The Molecular Mechanism Of Acetylcholinesterase Regulating The Inflammation In Diabetic Retinopathy

Objective:In the present study,we sought to determine whether AChE regulates retinal inflammation during DR and explore the molecular mechanism of its regulation.Method:(1)In our study,Diabetes was induced by intraperitoneal injection of streptozotocin in male C57BL/6J mice.Donepezil,the AChE inhibitor was administrated by rodent diet.The experiment was divided intoVehicle+Non-DM,Vehicle+DM,Donepezil+Non-DM and Donepezil+DM.Retinal expression of ICAM-1,Albumin and ZO-1 in each group was determined by western-blot analysis.The expression of ICAM-1,GFAP and VEGF in each group was detected by immunofluorescence staining.Concanavalin A lectin perfusion was utilized to evaluate retinal vascular leukostasis.Claudin5 was detected by retinal stain.Then,Diabetes was induced by intraperitoneal injection of streptozotocin in both AChE knockdown(AChE+/-)and wild type(AChE+/+)mice.The experiments were divided into AChE+/+ +Non-DM,AChE+/+ +DM,AChE+/-+Non-DM and AChE+/-+DM.Retinal expression of ICAM-1 and ZO-1 was determined by western-blot analysis.The expression of ICAM-1,GFAP and VEGF also was detected by immunofluorescence staining.Diabetic retinal vascular leakage was measured by Evans blue permeability assay.Claudin5 was detected by retinal stain.(2)Establishment of DM animal model,and diabetic mice were fed with donepezil.Retinal expression of NLRP3 inflammation related protein was determined by western-blot analysis.The expression of Caspase-1 in each group was detected by immunofluorescence staining.(3)Further,Diabetes was induced by intraperitoneal injection of streptozotocin in both Caspase-1 knockdown(Caspase-1+/-)and wild type(Caspase-1+/+)mice.The expression of IL-1 beta,ICAM-1,GFAP and Albumin was determined by western-blot analysis.Concanavalin A lectin perfusion was utilized to evaluate retinal vascular leukostasis.Results:(1)AChE were markedly upregulated in diabetic retinas,in parallel with higher ICAM-1 expression,increased retinal leukostasis,leakage and activated theretinal glia cells.Pharmaceutical inhibition of AChE by donepezil or Genetic knockdown of AChE significantly reduced diabetes-instigated retinal ICAM-1,Albumin and ZO-1 expression and mitigated retinal microvasculopathy such as vascular leukostasis,hyperpermeability,capillary degeneration and inhibits the activation of glial cells.(2)Suppression of AChE by Donepezil significantly inhibited diabetes-induced NLRP3 related protein upregulated.(3)knockdown Caspase-1 can reverse the increase of IL-1 beta,ICAM-1,GFAP and Albumin in diabetic mice and decrease the retinal leukocyte adhesion.Conclusion: Taken together,these result suggest that AChE can regulate retinal inflammation in DR,at least,partially through activating the NLRP3-Caspase-1inflammatory corpuscle,which provides a new theoretical basis for the future prevention and treatment of DR and provides new drug targets.