## 2 Biomarkers and Signalling Pathways Associated with the Degenerated Intervertebral Disc  
The literature associates IVD degeneration with dysregulated quantities of inflammatory cytokines produced by infiltrating macrophages, neutrophils, T cells, as well as disc cells [38]. During disc degeneration, the infiltration of mast cells and macrophages is enhanced by the vascularisation of the AF, which leads to amplified propagation of inflammatory signals, consequently, leading to LBP [39]. The inflammatory molecules secreted in the IVD, specifically prostaglandin E2 (PGE2), interferon-gamma (IFN-\(\gamma\)), tumour necrosis factor-alpha (TNF-\(\alpha\)), and interleukin one alpha/beta (IL-1\(\alpha\)/\(\beta\)), interleukin (IL)-17, IL-10, IL-6, IL-8, IL-2, IL-4, [38], stimulate the breakdown of the extracellular matrix (ECM), cell senescence, autophagy and apoptosis of the disc cells [38; 40; 41]. Pain sensation was also correlated with up-regulated nitric oxide and nitric oxide synthase [39]. Furthermore, the induced pro-inflammatory cytokines were found to stimulate the expression of neurogenic molecules, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which may support the ingrowth of nerves as well as enhance nociception in dorsal root ganglia (DRG)s [38; 41]. Hence, chronic inflammation prompts permanent structural and biochemical changes in the IVD comprising ECM degradation, vascularisation and innervation that ultimately leads to LBP [42]. Increased chemokine expression, especially C-C motif ligand 2 (CCL2), has been reported in the degenerated IVD [43]. Cultured AF cells from Thompson-graded discs of grade II-III were able to stimulate higher expression of CCL2 upon treatment with IL-1\(\beta\) than TNF\(\alpha\) unlike AF cells from grades IV-V which showed a similar response to IL-1\(\beta\) and TNF\(\alpha\) treatment in terms of CCL2 expression [44].  
Different key dysregulated genes and proteins were discovered in the degenerated human AF such as interferon-stimulated genes (ISGs), which are interferon-induced proteins with tetratricopeptide repeats (IFIT)1, IFITC2, IFITC3 as well as Insulin-like growth factor-binding protein 3 (IGFBP3) [12]. Overexpression of the anti-proliferative IFIT3 protein and the pro-apoptotic IGFBP3 can negatively regulate the cell fate directly or indirectly, to induce growth arrest and apoptosis in AF cells [45; 46; 47; 48]. IFIT3 can act as an anti-proliferative molecule through inducing the cyclin-dependent kinase inhibitors, such as cyclin-dependent kinase inhibitor 1A and cyclin-dependent kinase inhibitor 1B which interact with cyclin dependent kinases (CDK)s in the G1 phase and prevent the cell from proceeding into the S phase of the cell cycle [45]. IFN\(\alpha\) was also able to induce IGFBP3 through the signal transducer, and activator of transcription 1 (STAT1) via cascades that have Insulin-like growth factor (IGF)-related anti-proliferative properties or IGFBP3 directly or indirectly induced pro-apoptotic consequences [46; 47; 48]. IGFBP3 is a vital controller of IGF availability that inhibits IGF activity, which is associated with osteoarthritis [49]. Moreover, IGFBP3 can sensitize cells to apoptosis indirectly through TNF\(\alpha\) and IFN\(\gamma\)[50; 51]. Several signalling pathways have also been identified in the disc as being aberrantly regulated, which are illustrated in Table 1.