Role of TGF-β1-mediated epithelial-mesenchymal transition in the pathogenesis of tympanosclerosis

Abstract

The present study aimed to explore the role of TGF-β1-mediated epithelial-mesenchymal transition (EMT) in the pathogenesis of tympanosclerosis. Sprague Dawley rats were injected with inactivated Streptococcus pneumoniae suspension to establish a rat model of tympanosclerosis. The rats were sacrificed 8 weeks after the model was established. H&E and von Kossa staining was used to observe the morphological changes of middle ear mucosa. Western blotting was used to detect the expression of TGF-β1 and EMT-associated proteins in the mucosa samples. Middle ear mucosal epithelial cells of rats were collected to establish a primary culture. The cultured cells were stimulated with TGF-β1 and the expression of EMT-associated proteins was detected by western blotting and immunofluorescence. In addition, the cells were treated with TGF-β receptor type I/II inhibitor and the expression level of EMT-associated proteins was detected by western blotting. Sclerotic lesions appeared on 72.4% of tympanic membranes, and marked inflammation, inflammatory cell infiltration and fibrosis were found in the middle ear mucosa of rat models of tympanosclerosis. In middle ear mucosa of rats with tympanosclerosis, the expression of mesenchymal cell markers increased and that of epithelial cell markers decreased compared with the control group. TGF-β1 stimulated the activation of the EMT pathway in middle ear mucosal epithelial cells, resulting in an increased expression of fibronectin and N-cadherin. In addition, a decreased expression level of EMT-associated proteins was observed when TGF-β1 was inhibited. In conclusion, the present study indicated that TGF-β1-mediated EMT may play an important role in the pathogenesis of tympanosclerosis.

Keywords: N-cadherin; TGF-β1; epithelial-mesenchymal transition; fibronectin; tympanosclerosis.

Figure 1

Otomicroscopic observations at week 8. (A) Representative left ear used as the control. The tympanic membrane was intact and transparent without calcification. (B) Representative right ear used as the experimental model. The tympanic membrane showed obvious turbidity and sclerotic plaques (arrow) at the tympanic ring. Original magnification, x20.

Figure 2

H&E staining observations. (A) Representative control group image. Morphology and structure of mucosa (arrow) were normal and without inflammation. (B) Representative experimental group image. Mucosa was markedly thickened with inflammatory cell infiltration (triangle) and fibrous tissue hyperplasia (arrow). Original magnification, x200.

Figure 3

Von Kossa staining observations. (A) Representative control group image. No calcium deposition was found. (B) Representative experimental group image. Mucosa was markedly thickened with scattered calcium deposits (arrows). Original magnification, x400.

Figure 4

Detection of protein expression by western blotting. (A) Western blotting results for E-cadherin, TGF-β1, snail, N-cadherin and fibronectin in the TS and CT groups. (B) Semi-quantitative analysis of the expression levels of E-cadherin, TGF-β1, snail, N-cadherin and fibronectin in the TS and CT groups. ^*P<0.05 and ^**P<0.01 vs. CT. (C) Western blotting results for EMT-associated proteins after treatment with TGF-β1 or co-treatment with TGF-β1 and LY2109761. (D) Semi-quantitative analysis of the expression levels of EMT-associated proteins after TGF-β1 stimulation or co-treatment with TGF-β1 and LY2109761. ^**P<0.01 as indicated. CT, control; TS, tympanosclerosis.

Figure 5

Immunofluorescence staining for vimentin. Nuclei were stained blue using DAPI. The expression levels of vimentin were upregulated following TGF-β1 stimulation. ^*P<0.05 vs. CT. Original magnification, x200.