Nrf2 Inhibits NLRP3 Inflammatory Activation By Regulating The Trx1/TXNIP Complex In Dry AMD

ObjectiveAge-related macular degeneration（AMD）mainly occurs in middle-aged and elderly people over the age of 50,with the onset of both eyes in succession or at the same time,eventually leading to blurred central vision,decreased vision and deformation of objects,among which chronic inflammation is an important mechanism of AMD.Our previous research showed that mi R-27a was up-regulated in the blood of AMD patients,and Thioredoxin Interacting was identified through RNA databases Protein（TXNIP）is the downstream gene of mi R-27a,and has complex form with Thioredoxin（Trx1）in cells.Meanwhile,it has been documented that nuclear transcription factor（Nrf2）has antioxidant effect in AMD patients.Therefore,this study investigated the mechanism of Nrf2 signaling pathway in regulating TXNIP/Trx1 hydrogen peroxide induced oxidative damage model of retinal pigment epithelial cells and C57BL/6 mice retinal blue light LED induced light damage model,and provided a new idea for clinical researchMethods1.Establishment of oxidative stress cell model:ARPE-19 cells were cultured in vitro,and treated with different concentrations of H₂O₂（0μM,100μM,200μM,300μM,400μM,500μM）for 1h,1.5h and 2h.The proliferation ability of ARPE-19 cells was detected by CCK-8 assay to determine the concentration and time of H₂O₂;2.The changes of reactive oxygen species（ROS）levels in ARPE-19 cells induced by 100μM H₂O₂ were detected by DCFH-DA fluorescent probe after 1h;3.Immunofluorescence staining was used to detect the localization and level changes of TXNIP under the condition of oxidative injury;4.The effect of TXNIP on H₂O₂-induced migration of ARPE-19 cells was determined by scratch test;5.Quantitative real time polymerase chain reaction（q-PCR）and Western blot（WB）were used to determine the expression of Nrf2,TXNIP,Trx1 and inflammation-related factors and proteins;6.He staining was used to examine the changes in retinal thickness,ERG was used to detect the retinal functional status,and the establishment of the retinal oxidation model of light-damaged mice was evaluated:C57BL/6 mice were exposed to 460nm blue LED light with 3000Lux intensity for 2h,and the changes in retinal thickness and functional status were measured;7.The m RNA and protein expressions of Nrf2,TXNIP and Trx1 were measured by q PCR and WB under the condition of light injury in the retina of mice;8.Changes in retinal function under light injury model:Changes in retinal function of mice after intravitreal injection of tert-butylhydroquinone（t BHQ）activator activated Nrf2 and transfection with lentivirus Trx1,followed by Blue LED for 2h and ERGdetection 5 days later.9.Under the light injury model,retinal pathological changes:Nrf2 was activated by intravitreal injection of t BHQ activator and transfected with lentivirus Trx1,followed by Blue LED for 2h and HE detection for 5d later.Results1.CCK-8 results showed that the optimal concentration and time of H₂O₂ were 100μM for 1h intervention;2.Flow cytometry analysis showed that the concentration of H₂O₂（0-500μM）was directly proportional to the ROS production in a certain range after the intervention of ARPE-19 cells;3.Cell immunofluorescence results showed that TXNIP increased in cytoplasm and decreased in nucleus after 1h intervention with 100μM H₂O₂;4.The cell migration experiment showed that the cell migration rate in the H₂O₂treatment group was decreased compared with the normal group,while the cell migration rate in the si-Trx1 group was decreased more obviously;5.Effects of Nrf2,TXNIP and Trx1 on anti-oxidative stress capacity and inflammatory factors of ARPE-19 cells:Nrf2 was activated by 30μM t BHQ and si-Trx1 was transfected into AREP-19 cells.According to q PCR and WB results,the expression of Nrf2 and Trx1 were decreased in the H₂O₂-treated group,and the expression of TXNIP in the cytoplasm was increased in the H₂O₂-treated group compared with the control group.The expression of inflammation-related factors and proteins IL-18,IL-1β,caspase1 and NLRP3 increased;Compared with the H₂O₂ treatment group,the expression of Nrf2 increased in the t BHQ-activated group,and the expression of TXNIP,IL-18,IL-1β,caspase1,and NLRP3 decreased.The expressions of TXNIP,IL-18,IL-1β,caspase1 and NLRP3 in si-Trx1+t BHQ activated group were further increased.;6.The results of HE staining showed that the retinal layers of control mice were clearly demarcated,closely arranged,with regular cell morphology and hyperchromatic nucleus.The boundary between the retinal layers was not clear and invaded each other after blue light irradiation,and the ONL layer thickness became thinner.The results of ERG showed that the amplitudes of a and b waves in the retina of mice exposed to blue light decreased significantly;7.According to the q PCR and WB results,compared with the control group,the expression of Nrf2 and Trx1 were decreased in the blue light group,while the expression of cytoplasmic TXNIP was increased,and the expression of inflammatory factors TXNIP,IL-18,IL-1β,caspase1 and NLRP3 were increased.Compared with the blue LED irradiation group,the expressions of IL-18,IL-1β,caspase1 and NLRP3 were decreased in the t BHQ-activated group,while the expressions of TXNIP,inflammation-related factors and proteins IL-18,IL-1β,caspase1 and NLRP3 were further increased in the si-Trx1+t BHQ-activated group;8.ERG results showed that blue light irradiation after intravitreal injection of t BHQ resulted in orderly retinal structure compared with Blue LED irradiation group.However,after transfection with sh Trx1,the retinal structure was more disorderly,and the ONL layer thinning was further deepened and more obvious.9.The results showed that the retinal a wave and b wave amplitude were slightly increased after invitreous injection of t BHQ compared with the Blue LED irradiation group.However,after transfection with Trx1 sh RNA,the amplitude of a wave and b wave decreased more obviously.Conclusion1.The oxidative stress cell model of ARPE-19 cells was successfully established after treatment with 100μM H₂O₂ for 1h.2.C57BL/6 mice were induced by 3000Lux 460nm blue LED light.After 5 days,the oxidative stress model of light injury retina of mice was successfully established;3.Nrf2 exhibited antioxidant and inflammatory effects in the H₂O₂-induced cell stress model and in the blue-light LED-induced retinal oxidative stress model of mice;4.In H₂O₂-induced cell stress model and blue LED light-induced mouse retina oxidative stress model,TXNIP in the cell nucleus transferred to the cytoplasm,activated NLRP3,and aggravated retinal injury,and Trx1 knockout promoted this process.