Salidroside mitigates experimental colitis through cyclic adenosine monophosphate pathway activation and suppression of enteric glial cell responses

Figure 1 Salidroside alleviated the clinical symptoms of dextran sulfate sodium salt-induced colitis. A: Schematic of the experimental procedures; B: Chemical structure of salidroside (Sal); C: Changes in body weight of mice; D: Changes in body weight loss of mice; E: Comparison of colon length in each group of mice; F: Hematochezia observed in mice from each group: Control group (F1); Dextran sulfate sodium salt (DSS)-induced colitis (DSS) group (F2); DSS-induced colitis + Sal treatment (DSS + Sal) group (F3); G: Disease activity index scores after Sal treatment. Data are shown as mean ± SEM (n = 8), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 1D and F and two-way analysis of variance with Šídák’s multiple comparisons test for Figure 1C and H. ^aP < 0.05. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside.

Figure 2 Salidroside attenuated colonic injury in dextran sulfate sodium salt-induced colitis mice. A: Results of hematoxylin-eosin staining of mouse: Control group (A1); Dextran sulfate sodium salt (DSS) group (A2); DSS + salidroside (Sal) group (A3); The magnified images show the corresponding black rectangle areas in greater detail (A4-A6); B: Results of alcian blue-periodic acid Schiff staining of mouse: Control group (B1); DSS group (B2); DSS + Sal group (B3); The magnified images show the corresponding black rectangle areas in greater detail (B4-B6); C: Histological index scores; D: The number of goblet cells in each crypt. Data are shown as mean ± SEM (n = 8), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 2C and D. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside.

Figure 3 Salidroside inhibited enteric glial cells activation in the myenteric plexus and submucosal plexus of dextran sulfate sodium salt-induced colitis mice. A-F: Immunofluorescent histochemistry double-staining of mouse colonic myenteric plexus (MP) (A-C) and submucosal plexus (SP) (D-F). The magnified images show the corresponding white square areas in greater detail. The white arrows indicate glial fibrillary acidic protein-positive enteric glial cells (EGCs): Control group (A and D); Dextran sulfate sodium salt (DSS) group (B and E); DSS + salidroside group (C and F); G: The number of Hu C/D positive enteric neurons in the MP subjected to quantitative analysis; H: The area exhibiting apparent overlap of EGCs processes with the neurons in the MP analyzed quantitatively; I: The number of Hu C/D positive enteric neurons in the SP subjected to quantitative analysis; J: The area exhibiting apparent overlap of EGCs processes with the neurons in the SP analyzed quantitatively. Data are shown as mean ± SEM (n = 4), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 3G-J. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside; EGC: Enteric glial cell; DAPI: 4’,6-diamidino-2-phenylindole; GFAP: Glial fibrillary acidic protein.

Figure 4 Salidroside protected intestinal mucosal barrier and attenuated inflammation in dextran sulfate sodium salt-induced colitis mice. A and B: Immunofluorescent histochemical staining of mouse colonic mucosa with occludin (A) and zonula occludens-1 (ZO-1) (B): Control group (A1-A4 and B1-B4); Dextran sulfate sodium salt (DSS) group (A5-A8 and B5-B8); DSS + salidroside (Sal) group (A9-A12 and B9-B12). The magnified images show the corresponding white square areas in greater detail; C and D: The relative fluorescence intensities of occludin (C) and ZO-1 (D) expression was subjected to quantitative analysis; E: Western blotting analysis was conducted to assess the expression levels of glial fibrillary acidic protein (GFAP), occludin, and ZO-1 in the colon of the mice; F-H: Quantitative analysis was carried out for the expression levels of GFAP (F), occludin (G), and ZO-1 (H) proteins; I: The ultrastructural characteristics of the colonic mucosal epithelium. The black triangle indicates the tight junctions between the epithelial cells of the mucosa: Control group (I1); DSS group (I2); DSS + Sal group (I3); The magnified images show the corresponding black square areas in greater detail (I4-I6); J: Intestinal permeability was detected by measuring serum fluorescein isothiocyanate-dextran levels; K-N: Comparison of interleukin (IL)-1β (K), IL-6 (L), tumor necrosis factor-α (M) and nuclear factor kappa-B (N) expression levels in peripheral blood of the three groups of mice. Data are shown as mean ± SEM (n = 4), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 4C, D, F-H, and J-N. ^aP < 0.05. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside; DAPI: 4’,6-diamidino-2-phenylindole; ZO-1: Zonula occludens-1; GFAP: Glial fibrillary acidic protein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; FITC: Fluorescein isothiocyanate; IL: Interleukin; TNF: Tumor necrosis factor; NF-κB: Nuclear factor kappa-B.

Figure 5 Network pharmacology predicted the targets of salidroside in alleviating ulcerative colitis, and transcriptomic analysis identified differentially expressed genes in the colon of ulcerative colitis patients. A: Venn diagram of potential targets of salidroside (Sal) in ameliorating ulcerative colitis (UC); B: Protein-protein interaction network of the 52 common targets of Sal and UC; C: Gene Ontology (GO) functional enrichment analysis of Sal in ameliorating UC; D: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of Sal in improving UC; E: Volcano plot displayed differentially expressed genes in the colon of UC patients compared with healthy controls; F: GO functional enrichment analysis of downregulated and upregulated genes in the colon of UC patients and healthy controls; G and H: KEGG pathway enrichment analysis of downregulated (G) and upregulated (H) genes in the colon of UC patients vs healthy controls. I: Gene set enrichment analysis of the cyclic adenosine monophosphate signaling pathway in UC patients compared with healthy controls. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; IL: Interleukin; EGFR: Epidermal growth factor receptor; BP: Biological process; CC: Cellular component; MF: Molecular function; cAMP: Cyclic adenosine monophosphate; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; TNF: Tumor necrosis factor; AGE-RAGE: Advanced glycation end products-receptor for advanced glycation end products; PPAR: Peroxisome proliferator-activated receptor.

Figure 6 Salidroside alleviated the clinical symptoms and colonic injury of dextran sulfate sodium salt-induced colitis by activating the cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-response element binding protein signaling pathway. A: Schematic of the experimental procedures; B: Changes in body weight of mice; C: Disease activity index scores; D: Comparison of colon length in each group of mice; E: Results of hematoxylin-eosin staining of mouse; F: Results of alcian blue-periodic acid Schiff staining of mouse. Control group (E1 and F1); Dextran sulfate sodium salt (DSS) group (E2 and F2); DSS + salidroside (Sal) group (E3 and F3); DSS + Sal combined with the protein kinase A inhibitor H89 (DSS + Sal + H89) group (E4 and F4); The magnified images show the corresponding black rectangle areas in greater detail (E5-E8 and F5-F8); G: Histological index scores; H: The number of goblet cells in each crypt; I: Intestinal permeability was detected by measuring serum fluorescein isothiocyanate-dextran levels. Data are shown as mean ± SEM (n = 8), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 6E, H-J and two-way analysis of variance with Šídák’s multiple comparisons test for Figure 6B and C. ^aP < 0.05. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside; FITC: Fluorescein isothiocyanate.

Figure 7 Salidroside inhibited enteric glial cells activation in the myenteric plexus and submucosal plexus through cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-response element binding protein signaling and reduced intestinal inflammation in dextran sulfate sodium salt-induced colitis. A-H: Immunofluorescent histochemistry double-staining of mouse colonic myenteric plexus (MP) (A-D) and submucosal plexus (SP) (E-H). The magnified images show the corresponding white square areas in greater detail. The white arrows indicate glial fibrillary acidic protein-positive enteric glial cells (EGCs). Control group (A and E); Dextran sulfate sodium salt (DSS) group (B and F); DSS + salidroside (Sal) group (C and G); DSS + Sal + H89 group (D and H); I: The number of Hu C/D positive enteric neurons in the MP was subjected to quantitative analysis; J: The area exhibiting apparent overlap of EGCs processes with the neurons in the MP; K: The number of Hu C/D positive enteric neurons in the SP was subjected to quantitative analysis; L: The area exhibiting apparent overlap of EGCs processes with the neurons in the SP; M-P: Comparison of interleukin (IL)-1β (M), IL-6 (N), necrosis factor-α (O) and nuclear factor kappa-B (P) expression levels in peripheral blood of the four groups of mice. Data are shown as mean ± SEM (n = 4), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 7I-P. ^aP < 0.05. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside; EGC: Enteric glial cell; IL: Interleukin; TNF: Tumor necrosis factor; NF-κB: Nuclear factor kappa-B; DAPI: 4’,6-diamidino-2-phenylindole; GFAP: Glial fibrillary acidic protein.

Figure 8 Salidroside protected the intestinal mucosal barrier in dextran sulfate sodium salt-induced colitis mice by inhibiting enteric glial cells activation. A: The ultrastructural characteristics of the colonic mucosal epithelium. The black triangle indicates the tight junctions between the epithelial cells of the mucosa. Control group (A1); Dextran sulfate sodium salt (DSS) group (A2); DSS + salidroside (Sal) group (A3); DSS + Sal + H89 group (A4); The magnified images show the corresponding black square areas in greater detail (A5-A8); B: Western blotting analysis was conducted to assess the expression levels of glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor (GDNF) in the colon of the mice; C: Western blotting analysis was conducted to assess the expression levels of protein kinase A (PKA) and phosphorylated PKA (p-PKA) in the colon of the mice; D-G: Quantitative analysis was carried out for the expression levels of GFAP (D), GDNF (E), PKA (F), and p-PKA/PKA (G) proteins; H: Western blotting analysis was conducted to assess the expression levels of cyclic adenosine monophosphate-response element binding protein (CREB) and phosphorylated CREB (p-CREB) in the colon of the mice; I and J: Quantitative analysis was carried out for the expression levels of CREB (I) and p-CREB/CREB (J) proteins. Data are shown as mean ± SEM (n = 4), P values were calculated using one-way analysis of variance with Tukey’s test for Figure 7D-G, I and J. ^aP < 0.05. ^bP < 0.01. DSS: Dextran sulfate sodium salt; Sal: Salidroside; GFAP: Glial fibrillary acidic protein; GDNF: Glial cell line-derived neurotrophic factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PKA: Protein kinase A; p-PKA: Phosphorylated protein kinase A; CREB: Cyclic adenosine monophosphate-response element binding protein; p-CREB: Phosphorylated cyclic adenosine monophosphate-response element binding protein.

Figure 9 Schematic representation of the therapeutic mechanism of salidroside in dextran sulfate sodium salt-induced colitis in mice. Salidroside treatment mitigates colitis and restores intestinal mucosal barrier integrity, primarily by activating the cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-response element binding protein signaling pathway. This activation inhibits enteric glial cells activation, limits proinflammatory cytokine release, and enhances glial cell line-derived neurotrophic factor secretion. Ultimately, these effects attenuate intestinal inflammation, preserve barrier function, and reduce colonic injury. ZO-1: Zonula occludens-1; cAMP: Cyclic adenosine monophosphate; GFAP: Glial fibrillary acidic protein; GDNF: Glial cell line-derived neurotrophic factor; EGC: Enteric glial cell; IL: Interleukin; TNF: Tumor necrosis factor; NF-κB: Nuclear factor kappa-B; DSS: Dextran sulfate sodium salt; PKA: Protein kinase A.