| Background:Diabetic retinopathy(DR)is the most common complication of diabetes mellitus,which is often charactized by the injury of retinal vascular microcirculation,retinal exudation,retinal hemorrhage,fibrous proliferation and neovascularization.With the ageing of the population in our country,the incidence of DR rises year by year and it has now become one of the major blinding eye disease.With the onset of diabetes age is getting smaller and smaller,the onset age of DR tends to be younger.Although the pathogenesis of DR is not entirely clear untill now,the scholars recognize that DR is a chronic disease on the pathological basis of glucose metabolism disorder,causing the biological changes of ocular tissues,nerves and retinal vascular microcirculation,resulting in eye nutrition and visual function impairment.Studies have shown that inflammation is accompanied by the development of the whole process of DR.The injury of retinal vascular endothelial cell induced by inflammation can occur in the early stage of DR.RECs are the structural basis of retinal blood vessels and have very important physiological functions.RECs are metabolically active and are able to directly sense changes in metabolites in the blood.When RECs encounter hyperglycemia or hypertension,especially chronic hyperglycemia or repeated fluctuation of blood pressure,cell dysfunction,irreversible damage and even death will occur.As the first line of defense of human body,NLRP3 inflammasome belongs to the innate immune system and plays an important role in immune surveillance.The role of NLRP3 inflammasome activation in the pathogenesis of DR has become a hotspot.Mcc950,a diarylsulfonylurea-containing compound known to be the specific inhibitor of NLRP3 inflammasome activation,can effectively suppresse the inflammatory response in a variety of autoimmune diseases,but the role of Mcc950 in the model of sterile immune disease caused by glucose metabolism disorder has not been discussed still now.Purpose:To study the protective effect of Mcc950 on high glucose induced human retinal vascular endothelial cell injuryMethods:Chapter 1:to study whether NLRP3 inflammasome can activate in the retinal vascular endothelial cells in proliferative membrane from patients with diabetic retinopathy.Clinical sample collection and grouping:normal retina group and proliferative membrane group.Immunofluorescence and Western Blot were used to detect the expression of NLRP3 inflammasome associated proteins.Chapter 2:to investigate the effect of Mcc950 on high glucose-induced retinal vascular endothelial cell injury.HRECs culture and grouping:control group,high glucose group,Mcc950 intervention group.CCK8 was used to detect the effect of Mcc950 and high glucose on cell proliferation and viability,and the changes of cell proliferation and viability induced by high glucose after Mcc950 intervention.Elisa assay was used to explore the concentration level of IL-1β after simply high glucose stimulation and Mcc950 intervention after high glucose stimulation,respectively.Immunofluorescence was used to observe the changes of inflammatory factors expression in HRECs with or without Mcc950 intervention.The ratio of apoptosis after high glucose stimulation or Mcc950 intervention was detected by TUNEL assay.RT-PCR was used to explore the transcription expression of NLRP3 inflammasome associated inflammatory factors.The protein expression of NLRP3,ASC,pro-Caspasel,Caspasel,pro-IL-1β and IL-1β in HRECs induced by high glucose or Mcc950 intervention were compared by western blot.The role of Mcc950 in the interaction of NLRP3 and NEK7 was verified by Immunoprecipitation.Results:CCK8 results showed:Cell proliferation increased when the high glucose concentration was at 15mM,while it was significantly decreased when the high glucose concentration increased to 30 or 50mM.Cell activity was not significantly changed when the Mcc950 concentration was at 0.1,1,or 10μM.Slight decrease of cell activity was found when the Mcc950 concentration reached to 100 M,but there was no statistical difference.In the 30mM high glucose environment,the cell activity of HRECs were protected by Mcc950(0.1,1,10,100μM)addition compared to simply high glucose group(P<0.05).Elisa results showed:The concentration of IL-1β released in the cell supernatant increased with the prolongation of high glucose stimulation.In addition,the concentration of IL-1β was significantly reduced in the Mcc950 pretreated group(0.1,1,10,100μM)compared to the high glucose group(P<0.05).RT-PCR results showed:The mRNA expression of NLRP3,Caspase-1 and IL-1βincreased after high glucose stimulation,but there was no statistically significant change after the treatment of Mcc950(P<0.05).Western Blot results showed:The protein expression of NLRP3,Caspase-1 and IL-1βwere increased in proliferation membranes compared with normal retina tissues.In vitro experiment,the expression of NLRP3,pro-Caspase,Caspase 1,pro-IL-1β and IL-1β in HRECs induced by high glucose were remarkably higher than that of normal control group.Yet,compared with the high glucose group,NLRP3,pro-Caspasel,pro-IL-1β protein expression was still higher in Mcc950 treatment group,while Caspase-1,IL-1β protein expression were significantly reduced(P<0.05).Immunofluorescence results showed:There was obvious co-staining of NLRP3 and IL-1β in CD31-labeled retinal vascular endothelial cells in proliferation membranes.compared with normal retina tissues.A strong double-labelling of NLRP3 and IL-1βwere found in the high glucose group while Mcc950 effectively reversed the situation to the normal state.TUNEL assay results showed:The apoptotic rate of HRECs induced by high glucose was much higher than Mcc950 treatment group.Immunoprecipitation results showed:The combination of NLRP3 and NEK7 in the case of high glucose stimulation was significantly higher than that in the control group,while the treatment of Mcc950 reduced the interaction between NLRP3 and NEK7.Conclusion:NLRP3 inflammasome activation can be found in HRECs induced by high glucose.However,Mcc950 can effectively reduce the dysfuction of HRECs through interrupting Nek7-NLRP3 pathway.Our study provided an important clue to develop pharmaceutical approach for the future treatment of DR. |
