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Fig. 13 Inhibition of TGFβ1 signaling or DNA methylation in ATP7B-KO mice decreases liver N2-neutrophil polarization and improves liver pathology. (A) Representative immunohistochemical staining images of anti-TGFβ1 in wild-type and ATP7B-KO mice livers. Scale bar: 50 μm. (B) qPCR of gene expression changes in Dnmt1, Dnmt3a, Dnmt3b, Scos3, and Stat3 in wild-type and ATP7B-KO mice liver neutrophils. n = 3 sets of neutrophils pooled from 2∼3 mice. ∗∗P < .01. (C) Flow cytometric analysis of N1 (CD11b+Ly6G+NOS2+) and N2 (CD11b+Ly6G+CD206+) neutrophil population in total CD11b+Ly6G+ cells derived from the livers of wild-type and ATP7B-KO mice treated with/without SM16 or 5-aza. (D) Flow cytometric quantification of CD11b+Ly6G+ neutrophils from wild-type and ATP7B-KO mice livers treated with/without SM16 or 5-aza. (E) Flow cytometric quantification of liver N1 and N2 neutrophils from wild-type and ATP7B-KO mice treated with/without SM16 or 5-aza. (D and E) n = 6 mice/group, unpaired 2-tailed t test. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Serum activities of (F) ALT, (G) AST, (H) HDL-C, and (I) TChol in wild-type and ATP7B-KO mice treated with/without SM16 or 5-aza. n = 6 mice/group. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. (J) Liver body ratio in wild-type and ATP7B-KO mice treated with/without SM16 or 5-aza. n = 6 mice/group. ∗∗∗P < .001. (K) Liver Cu content in wild-type and ATP7B-KO mice treated with/without SM16 or 5-aza. n = 4 mice/group. ∗∗∗P < .001. (L) Representative H&E images in liver sections from wild-type and ATP7B-KO mice treated with/without SM16 or 5-aza. Scale bar: 50 μm. WT, wild-type.