Dendrobium officinale polysaccharides attenuates spasmolytic polypeptide-expressing metaplasia via dual modulation of gastric epithelial cell fate and macrophage polarization

Figure 1 The Dendrobium officinale polysaccharides effects on mouse body weight, pathological improvement, mucus types and levels of interleukin-13 and interleukin-33 on spasmolytic polypeptide-expressing metaplasia model. A: Mouse body weight; B: Pathological improvement; C: Mucus types; D and E: Levels of interleukin-13 and interleukin-33. The data are represented as mean ± SD, n = 6. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; IL: Interleukin.

Figure 2 Analysis of parietal, chief cell and spasmolytic polypeptide-expressing metaplasia markers. A: Immunofluorescence (IF) analysis of the parietal cell marker ATP4α; B: IF analysis of the chief cell marker Mist1; C: Statistical analysis of IF results (ATP4α) by using ImageJ software; D: Statistical analysis of IF results (Mist1) by using ImageJ software; E and F: IF analysis of spasmolytic polypeptide-expressing metaplasia markers trefoil factor 2 and GS II; G and H: Statistical analysis of IF results (TFF2 and GS II) by using ImageJ software; I and J: Real-time polymerase chain reaction analysis of ATP4α, Mist1, TFF2 and Gpx2. The data are represented as mean ± SD, IF analysis, n = 3. Real-time polymerase chain reaction analysis, n = 6. ^aP < 0.05, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides.

Figure 3 The co-expression of aquaporin 5 and Ki67, gastric intrinsic factor and Ki67 in mouse gastric mucosal cells. A: Immunofluorescence (IF) analysis the co-expression of aquaporin 5 (AQP5) and Ki67; B: Statistical analysis of IF results (Ki67+ AQP5+) by using ImageJ software; C: Real-time polymerase chain reaction analysis of AQP5; D: IF analysis of the co-expression of gastric intrinsic factor and Ki67; E: Statistical analysis of IF results (gastric intrinsic factor + AQP5+) by using ImageJ software. ^aP < 0.05, ^cP < 0.001, vs control; ^dP < 0.05, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; AQP5: Aquaporin 5; GIF: Gastric intrinsic factor.

Figure 4 Analysis of M2-macrophages in gastric tissue by immunofluorescence and real-time polymerase chain reaction. A: Immunofluorescence (IF) analysis of the M2 macrophage polarization marker F4/80+ CD163+; B: Statistical evaluation of the IF results (F4/80+ iNOS+) conducted using Image J 6.0 software; C: IF analysis of the M1 macrophage polarization marker F4/80+ iNOS+; D: Statistical evaluation of the IF results (F4/80+ CD163+) conducted using Image J 6.0 software; E and F: Real-time polymerase chain reaction analysis of CD206 and CD163 expression levels. The data are represented as mean ± SD, Sample sizes: IF analysis, n = 3; real-time polymerase chain reaction analysis, n = 6. ^aP < 0.05, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides.

Figure 5 Western blot analysis of M2 macrophage polarization and the IL-13/STAT6 signaling pathway. A: Western blot analysis of M2 macrophage polarization markers; B: Relative protein quantification (CD206, ARG1, CD163, iNOS) performed using Image Lab 6.0; C: Western blot analysis of interleukin-13 and STAT6 protein expression; D: Relative protein quantification (interleukin-13, p-STAT6/STAT6) performed using Image Lab 6.0. The data are represented as mean ± SD, n = 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; IL: Interleukin.

Figure 6 The effect of Dendrobium officinale polysaccharides on IL-13/STAT6 signal pathway on bone marrow-derived macrophages cell. A: Flow cytometry analysis of M2 macrophage polarization; B: Flow cytometry result analysis by flowjo software; C: Genes expression of macrophage marker and interleukin (IL)-13, IL-13Ra1, IL-13Ra2, ST2 by real-time polymerase chain reaction; D: Protein expression of macrophage marker; E: Relative protein quantification (macrophage marker) performed using Image Lab 6.0; F: Protein expression of IL-13, STAT6 and p-STAT6; G: Relative protein quantification (IL-13, STAT6 and p-STAT6) performed using Image Lab 6.0. The data are represented as mean ± SD, n = 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; M-DOP: Middle-dose Dendrobium officinale polysaccharides; IL: Interleukin.

Figure 7 Molecular mechanism of Dendrobium officinale polysaccharides in inhibiting spasmolytic polypeptide-expressing metaplasia. DOP: Dendrobium officinale polysaccharides; IL: Interleukin; BMDMs: Bone marrow-derived macrophages; SPEM: Spasmolytic polypeptide-expressing metaplasia; TFF2: Trefoil factor 2; AQP5: Aquaporin 5.