Herbs-Partitioned Moxibustion Combined with Acupuncture Inhibits TGF- β 1-Smad-Snail-Induced Intestinal Epithelial Mesenchymal Transition in Crohn's Disease Model Rats

Abstract

Crohn's disease may cause excessive damage and repair in the intestinal epithelium due to its chronic relapsing intestinal inflammation. These factors may initiate the TGF-β 1-Smad pathway to activate the transcription factor of Snail, and the Snail-mediated pathway promotes the transformation of intestinal epithelial cells to mesenchymal cells, leading to intestinal fibrosis. Acupuncture and moxibustion have been demonstrated to prevent intestinal fibrosis in Crohn's disease. However, it is not clear whether acupuncture and moxibustion can inhibit intestinal epithelial mesenchymal transformation in Crohn's disease by affecting the TGF-β 1-Smad-Snail pathway. This study indicated that abnormal increased expressions of TGFβ1, TβR2, Smad3, and Snail were significantly downregulated by herbs-partitioned moxibustion at Tianshu (ST25) and Qihai (RN6) and acupuncture at Zusanli (ST36) and Shangjuxu (ST37). In addition, protein and mRNA levels of E-cadherin, the epithelial cell marker, were significantly increased. Protein and mRNA levels of fibronectin, the mesenchymal cell marker, were decreased in the intestinal tissue. Moreover, the number of mesenchymal cells in the intestinal mucosa can be reversely transformed to intestinal epithelial cells. Therefore, herbs-partitioned moxibustion combined with acupuncture can prevent intestinal epithelial mesenchymal transition by inhibiting abnormal expression of TGFβ1, TβR2, Smad3, and Snail in the TGF-β1-Smad-Snail pathway in Crohn's disease.

Figure 1

Representative histological observation from a light microscope of the rat colonic epithelial tissue sections stained with HE (magnification×100). (a) Normal control; (b) model control; (c) salicylazosulfapyridine; (d) acupuncture; (e) herbs-partitioned moxibustion; (f) herbs-partitioned moxibustion combined with acupuncture. Arrowheads: (1) multiple superficial and small ulcers; (2) fibrous tissue; (3) congestion and edema; (4) inflammatory cells; (5) epithelial proliferation and repair; (6) lymphatic dilatation; (7) intestinal wall of lymphatic tissue proliferation.

Figure 2

The protein expression levels of TGF-β1, TβR1, TβR2, Smad3, and Snail and the corresponding GAPDH were determined using Western blot analysis. The following histogram shows the quantitative densitometry of the blots in each group (n= 10 for each group). White space was used to make explicit for the grouping of blots cropped from different parts of the same gel or from different gels. Values are the means ± SD. In all panels, ###P<0.001, ##P<0.01, and #P<0.05 vs. NC. △△△P<0.001 vs. MC. ▲▲▲P <0.001, ▲▲P <0.01, and ▲P <0.05 vs. HPM+AC.

Figure 3

The protein expression levels of E-cadherin and fibronectin and the corresponding GAPDH were determined using Western blot analysis. The following histogram shows the quantitative densitometry of the blots in each group (n= 10 for each group). White space was used to make explicit for the grouping of blots cropped from different parts of the same gel or from different gels. Values are the means ± SD. In all panels, ### P<0.001, ##P<0.01, and #P<0.05 vs. NC. △△△P<0.001, △△P<0.01, and △P<0.05 vs. MC. ▲▲▲P<0.001 vs. HPM + AC.

Figure 4

Graphic representation shows the expression levels of E-cadherin mRNA and fibronectin mRNA analyzed by quantitative RT-PCR (n=10, for each group). Values are the means ± SD. ##P<0.01 vs. NC. △△P <0.01 vs. MC. ∗∗P <0.01 vs. SASP. ▲▲P <0.01 vs. HPM+AC.

Figure 5

Coexpression of Snail and E-cadherin in the rat intestinal epithelial tissues from the NC, MC, SASP, AC, HPM, and HPM+AC groups (n=10, for each group). (a) Normal control; model control; (c) salicylazosulfapyridine; (d) acupuncture; (e) herbs-partitioned moxibustion; (f) herbs-partitioned moxibustion combined with acupuncture. (g) The histogram shows the coexpression of Snail and E-cadherin in each group (n= 10, for each group). Values are the means ± SD. In all panels, ### P<0.001 and ##P<0.01 vs. NC. △△△P<0.001, △△P<0.01, and △P<0.05 vs. MC. ▲▲P <0.01 and ▲P <0.05 vs. HPM+AC. Scale bar ×200. Labelled for Snail (red), E-cadherin (green), Snail, and E-cadherin coexpression (yellow).

Figure 6

Coexpression of Snail and fibronectin in the rat intestinal epithelial tissues from the NC, MC, SASP, AC, HPM, and HPM+AC groups (n=10 for each group). (a) Normal control; model control; (c) salicylazosulfapyridine; (d) acupuncture; (e) herbs-partitioned moxibustion; (f) herbs-partitioned moxibustion combined with acupuncture. (g) The histogram shows the coexpression of Snail and E-cadherin in each group (n= 10, for each group). Values are the means ± SD. In all panels, ###P<0.001, ##P<0.01, and #P<0.05 vs. NC. △△△P<0.001, △△P<0.01, and △P<0.05 vs. MC. ▲P<0.05 vs. HPM+AC. Scale bar ×200. Labelled for Snail (red), fibronectin (green), Snail, and fibronectin coexpression (yellow).