Integrative study reveals NR1D1 mediates Hedyotis diffusa’s antifibrosis via hypoxia inducible factor-1/ammonia axis

Figure 1 Identification of the active ingredients of Hedyotis diffusa injection. A: Total ion chromatograms of Herba Hedyotidis medicated liver: Positive ion mode and negative ion mode; B: Chemical formula structure of the 6 main components.

Figure 2 Network pharmacology analysis. A: Venn analysis; B: Protein-protein interaction topology analysis; C: Component-disease-target network diagram; D: Kyoto Encyclopedia of Genes and Genomes enrichment analysis. HDI: Hedyotis diffusa injection; HIF: Hypoxia inducible factor.

Figure 3 Hedyotis diffusa injection can ameliorate carbon tetrachloride-induced liver fibrosis in mice. A: Hematoxylin-eosin; B: Masson; C: Sirius red staining; D: Α-smooth muscle actin immunohistochemistry; E: Serum biochemical indices of hepatic fibrosis and liver injury. P value listed, compared with carbon tetrachloride group. HE: Hematoxylin-eosin; HDI: Hedyotis diffusa injection; HD: Hedyotis diffusa; L: Low; M: Median; H: High; CCl₄: Carbon tetrachloride; IHC: Immunohistochemistry; SMA: Smooth muscle actin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase; LDH: Lactate dehydrogenase; TBIL: Total bilirubin; LN: Laminin; HA: Hyaluronidase; PC-III: Procollagen-III; IV-C: Type IV collagen.

Figure 4 Hedyotis diffusa injection ameliorates liver fibrosis by influencing circadian rhythm, hypoxia inducible factor-1 signaling pathway and urea metabolic process. A and B: GSE990887 dataset analysis; C and D: GSE33650 dataset analysis; E: Volcano analysis of proteomics in mice; F: Venn analysis of the differentially expressed proteins in these two groups: Control vs carbon tetrachloride (CCl₄) and CCl₄vs Hedyotis diffusa injection; G and H: Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. KEGG: Kyoto Encyclopedia of Genes and Genomes; TGF: Transforming growth factor; HIF: Hypoxia inducible factor; PPAR: Peroxisome proliferators-activated receptor; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; TCA: Tricarboxylic acid cycle; mTOR: Mammalian target of rapamycin; TNF: Tumor necrosis factor; AMPK: Adenosine 5’-monophosphate-activated protein kinase; Th17: T helper 17 cell.

Figure 5 Hedyotis diffusa injection regulates the hepatic hypoxia inducible factor-1 signaling pathway and urea cycle through the circadian rhythm gene NR1D1. A: The expression changes of circadian rhythm genes in mouse liver tissue before and after Hedyotis diffusa injection treatment using real-time polymerase chain reaction; B: The protein expression of hypoxia inducible factor (HIF)-1α, NR1D1, and BMAL1 analyzed by Western blot; C: Detection of urea cycle-related enzyme levels in liver tissue by enzyme-linked immunosorbent assay (ELISA); D: Detection of urea and ammonia levels in liver tissue by ELISA; E: Effect of NR1D1 on HIF-1α protein expression detected by Western blot; F: Effect of NR1D1 on carbamoyl phosphate synthetase 1, urea, and ammonia levels detected by ELISA. HDI: Hedyotis diffusa injection; CCl₄: Carbon tetrachloride; ZT: Zeitgeber time; mRNA: Messenger RNA; HIF: Hypoxia inducible factor; CPS1: Carbamoyl phosphate synthetase 1; Otc: Ornithine transcarbamylase; Ass1: Argininosuccinate synthase 1; Asl: Argininosuccinate lyase; Arg-1: Arginase 1; KD: Knockdown; OE: Overexpression.

Figure 6 NR1D1 is the primary target of Hedyotis diffusa injection for its anti-hepatic fibrosis effects. A: Hematoxylin-eosin, Masson, Sirius red and α-smooth muscle actin staining. Scale bar: 50 μm; B: Serum biochemical indices of liver injury; C: Serum biochemical indices of hepatic fibrosis. P value listed, compared with each group. SMA: Smooth muscle actin; HE: Hematoxylin-eosin; HDI: Hedyotis diffusa injection; CCl₄: Carbon tetrachloride; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase; LDH: Lactate dehydrogenase; TBIL: Total bilirubin; LN: Laminin; HA: Hyaluronidase; PC-III: Procollagen-III; IV-C: Type IV collagen; KD: Knockdown.

Figure 7 Abnormally elevated ammonia levels promote LX2 cell activation. A: Ammonia level test by enzyme-linked immunosorbent assay kit; B: Immunofluorescence detection of α-smooth muscle actin expression; C: Laser confocal analysis of mitochondrial morphology. P value listed, compared with each group. CTRL: Control; NH₄Cl: Ammonium chloride; SMA: Smooth muscle actin; DAPI: 4’,6-diamidino-2-phenylindole.