Narfl Deletion Induced zebrafish Endothelial Dysfunction by Upregulating the Iron Level and Production of Reactive Oxygen Species and Lipid Peroxidation. A) The Micro Zebra Lab system detects the blood flow of zebrafish, the red asterisk part detects the line speed, and the red box part detects the average speed. B–E) The mean blood flow velocity (B, D) and mean linear velocity (C, E) of narfl−/−zebrafish were not significantly different from those of wild type zebrafish at 3–5 dpf, significantly higher at 6 dpf, significantly lower at 7–11 dpf, and significantly lower at 11–13 dpf than those of wild type (n = 5). F) DCFH‐DA probe was used to detect oxidative stress in 5 dpf zebrafish (n = 30). The stronger the green fluorescence, the higher the oxidative stress level; the magnification was 200×; Compared with wild zebrafish, the narfl−/− zebrafish fluorescence intensity was significantly enhanced. G) DPPP probe was used to detect lipid peroxidation levels in 5 dpf zebrafish (n = 30). The stronger the purple fluorescence, the higher the lipid peroxidation level. H) BODIPY 493/503 probe was used to detect neutral lipid levels (n = 30), with stronger green fluorescence indicating higher lipid levels. I) Determination of iron level in zebrafish (n = 5) by colorimetric method; the results showed that the iron level in narfl−/− zebrafish was significantly higher than that in wild type (p <0.001), and Fe3+ was also increased. J) Quantitative results of detection of cytoplasmic cis‐aconitase activity in zebrafish (n = 5), the results showed that the cytoplasmic ciaconitase activity of narfl−/− zebrafish was significantly reduced. K) Quantitative detection of glutathione and glutamine levels in zebrafish. The results showed that glutathione and glutamine levels in narfl−/− zebrafish were significantly reduced. L) The results of mitochondrial respiration integration of 11 wild type and 8 narfl−/− zebrafish in two groups, which indicated that the deletion of the narfl gene led to a decrease in mitochondrial respiratory function of zebrafish. M) qRT‐PCR was used to verify the P450 family genes with differences in RNA sequencing, and the results showed that cyp2p8, cyp3c3, cyp8b2, and cyp2n13 were significantly decreased in narfl−/− zebrafish. cyp2×7, cyp2k8, and cyp2v1 were significantly increased, while there was no significant difference in other genes. N) In situ hybridization was used to detect 5 dpf wild type and narfl−/− zebrafish, as well as the results of treatment with CYP2P8‐specific activator ophiopogon D. The locations indicated by the black arrows showed the sites with positive cyp2p8 probe signals. O) The expression of CYP2J2 in adjacent lung tissue and in the progenitor with diffuse pulmonary malformation was detected by immunohistochemistry, CYP2J2 is expressed in the cytoplasm and decreased expression of CYP2J2 was found in progenitors with down‐regulated NARFL expression. Statistical significance was assessed using one‐way ANOVA with Tukey's multiple comparison correction. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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