Prostaglandin E2 Promotes Diabetic Retinopathy By Inducing NLRP3-ASC Inflammasome Activation Via EP2 Receptor

Background:Diabetic retinopathy(DR)is one of the most common and devastating microvascular consequences of diabetes mellitus,which makes it one of the major causes of blindness among eye diseases in the global world.Early pathological changes of DR include increased vascular permeability,leakage,inflammation,and destruction of the blood-retinal barrier.Capillary degeneration,endothelial cell hyper proliferation,and neovascularization,these vascular pathological events will further lead to the vitreous hemorrhage and retinal detachment,which eventually cause blindness.According to the literature,RMECs dysfunction resulted from hyperglycemia contributes substantially to the pathogenesis of vascular leak in early DR,as well as neovascularization in the late stage of DR.Previous studies have revealed that retinal microvascular endothelial cells(RMECS)dysfunction caused by hyperglycemia is one of the main causes of early DR vascular leakage and advanced DR angiogenesis.In fact,the pathogenic mechanisms for hyperglycemia-induced RMECs dysfunction are very complicated,and inflammatory response is a major contributor.The innate immune system involves inflammasome as an important member,of which the NLRP3(nucleotide-binding domain and leucine-rich repeat protein 3)inflammasome has been widely and thoroughly studied.NLRP3 is activated by recognition of pathogen-associated molecular patterns(PAMPs)or host-derived danger-associated molecular patterns(DAMPs),which further activates Caspase-1;Pro-IL-1β is then spliced to its activated form IL-1β by the activated Caspase-1 and then lead to the inflammatory cascade.NLRP3 inflammasome plays an important role in the pathogenesis of many diseases.Current clinical and animal researches indicate that the activation of NLRP3 is closely related to the pathogenesis of DR,while its underlying mechanisms remain incompletely understood.Our prior studies have found that,with the stimuli of hyperglycemia or LPS,NLRP3 inflammasomes in RMECs could be activated and its effectors IL-1β could lead to inflammatory response and apoptosis of RMECs through induced oxidative stress and nitration reaction eventually lead to vascular dysfunction.Prostaglandin E2(PGE2)is a metabolic product of arachidonic acid catalyzed by Cyclooxygenase(COX),a fundamental inflammatory factor,which is related to the development and progress of many disorders.PGE2 imparts its effect through EProstanoid Receptor(EPR)on cell surface membranes;EPRs,a family of G ProteinCoupled Receptor,have 4 different subtypes: EP1 R,EP2R,EP3 R,EP4R.The expression of PGE2 was abnormally elevated in the vitreous humor of DR patients,and strongly corresponded to the pathological state of DR;however,its detailed regulatory mechanism remained unclear.Some studies indicated that the activation and regulation of NLRP3 inflammasome probably mediated the effects of the PGE2/EPR signaling pathway in the process of infection,inflammation,autoimmune disease and the growth of tumor.Our previous study discovered that 4 different types of EPR all expressed in RMECs;the level of transcription and translation of COX2,EP2 R and EP4 R in RMECs increased significantly,compared to the control,with the stimuli of hyperglycemia for 72 h.PGE2 and its EP receptor agonists can significantly promote the expression of NLPR3,IL-1β mRNA levels in RMECS.However,only EP2 receptor inhibitors can effectively inhibit the promotion of NLRP3 and active molecules by high glucose,PGE2 and EP2 R agonists,suggesting that EP2 R may play a leading role in the inflammatory injury of PGE2-induced RMECs in the development of DR.But its detailed molecular mechanisms for the regulation of NLRP3 inflammasomes in RMECs need to be further illustrated.Objective: 1.To explore the effect of PGE2/EP2 R signaling pathway on RMECs injury and microvascular dysfunction in STZ-induced diabetic rats.2.To investigate whether PGE2/EP2 R participates in the activation of NLRP3 inflammasomes in DR 3.To elucidate the regulate mechanism of PGE2/EP2 R signaling pathway on NLRP3 inflammasomes in RMECs Methods: 1.Animals and treatments: Eight-week-old homozygous male Sprague Dawley rats(100 of 120 individuals)were randomly selected,and diabetes was induced with injection of streptozotocin among them.The unselected rats were arranged as the control group.The STZ-treated rats with diabetes were then randomly divided into five groups,as follows: untreated STZ;STZ + PGE2;STZ +Butaprost(a PGE2/EP2 R agonist);STZ + AH6809(an EP2 R antagonist)and STZ + DMSO.Rats were anaesthetized,and a dose about 6μl of the designated mixture was delivered into the vitreous cavity.Rats received an intravitreal injection every 3 weeks in order to maintain the plasma concentration.2.Immunofluorescence and Western Blot were performed to observe the expression of EP2 R in rat retina.The retinas stained with Periodic acid–Schiff(PAS)were mounted onto glass slides,which allowed the observation of their vasculature and pericyte loss;the permeability of the retinal vascular leak was quantified with fluorescence angiography using Evans blue;lectin labelling of the adherent retinal leucocytes was performed to examine the perivascular inflammation;the Spectral domain optical coherence tomography(OCT)was performed,along with microscopic view in H&E staining,to observe the morphological and histologic changes in retinas;the ultrastructure of the retina tissues was inspected using a transmission electron microscope;TUNEL detected the apoptosis of retinal cells;immunofluorescence analysis was performed to localize and quantify the expression of target proteins in retinas,including EP2 R.3.Treat RMECs with PGE2,Butaprost(EPR2 agonist),AH6809(EPR2 inhibitor),H89(PKA inhibitor),SQ22536(cAMP inhibitor)respectively,and then conduct the real-time PCR,Western Blot,ELISA and co-immunoprecipitation to quantify the variance of NLRP3,Caspase1,IL-1 and downstream nuclear translocation factor;Treat RMECs with different concentrations of IL-1β(0、0.1、1、10ng/mL)for 24 h,and then measure Caspase-3 by Western Blot and test the apoptosis rate when using IL-1β(10ng/mL).Results: 1.EP2 R was expressed in SD rat retina and in vitro RMECs.Compared with normal SD rats,STZ significantly up-regulated the expression of EP2 R.2.Intravitreal injection of PGE2 and Butaprost significantly accelerated retinal edema,vascular leakage,leukocyte adhesion,endothelial cell apoptosis and other retinal micro-angiopathy in STZ induced diabetic rats.However,this response was improved in diabetic rats treated with the EP2 R inhibitor AH6809.3.PGE2/EP2 R signaling pathway promotes NLRRP3 inflammasomes activation in DR retina.PGE2 is coupled to EP2 R via Gαs subunit,activates cAMP/PKA signaling pathway to up-regulate NLRP3 and pro-IL-1β transcription,and promotes NLRP3 inflammasomes.Activation.