Therapeutic efficacy of a combined sage and bitter apple phytopharmaceutical in chronic DSS-induced colitis - PubMed (original) (raw)
Therapeutic efficacy of a combined sage and bitter apple phytopharmaceutical in chronic DSS-induced colitis
Maximilian Hoffmann et al. Sci Rep. 2017.
Abstract
Inflammatory bowel diseases are multifactorial disorders of the gastrointestinal tract with rising incidence worldwide. Current standard therapies are only partially effective and often show severe adverse effects. Thus, novel, more efficient and well-tolerated therapeutic options are urgently needed. We have studied the therapeutic potential of a phytopharmaceutical combining sage and bitter apple (SBA) in the mouse model of chronic dextran sulfate sodium (DSS) colitis. SBA represents a traditional medicine against diarrhea and was shown to exhibit anti-inflammatory effects in vitro. In the chronic DSS colitis model SBA treatment significantly reduced clinical symptoms in a dose-dependent manner. The positive therapeutic effect of SBA was characterized by a decreased histopathological score indicating tissue healing. Moreover, the number of neutrophils as well as the expression of the neutrophil-recruiting chemokine CXCL-1/KC in the colon tissue was significantly reduced, whereas the recruitment of macrophages was induced. Also, the expression of inflammatory markers was significantly suppressed, while the expression of the anti-inflammatory cytokine interleukin-10 was induced in colon tissue following treatment with SBA. Phytopharmaceuticals are increasingly recognized as potential therapeutics in IBD. Thus, based on the results from this study, SBA can be considered as an alternative or supplementary option for IBD therapy.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Figure 1
SBA reduced clinical symptoms in chronic DSS colitis. Chronic colitis was induced in BALB/c mice by feeding of 2% DSS in drinking water for 7 days followed by 10 days of 1% DSS and another 12 days of 2% DSS. Animals were treated from days 1–10 and 18–22 with 6-TG (1 mg/kg) as positive control (a,e,i) or at days 1, 3, 5, 7, 9, 18, 20, 22 with different concentrations of SBA: 100 mg/kg (b,f,j); 10 mg/kg (c,g,k); or 1 mg/kg (d,h,l). Animals treated orally with PBS from days 1-10 and 18–22 were used as vehicle control. Clinical score (a–d), stool-consistency-blood score (e–h), and body weight (i–l) were evaluated daily and are shown as mean ± SEM for 6–10 animals per group. Statistics: Two-way ANOVA followed by a Bonferroni’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control group.
Figure 2
SBA ameliorated pathology of the colon tissue. Chronic colitis was induced in BALB/c mice by feeding of 2% DSS in drinking water for 7 days followed by 10 days of 1% DSS and another 12 days of 2% DSS. Animals were treated orally from days 1–10 and 18–22 with 6-TG (1 mg/kg) as positive control or at days 1, 3, 5, 7, 9, 18, 20, 22 with different concentrations of SBA: 100 mg/kg; 10 mg/kg; or 1 mg/kg. Animals treated orally with PBS from days 1–10 and 18–22 were used as vehicle control. The colon length was determined as a marker for the extent of tissue inflammation and fibrosis (a). The histopathological score was determined based on hematoxylin-eosin-stained cross sections of the distal colon (b). The median of individual data points is indicated. Statistics: One-Way ANOVA with Dunnett’s multiple comparison test; number of animals per group = 6–10; *p < 0.05; **p < 0.01 versus vehicle control group.
Figure 3
SBA altered infiltration of inflammatory cells into the colon tissue. Chronic colitis was induced in BALB/c mice by feeding of 2% DSS in drinking water for 7 days followed by 10 days of 1% DSS and another 12 days of 2% DSS. Animals were treated orally from days 1–10 and 18–22 with 6-TG as positive control (1 mg/kg) or at days 1, 3, 5, 7, 9, 18, 20, 22 with different concentrations of SBA: 100 mg/kg; 10 mg/kg; or 1 mg/kg. Animals treated orally with PBS from days 1–10 and 18–22 were used as vehicle control. Cross sections of the distal colon were stained for the macrophage marker F4/80, the T cell markers CD3 and CD4, the inflammatory markers COX-2 and MPO, and for IgA; scale bars: 500 µm (a). Stained cross sections (2 sections/mouse) were digitized with a slide scanner (Zeiss AxioScan.Z1) and expression was quantified using the ImageJ software program and is shown as marker-positive area relative to the DAPI-positive area (b–g). The median of individual data points is indicated. Statistics: One-Way ANOVA with Dunnett’s multiple comparison test; number of animals per group = 6–10; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 versus vehicle control group.
Figure 4
SBA restored number but not function of goblet cells. Chronic colitis was induced in BALB/c mice by feeding of 2% DSS in drinking water for 7 days followed by 10 days of 1% DSS and another 12 days of 2% DSS. Animals were treated orally from days 1–10 and 18–22 with 6-TG as positive control (1 mg/kg) or at days 1, 3, 5, 7, 9, 18, 20, 22 with different concentrations of SBA: 100 mg/kg; 10 mg/kg; or 1 mg/kg. Animals treated orally with PBS from days 1–10 and 18–22 were used as vehicle control. Cross sections of the distal colon were stained with periodic-acid-Schiff reagent and counterstained with hematoxylin; scale bars: 20 µm (a). Stained cross sections (2 sections/mouse) were digitized with a slide scanner (Zeiss AxioScan.Z1) and expression was quantified using the ImageJ software program and is shown as PAS-positive area relative to the DAPI-positive area (b). The number of goblet cells per crypt was counted manually (c). The median of individual data points is indicated. Statistics: One-Way ANOVA with Dunnett’s multiple comparison test; number of animals per group = 6–10; ***P < 0.001; ****P < 0.0001 versus vehicle control group.
Figure 5
SBA restored expression of tight junction proteins. Chronic colitis was induced in BALB/c mice by feeding of 2% DSS in drinking water for 7 days followed by 10 days of 1% DSS and another 12 days of 2% DSS. Animals were treated orally from days 1–10 and 18–22 with 6-TG as positive control (1 mg/kg) or at days 1, 3, 5, 7, 9, 18, 20, 22 with different concentrations of SBA: 100 mg/kg; 10 mg/kg; or 1 mg/kg. Animals treated orally with PBS from days 1–10 and 18–22 were used as vehicle control. Cross sections of the distal colon were stained for claudin (CLDN)1 (a) and CLDN4 (b), Zonula occludens protein (ZO)-1 (c) and occludin (OCLN) (d). Stained cross sections (2 sections/mouse) were digitized with a slide scanner (Zeiss AxioScan.Z1) and expression was quantified using ImageJ software program and is shown as TJ protein-positive area relative to the DAPI-positive area. The median of individual data points is indicated. Statistics: One-Way ANOVA with Dunnett’s multiple comparison test; number of animals per group = 1–10; *P < 0.05; **P < 0.01; ****P < 0.0001 versus vehicle control group. Representative images of CLDN1 expression in the colon tissue; scale bars: 500 µm (e).
Figure 6
SBA reduced expression of inflammatory markers in the colon tissue. Chronic colitis was induced in BALB/c mice by feeding of 2**%** DSS in drinking water for 7 days followed by 10 days of 1**%** DSS and another 12 days of 2**%** DSS. Animals were treated orally from days 1–10 and 18–22 with 6-TG as positive control (1 mg/kg) or at days 1, 3, 5, 7, 9, 18, 20, 22 with different concentrations of SBA: 100 mg/kg; 10 mg/kg; or 1 mg/kg. Animals treated orally with PBS from days 1–10 and 18–22 were used as vehicle control. Concentrations of CXCL1/KC (a), IL-1β (b) and IL-10 (c) were determined in the supernatant of homogenized tissue samples of the distal colon by ELISA. The median of individual data points is indicated. Statistics: One-Way ANOVA with Dunnett’s multiple comparison test; number of animals per group = 6–10; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 versus vehicle control group.
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