Qingchang Suppository Ameliorates Colonic Vascular Permeability in Dextran-Sulfate-Sodium-Induced Colitis

Abstract

Ulcerative colitis (UC), with a long course and repeated attack, severely affects patient's life quality and increases economic burden all over the world. However, the concrete causes and mechanisms of UC are still unclear, but it is generally considered that many factors participate in this process. Qingchang Suppository (QCS) has been used in treating rectitis and colitis for about 30 years in Shanghai, China. Its satisfactory clinical effects have been proved. The aim of this study is to investigate the effect and mechanisms of QCS on colonic vascular endothelial barrier in dextran sulfate sodium (DSS)-induced colitis. The results indicated that increased vascular permeability (VP) appeared earlier than increased intestinal epithelial permeability (EP) in the process of DSS-induced colitis. QCS attenuated colonic tissue edema, vascular congestion and inflammatory cell infiltration. QCS inhibited the elevation of MPO, TNF-α, and IL-6 levels in colon tissues and alleviated the microvascular damage induced by DSS. QCS also improved colonic hypoxia and decreased the expression of VEGF, HIF-1α, and iNOS. These results revealed that QCS can reduce colonic VP and can improve vascular endothelial barrier function maybe by regulating the VEGF/HIF-1α signaling pathway.

Keywords: epithelial permeability; qingchang suppository; ulcerative colitis; vascular endothelial barrier; vascular permeability.

Figure 1

Typical chromatogram of QCS in negative ion mode by LC-ESI-MS.

Figure 2

Dynamic pathological changes of the distal colon in DSS models of UC. Paraffin sections of the distal colon were stained with HandE (A: DSS 0 d; B: DSS 1d; C: DSS 2 d; D: DSS 3 d; E: DSS 4 d; F: DSS 5 d) (HandE staining, × 100). DAI (G) was assessed daily and colon length was measured on day 5 (H). Data were expressed as mean ± SD (n = 8–10), ^*P < 0.05, ^**P < 0.01 vs. control group or vs. 0 d group.

Figure 3

DSS administration increased colonic VP that preceded increased colonic EP in rats. (A) Quantitative measurement of VP by extracting extravasated Evans blue in the colonic mucosa. (B) Quantitative measurement of colonic epithelial permeability by determining serum concentration of FITC–dextran. Both VP and EP in the same animal were measured. n = 8. ^*P < 0.05; ^**P < 0.01 vs. control group.

Figure 4

Effects of QCS on histopathological changes, DAI and MPO activity in colon of rats with DSS-induced colitis. (A) Control; (B) DSS model; (C) QCS 0.36 g/kg; (D) QCS 0.72 g/kg; (E) QCS 1.44 g/kg; (F) SASP 0.135 g/kg; (HandE staining, × 100); (G) histological score; and (H) The disease activity index (DAI) was determined by combining scores of body weight loss, stool consistency, and occult blood, The final result was expressed as the average of the three. (I) MPO activity. DSS administration was performed in all groups except the control group. QCS and SASP were administered to rats each day after DSS treatment. All rats were killed on day 3 after DSS administration, three sections from each animal tissue were scored, colonic tissue damage was evaluated by histopathological analysis (HandE staining). MPO activity in colonic tissue was determined. Data were expressed as mean ±SD (n = 8), ^*P < 0.05, ^**P < 0.01 vs. model group; ^##P < 0.01, vs. control group.

Figure 5

Effects of QCS on VP in colon of rats with DSS-induced colitis. Quantitative measurement of VP. Data were expressed as mean ±SD (n = 8), ^*P < 0.05, ^**P < 0.01 vs. model group; ^##P < 0.01, vs. control group.

Figure 6

Effects of QCS on DSS-induced ultrastructural pathology changes in colon tissue. Histopathological examination of colon tissues by TEM (4,200 ×), n = 5. (A) Control; (B) DSS model; (C) QCS 0.36 g/kg; (D) QCS 0.72 g/kg; (E) QCS 1.44 g/kg; (F) SASP 0.135 g/kg. Red blood cells (white triangles). Platelet aggregation (red arrow). The microvascular basement membrane and endothelium (white arrow).

Figure 7

Effects of QCS on DSS-induced colon tissues hypoxia. Visualization of hypoxia in colonic mucosa by Hypoxyprobe-1 staining. Images are representative of three tissue slices (brown staining, × 100). (A) Control; (B) DSS model; (C) QCS 0.36 g/kg; (D) QCS 0.72 g/kg; (E) QCS 1.44 g/kg; (F) SASP 0.135 g/kg. (G) Quantified data of hypoxia in colonic mucosa. The final result was expressed as integral optical density (IOD)/ area of effective statistical. (n = 6/group). ^**P < 0.01; ^***P < 0.001, vs. model group; ^#P < 0.05, vs. control group.

Figure 8

Effects of QCS on DSS-induced production of TNF-α and IL-6 and expression of VEGF, HIF-1α and iNOS in colonic tissue. (A) Concentration of TNF-α in colonic tissue; (B) concentration of IL-6 in colonic tissue. (C,D) Protein expression of VEGF, HIF-1α, and iNOS. β-Actin levels were used as loading controls. Results are expressed as mean ±SD; n = 5, ^*P < 0.05; ^**P < 0.01, vs. model group; ^#P < 0.05; ^##P < 0.01, vs. control group.