1-KO mice compared with those in C57BL/6 mice just after alkali burn injury (N = nine). D The DCFDA ROS assay revealed that AIP1 KO notably elevated ROS production right after alkali burn injury compared with that inside the manage group (scale bar: one hundred m). E RT PCR analysis of AIP1 gene knockout efficiency. Deletion of AIP1 substantially enhanced the reduction in NLRP6 and elevation in NOX4, NLRP3 and VEGFa induced by alkali burn injury (N = 3). F Western blot analysis showed that AIP1 deletion significantly increased the reduction in NLRP6 and elevation in NOX4 and NLRP3 induced by alkali burn injury (N = 3). G Immunofluorescence staining displaying that deletion of AIP1 drastically increased the elevation in VEGFa induced by alkali burn injury (scale bar: 50 m; magnification: 400). Error bars represent the imply SD, and comparisons had been performed using one-way ANOVA. P 0.05, P 0.01, P 0.001, and P 0.Li et al. Cell Communication and Signaling(2022) 20:Page 7 ofand protein levels (Fig. 2E, F). AIP1 deletion drastically increased the elevation in NOX4 and NLRP3 induced by alkali burn injury at each the mRNA and protein levels (Fig. 2E, F). In addition, AIP1 deletion drastically improved the elevation in VEGFa induced by alkali burn injury at both the mRNA and protein levels (Fig. 2E, G).AIP1 overexpression decreases neovascularization, ROS production, and NOX4 expression and alleviates the imbalance in NLRP3 activation and NLRP6 suppressionTo confirm the function of AIP1 in alkali burns, we compared corneal neovascularization in response to alkali burns in mice injected with Ad-AIP1-GFP inside the anterior chamber and mice administered Ad-GFP. AIP1 gene overexpression was confirmed by western blotting, which showed that AIP1 was drastically upregulated in AIP1overexpressing mice compared with that in control mice (Fig.ADAM12 Protein Molecular Weight 3A).HGF Protein Gene ID Slit-lamp photos and corneal whole-mount staining revealed that AIP1 overexpression notably decreased neovascularization compared with that in the control group (Fig.PMID:29844565 3B, C). The corneas of AIP1-overexpressing mice and handle mice were each very transparent and absolutely free of neovascularization (Fig. 3B). The corneal opacity, neovessel size, and vessel size scores had been lower in AIP1-overexpressing mice than in control mice soon after alkali burn injury (Fig. 3D). The DCFDA ROS assay revealed that AIP1 overexpression notably decreased the ROS production observed immediately after alkali burn injury compared with that inside the handle group (Fig. 3E). AIP1 overexpression drastically abrogated the reduction in NLRP6 induced by alkali burn injury at each the mRNA and protein levels (Fig. 3F, G). AIP1 overexpression drastically decreased the elevation in NOX4 and NLRP3 induced by alkali burn injury at both the mRNA and protein levels (Fig. 3F, G). In addition, AIP1 overexpression drastically decreased the elevation in VEGFa induced by alkali burn injury at each the mRNA and protein levels (Fig. 3F, H). Immunoprecipitation evaluation additional showed that AIP1 could bind straight to NOX4 in 293 T cells (Fig. 3I), suggesting that AIP1 may well type a complex with NOX4 and reduce the activities of NOX4.The NOX4 inhibitor GLX351322 reverses the imbalance in NLRP3 activation and NLRP6 suppressionwere very transparent and absolutely free of neovascularization (Fig. 4A). The corneal opacity, neovessel size, and vessel size scores were reduce within the GLX351322 eye drop groups than inside the manage groups just after alkali burn injury (Fig. 4C). Inside the manage groups.