Discussion on the effect of Honghua Qinggan Shisanwei Wan on regulating macrophage polarization and alleviating liver injury

Figure 1 Cell and animal experiments confirm that Honghua Qinggan Shisanwei Wan is not hepatotoxic. A: Determination of body weight of mice; B: Aspartate aminotransferase assay in mouse serum; C: Alanine aminotransferase assay in mouse serum; D: The liver tissue was examined using hematoxylin and eosin staining (200 ×); E: The liver histopathology was analyzed by Masson staining (200 ×). Con: Control; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; NS: Not significant.

Figure 2 Protective effect of Honghua Qinggan Shisanwei Wan against carbon tetrachloride-induced liver injury in mice. A: Mouse body weight alterations following acute liver injury induced by carbon tetrachloride; B: Alanine aminotransferase assay in mouse serum; C: Aspartate aminotransferase assay in mouse serum; D: The liver histopathology was analyzed by hematoxylin and eosin staining (200 ×); E: The liver histopathology was analyzed by Masson staining (200 ×); F: The tumor necrosis factor-α/C-C motif chemokine ligand 2/interleukin-1β/interleukin-6 expression of liver tissue in the liver injury; G: The expression of tumor necrosis factor-α in liver injury (200 ×). The regions indicated by arrows represent the injured areas of liver tissue and the corresponding positive expression regions of the gene. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, NS: Not significant. ALT: Alanine aminotransferase; CCL2: C-C motif chemokine ligand 2; CCL₄: Carbon tetrachloride; HHQG: Honghua Qinggan Shisanwei Wan; AST: Aspartate aminotransferase; TNF-α: Tumor necrosis factor-α; IL: Interleukin; Con: Control.

Figure 3 Protective effect of Honghua Qinggan Shisanwei Wan against carbon tetrachloride-induced liver injury in mice. A: Gene Ontology enrichment of differential gene in model group and tr Honghua Qinggan Shisanwei Wan (HHQG) hepatoprotective group on the 2^nd day; B: Kyoto Encyclopedia of Genes and Genomes enrichment of differential gene in model group and HHQG hepatoprotective group on the 2^nd day; C: Heatmap of differentially expressed genes associated with macrophage polarization. Carbon tetrachloride (CCl₄) was the model group, which included three different samples: CCl₄-1, CCl₄-2, and CCl₄-3; HHQG was the HHQG group, which included three different samples: HHQG-1, HHQG-2, and HHQG-3. PPAR: Peroxisome proliferator-activated receptor; HIF: Hypoxia-inducible factor; MAPK: Mitogen-activated protein kinase; GTP: Guanosine triphosphate; BP: Biological process; CC: Cellular component; MF: Molecular function; CCl₄: Carbon tetrachloride; HHQG: Honghua Qinggan Shisanwei Wan.

Figure 4 Honghua Qinggan Shisanwei Wan affects macrophage infiltration and anti-inflammatory factor expression in carbon tetrachloride-induced liver injury. A: The F4/80 expression of liver tissue in the liver injury; B: The expression of F4/80 in liver injury; C: The C-X3-C motif chemokine receptor 1/transforming growth factor-β/YM-1/interleukin-10 expression of liver tissue in the liver injury (400 ×); D: The expression of YM-1 in liver injury (400 ×); E: The expression of transforming growth factor-β in liver injury (400 ×). The regions indicated by arrows represent the injured areas of liver tissue and the corresponding positive expression regions of the gene. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, NS: Not significant. Con: Control; CCl₄: Carbon tetrachloride; HHQG: Honghua Qinggan Shisanwei Wan; CX3CR1: C-X3-C motif chemokine receptor 1; TNF-α: Tumor necrosis factor-α; IL: Interleukin.

Figure 5 The effect of Honghua Qinggan Shisanwei Wan on the expression of macrophage markers and peroxisome proliferator-activated receptor-γ in liver tissue. A: The expression of T-cell immunoglobulin and mucin domain-4 in mouse liver tissue by immunohistochemistry (IHC) (200 ×); B: The expression of CD11b in mouse liver tissue by IHC (200 ×); C: The expression of peroxisome proliferator-activated receptor-γ in mouse liver tissue by IHC (200 ×). The regions indicated by arrows represent the injured areas of liver tissue and the corresponding positive expression regions of the gene. Con: Control; CCl₄: Carbon tetrachloride; HHQG: Honghua Qinggan Shisanwei Wan.

Figure 6 Honghua Qinggan Shisanwei Wan affects macrophage polarization and nuclear receptor subfamily 4 group A member 1/nuclear receptor subfamily 4 group A member 2 expression in carbon tetrachloride-induced liver injury. A: The nuclear receptor subfamily 4 group A member 1 (NR4A1)/NR4A2 expression of liver tissue in the liver injury; B: The expression of NR4A1 in liver injury (200 ×); C: The expression of NR4A2 in liver injury (200 ×); D: The expression of NR4A1 in mouse liver tissue by immunohistochemistry (200 ×); E: The expression of NR4A2 in mouse liver tissue by immunohistochemistry (200 ×). The regions indicated by arrows represent the injured areas of liver tissue and the corresponding positive expression regions of the gene. ^aP < 0.05, ^cP < 0.001, ^dP < 0.0001, NS: Not significant. Con: Control; CCl₄: Carbon tetrachloride; HHQG: Honghua Qinggan Shisanwei Wan; NR4A1: Nuclear receptor subfamily 4 group A member 1.

Figure 7 Honghua Qinggan Shisanwei Wan affects M1 and M2 macrophage polarization and expression of its associated factors. A: Cell counting kit-8 was used to detect the effects of different concentrations of Honghua Qinggan Shisanwei Wan (HHQG) on the viability of RAW264.7 cells; B: MRNA expression of inducible nitric oxide synthase, tumor necrosis factor-α, C-C motif chemokine ligand 2, and interleukin-6 in the M1 macrophage polarization model; C: MRNA expression of C-X3-C motif chemokine receptor 1, YM-1, transforming growth factor-β, interleukin-10 in the M1 macrophage polarization model; D: The inhibitory effect of HHQG on the expression of inducible nitric oxide synthase and pro-inflammatory factors in M1-type macrophage markers; E: The promoting effect of HHQG on the expression of M2 macrophage marker YM-1 and anti-inflammatory factors. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, NS: Not significant. OD: Optical density; iNOS: Inducible nitric oxide synthase; Con: Control; HHQG: Honghua Qinggan Shisanwei Wan; TNF-α: Tumor necrosis factor-α; CCL2: C-C motif chemokine ligand 2; IL: Interleukin; CX3CR1: C-X3-C motif chemokine receptor 1; TGF: Transforming growth factor.

Figure 8 The effect of Honghua Qinggan Shisanwei Wan on the secretion levels of M1/M2 phenotype-related factors in macrophages. A: The inhibitory effect of Honghua Qinggan Shisanwei Wan on the expression of inducible nitric oxide synthase, a marker of M1-type macrophages, and the secretion of pro-inflammatory factors; B: The promoting effect of Honghua Qinggan Shisanwei Wan on the expression of YM-1, a marker of M2-type macrophages, and the secretion of anti-inflammatory factors. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, NS: Not significant. TNF-α: Tumor necrosis factor-α; Con: Control; HHQG: Honghua Qinggan Shisanwei Wan; IL: Interleukin; CCL2: C-C motif chemokine ligand 2; iNOS: Inducible nitric oxide synthase; TGF: Transforming growth factor; CX3CR1: C-X3-C motif chemokine receptor 1; Arg1: Arginase1.

Figure 9 Honghua Qinggan Shisanwei Wan dose-dependently induces the expression of nuclear receptor subfamily 4 group A member 1/nuclear receptor subfamily 4 group A member 2 receptors nuclear receptors in macrophages. A: The effect of Honghua Qinggan Shisanwei Wan on the expression of nuclear receptor subfamily 4 group A member 1/nuclear receptor subfamily 4 group A member 2 receptors nuclear receptors in M1-type macrophages; B: The effect of Honghua Qinggan Shisanwei Wan on the expression of nuclear receptor subfamily 4 group A member 1/nuclear receptor subfamily 4 group A member 2 receptors nuclear receptors in M2-type macrophages. ^aP < 0.05, ^cP < 0.001, ^dP < 0.0001, NS: Not significant. NR4A1: Nuclear receptor subfamily 4 group A member 1; Con: Control; HHQG: Honghua Qinggan Shisanwei Wan.

Figure 10  Results of molecular docking between key compounds of Honghua Qinggan Shisanwei Wan and nuclear receptor subfamily 4 group A member 1/nuclear receptor subfamily 4 group A member 2 receptors. NR4A: Nuclear receptor subfamily 4 group A.