Advanced oxidation protein products induce annulus fibrosus cell senescence through a NOX4-dependent, MAPK-mediated pathway and accelerate intervertebral disc degeneration

Abstract

Background: Intervertebral disc degeneration (IVDD) is closely associated with senescence. Annulus fibrosus (AF) cell senescence is a crucial driver of AF tissue tearing and fissures, thereby exacerbating IVDD. Increased advanced oxidative protein products (AOPPs) were found in human degenerative discs and aged rat discs and may be involved in IVDD. This study aimed to explore the mechanism of AOPPs-induced senescence in AF cells.

Methods: The pathological effects of AOPPs in vivo were investigated using a rat lumbar disc persistent degeneration model and a rat caudal disc puncture model. Rat primary AF cells were selected as in vitro models, and AOPPs were used as direct stimulation to observe their pathological effects. Setanaxb (NOX1/4 inhibitor), apocynin (NADPH oxidase inhibitor) and adenovirus (ADV) packed NADPH oxidase 4 (NOX4) specific shRNAs were used for pathway inhibition, respectively. Finally, adeno-associated viruses (AAVs) packed with NOX4-specific blocking sequences were used to inhibit the in vivo pathway.

Results: AOPPs accumulated in the rat lumbar and caudal degenerative discs. Intra-discal loading of AOPPs up-regulated the expression of NOX4, p53, p21, p16, IL-1β, and TNF-α, ultimately accelerating IVDD. Exposure of AOPPs to AF primary cells up-regulated NOX4 expression, induced phosphorylation of mitogen-activated protein kinases (MAPK), triggered senescence and increased IL-1β and TNF-α. Apocynin, setanaxib, and ADV pre-cultured AF cells abrogated AOPPs-induced senescence. AAV-mediated inhibition of NOX4 expression in vivo reduced the expression of p53, p21, p16, IL-1β and TNF-α in vivo and delayed IVDD.

Conclusions: AOPPs induced AF cell senescence through a NOX4-dependent and MAPK-mediated pathway.

Keywords: Advanced oxidized protein products; Inflammation associated with senescence; Intervertebral disc degeneration; Senescence.

Figure 1. AOPPs accumulate in IVDD, up-regulate the expression of NOX4, senescence markers, and their associated inflammatory proteins, and accelerate IVDD.

(A) MRI of a persistent lumbar degeneration model showed significant disc degeneration. (B) Immunofluorescence of AOPPs showed massive accumulation of AOPPs in AF tissues at two months of degenerative discs. Scale bar: 25 µm. (C) The DMMB assay of the rat lumbar discs showed a gradual decrease in GAG content after surgery, suggesting IVDD. (D) Western blot analysis of lumbar discs showed that albumin and AOPPs accumulated in IVDD, up-regulating NOX4, senescence markers p53, p21, p16, and senescence-associated inflammatory proteins IL-1β and TNF-α expression. (E) Western blot analysis of the caudal discs showed that albumin and AOPPs accumulated in IVDD up-regulated the expression of NOX4, senescence markers, and senescence-associated inflammatory proteins. (F) X-rays of Co6/7 and Co8/9 showed lower DHI in the puncture + intra-discal AOPPs group. (G, H, I) Immunohistochemical staining of AF tissue for p16 and NOX4 was positive in the punctured discs and much higher after stimulation with exogenous AOPPs. Scale bars: 100 µm and 200 µm. (J) DMMB measurements of rat lumbar discs showed a decrease in GAG content in all puncture groups, indicating IVDD in all puncture groups, with the lowest content and most severe degeneration in the intra-discal injection AOPPs group. Sham, sham surgery. IP, intra-peritoneal injection. N = 6 per group. *P < 0.05 compared to Sham and IP for AOPPs. ** P < 0.01 compared to Sham and IP for AOPPs. #P < 0.05 compared to the other five groups. All experiments were repeated at least three times. Error bars represent standard errors.

Figure 2. AOPPs up-regulate NOX4 expression, induce senescence in AF cells, and increase the secretion of senescence-associated inflammatory proteins.

(A) FACS analysis of AF cells treated with AOPPs (50–400 µg/mL, 24 h) induced G1 phase arrest. (B) SA-β-gal staining of AF cells treated with AOPPs (50–400 µg/mL, 24 h). AOPPs increased the proportion of senescent AF cells. Scale bar: 100 µm. (C) AOPPs significantly increased the expression of NOX4, p53, p21, p16, IL-1β and TNF-α in AF cells. *P < 0.05 compared to the control. **P < 0.01 compared to the control. Error bars represent standard errors.

Figure 3. AOPPs up-regulate NOX4 expression, induce senescence in AF cells, and increase the secretion of senescence-associated inflammatory proteins.

(A) FACS analysis of AF cells treated with AOPPs (200 µg/mL, 3–48 h) to induce G1 phase arrest. (B) SA-β-gal staining of AF cells treated with AOPPs (200 µg/mL, 3–48 h). AOPPs increased the proportion of senescent AF cells. Scale bar: 100 µm. (C) AOPPs significantly increased the expression of NOX4, p53, p21, p16, IL-1β and TNF-α in AF cells. *P < 0.05 compared to the control. **P < 0.01 compared to the control. Error bars represent standard errors.

Figure 4. The AOPPs-induced senescence of AF cells was mediated by NADPH oxidase.

(A) FACS analysis of AF cells pre-incubated with GKT (20 µM) and APO (100 µM) prevented AOPPs-induced G1 phase arrest. (B) SA-β-gal staining of AF cells pre-incubated with GKT (20 µM) and APO (100 µM) showed restoration of AOPPs-induced senescence. Scale bar: 100 µm. (C) Expression of NOX4, p53, p21, p16, IL-1β and TNF- α was down-regulated in response to inhibitor treatment. *P < 0.05 compared to the control. **P < 0.01 compared to the control. #P < 0.05 compared to the AOPPs group. Error bars represent standard errors.

Figure 5. AOPPs induce AF cell senescence by activating phosphorylation of MAPK.

(A–C) AOPPs (200 µg/mL, 24 h) significantly increased phosphorylation of p38, ERK1/2 and JNK in AF cells. (D–F) p38 MAPK inhibitor (SB202190, 10 µM), ERK1/2 inhibitor (FR180204, 10 µM) and JNK inhibitor (SP600125, 10 µM) AOPPs-induced (200 µg/mL, 24 h) senescence in AF cells. (G–I) Activation of AOPPs-induced (200 µg/mL, 24 h) MAPK phosphorylation was ameliorated by GKT (20 µM) and APO (100 µM). *P < 0.05 compared to the control. ** P < 0.01 compared to the control. #P < 0.05 compared to the AOPPs group. Error bars represent standard errors.

Figure 6. AOPPs induce AF cell senescence via the NOX4-MAPK pathway.

(A) NOX4-specific shRNAs significantly inhibited AOPPs-induced (200 µg/mL, 24 h) G1 phase arrest. (B) NOX4-specific shRNAs significantly reduced AOPPs-induced (200 µg/mL, 24 h) SA-β-gal staining of positive cells. Scale bar: 100 µm. (C) NOX4 shRNA-1 only blocked the expression of p53, p21, and the hyperphosphorylation levels of p38, ERK, and JNK, but not the expression of p16, IL-1β, and TNF-α. (D) AOPPs-induced (200 µg/mL, 24 h) up-regulation of NOX4, p53, p21, p16, IL-1β, and TNF-α, and hyperphosphorylation levels of p38, ERK, and JNK were reduced after transfection with NOX4-specific blockade of shRNA-2 sequences. *P < 0.05 compared to NC. **P < 0.01 compared to NC. #P < 0.05 compared to NC+AOPPs. Error bars represent standard errors.

Figure 7. Blocking NOX4 expression in vivo by AAV delays IVDD.

(A) MRI showed that blocking NOX4 expression by AAV reduced the Pfirrmann grade of disc degeneration caused by RSA and AOPPs. *P < 0.05. **P < 0.01. Error bars represent the interquartile range. (B, C) HE staining and saffron O/fast green staining showed less disc destruction caused by AOPPs and RSA in discs with blocked NOX4 expression and correspondingly decreased disc injury scores. **P < 0.01 and #P < 0.05 compared with the other four groups. (D) Western blot analysis showed that blocking NOX4 expression reduced the expression of p53, p21, p16, IL-1β and TNF-α in disc tissue injected with AAV. (E, F) Immunofluorescence assay showed that blocking NOX4 expression reduced the expression of p53, p21, p16, p-p38, p-ERK, p-JNK, IL-1β and TNF-α. (G) Diagram of AOPPs-induced NOX4, p53, p21, p16, IL-1β and TNF-α expression. #P < 0.05 compared to the AAV-NC+RSA and AAV-NC+AOPP groups. *P < 0.05. **P < 0.01. Error bars represent standard errors.