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

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.