| Purposes:Under pathological conditions,medium and high concentrations of reactive oxygen species(ROS)induce cell apoptosis or even necrosis through oxidative stress.ROS are also intrinsically correlated with cell signal transduction.Experimental studies showed that NADPH oxidase 4(NOX4)expression increased after corneal alkali burn in mice,which accelerated the formation of corneal neovascularization.We know ROS can come from NOX4.Studies have shown that ROS regulate inflammasomes after corneal alkaline burn in mice,leading to imbalance of NLRP3 and NLRP6 expression,which further promotes the expression of cytokines.The purpose of this study was to investigate the relationship between ROS regulated NLRP3 and NLRP6 and corneal neovascularization in mice with alkali burn.AIP1(apoptosis signal-regulating kinase1-interacting protein 1)is a new member of the Ras GTPase-activating protein family.Recent studies have shown that AIP1 can not only inhibit tumor development as a tumor suppressor gene,but also participate in inflammatory induction of neovascularization through a variety of signaling pathways.We further studied the regulatory function of AIP1 in corneal neovascularization.The mechanism of AIP1,ROS and inflammasome in the corneal alkali burn murine model was studied.Methods:Alkali burn was administrated on the right eye with Whatman filter paper(2 mm diameter)soaked in 1 N Na OH solution.Mice corneas were monitored and recorded by slit lamp microscopy at 4 different time points after alkali burn(Day 3,Day 7,Day10,and Day 14).After alkali burns,10μM GLX351322,a NOX4 inhibitor,or 10μM DMSO solution was applied to the eyes 4 times a day for 10 days,respectively.The corneas of the right eye of AIP1 knockout mice and wild-type mice were burned by alkali respectively.Adenovirus encoding AIP1 and GFP or GFP(2.54×?10~8 PFU)was injected into right anterior chamber of mice two days before the alkali burn.Mice corneas were monitored and recorded by slit lamp microscopy at Day 10 after alkali burn.The state of corneal neovascularization was recorded by slit-lamp.AIP1,NOX4,NLRP3 and NLRP6 and their associated inflammatory cytokines and neovascularization associated proteins were detected by RT-PCR and western-blot.DCFDA staining was used to determine the amount of reactive oxygen species(ROS)produced after alkali burn treatment of the mouse cornea to evaluate the oxidative stress of the mouse cornea under different treatment conditions.The changes of corneal neovascularization were observed by corneal whole mount staining.Results:In the corneal alkali burn murine model,the expression of NLRP3/NLRP6 was unbalanced,with increased expression of NLRP3 and decreased expression of NLRP6.It also caused increased expression of clv-casp-1,ASC,clv-IL-1βand VEGFA.In the corneal alkali burn murine model,the NOX4 inhibitor GLX351322 eye drops can reverse the NLRP3/NLRP6 imbalance by reducing ROS expression.It can also reduce the expression of clv-casp-1,ASC,clv-IL-1β,and the expression of neovascularization related protein,VEGFA,thereby reducing neovascularization.The activation of NOX4is due to decreased expression of AIP1 in the murine corneas of alkali burned.After corneal alkali burn in mice,the expression of NOX4 in AIP1 knockout mice increased,the imbalance of NLRP3/NLRP6 exacerbated,and the corneal neovascularization increased significantly compared with the control group.These results were reversed by injecting adenovirus encoding AIP1 and GFP into right anterior chamber of mice.After alkali burn,the expression of NOX4 and ROS decreased,the imbalance of NLRP3/NLRP6 was reversed,and neovascularization decreased significantly in the cornea of mice which received adenovirus encoding AIP1 and GFP in the anterior chamber compared with the control group.Conclusions:AIP1/NOX4/NLRP3 and NLRP6 can directly regulate corneal inflammation and corneal neovascularization after alkali burn.Based on the pathogenesis of corneal neovascularization under alkali burn,it is expected to provide new therapeutic strategies for patients. |
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