Huanglian Jiedu Decoction ameliorates DSS-induced colitis in mice via the JAK2/STAT3 signalling pathway

Abstract

Background: Ulcerative colitis (UC) is an intestinal disease which was characterized by intestinal inflammation, mucosal injury and fibrosis. In this paper, the effect of Huanglian Jiedu Decoction (HJD), a well-known traditional Chinese medicine with significant anti-inflammatory effect, on dextran sulphate sodium (DSS)-induced UC in mice and inhibition of JAK2/STAT3 pathway were investigated.

Methods: BALB/c mice were randomly divided into 6 groups: HJD group (high, medium and low dose), USAN group, UC group, and control group. UC in mice were induced through free access to 3% DSS solution. After being treated with HJD for 8 days, all animals were sacrifice. Pathological examination of colonic specimen was performed by H&E staining. Cytokines (TNF-α, IL-6, and IL-1β) in colon were assayed by ELISA and immunofluorescence, MPO in colon and ATT in serum were detected by ELISA. Moreover, mice in HJD group and UC group were treated with AG490 to inhibit the expression of JAK2 protein, then the expression of JAK2 and STAT3 protein in colon was determined by western blotting and immunofluorescence staining. Furthermore, KI67 in colon was examined by immunohistochemistry, and apoptosis was detected by TUNEL staining, and collagen deposition was assayed by Masson staining after JAK2/STAT3 pathway in UC mice was inhibited by HJD.

Results: After mice being treated with HJD, the symptoms (weight loss and haematochezia) of UC were alleviated, and the contents of inflammatory cytokines (TNF-α, IL-6 and IL-1β) and MPO in colon were significantly decreased. The expression of JAK2 and STAT3 protein was reduced after administration with HJD. After JAK2/STAT3 pathway being inhibited with HJD, the cell apoptosis, collagen deposition and immunoreactivity of macrophage in colon were significantly reduced, but the expression of Ki67 was markedly enhanced in both UC group and HJD group compare with control group.

Conclusions: HJD treatment can alleviate intestinal mucosal damage and has the protective effect on UC by downregulating JAK2 and STAT3 expression to reduce inflammation via JAK2/STAT3 pathway.

Keywords: Dextran sulphate sodium; Huanglian Jiedu Decoction; JAK2/STAT3; Ulcerative colitis.

Fig. 1

Protective effect of HJD against DSS-induced UC in mice. a Body weight. b DAI. c CMDI. d Histology score of the colon calculated by H&E staining. e Colon macroscopic damage and length. f Image of H&E staining obtained at 20× magnification in the colon. All of the data are expressed as mean ± SD (n = 10), **P < 0.01 compared with UC group, and ^##P < 0.01 compared with control group. Scale bar = 50 μm

Fig. 2

HJD downregulated the contents of TNF-α, IL-6, and IL-1β in colons and increased ATT content in serum. a–e Contents of TNF-α, IL-1β, IL-6, MPO and ATT. f–i Immunofluorescence staining of TNF-α, IL-1β and IL-6 in colon. *P < 0.05, **P < 0.01 compared with UC group, and ^##P < 0.01 compared with control group. Scale bar = 50 μm

Fig. 3

The effects of HJD and AG490 on UC in mice. a Body weight. b DAI. c CMDI. d Histology score of the colon calculated by H&E staining. e Macroscopic damage and length of colon. f Image of H&E staining obtained at 20× magnification. All of the data are expressed as mean ± SD (n = 10), **P < 0.01 compared with UC group; ^##P < 0.01 compared with control group. Scale bar = 50 μm

Fig. 4

Effect of HJD on the protein expression of JAK2 and STAT3 in colon. a Western blotting of JAK2 and STAT3 protein. b Semi-quantitative analysis of JAK2 and STAT3 proteins. c, d Immunofluorescence staining of JAK2 and STAT3 co-localized in control group, UC group, AG490 group, AG490 + HJD group and HJD group. All data are expressed as mean ± SD, **P < 0.01 compared with UC group, and ^##P < 0.01 compared with control group. Scale bar = 50 μm

Fig. 5

Effects on apoptosis, Ki67 expression and macrophages in colonic mucosa after HJD pre-treatment. a F4/80 by immunofluorescence, Ki67 expression by IHC staining, and apoptosis by TUNEL staining. b AOD of Ki67. c AOD of apoptosis. All of the data were expressed as mean ± SD. d Semi-quantitative analysis of immunofluorescence staining for F4/80 co-localized in UC group, control group and HJD group. All images were obtained at a magnification of 20× . **P < 0.01 compared with UC group, and ^#P < 0.05, ^##P < 0.01 compared with control group. Scale bar = 50 μm

Fig. 6

Effects on collagen expression in colonic mucosa of UC in mice after HJD pre-treatment. a Collagen deposition by Masson staining (blue). b IOD analysis. All of the data are expressed as mean ± SD. Images were obtained at a magnification of 20× or 40× . **P < 0.01 compared with UC group. Scale bar = 50 μm