Smurf1 Facilitates Oxidative Stress and Fibrosis of Ligamentum Flavum by Promoting Nrf2 Ubiquitination and Degradation

Abstract

Lumbar spinal stenosis (LSS), which can lead to irreversible neurologic damage and functional disability, is characterized by hypertrophy and fibrosis in the ligamentum flavum (LF). However, the underlying mechanism is still unclear. In the current study, the effect of Smurf1, a kind of E3 ubiquitin ligase, in promoting the fibrosis and oxidative stress of LF was investigated, and its underlying mechanism was explored. The expression of oxidative stress and fibrosis-related markers was assessed in the tissue of lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). Next, the expression of the top 10 E3 ubiquitin ligases, obtained from Gene Expression Omnibus (GEO) dataset GSE113212, was assessed in LDH and LSS, and confirmed that Smurf1 expression was markedly upregulated in the LSS group. Furthermore, Smurf1 overexpression promotes the fibrosis and oxidative stress of LF cells. Subsequently, NRF2, an important transcription factor for oxidative stress and fibrosis, was predicted to be a target of Smurf1. Mechanistically, Smurf1 directly interacts with Nrf2 and accelerates Nrf2 ubiquitination and degradation. In conclusion, the current study suggests that Smurf1 facilitated the fibrosis and oxidative stress of LF and induced the development of LSS by promoting Nrf2 ubiquitination and degradation.

Figure 1

Fibrosis was upregulated in LF tissues from HLF patients. (a) Magnetic resonance imaging (MRI) shows axial views of the lumbar spinal canal in the LDH and HLF patients. (b) Collagen I, Collagen III, and α-SMA mRNA levels in the LDH and HLF patients were assessed by qRT-PCR (n = 27). ^∗∗∗p < 0.001. (c, d) Collagen I, Collagen III, and α-SMA protein expression in the LDH and HLF patients was assessed by western-blot and IHC (n = 10). ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 2

Oxidative stress was upregulated in LF tissues from HLF patients. (a) The ROS levels and malondialdehyde (MDA) were higher in the HLF group compared with the LDH group. (n = 27). ^∗∗p < 0.01. (b) The glutathione (GSH) content, and superoxide dismutase (SOD) activity were decreased in the HLF group compared with the LDH group (n = 27). ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 3

Smurf1 was upregulated in LF tissues from HLF patients. (a, b) The mRNA expression of the top ten E3 ligases was assessed by qRT-PCR array in the HLF and LDH group (n = 27). ^∗p < 0.05. ^∗∗∗p < 0.001. (c) Western blotting and semiquantification for Smurf1 expression in the HLF and LDH groups (n = 10). ^∗∗∗p < 0.001. (d) Immunohistochemistry for Smurf1 expression in the HLF and LDH groups.

Figure 4

Smurf1 facilitated the fibrosis and oxidative stress of LF cells. (a, b) the transfection efficiency of Smurf1 was analyzed by qRT-PCR and western-blot. ^∗∗∗p < 0.001. (c, d) Western blotting and semiquantification for Collagen I, Collagen III, and α-SMA mRNA levels in the LF cells with or without Smuf1-OE). ^∗∗p < 0.01. ^∗∗∗p < 0.001. (e) The ROS levels and MDA content was assessed in the LF cells with or without Smuf1-OE. ^∗p < 0.05. ^∗∗p < 0.01. (f) the Glutathione (GSH) content, and Superoxide dismutase (SOD) activity was assessed in the LF cells with or without Smuf1-OE. ^∗p < 0.05. ^∗∗p < 0.01.

Figure 5

Nrf2 is the target of Smurf1. (a) Nrf2 mRNA level in the LDH and HLF patients was assessed by qRT-PCR (n = 27). (b) Western blotting and semiquantification for Nrf2 level in the LDH and HLF patients (n = 10). ^∗∗p < 0.01. (c) qRT-PCR was used to determine the level of Nrf2 mRNA in LF cells with or without Smuf1-OE. (d) Western blotting and semiquantification for Collagen I, Collagen III, and α-SMA mRNA level in the LF cells with or without Smuf1-OE. ^∗∗∗p < 0.001.

Figure 6

Smurf1 promotes the ubiquitination and degradation of Nrf2. (a) Smurf1 directly interacts with Nrf2 was assessed by CO-IP. (b, c) Western blot analysis of Nrf2 protein stability in Smurf1-OE LF cells treated with 25 ug/ml CHX for various times. ^∗∗p < 0.01. (d) Cell lysates from control and Smurf1-OE LF cells was immunoprecipitated with anti-Nrf2 antibody, then assesed by western blot using antiubiquitin antibody.