Acidic pH promotes intervertebral disc degeneration: Acid-sensing ion channel -3 as a potential therapeutic target

Abstract

The aetiology of intervertebral disc (IVD) degeneration remains poorly understood. Painful IVD degeneration is associated with an acidic intradiscal pH but the response of NP cells to this aberrant microenvironmental factor remains to be fully characterised. The aim here was to address the hypothesis that acidic pH, similar to that found in degenerate IVDs, leads to the altered cell/functional phenotype observed during IVD degeneration, and to investigate the involvement of acid-sensing ion channel (ASIC) -3 in the response. Human NP cells were treated with a range of pH, from that of a non-degenerate (pH 7.4 and 7.1) through to mildly degenerate (pH 6.8) and severely degenerate IVD (pH 6.5 and 6.2). Increasing acidity of pH caused a decrease in cell proliferation and viability, a shift towards matrix catabolism and increased expression of proinflammatory cytokines and pain-related factors. Acidic pH resulted in an increase in ASIC-3 expression. Importantly, inhibition of ASIC-3 prevented the acidic pH induced proinflammatory and pain-related phenotype in NP cells. Acidic pH causes a catabolic and degenerate phenotype in NP cells which is inhibited by blocking ASIC-3 activity, suggesting that this may be a useful therapeutic target for treatment of IVD degeneration.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Acidic pH reduces NP cell proliferation and increases cell death.

NP cells (n = 3) were cultured in DMEM medium with a pH of 7.4, 7.1, 6.8, 6.5 and 6.2, for 0 days (baseline control) or 7 days and (a) Stained with LIVE/DEAD viability stain. Viable and non-viable cells stained green and red, respectively. (b) To assess proliferation and/or cell death, a Pico Green assay was used to quantify the total amount of DNA from cells cultured at different pH and normalised to baseline control (0 day of culture at pH 7.4). *Indicates p ≤ 0.05. Scale bar represents 100 μm.

Figure 2. Acidic pH induces increases in proinflammatory and pain-related markers in NP cells.

(a) Gene expression of IL -1β, IL -6, NGF and BDNF relative to MRPL19 by NP cells (n = 3) cultured at pH 7.1, 6.8 and 6.5 for 7 days and normalised to gene expression at pH 7.4. (b) Protein expression (determined by ELISA) of IL -1β, IL -6, NGF and BDNF by NP cells (n = 3) cultured for 7 days at pH 7.1 and 6.5, normalised to expression levels at pH 7.4. *Indicate p ≤ 0.05.

Figure 3. Low pH leads to a decrease in the anabolic protein aggrecan.

(a) Gene expression of AGC relative to MRPL19 following culture of NP cells (n = 3) at pH 7.4, 7.1, 6.8 and 6.5 for 7 days and normalised to gene expression at pH 7.4. (b) Western blot for aggrecan protein expression following culture of NP cells (n = 3) at pH 7.4, 7.1 and 6.5 for 7 days and normalised to GAPDH. *Indicate p ≤ 0.05. (c) Immunofluorescence for AGC protein (green) and cell nuclei (DAPI; blue) of NP cells cultured at pH 7.4, 7.1 and 6.5.

Figure 4. Acidic pH induces a more catabolic phenotype in NP cells.

Gene expression of (a) matrix proteins, including VCAN, COL1 and COL2A1, (b) matrix degrading enzymes, including MMP-3, ADAMTS-4 and -5, and TIMPs-1, -2 and -3, by NP cells (n = 3) cultured at pH 7.1, 6.8 and 6.5 for 7 days and normalised to MRPL19 and gene expression at pH 7.4. *Indicate p ≤ 0.05.

Figure 5. Low pH regulates acid-sensing ion channels (ASICs) in NP cells.

(a) Gene expression of ASICs-1, -2 and -3 by NP cells (n = 3) cultured at pH 7.1, 6.8 and 6.5 for 7 days and normalised to MRPL19 and gene expression at pH 7.4. (b) Immunofluorescence for ASIC -3 (green) and cell nuclei (DAPI; blue) in NP cells cultures at pH 7.4, 7.1 and 6.5 for 7 days. (c) Western blot for ASIC-3 protein expression in NP cells (n = 3) cultured at pH 7.1 and 6.5 for 7 days relative to GAPDH and protein expression to pH 7.4. *Indicate p ≤ 0.05.

Figure 6. Inhibition of ASIC-3 prevents the pro-inflammatory/pain-related neurogenic response in NP cells exposed to an acidic environment.

Expression of proteins assessed using (a) ELISA for pro-inflammatory cytokines IL -1β and IL -6, as well as the pain-related neurogenic factors NGF and BDNF, and assessed using (b) western blot for aggrecan protein expression by NP cells (n ≥ 3) treated with carrier alone (PBS) or APETx2 (inhibitor of ASIC-3) at 100 nM cultured at pH 7.4 and 6.5 for 7 days and normalised to protein expression levels at pH 7.4. *Indicate p ≤ 0.05.