Nrf2 inhibits epithelial-mesenchymal transition by suppressing snail expression during pulmonary fibrosis

Abstract

Epithelial-mesenchymal transition (EMT) is a phenotype conversion that plays a critical role in the development of pulmonary fibrosis (PF). It is known that snail could regulate the progression of EMT. Nuclear factor erythroid 2 related factor 2 (Nrf2), a key regulator of antioxidant defense system, protects cells against oxidative stress. However, it is not known whether Nrf2 regulates snail thereby modulating the development of PF. Here, bleomycin (BLM) was intratracheally injected into both Nrf2-knockout (Nrf2^-/-) and wild-type mice to compare the development of PF. Rat type II alveolar epithelial cells (RLE-6TN) were treated with a specific Nrf2 activator sulforaphane, or transfected with Nrf2 and snail siRNAs to determine their effects on transforming growth factor β1 (TGF-β1)-induced EMT. We found that BLM-induced EMT and lung fibrosis were more severe in Nrf2^-/- mice compared to wild-type mice. In vitro, sulforaphane treatment attenuated TGF-β1-induced EMT, accompanied by the down-regulation of snail. Inversely, silencing Nrf2 by siRNA enhanced TGF-β1-induced EMT along with increased expression of snail. Interestingly, when snail was silenced by siRNA, sulforaphane treatment was unable to reduce the progression of EMT in RLE-6TN cells. These findings suggest that Nrf2 attenuates EMT and fibrosis process by regulating the expression of snail in PF.

Figure 1. Relationship between Nrf2 and EMT in BLM-induced PF.

Nrf2 knockout mice and wild-type mice were treated with BLM (4.5 mg/kg) or saline on days 7, 14 and 28. (A): Pulmonary tissue sections were stained with H&E and Masson’s trichrome to detect the histopathological structure and collagen accumulation respectively. Scale bars represent 20 μm. (B): Relative protein expression of Nrf2, E-cadherin, α-SMA and SPC was measured by IHC. Scale bars represent 20 μm. Histogram bars represent means ± SD (n = 4–6 per group). **P < 0.01 compared with WT saline group, ^##P < 0.01 compared with Nrf2^−/− saline group, ⁺P < 0.05, ⁺⁺P < 0.01 compared with WT BLM group.

Figure 2. Relationship between Nrf2 and EMT in BLM-induced PF.

Lung tissues were subjected to Western blot analysis for Nrf2, E-cadherin, SPC, α-SMA, vimentin and snail. The representative bands were obtained from different gels for repeated experiments. β-actin was used as an internal reference for relative quantification. Data represent the mean ± SD (n = 3–4 per group), **P < 0.01 compared with WT saline group; ^##P < 0.01 compared with Nrf2^−/− saline group, ⁺P < 0.05, ⁺⁺P < 0.01 compared with WT BLM group.

Figure 3. Activating Nrf2 attenuated TGF-β1-induced EMT in RLE-6TN cells.

(A) Rats RLE-6TN cells were treated with SFN (0–10 μmol/L) for 24 h and the nuclear expression of Nrf2 was measured by Western blot. The representative bands were obtained from different gels for repeated experiments. Histone H3 was used as an internal reference for relative quantification. Data were expressed as mean ± SD (n = 3–4 per group), ⁺⁺P < 0.01 compared with vehicle group. (B): Cells were cultured in the absence or presence of SFN (1 μmol/L) for 24 h, and then treated with TGF-β1 (5 ng/ml) for 24 h. Cell lysates were collected and the relative proteins were determined by Western blot. The representative bands were obtained from different gels for repeated experiments. β-actin was used as an internal reference for relative quantification. Data were expressed as mean ± SD (n = −4 per group). **P < 0.01 compared with vehicle group; ^#P < 0.05, ^##P < 0.01 compared with TGF-β1 group.

Figure 4. Silencing Nrf2 enhanced TGF-β1-induced EMT in RLE-6TN cells.

Nrf2 siRNA were transfected in cells before stimulated with TGF-β1 for 24 h, then cell lysates were collected and the relative proteins were determined by Western blot. The representative bands were obtained from different gels for repeated experiments. β-actin was used as an internal reference for relative quantification. Data were expressed as mean ± SD (n = 3–4 per group), **P < 0.01 compared with vehicle group; ^##P < 0.01 compared with TGF-β1 group.

Figure 5. Nrf2 inhibited the development of EMT via suppressing the expression of snail.

Snail siRNA were transfected in RLE-6TN cells, after 6 h incubation, the cells were treated with SFN for 24 h, followed by stimulation with TGF-β1 for 24 h. Cell lysates were collected and the relative proteins were determined by Western blot. The representative bands were obtained from different gels for repeated experiments. β-actin was used as an internal reference for relative quantification. Data were expressed as mean ± SD (n = 3–4 per group), **P < 0.01 compared with vehicle group; ⁺⁺P < 0.01 compared with TGF-β1 group; ^##P < 0.01 compared with TGF-β1+SFN group.

Figure 6. Nrf2 inhibited the development of EMT via suppressing the expression of snail.

Nuclear snail protein level was assessed by Western blot in lung tissues. The representative bands were obtained from different gels for repeated experiments. Histone H3 was used as a loading control, and data were expressed as mean ± SD (n = 3–4 per group), **P < 0.01 compared with WT saline group; ^##P < 0.01 compared with Nrf2^−/− saline group, ⁺P < 0.05, ⁺⁺P < 0.01 compared with WT BLM group.

Figure 7. Nrf2 inhibited the development of EMT via suppressing the expression of snail.

(A) The nuclear and cytoplasm expression of snail was assessed by Western blot after silencing Nrf2. (B) The nuclear and cytoplasm expression of snail was assessed by Western blot after activating Nrf2. The representative bands were obtained from different gels for repeated experiments. The densitometry values were normalized to β-actin or histone H3, respectively. Data were expressed as mean ± SD (n = 3–4 per group), **P < 0.01 compared with vehicle group; ^##P < 0.01 compared with TGF-β1 group.

Figure 8. Nrf2 protected against the development of EMT by suppressing snail expression during pulmonary fibrosis.