The Mechanism Of Drp1 Mediated Mitochondrial Division And NADPH Oxidase Involved In Alkali Burn-induced Corneal Iniury

Objective:In the present study,we investigated the mechanism of Drp1 mediated mitochondrial division and NADPH oxidase in the development of corneal tissue injury induced by alkali burn,thus providing a theoretical basis and experimental basis for the clinical exploration of the new target for the treatment of corneal alkali burn.Method:The model of alkali burn was established in right eye of 6-8 week C57 mice.q PCR and western blot were used to detect the level of the transcriptional and phosphorylation of mitochondrial regulatory protein and NADPH oxidase in corneal tissue.After alkali burn of cornea,the expression of Drp1 was inhibited by Drp1 inhibitor,and the expression of NADPH oxidase was detected by Western blot to fund out the relationship between Drp1 and NADPH oxidase.Using gene silencing method and inhibitor to inhibit Drp mediated mitochondrial division and NADPH oxidase activity,ROS was detected by Dihydroethidium,and the inflammatory factors(TNF-αIL-1β,IL-6)in each group was detected by q PCR.Western blot detected the level of phosphorylation of IκBα to verify the activation of NF-κB signaling pathway.After alkali burn of cornea,Drp1 inhibitor Mdivi-1 and NADPH oxidase inhibitor Apo were used in individual or combination with two inhibitors.Cardiac perfusion fluorescein FITC-dextran was used to detect the formation of neovascularization in corneal;q PCR method was used to detect the expression of inflammatory factors(TNF-α,IL-1β,IL-6)in each group.Results:The results of western blot showed that the level of Drp1,Nox2 and Nox4 protein in the alkali burn group was significantly higher than that in the normal group,and the time and peak of Drp1 increased earlier than that of the NADPH oxidase group,while the expression level of mitochondrial fusion protein Mfn2 and OPA1 protein decreased significantly.The level of m RNA in Drp1 was also increased significantly after alkali burn.Immunofluorescence results showed that the phosphorylation level of Drp1 of corneal alkali burn was upregulated in the early moment.Drp1 inhibitor Mdivi-1 can significantly reduce the expression of NOX-2and NOX-4 after corneal alkali burn,indicating that Drp mediated mitochondrial division and NADPH oxidase activity.After alkali burn,the ROS,the inflammatory factor and the phosphorylation of IκBα is obviously up-regulated,indicating that alkali burn activates the NF-κB pathway and also causes oxidative stress and inflammation.Gene silencing and Mdivi-1 inhibition of Drp1 can effectively prevent the reactions.Drp1 inhibitor Mdivi-1 and NADPH oxidase inhibitor Apo can inhibit the production of inflammatory reaction and corneal neovascularization after corneal alkali burn,and the combination of the two inhibitors has synergistic effect.Conclusion:Drp1 mediated mitochondrial division and NADPH oxidase participate in corneal alkali burn.Which provides a new therapeutic target and theoretical basis for the better treatment of corneal alkali burn in the future.