Fig 2: Genetic deletion of the Ncf1 gene in neutrophils ameliorates chronic-plus-binge ethanol-induced liver ROS, inflammation, steatosis, and injury.WT and Ncf1Lyz–/– mice were fed an ethanol diet for 10 days, followed by gavage of ethanol, and were euthanized 9 hours later. (A) Serum ALT levels and circulating neutrophils were measured (WT, n = 14; KO, n = 12). (B) Liver tissues were subjected to H&E staining, TG measurement (WT, n = 12; KO, n = 12), and IHC staining (WT, n = 8; KO, n = 6) with antibodies against MDA, HNE, MPO, and F4/80. Representative images are shown on the left. Liver TG levels were quantified and are shown on the right. Quantification of the area positive for MDA or HNE staining is shown on the right. Scale bars: 200 µm (H&E row), 100 µm (additional rows). Quantification of number of MPO+ cells, percentage of F4/80+ area, and F4/80 clusters are on the right. Values in A and B represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (C and D) Heatmap illustrations of the expression profiles of genes involved in inflammation (C) and fibrosis (D) (WT, n = 6; KO, n = 6). P values are indicated. Statistical significance was assessed using 2-tailed Student’s t test for comparing 2 groups (A and B) and 1-way ANOVA followed by Tukey’s post hoc test for multiple groups (C and D).

Fig 3: The impacts of calcitriol on protein expression and activity of Nox in the hypertensive brain. Representative Western blot (A) and statistical graphs of protein expression of Nox2 (B), p-p47phox (C), p47phox (D) and Nox4 (E), and total Nox activity (F). 100 ng/kg calcitriol was administrated daily for 6 weeks in both control (Con) and SHR animals. Data are means ± SD (n = 7). **p < 0.01 compared to control group. +p < 0.05, ++p < 0.01 compared to SHR group.

Fig 4: The impacts of calcitriol treatment on Nox signaling and cellular superoxide generation in microglia BV2 cells. Representative western blots (A) and statistical graphs of protein expression of Nox2 (B), Nox4 (C), p-p47phox (D) and p47phox (E). NADPH oxidase activity (F) and ROS generation (DCF fluorescence intensity) (G). 1 µM calcitriol was pre-treated for 30min before exposure to 100 nM Ang II for 24 h. 1 µM A779 was added simultaneously with Ang II to block MasR. Data are means ± SD (n = 6). **p < 0.01 compared to control group. ++p < 0.01 compared to Ang II treated group. ##p < 0.05, ##p < 0.01 compared to Ang II + Calcitriol treated group.

Fig 5: Hepatic levels of p47phox/NCF1 are elevated and correlated with the quantity of intrahepatic neutrophils in SAH patients.(A) Hepatic NCF1 levels were compared among healthy control (n = 7) and Neulo (n = 5) and Neuhi (7) groups, based on RNA-Seq data. (B) Liver tissues from SAH patients were subjected to immunofluorescence staining of MPO and NCF1. Representative images are shown. Scale bars: 25 µm. (C and D) Correlation analyses were performed between NCF1 and inflammation-related genes or oxidative stress–related genes based on RNA-Seq data (n = 13). Values in the x and y axes represent relative fragments per kilobase of exon per million mapped fragments (FPKM) from RNA-Seq data. Data in A are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical significance was assessed using 1-way ANOVA followed by Tukey’s post hoc test for multiple groups (A) and Pearson’s correlation analysis (C and D).