration of Naringin for 2h before induction of high glucose.The cells were cultured for optimal cell culture time.The total protein was extracted and quantified,and the samples were used for subsequent experiments.The changes of superoxide dismutase?SOD?, catalase?CAT?and glutathione?GSH?in the samples were examined by using the kit.The effects of Naringin on high glucose-induced rMC-1 in the presence of oxidative stress.?Interleukin-1?,IL-1??,interleukin-6?IL-6?and tumor necrosis factor-??Tumor necrosis factor-1?in the supernatant of each group were measured by ELISA.?,TNF-?),and to evaluate the effect of Naringin on hyperglycemia-induced rMC-1 inflammatory response.Animals were taken from the experimental study of the third set of histological slices,Immunohistochemistry was used to detect the expression of NF-Kb p65 in the retina of each group.The nuclear transfer of NF-?B p65 in each group of rMC-1 was observed by immunofluorescence.The nucleoprotein and cytoplasmic protein of rMC-1 were extracted and quantified by Western Blot.The expression of NF-?B p65 in the nucleus and cytoplasm of rMC-1 in each group further revealed that NF-?B pathway is a possible mechanism for Naringin intervention in DR.Results:1.Effects of Naringin on body weight and blood glucose in rats:There was no significant difference in body weight and blood glucose between high dose Naringin group and control group after 12W?P>0.05?.Compared with the control group,the weight of the diabetic model group?diabetic group and diabetes mellitus+low,medium and high dose Naringin group?was significantly reduced and the blood glucose was significantly increased?P<0.01?.There was no statistical difference in body weight and blood glucose between diabetes mellitus and low dose Naringin rats?P>0.05?.With the increase of Naringin dose,the increase in body weight?P<0.05?and blood glucose?P<0.05?was higher in diabetes mellitus and middle dose Naringin rats than in diabetic group.Diabetic+high dose Naringin rats compared with the diabetic group,significantly increased body weight?P<0.01?;blood glucose decreased?P<0.05?.Compared with diabetes mellitus+low dose Naringin group,the weight gain?P<0.05?and blood glucose decreased?P<0.05?.2.Effects of Naringin on retinal ganglion cells:There was no statistical difference in the thickness and number of retinal ganglion cells in the high dose Naringin group compared with the control group at 12W?P>0.05?.Compared with the control group,the thickness of the retinal ganglion cells and the number of cells were significantly decreased?P<0.01?,while the diabetic and high dose Naringin group compared with the control group,the retinal ganglion cells Layer thickness and cell number were not significantly reduced?P<0.05?.With the increase in dose of Naringin,the thickness and number of retinal ganglion cells in the diabetic+low dose Naringin group were not statistically different from those in the diabetic group?P>0.05?.Compared with diabetes mellitus group,the thickness of retinal ganglion cells increased?P<0.05?,and the number of cells was not statistical significant?P>0.05?.Compared with diabetes mellitus group,diabetic retinopathy?P<0.01?,and the number of cells in the retinal ganglion cells increased?P<0.01?,and the number of cells in the diabetic group was significantly higher than that in the diabetic and low dose Naringin group?P<0.05?.3.Effects of Naringin on retinal Müller cells:After 12 weeks of administration,GFAP-positive red fluorescence staining and fluorescence were weak in the nerve fibers and ganglion cells in the control and high Naringin groups.In the diabetic group,the GFAP fluorescence intensity was significantly enhanced in the nerve fiber layer,the ganglion cell layer and the intrathecal layer,and the long protrusions were filamentous fluorescence or even through the inner and outer nuclear layers,showing the tendency of the Müller cells to penetrate the retina The Diabetes+low,medium and high dose of Naringin group compared with diabetic group,GFAP fluorescence intensity decreased to varying degrees,and diabetes+high dose Naringin group weakened the most obvious.4.MTT assay was used to detect the proliferation of rMC-1:There was no statistical difference in cell proliferation between the two groups?P>0.05?.Compared with the control group,the proliferation of rMC-1 was significantly increased?P<0.01?,and the inhibitory effect on the proliferation of rMC-1 was higher with the increase of Naringin dosage.The proliferation activity of rMC-1was significantly decreased in high glucose+Naringin?50?M?,high glucose+Naringin?100?M?group compared with high glucose group?P<0.01?.The proliferation of rMC-1 was decreased?P<0.05?in high glucose+Naringin?1?M,5?M,10?M?group and high glucose group?P<0.05?,while high glucose+ Naringin?25?M,50?M,100?M??P<0.01?,and the proliferation of rMC-1 was significantly decreased?P<0.01?.The proliferation of rMC-1 in the adjacent high glucose+Naringin?25?M?group was not significantly increased or decreased?P>0.05?compared with the high glucose group at 48 h,while the high glucose+Naringin?50?M??P<0.01?.Therefore,the optimal concentration of Naringin was 50?M,and the optimal cell culture time was 48h,which was used in the following cell trial.5.Effects of Naringin on hyperglycemia-induced rMC-1 oxidative stress:The expression of SOD,CAT and GSH in rMC-1 protein of Naringin group was not statistically different from that of control group?P>0.05?.Compared with the control group,the expression of SOD,CAT and GSH in the high glucose group was significantly decreased?P<0.01?.The expression of GSH,SOD and CAT increased in high glucose+Naringin group compared with high glucose group?P<0.05?.6.Effects of Naringin on hyperglycemia-induced rMC-1 inflammatory response:The expression of TNF-?,IL-1?and IL-6 in the supernatant of rMC-1in Naringin group was not statistically different from that in control group ?P>0.05?.The expression of TNF-?,IL-1?and IL-6 in the high glucose group was significantly higher than that in the control group?P<0.01?.The expression of TNF-?,IL-1?and IL-6 was decreased in high glucose+Naringin group compared with high glucose group?P<0.05?.7.Immunohistochemistry was used to determine whether NF-?B p65 was expressed in the retina of each group:NF-KB p65 was only expressed in the nucleus and cytoplasm of retinal tissue in the control and high dose Naringin groups.In the diabetic model group?diabetic group and diabetes mellitus,low,medium and high dose of Naringin group?retinal nerve fiber layer,ganglion cell layer and inner mesh layer of the nucleus and cytoplasm were granular,brown expression.The expression of diabetes mellitus was the most obvious,and the expression of Naringin increased gradually with the increase of Naringin dosage.8.Immunofluorescence was used to observe the nuclear translocation of NF-?B p65 in each group rMC-1.The expression of NF-?B p65 in the control group and Naringin group rMC-1 was mainly expressed in the cytoplasm.In the high glucose group rMC-1,the expression of NF-?B p65 was decreased and the expression of NF-?B p65 was significantly increased Compared with the high glucose group,the expression of NF-?B p65 increased in the Naringin group and decreased in the Naringin group.9.Western Blot clarifies the changes of NF-?B p65 expression in nucleus and cytoplasm of rMC-1 in each group:NF-?B p65 nuclear expression results show:NF-?B p65 in the control group and Naringin group rMC-1 nuclear expression was low,the difference was not statistically significant?P>0.05?.The expression of NF-?B p65 in the high glucose group was significantly higher than that in the control group?P<0.01?.The expression of NF-?B p65 was decreased in the high glucose+Naringin group compared with the high glucose group?P<0.05?.The results of cytoplasmic expression showed:NF-?B p65 in the control group and Naringin group rMC-1 cytoplasm expression were high,the difference was not statistically significant?P>0.05?.The expression of NF-?B p65 in the high glucose group was significantly lower than that in the control group?P<0.01?.The expression of NF-?B p65 in the high glucose+Naringin group was higher than that in the high glucose group?P<0.05?.Conclusions:1.High dose of Naringin had no toxic and side effects on body weight,blood glucose,retinal ganglion cells and Müller cells in normal rats,but could increase the body weight of type 1 diabetic rats,decrease blood glucose,inhibit the proliferation of Müller fine,protect retinal ganglion Cells,and with the use of Naringin dose increases,the more obvious the protective effect.2.Naringin has no toxic side effects on normal rMC-1,but can u p-regulated the activity of antioxidant enzymes SOD,CAT and GSH in rMC-1 induced by high glucose,decrease the overexpression of inflammatory factors IL-1?,IL-6 and TNF-?,and inhibit the expression of rMC-1 Excessive keratinization,early intervention in the treatment of diabetic retinopathy play a positive role.3.Naringin had no effect on the expression of NF-?B p65 in the retina of normal rats and had no effect on the expression of NF-?B p65 in the retinal tissue of diabetic group,but also had no effect on the expression of NF-?B p65in normal Müller cells Expression,but can down-regulate the expression of NF-?B p65 in the retina of diabetic group and inhibit the nuclear translocation of NF-?B p65 in rMC-1 induced by high glucose,thereby reducing nuclear expression.Suggesting that NF-?B pathway is involved in the protective effect of Naringin on diabetic retinopathy. |
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