Figure 1.
The liverwort plant Dumortiera hirsuta and chemical structure of Riccardin D.
Figure 2.
Riccardin D prevented spontaneous intestinal polyposis in APCMin/+ mice.
(A and B): Representative pictures of distal small intestinal and colon polyps; (C): The number of polyps per mouse in different parts of small intestines; (D): The size distribution of polyps in different part of small intestines; (E): The number of polyps per mouse in colons; (F): The size distribution of polyps in colons. *, p<0.001; #, p<0.01; $, p<0.05 versus control. (G): Histological analysis of intestinal polyps. Sections of small intestine and colon from control or Riccardin D-treated mice were stained with hematoxylin and eosin (H&E) and analyzed under microscopy. a, Normal histology of small intestinal mucosa (100×). b, Small intestine of control mice showed an area of villus effacement and formation of a polyp with dysplastic glands. The central part of the polyp showed surface erosion and necrosis (40×). c, Riccardin D-treated small intestine showed an area of epithelial hyperplasia without obvious dysplasia. The villus architecture in polypoid area is well preserved (40×). d, Normal histology of colonic mucosa (100×). e, Colon from control mice showed a large pedunculated tubular adenoma (40×). f, Colon in mucosa of Riccardin D-treated mice showed well preserved crypt architecture with normal surface maturation. The surface epithelium showed minimal evidence of hyperplasia (40×).
Table 1.
The number of polyps per mouse in different parts of small intestine and colon.
Table 2.
The size distribution of polyps per mouse in different part of small intestine and colon.
Figure 3.
Riccardin D decreased expression of β-catenin and cyclin D1 in intestinal polyps.
Intestinal sections were processed for β-catenin and cyclin D1 immunohistochemistry staining. Intestinal sections of control and Riccardin D-treated mice showed brown-colored PCNA-positive (A) and cyclin D1-positive cells (B) in polyps on small intestines and colons (400×). Bar represents the mean ± S.D of six animals. *, p<0.001 versus control.
Figure 4.
Riccardin D suppressed inflammation by decreasing COX-2 and NF-κB in intestinal polyps.
(A): The inhibition of NF-κB and p-NF-κB Ser536 in intestinal polyps of Riccardin D-treated mice (n = 3). (B): The inhibition of TNF-α in intestinal polyps of Riccardin D-treated mice (n = 3). (C): The inhibition of COX-2 expression in polyps on small intestines and colons of Riccardin D-treated mice. The bars indicate mean ± S.D. (n = 6). (D): The decrease of COX-2 mRNA in polyps on small intestines as estimated by RT-PCR analysis (n = 3). (E): The content of PGE2 in small intestinal polyps was decreased in Riccardin D-treated mice as measured by ELISA analysis. The bars indicate mean ± S.D. (n = 3). *, p<0.001 versus control.
Figure 5.
Riccardin D decreased proliferation and induced apoptosis in intestinal polyps.
(A and B): Intestinal polyps of control and Riccardin D-treated mice showed brown-colored PCNA-positive (A) and TUNEL-positive (B) cells in polyps (400×). Quantitative data for proliferation and apoptosis were determined as number of PCNA-positive, TUNEL-positive cells×100/total number of cells, respectively. The bars represent mean ± S.D. of six animals. *, p<0.001 versus control. (C and D): The alteration of apoptotic proteins in polyps on small intestine as estimated by western blotting analysis. (C): The levels of caspase-9, caspase-3 and cleaved PARP in intestinal polyps were increased by Riccardin D (n = 3). (D): Bcl-2 was decreased and Bax was increased in intestinal polyps in Riccardin D-treated mice (n = 3).
Figure 6.
Riccardin D inhibited angiogenesis in intestinal polyps.
(A): Immunohistochemical staining of CD34 for analysis of microvessel density (MVD) in polyps of 2–3 mm on small intestines and colons. MVD in polyps of different intestine segments was significantly inhibited by Riccardin D. (B): The expression of VEGF in polyps was inhibited by Riccardin D as estimated by immunohistochemical staining assay. The bars represent mean ± S.D. of six animals. *, p<0.001 versus control. (C): Riccardin D decreased the expression of FGF-2 in intestinal polyps as estimated by western blotting assay (n = 3).
Figure 7.
Summary for the pathways inhibited by Riccardin D in APCMin/+ mice intestinal polyps.