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. 2020 Jun 19;10(1):9972.
doi: 10.1038/s41598-020-65306-4.

Sanguisorba officinalis L. derived from herbal medicine prevents intestinal inflammation by inducing autophagy in macrophages

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Sanguisorba officinalis L. derived from herbal medicine prevents intestinal inflammation by inducing autophagy in macrophages

Asuka Yasueda et al. Sci Rep. .

Abstract

Disturbed activation of autophagy is implicated in the pathogenesis of inflammatory bowel disease. Accordingly, several autophagy-related genes have been identified as Crohn's disease susceptibility genes. We screened the autophagy activators from a library including 3,922 natural extracts using a high-throughput assay system. The extracts identified as autophagy activators were administered to mice with 2% dextran sodium sulfate (DSS). Among the autophagy inducers, Sanguisorba officinalis L. (SO) suppressed DSS-induced colitis. To identify the mechanism by which SO ameliorates colitis, epithelial cell and innate myeloid cells-specific Atg7-deficient mice (Villin-cre; Atg7f/f and LysM-cre; Atg7f/f mice, respectively) were analyzed. SO-mediated inhibition of colitis was observed in Villin-cre; Atg7f/f mice. However, SO and a mixture of its components including catechin acid, ellagic acid, gallic acid, and ziyuglycoside II (Mix4) did not suppressed colitis in LysM-cre; Atg7f/f mice. In large intestinal macrophages (Mφ) of Atg7f/f mice, SO and Mix4 upregulated the expression of marker genes of anti-inflammatory Mφ including Arg1, Cd206, and Relma. However, these alterations were not induced in LysM-cre; Atg7f/f mice. These findings indicate that SO and its active components ameliorate DSS-induced colitis by providing intestinal Mφ with anti-inflammatory profiles via promotion of Atg7-dependent autophagy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
An autophagy activator SO ameliorated DSS-induced colitis. C57BL/6J mice were administered either SO or vehicle with (n = 14) or without (n = 7) 2% DSS for 6 days. (a) Body weight changes (means ± SD). (b) Colon length 7 days after starting DSS administration (means ± SD). (c) Representative histopathological images of distal colons (left) and histopathological score (right) (means ± SD from three independent experiments). Data were analyzed by Dunnet’s test, *p < 0.05. Bar; 100 μm. (d) Representative images of TUNEL staining of the colon 6 days after DSS administration. All data are representative of three independent experiments. (e) Concentration of FITC-dextran in the plasma (n = 5 in each group).
Figure 2
Figure 2
SO did not induce autophagy in large intestinal epithelial cells in the steady state. (a) Activation of autophagy by SO in colonic epithelial cells was analyzed by measuring the ratio of LC3-II to LC3-I that detected by western blot analysis using the indicated antibodies. Conversion of LC3 in colonic epithelial cells from C57BL/6J mice administered SO in drinking water for 7 days. The ratio of LC3-II to LC3-I was measured with Image studio lite ver. 5.2 (Li-Cor Bioscience, Nebraska, USA). Full-length blots are shown in Supplementary Fig. S10a. (b,c) Expression levels of the indicated genes in large intestinal epithelial cells from mice treated or untreated with SO in the steady state. Data are mean ± SD from three independent experiments. Data were analyzed by unpaired t-test. n.s., not significant. (d) Conversion of LC3 in colonic epithelial cells from C57BL/6J mice administered DSS with or without SO in drinking water for 7 days. *P < 0.05. Full-length blots are shown in Supplementary Fig. S10b. All data are representative of three independent experiments. (e) Expression of LC3B in the colon of C57BL/6J mice administered 2% DSS with or without SO in drinking water for 7 days were analyzed with immunohistochemistry using anti-LC3B antibody (upper). Representative images of H&E staining (bottom). Data are representative of three independent experiments. Bars; 100 μm.
Figure 3
Figure 3
SO-induced autophagy in epithelial cells did not affect intestinal inflammation. Atg7f/f and Atg7ΔIEC mice were administered 2% DSS with or without SO in drinking water for 7 days. (ac) Weight changes (a), colon length (b), (c) representative colon sections (upper), and histological score of the colon (bottom) (mean values ± SD). Data are of two independent experiments. Data were analyzed by un-paired t-test. n = 5 in each group. *p < 0.05. n.s, not significant. Bars; 100 μm.
Figure 4
Figure 4
SO induced autophagy in bone marrow-derived Mφ (BMMφ) and intestinal Mφ. (a) Expression of the indicated genes in colonic lamina propria cells from mice treated with or without SO in the steady state. Data are mean ± SD of three independent experiments. Data were analyzed by unpaired t-test, *p < 0.05. n.s., not significant. (b) CYTO-ID staining in BMMφ treated with or without SO for 24 hours. (c) BMMφ were cultured in the presence or absence of SO for 24 hours. The immunoblotting was performed with the indicated antibodies. The graphs show the ratio of LC3-II to LC3-I. Full-length blots are shown in Supplementary Fig. S10c. (d) C57BL/6J mice administered 2% DSS with or without SO in drinking water for 7 days and colonic lamina propria cells were isolated. The autophagosome formation stained by CYTO-ID in macrophages, eosinophils, dendric cells and neutrophils were analyzed with flow cytometry. Data are representative of two independent experiments.
Figure 5
Figure 5
SO did not prevent intestinal inflammation in Atg7LysM-cre mice. Atg7f/f and Atg7LysM-cre mice were administered 2% DSS with or without SO for 7 days. (ac) Weight changes (a), colon length (b), representative colon sections (upper), and histological score of the colon (bottom) (c) (mean values ± SD). *p < 0.05. n.s, not significant. n = 6 in each group. Bars; 100 μm. Data are of two independent experiments. Data were analyzed by un-paired t-test.
Figure 6
Figure 6
Promotion of Atg7-dependent autophagy by SO altered gene expression patterns in Mφ during DSS-induced colitis. (ac) CD11b+ cells from the large intestinal lamina propria of Atg7f/f and Atg7LysM-cre mice administered 2%DSS with or without SO for 6 days were analyzed expression the indicated genes. Data are mean ± SD of three independent experiments. Data were analyzed by unpaired t-test, *p < 0.05. n.s., not significant.
Figure 7
Figure 7
Minimal therapeutic effect of the mixture of SO components including CA, GA, EA, ZY (Mix4) on suppression of DSS-induced colitis in Atg7LysM-cre mice. Atg7f/f and Atg7LysM-cre mice were administered 2% DSS with or without Mix4 for 7 days. (a) Weight changes, (b) colon length, (c) representative colon sections (upper), and histological score of the colon (bottom). Data are mean ± SD of five mice in each group. *p < 0.05. n.s., not significant. Bars; 100 μm. Data are of two independent experiments.

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