Targeting clock-controlled gene Nrf2 ameliorates inflammation-induced intervertebral disc degeneration

Abstract

Background: Intervertebral disc (IVD) is a highly rhythmic tissue, which experiences a diurnal cycle of high/low mechanical loading via the changes of activity/rest phase. There are signs that disruption of the peripheral IVD clock is related to the process of intervertebral disc degeneration (IDD). However, it is still unclear whether inflammation could disturb the IVD clock and thus induce the process of IDD.

Methods and results: In this study, we used IL-1β, a commonly used inflammatory factor, to induce IDD and found that the IVD clock was dampened in degenerated human nucleus pulposus specimens, rat nucleus pulposus (NP) tissues, and cells. In this study, we found that the circadian clock of NP cells was totally disrupted by knockdown of the core clock gene brain and muscle arnt-like protein-1 (Bmal1), which thus induced the dysfunction of NP cells. Next, we explored the mechanism of dampened clock-induced IDD and found that knockdown of Bmal1 decreased the expression of nuclear factor erythroid2-related factor 2 (NRF2), a downstream target gene of Bmal1, and increased inflammatory response, oxidative stress reaction, and apoptosis of NP cells. In addition, NRF2 activation attenuated the dysfunction of NP cells induced by the dampened IVD clock and the degenerative process of NP tissues in an organotypic tissue-explant model.

Conclusions: Taken together, our study extends the relationship between peripheral clock and IVD homeostasis and provides a potential therapeutic method for the prevention and recovery of IDD by targeting the clock-controlled gene Nrf2.

Keywords: Bmal1; Circadian rhythms; Inflammation; Intervertebral disc degeneration; NRF2.

Fig. 1

The BMAL1 expression of NP specimens in human donors, rat NP tissues, and cells. a, b Immunohistochemical staining images and quantification of BMAL1 in human NP tissues from the grade II and grade V groups. We selected 3 random fields in per individual for quantitative analysis (scale bar: 250 μm). n = 3, **P < 0.01. c, d Immunofluorescence staining images and quantification of BMAL1 in human NP tissues from the Grade II and Grade V groups. We selected 3 random fields in per individual for quantitative analysis (scale bar: 125 μm). n = 4, **P < 0.01. e, f Immunofluorescence staining images and quantification of BMAL1 in rat NP tissues from Control and puncture groups cultured 0 days, 7 days, and 14 days (scale bar: 500 μm). n = 3, NS, P>0.01, **P < 0.01. g The mRNA levels of Bmal1 and Clock in control and IL-1β. n = 3, *P<0.05, **P < 0.01

Fig. 2

Knockdown of Bmal1 led to the degeneration of NP cells in vitro. a The mRNA levels of Bmal1 in the control and si-Bmal1 groups. b The mRNA levels of Acan and Mmp13 in the control and si-Bmal1 groups. n = 3, *P<0.05, **P < 0.01. c–f Immunofluorescence staining images and quantification of Aggrecan and MMP13 in the control and si-Bmal1 groups (scale bar: 25 μm). n = 3, **P < 0.01

Fig. 3

Si-Bmal1 decreased the expression of NRF2 and increased the ROS, inflammatory response, and apoptosis. a, b Immunofluorescence staining images and quantification of NRF2 in the control and si-Bmal1 groups (scale bar: 25 μm). n = 3, **P < 0.01. c, d DCFDA staining images and quantification in the control and si-Bmal1 groups. n = 3, **P < 0.01. e The mRNA levels of Il-1β, Tnf-α, and Il-6 in the control and si-Bmal1 groups. n = 3, *P<0.05, **P < 0.01. f, g TUNEL staining and quantification in the control and si-Bmal1 groups (scale bar: 25 μm). n = 3, **P < 0.01

Fig. 4

SFN attenuated si-Bmal1-induced the dysfunction of NP cells. The NP cells were divided into four groups, with no treatment, incubated with 5 μM SFN, si-Bmal1, and si-Bmal1 + 5 μM SFN. a Western blotting of BMAL1, NRF2, Aggrecan, and MMP13 protein expression (normalized to GAPDH expression). b Immunofluorescence staining of NRF2, Aggrecan, and MMP13 staining in Control, SFN, si-Bmal1, and si-Bmal1 + 5 μM SFN group (scale bar: 25 μm). c The levels of TNF-α and IL-1β protein. n = 3, NS, not significant difference, *P<0.05, **P < 0.01. d, e DCFDA staining images and quantification (scale bar: 50 μm). f, g TUNEL staining images and quantification (scale bar: 25 μm)

Fig. 5

SFN attenuated the degeneration of rat NP tissues in an organotypic tissue-explant model. The NP tissues were divided into four groups, control, needle-punctured, needle-punctured + 5 μM SFN, and needle-punctured + 10 μM SFN. a Immunofluorescence staining of BMAL1 in rat NP tissues cultured 7 days and 14 days (scale bar: 500 μm). b Immunofluorescence staining of NRF2 in rat NP tissues cultured for 7 days and 14 days (scale bar: 500 μm). c HE and safranin O staining of the rat IVDs cultured for 7 days and 14 days (scale bar: 500 μm)