| Objective:Diabetic retinopathy(DR)is one of the most common complications of diabetes mellitus,and its pathogenesis is closely related to oxidative stress and inflammation.Mangiferin,the main active ingredient in the Chinese medicine Zhi Mu,has strong anti-inflammatory and antioxidant effects,but its protective effect on DR is not clear.In this study,we intend to investigate the protective effects of mangiferin on retinal damage caused by Müller cell activation in model of DR,and the role played by ERK/NF-κB signaling pathway in it,to provide an experimental basis for mangiferin treatment of DR.Methods: Part I: SD rats were divided into 4 groups: control group,diabetic group,mangiferin low dose group,mangiferin high dose group.Except for the control group,the rats in other groups were injected intraperitoneally with streptozocin(STZ)to establish the diabetes mellitus model.The general status and lens cloudiness of the rats in each group were observed,and the structural changes of the retinal tissue were examined by HE staining.PAS staining was used to observe the morphological changes of retinal vessels.Evans blue was used to detect the leakage of retinal barrier in rats.Western blot and immunofluorescence staining were used to detect the expression of tight junction proteins Claudin-1,Ocludin and ZO-1 in the retinal tissues of each group.Part II: Animal grouping and treatment as in Part I.To observe the effects of mangiferin on Müller cell activation and retinal tissue inflammation and oxidative stress in diabetic rats.Western blot and immunofluorescence staining were used to detect the expression of GFAP and GS in the retinal tissue of rats in each group.SOD,CAT and MDA kits were used to detect oxidative stress in the retinal tissue.RT-q PCR was used to detect the expression of inflammatory factors TNF-α,IL-1β and IL-6 m RNA in the retinal tissue of rats in each group.The levels of TNF-α,IL-1β and IL-6 in the retinal tissues were measured by ELISA.The expression of p-ERK,ERK,p-p65 and p65 protein in the retinal tissues of rats was detected by Western blot.Part III: A cell injury model was established using high glucose treatment of rat retinal Müller cells(r MC-1).CCK8 kit was used to detect changes in cell viability in each group.The expression of GS in Müller cells was detected by immunofluorescence,and the oxidative stress in Müller cells was detected by SOD,CAT and MDA kits.The m RNA expression of inflammatory factors TNF-α,IL-1β and IL-6 was detected by RT-q PCR,and TNF-α,IL-1β and IL-6 levels were detected by ELISA.Western blot was performed to detect the expression of p-ERK,ERK,p-p65,p65 protein in each group of cells.The supernatants of Müller cells from different treatment groups were collected and used to culture human umbilical vein vascular endothelial cells(HUVEC),and the integrity of the HUVEC barrier was detected by FITC-Dextran leakage assay.Western blot was used to detect the expression of the tight junction proteins Claudin-1,Occludin,and ZO-1 in each group of HUVEC.The level of reactive oxygen species in each group of HUVEC was detected using fluorescent probes.Results: 1.The diabetic rats showed signs of diabetes such as weight loss,hyperglycemia,increased food intake,water intake,and lens clouding began to appear in the sixth week.Mangiferin reduced the incidence of lens clouding.Rats with diabetes had thinner retinas,fewer ganglion cells,dilated capillaries,thicker lumen,and other pathological alterations to the retinal tissue.Treatment with mangiferin reduced the structural alterations in diabetic rats’ retinas.Retinal vascular degeneration with increased acellular vessels in diabetic rats and improved vascular morphology after mangiferin treatment.In diabetic rats,there was an increase in EB leakage,a reduction in the expression of the tight junction proteins Claudin-1,Occludin,and ZO-1,a disruption of the blood-retinal barrier,and an increase in permeability.Treatment with mangiferin boosted tight junction protein expression and decreased BRB permeability.2.In the retinal tissue of diabetic rats,GFAP expression was raised while GS expression was decreased,as demonstrated by Western blot and immunofluorescence staining.Mangiferin therapy reduced GFAP expression while increasing GS expression.In diabetic rats’ retinal tissues,SOD and CAT levels decreased and MDA levels increased;mangiferin therapy increased SOD and CAT levels while decreasing MDA content.In diabetic rats,levels of the inflammatory substances TNF-α,IL-1β,and IL-6 were increased,whereas mangiferin reduced these levels.In diabetic rats,the expression of p-ERK and p-p65 was increased,and mangiferin therapy reduced the activation of the ERK/NF-κB signaling pathway.3.After being exposed to high glucose,r MC-1 cells’ viability and GS expression declined.Intervention with mangaferin increased GS expression and r MC-1 cell viability.In the Müller cells of the high glucose group,inflammatory factors such TNF-α,IL-1β,and IL-6 were upregulated,along with the levels of p-ERK and p-p65.Mangiferin pretreatment had the same impact as the ERK signaling pathway inhibitor U0126 in that it suppressed inflammatory factor levels and prevented the activation of the ERK/NF-κB signaling pathway.The conditioned medium from high glucose treated Müller cell increased the leakage of HUVEC barrier,decreased expression of HUVEC tight junction proteins Claudin-1,Occludin,and ZO-1,and increased cellular ROS levels,and these changes were attenuated in the mangiferintreated group.Conclusion: 1.Mangiferin has protective effects on retinal tissue structure and blood retinal barrier in diabetic rats.2.Mangiferin can reduce inflammatory response and oxidative stress in diabetic rat retinal tissue,attenuate Müller cell activation,and inhibit activation of ERK/NF-κB signaling pathway.3.Mangiferin may reduce high glucose-induced Müller cell activation and attenuate BRB damage by activated Müller cells by inhibiting the ERK/NF-κB signaling pathway. |
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