Weiwei Decoction alleviates gastric intestinal metaplasia through the olfactomedin 4/nucleotide-binding oligomerization domain 1/caudal-type homeobox gene 2 signaling pathway

Figure 1 The olfactomedin 4/nucleotide-binding oligomerization domain 1/caudal-type homeobox gene 2 pathway is a characteristic of gastric intestinal metaplasia tissues. A: The representative images of Immunohistochemistry (IHC) staining for olfactomedin 4 (OLFM4), nucleotide-binding oligomerization domain 1 (NOD1), and caudal-type homeobox gene 2 (CDX2) in normal and intestinal metaplasia (IM) tissues; B: OLFM4, NOD1, and CDX2 immunoreactivity scores in normal and IM tissues were compared; C: Correlation of OLFM4 with CDX2, NOD1 with CDX2, and OLFM4 with NOD1 in gastric tissues by Spearman’s correlation analysis. ^aP < 0.01, ^bP < 0.001 vs control group. OLFM4: Olfactomedin 4; NOD1: Nucleotide-binding oligomerization domain 1; CDX2: Caudal-type homeobox gene 2; IM: Intestinal metaplasia.

Figure 2 Olfactomedin 4 exhibited direct binding and subsequent down-regulation of nucleotide-binding oligomerization domain 1, thereby sustaining the activation of caudal-type homeobox gene 2 and promoting the progression of intestinal metaplasia. A: Relative mRNA expressions of caudal-type homeobox gene 2 (CDX2), MUCIN 2 (MUC2), and villin 1 (VIL1) on stimulation of chenodeoxycholic acid (CDCA) in GES-1 cells (n = 3); B: Western blot (WB) detection of MUC2, VIL1, and CDX2 in GES-1 cells treated with CDCA (100 μM) for 24 h; C: Timeline of the cell experiments design for pathway regulatory verification; D: Short hairpin RNAs (shRNAs) targeting olfactomedin 4 (OLFM4) were transfected to GES-1 cells. Subsequently, the cells were treated with CDCA (100 μM) for 24 h. WB detection of OLFM4, nucleotide-binding oligomerization domain 1 (NOD1), CDX2, and MUC2; E: AGS cells were transfected with OLFM4 shRNAs. WB detection of OLFM4, NOD1, CDX2, MUC2, and VIL1; F: OLFM4-pcDNAs were transfected to GES-1 cells. WB detection of OLFM4, NOD1, CDX2, and VIL1; G: Co-immunoprecipitation detection of the interaction between OLFM4 and NOD1, and the interaction between OLFM4 and CDX2 in GES-1 cells; H: OLFM4 and NOD1 shRNAs were co-transfected to GES-1 cells. Subsequently, the cells were treated with CDCA (100 μM) for 24 h. WB detection of OLFM4, NOD1, CDX2, and MUC2. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group. OLFM4: Olfactomedin 4; NOD1: Nucleotide-binding oligomerization domain 1; CDX2: Caudal-type homeobox gene 2; MUC2: MUCIN 2; VIL1: Villin 1; CDCA: Chenodeoxycholic acid.

Figure 3 Weiwei Decoction improves gastric mucosal histological lesions. A: Timeline of the animal experiments design for Weiwei Decoction treatment; B: Representative hematoxylin and eosin-stained images of the gastric mucosa in each group (n = 3). Vacuolation changes (orange arrow), pyknotic and hyperchromatic nuclei (blue arrow); C: Representative Alcian blue-Periodic acid-Schiff-stained images of the gastric mucosa in each group (n = 3). Blue-stained cavities (yellow arrow). Scale bar: 100 μm. WWD: Weiwei Decoction; FA: Folic acid; HE: Hematoxylin and eosin; AB-PAS: Alcian blue-Periodic acid-Schiff.

Figure 4 Weiwei Decoction reduces the expression of intestinal markers KLF transcription factor 4, villin 1, and MUCIN 2 in intestinal metaplasia rats. A: Western blot detection of villin 1 (VIL1) and KLF transcription factor 4 (KLF4) in rat gastric mucosa; B: Relative protein expression of VIL1 and KLF4 (n = 4); C: Relative mRNA expression of MUCIN 2 (MUC2) and VIL1 in gastric mucosa (n = 5); D: Immunohistochemistry detection of MUC2, representative images of the gastric mucosa in each group. Scale bar: 100 μm; E: Quantitative analysis of MUC2 protein expression in gastric tissues (n = 3). ^aP < 0.01, ^bP < 0.001 vs control group; ^cP < 0.05, ^dP < 0.01, ^eP < 0.001 vs model group; ^fP < 0.05, ^gP < 0.01, ^hP < 0.001 vs folic acid group. VIL1: Villin 1; KLF4: KLF transcription factor 4; WWD: Weiwei Decoction; FA: Folic acid; MUC2: MUCIN 2.

Figure 5 Weiwei Decoction suppresses olfactomedin 4 expression and restores nucleotide-binding oligomerization domain 1 level, consequently reducing caudal-type homeobox gene 2 in intestinal metaplasia rats. A-C: Immunohistochemistry detection of olfactomedin 4 (OLFM4), nucleotide-binding oligomerization domain 1 (NOD1), and caudal-type homeobox gene 2 (CDX2), representative images of gastric mucosa in each group. Scale bar: 100 μm; D: Quantitative analysis of OLFM4, NOD1, and CDX2 protein expression in gastric mucosa (n = 3); E: Relative mRNA expression of OLFM4, NOD1, and CDX2 in gastric mucosa (n = 5). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group; ^gP < 0.05, ^hP < 0.01, ⁱP < 0.001 vs folic acid group. OLFM4: Olfactomedin 4; NOD1: Nucleotide-binding oligomerization domain 1; CDX2: Caudal-type homeobox gene 2; WWD: Weiwei Decoction; FA: Folic acid.

Figure 6 Weiwei Decoction-medicated serum attenuates olfactomedin 4 level and simultaneously strengthens olfactomedin 4 and nucleotide-binding oligomerization domain 1 binding, thereby enhancing nucleotide-binding oligomerization domain 1 expression and consequently mitigating caudal-type homeobox gene 2 in gastric cells. A: Timeline of the cell experiments design for Weiwei Decoction (WWD)-medicated serum treatment; B: GES-1 cells were treated with chenodeoxycholic acid (CDCA) for 24 h and then disposed to 10% (V/V) of the corresponding WWD-medicated serum for 48 h. WB detection of olfactomedin 4 (OLFM4), nucleotide-binding oligomerization domain 1 (NOD1), caudal-type homeobox gene 2 (CDX2), and MUCIN 2 (MUC2); C: Relative protein expression of OLFM4, NOD1, CDX2, and MUC2 in GES-1 cells (n = 3); D: AGS cells were disposed to 10% (V/V) of the corresponding WWD-medicated serum for 48 h. WB detection of OLFM4, NOD1, CDX2, MUC2, and villin 1 (VIL1); E: Relative protein expression of OLFM4, NOD1, CDX2, MUC2, and VIL1 in AGS cells (n = 3); F: GES-1 cells were treated with CDCA for 24 h and then disposed to 10% (V/V) of the WWD-H medicated serum for 48 h. Co-immunoprecipitation detection of the interaction between OLFM4 and NOD1, and the interaction between OLFM4 and CDX2 in GES-1 cells. ^aP < 0.05, ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs model group. OLFM4: Olfactomedin 4; NOD1: Nucleotide-binding oligomerization domain 1; CDX2: Caudal-type homeobox gene 2; WWD: Weiwei Decoction; MUC2: MUCIN 2; VIL1: Villin 1.

Figure 7 Weiwei Decoction inhibits cytokines and chemokines in gastric intestinal metaplasia. A: Enzyme-linked immunosorbent assay was utilized to detect interferon-gamma, interleukin (IL)-6, IL-17, macrophage chemoattractant protein-1, and macrophage inflammatory protein 1 alpha in intestinal metaplasia rats serum (n = 6); B: Relative mRNA expression of tumor necrosis factor alpha, IL-6, and IL-8 in GES-1 cells (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group; ^gP < 0.05, ^hP < 0.01, ⁱP < 0.001 vs folic acid group. IL: Interleukin; MIP: Macrophage inflammatory protein; MCP: Macrophage chemoattractant protein; IFN: Interferon; WWD: Weiwei Decoction; FA: Folic acid; TNF: Tumor necrosis factor.

Figure 8 A schematic model of the olfactomedin 4/nucleotide-binding oligomerization domain 1/caudal-type homeobox gene 2 pathway in gastric intestinal metaplasia cells and the mechanism of Weiwei Decoction in the treatment of gastric intestinal metaplasia through this pathway. OLFM4: Olfactomedin 4; NOD1: Nucleotide-binding oligomerization domain 1; CDX2: Caudal-type homeobox gene 2; VIL1: Villin 1; KLF4: KLF transcription factor 4; WWD: Weiwei Decoction; MUC2: MUCIN 2.