Protective Effects Of Sulforaphane On Diabetic Retinopathy:Activation Of Nrf2 Pathway And Inhibition Of NLRPp3 Inflammasome Formation

Background and Objectives: Diabetic retinopathy is one of the most common microvascular complications of diabetes,the incidence of which has been increasing in recent years.The pathogenesis of diabetic retinopathy is very complicated and has not been fully elucidated.Previous studies have shown that hyperglycemia increases oxidative stress in retinal tissues and capillaries,increases reactive oxygen species(ROS)in the retina,and damages the antioxidant defense system in diabetic patients.In addition,excessive ROS also increased the expression of proinflammatory factor and activated inflammatory response,which in turn aggravated the oxidative stress response and formed a vicious cycle of oxidative stress and inflammation.Sulforaphane(SFN)is an isothiocyanate rich in cruciferous vegetables including cabbage broccoli turnip and so on.It has strong anti-oxidation,anti-inflammatory and anti-tumor activities.SFN has been shown to have protective effects on diabetic nephropathy and cardiomyopathy,but it is not clear whether it has the same protective effect on diabetic retinopathy.The transcription of antioxidant genes and phase Ⅱ detoxification enzymes mediated by Nrf2/ARE signaling pathway is a key pathway in antioxidant mechanism.The changes of Nrf2/ARE signaling pathway in retinal cells after SFN intervention need further study.In addition,it is reported that the inflammatory bodies of nucleotide oligodeoxyribonucleotide domain like receptor protein 3(NLRP3)can be activated by oxidative stress to participate in the pathological process of DR.Therefore,it is necessary to find out whether SFN is involved in regulating the retinal Nrf2/ARE signaling pathway and the activity of NLRP3 inflammatory bodies in patients with DR,and to further study whether SFN can prevent the development of DR.It is helpful to evaluate the protective effect of SFN in diabetic retinopathy.Method: Intraperitoneal injection of streptozotocin(STZ)65 mg/kg,in male Sprague-Duler(SD)rats.Rats diagnosed with diabetes were divided into groups and injected with different doses of SFN(0.5 mg/kg/d and 1 mg/kg/d)for 12 weeks.Rat retinal Müller cells were exposed to 25 mmol/L glucose and 2.5 μmol/L SFN in vitro.The retinal tissues of rats were extracted and histologically analyzed by HE staining.Detection ofantioxidant GSH,SOD and CAT in retinal tissues and Müller cells by Kit method.We used ELISA kit to detect the changes of TNF-α,IL-6,IL-1β in retinal tissues and Müller cells.The expression of HO-1,NQO1 mRNA in retinal tissues and Müller cells was detected by Real-time PCR.We used EMSA method to detect the nuclear binding activity of Nrf2/ARE in retinal tissues.And then,Western blot was used to detect the expression of Nrf2 protein in retinal tissues and Müller cells,the expression levels of downstream target proteins HO-1 and NQO-1,and the changes of NLRP3,cleaved caspase-1 p20 and ASC,the components of inflammatory bodies in NLRP3.Finally,we further detected Müller cells silenced Nrf2 gene to verify whether the regulation of SFN on inflammatory bodies of NLRP3 was mediated by Nrf2 pathway.Results: SFN alleviated the pathological changes of retina,arranged the cells neatly and alleviated the vasodilation of diabetic rats.The count of ganglion cells showed that SFN increased the number of ganglion cells,especially in high dose SFN group.SFN significantly reduced the production of TNF-α,IL-6 and IL-1β,and increased the activities of antioxidant enzymes(GSH,SOD and CAT)in STZ rats and Müller cells induced by high glucose.In addition,SFN enhanced the aggregation of Nrf2 in the nucleus and increased the expression of HO-1 and NQO1,two major antioxidants downstream of Nrf2.At the same time,the expression of NLRP3,cleaved caspase-1 p20,ASC and IL-1βp17 was significantly increased in diabetic retinopathy induced by STZ,and decreased significantly after SFN intervention.Compared with the non-transfected control group,after silencing the Nrf2 gene,although treated with SFN,the inflammatory index of Müller cells was not decreased,the level of antioxidants was not increased,and the formation of inflammatory bodies of NLRP3 was not inhibited.Conclusions:SFN can reduce retinal inflammation and oxidative stress induced by high glucose by activating antioxidant Nrf2 signaling pathway and inhibiting the formation of NLRP3 inflammatory bodies,thus reducing the degree of diabetic retinopathy.