Protective Effect And Mechanism Of Inhibiting Acetylcholinesterase On Retinal Inflammation

Many pathophysiological processes of blindness are involved in inflammatory reaction,such as diabetic retinopathy,retinopathy of prematurity,uveitis,age-related macular degeneration and so on.Inflammation is a non-specific defense response,not only many molecular and functional changes,but also the occurrence of leukocyte recruitment and adhesion.In the acute phase,white blood cell recruitment can clear the harmful factors of invasion,usually resulting in tissue repair.In the acute phase,white blood cell recruitment can clear the harmful factors of invasion,usually resulting in tissue repair.However,if inflammation continues,leukocyte adhesion and long-term secretion of chemical mediators will damage tissue.Retinal inflammation often leads to retinal tissue edema,hemorrhage and exudation,which can cause eyesight to decrease at different extent.Mainly concentrated in the synaptic neural tissue,rapid hydrolysis of the neurotransmitter acetylcholine(ACh),acetylcholinesterase(AChE)can be used as a marker of systemic inflammation.AChE gene can start DNA binding sites,which may raise the nuclear transcription factor-Kappa B(NF-кВ)involved in inflammation process.AChE showed high expression on the inflammatory reaction in the many organs,tissues,cells,even in not obvious inflammatory reaction.A number of research suggest that AChE inhibitors have anti-inflammatory function on the cellular level and molecular level,inhibition of lymphocyte proliferation,pro-inflammatory cytokine production and cell of esterase activity.This study was to clarify the role of AChE and the effect of it deficiency in the retinal inflammation,and explore the possible mechanism.Using ARPE-19 cells,AChE gene knockout mice and C57BL/6J mice given AChE inhibitors,investigated the changes of AChE gene and protein level influence on retinal inflammation,and analyzed the relationship between the effect and NF-кВ classify single pathway.Study found that AChE take part in retinal inflammation and inhibition of AChE can reduce retinal inflammation.This effect may through the classic NF-кВ signal pathyway.It will help us to find new targets for intervention and treatment of retinal inflammation.Part ⅠThe effect of inhibiting acetylcholinesterase on retinal inflammationObjective: To investigate the effect of inhibiting AChE on retinal inflammation.Methods :(1)Cell experiment : Human retinal pigment epithelial cells(ARPE-19)were used in vitro.AChE inhibitor(Donepezil)and AChE si RNA inhibited the expression of AChE.Western blot and immunocytochemistry analysis of AChE and ICAM-1 level after treated with TNF-α(4 hours or 24 hours).(2)Animal experiment: ○1 C57 BL/6J mice with Donepezil(10 mg/kg)gavage or the same volume of 1XPBS for 3 days and one eye induced by intravitreal injection of TNF-α 1μl(50ng/ml)or 1XPBS 1μl.The retinas were divided into the Vehicle,the Vehicle + TNF-α,Donepezil and Donepezil + TNF-α.After 24 h,the mice were killed and the detection indexes were the same as the model one.○2 One eye of AChE+/-and AChE+/+ mice were treated with vitreous cavity injection of TNF-α 1μl(50ng/ml)or 1XPBS 1μl to induce retinal inflammation or as control.The retinas were divided into AChE+/+,AChE+/+ + TNF-α,AChE+/-and AChE+/-+ TNF-α.After 24 h,the mice were sacrificed and retinas or optic cups were taken out.The expression of inflammatory markers was detected by Western blot and immunofluorescence.Results :(1)Cell experiment: The expression of AChE and ICAM-1 were increased in ARPE-19 cells induced by TNF-α,but this tendency disappeared after using Donepezil or AChE si RNA,and the concentration of 10μM is the best.(2)Animal experiment: After treated with TNF-α,the level of AChE,ICAM-1,Albumin,CD11 b and leukocyte adhesion were decreased in AChE+/-mice and C57BL/6J mice with Donepezil gavage.Conclusions: Inhibition of AChE has protective effect on retinal inflammation.PartⅡ Study on the mechanisms of inhibition of AChE on the protection of retinal inflammation Objective: To investigate the mechanisms of inhibition of AChE on the protection of retinal inflammation.Methods :(1)Cell experiment:Cultured ARPE-19 cells,using AChE inhibitors(Donepezil)and AChE si RNA inhibition of AChE expression,western blot and immunocytochemistry were used to analyze the expression of NF-кВ classify single pathway related indexs after treated with TNF-α(5 minutes or 24 minutes).(2)Animal experiment: The same way as the first part,NF-кВ expression was detected by immunofluorescence.Results :(1)Cell experiment: Immunocytochemistry showed that NF-кВ expression was increased after treated with TNF-α for 30 minutes,and this phenomenon would reverse when pretreatment with Donepezil or AChE si RNA.Western blot analysis showed that the level of p-NF-кВ、p-IKK、p-IкВα were upregulated induced by TNF-α for 5 minutes or 30 minutes.However,above indexs were downregulated when using Donepezil or AChE si RNA,and the concentration of 10μM is the best.(2)Animal experiment: The expression of NF-кВ was reduced in C57BL/6J mice with Donepezil gavage and AChE+/-mice when treated with TNF-α.Conclusions: The protective effect of AChE on retinal inflammation may be by inhibiting the classic NF-кВ signal pathyway.