We classified and summarised the final list of 22 genes, their targeted drugs, and the signaling pathways involved.
一Genes, gene-targeted drugs, and gene-mediated signaling pathwaysassociated with the mechanism of LDD
1. Genes, gene-targeted drugs, and gene-mediated signaling pathwaysassociated with catabolism in discs.
Matrix metalloproteinases (MMPs) are enzymes responsible for the degradation of almost all extracellular matrix proteins (ECM). The over-expression of MMPs or tissue inhibitors of metalloproteinases (TIMPs) may disrupt the dynamic balance of the ECM.
MMP-1: Deng et al. demonstrated that MMP-1 expression is increased in LDD, with higher expression observed in more severe cases, whereas TIMP-1 expression was similarly expressed in both normal and degenerated discs . In terms of candidate drugs targeting genes, doxycycline calcium, doxycycline hyclate, and doxycycline hydrate are specific inhibitors of MMP1 and MMP13.
MMP-2: Rastogi et al. suggested that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target . In terms of candidate drugs targeting genes, tiludronic acid and captopril are special inhibitors of MMP-2.
MMP-9: Li et al. showed that the levels of IL-1α and MMP-9 in degenerated lumbar disc tissues are higher than normal levels, and the increasing levels are positively correlated with the disease condition . In terms of candidate drugs targeting genes, glucosamine is a special antagonist of MMP-9, and captopril is a special inhibitor of it.
MMP-3 and MMP-13: Ao et al. showed that 17β-estradiol protects nucleus pulposus cells from serum deprivation-induced apoptosis and regulates expression of MMP-3 and MMP-13 through promotion of autophagy . Furthermore, Wang et al. showed that BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration .
TIMP1: This gene belongs to the TIMP gene family. The proteins encoded by this gene family are natural inhibitors of the matrix metalloproteinases (MMPs), a group of peptidases involved in degradation of the extracellular matrix. In addition to its inhibitory role against most of the known MMPs, the encoded protein is able to promote cell proliferation in a wide range of cell types, and may also have an anti-apoptotic function. Transcription of this gene is highly inducible in response to many cytokines and hormones. Kwon et al. showed that IL-8, VEGF, MMP-1, and MMP-3 were significantly increased in both cell types during hypoxia, while VCAM, TIMP-1, and TIMP-2 were decreased .
2. Genes, gene-targeted drugs, and gene-mediated signaling pathwaysassociated with anabolism in discs.
IGF1: PInsulin-like growth factor 1 (IGF-1) and its receptor (insulin-like growth factor 1 receptor, IGF1R) can regulate the extracellular matrix synthesis and play a crucial role in maintaining the normal functions of the intervertebral disc. Li et al. showed that reduced expression of IGF1R leads to accelerated intervertebral disc degeneration in mice . Furthermore, Liu et al. demonstrated that IGF-1 activates PI3k/Akt signaling to antagonize lumbar disc degeneration .
ACAN: This gene is a member of the aggrecan/versican proteoglycan family. The encoded protein is an integral part of the extracellular matrix in cartilagenous tissue and it withstands compression in cartilage. Chen et al. showed that metformin was shown to promote the expression of anabolic genes such as Col2a1 and Acan expression while inhibiting the expression of catabolic genes such as Mmp3 and Adamts5 in nucleus pulposus cells .
SPARC: SPARC is a glycoprotein that has an important role in modulating interactions between cells and matrix. It influences remodeling, collagen fibrillogenesis, metalloproteinase expression, and cytokine expression. Gruber et al. showed that decreased presence of SPARC in disc cells of older subjects with disc degeneration and point to the importance of future studies designed to elucidate the unrecognized role of SPARC in disc remodeling, aging, and degeneration .
In conclusion, the above studies have shown that Lumbar disc degeneration (LDD) is a multi-factorial process characterized by phenotypic and genotypic changes. Prolonged imbalance between anabolism and catabolism in discs alters their composition resulting in progressive loss of proteoglycans and hydration leading to LDD. However, activation of autophagy could regulate this imbalance to inhibit LDD.
二Genes, gene-targeted drugs, and gene-mediated signaling pathwaysassociated withthe inflammatory-associated factors inLDD
The inflammatory-associated factors interleukin-1β(IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α) are widely reported to be associated with lumbar disc degeneration (LDD)：
IL1B: The protein encoded by this gene is a member of the interleukin 1 cytokine family. This cytokine is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. The induction of cyclooxygenase-2 (PTGS2/COX2) by this cytokine in the central nervous system (CNS) is found to contribute to inflammatory pain hypersensitivity. Zhang et al. showed that melatonin modulates IL-1β-induced extracellular matrix remodeling in human nucleus pulposus cells and attenuates rat intervertebral disc degeneration and inflammation . In terms of candidate drugs targeting genes, canakinumab and gallium nitrateI are special inhibitors of L1B.
Tumor Necrosis Factor-α(TNF-α): This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. Wang et al showed that TNF is a key contributor to intervertebral disc degeneration . Furthermore, Zuo et al. found that the inflammatory cytokine tumor necrosis factor-α (TNF-α) increased the level of intracellular reactive oxygen species (ROS) and caused cell senescence and osteogenic differentiation of cartilage endplate stem cells (CESCs), whereas rapamycin-induced autophagy protected CESCs from TNF-α-induced oxidative stress and cell senescence . In terms of candidate drugs targeting genes, certolizumab pegol, etanercept, golimumab, inamrinone, infliximab, lenalidomide, pirfenidone, pomalidomide, and thalidomide are special inhibitor of TNF. Besides, pentoxifylline is a special antibody of this gene.
IL6: This gene encodes a cytokine that functions in inflammation and the maturation of B cells. In addition, the encoded protein has been shown to be an endogenous pyrogen capable of inducing fever in people with autoimmune diseases or infections. The protein is primarily produced at sites of acute and chronic inflammation, where it is secreted into the serum and induces a transcriptional inflammatory response through interleukin 6 receptor, alpha. Lin et al. showed that the expression of microRNA-21 is abnormally high in the nerve root pain of the lumbar intervertebral disc, which can increase the IL-6 inflammatory response and reduce the capacity of cell autophagy . In terms of candidate drugs targeting genes, siltuximab is a specoial inhibitor of IL6.
IL-1a: The protein encoded by this gene is a member of the interleukin 1 cytokine family. This cytokine is a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. This cytokine is produced by monocytes and macrophages as a proprotein, which is proteolytically processed and released in response to cell injury, and thus induces apoptosis. Chen et al. showed that Interleukin-1 α (IL-1α) was thought to be involved in the pathogenesis of disc degeneration by increasing the production of extracellular matrix degradation enzymes and by inhibiting extracellular matrix synthesis . Furthermore, Li et al. showed that the levels of IL-1α and MMP-9 in degenerated lumbar disc tissues are higher than normal levels, and the increasing levels are positively correlated with the disease condition . In terms of candidate drugs targeting genes, rilonacept is special binder of IL1A and IL1B.
The anti-inflammatory cytokines IL-4 and IL-10：Hou et al. found that shDNMT1 significantly reduced levels of the pro-inflammatory cytokines TNFα, IL-1β and IL-6, significantly increased levels of the anti-inflammatory cytokines IL-4 and IL-10, significantly increased M2 macrophage polarization, significantly reduced cell apoptosis in the disc degeneration zone and significantly reduced LDD-associated pain . Furthermore, Hanaei et al. showed that the IL-10 SNPs were significantly associated with LDD in Iranian population, which proposes that genomic alterations of anti-inflammatory cytokines could lead to homeostasis imbalance in intervertebral discs and degenerative changes .
STAT3：The protein encoded by STAT3 is a member of the STAT protein family. In response to cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a crucial role in many cellular processes such as cell growth and apoptosis. In terms of candidate drugs targeting genes, acitretin is a specific inhibitor of this gene.
In conclusion, the inflammatory-associated factors could accelerate disc degeneration by inducing MMPs to degrade the extracellular matrix, but activation of autophagy could reduce inflammation.
三Four significant signaling pathways are associated with autophagy in LDD.
1. Three significant signaling pathways, which reduce apoptosis and neuroinflammation by enhancing autophagy, are associated with autophagy in LDD
AMPK signaling pathway:
Jiang et al. showed that activation of autophagy via ca(2+)-dependent AMPK/mTOR pathway in rat notochordal cells is a cellular adaptation under hyperosmotic stress . In addition, Wang et al. showed that resveratrol attenuated TNF-α-induced MMP-3 expression in human nucleus pulposus cells by activating autophagy via AMPK/SIRT1 signaling pathway . What’s more, Zhang et al. showed that naringin could reduce the incidence of oxidative stress-induced apoptosis in nucleus pulposus cells and promoted the expression of autophagy markers LC3-II/I and beclin-1. Further study showed that autophagy regulation of naringin may be related to AMPK signaling .
mTOR signaling pathway:
Jiang et al. showed that glucosamine can activate autophagy via the mTOR-dependent pathway to protect nucleus pulposus (NP) cells treated with IL-1βor hydrogen peroxide (100um H2O2) . Furthermore, Yurube et al. showed that resident disc cells may utilize autophagy and mTOR signaling to cope with harsh low-nutrient conditions, such as low glucose, low oxygen, and low pH .
PI3K-Akt signaling pathway:
Li et al. showed that compression stress Induces nucleus pulposus cell autophagy by inhibition of the PI3K/AKT/mTOR pathway and activation of the JNK pathway . Besides, Gao et al. showed that resveratrol enhances matrix biosynthesis of nucleus pulposus cells through activating autophagy via the PI3K/Akt pathway under oxidative damage（100um H2O2）. Furthermore, Guo et al. showed that Moracin M inhibits lipopolysaccharide-induced inflammatory responses in nucleus pulposus cells via regulating PI3K/Akt/mTOR phosphorylation .
In conclusion, nutrient deprivation, hyperosmotic stress, compression stress, inflammatory-associated factors, and oxidative stress can activate autophagy through different pathways, which can play a positive role after activation. These positive effects include reducing apoptosis, reducing catabolism by inhibition of matrix metalloproteinases, enhancing the matrix biosynthesis of nucleus pulposus cells, and inhibiting inflammatory responses in nucleus pulposus cells. Three significant genes, AKT1, PIK3CA, and TNFSF10, are involved in these signaling pathways.
AKT1: In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. In terms of candidate drugs targeting genes, arsenic trioxide is a special inducer of AKT1, but everolimus and nelfinavir are its inhibitor.
PIK3CA: Phosphatidylinositol 3-kinase is composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. The protein encoded by this gene represents the catalytic subunit, which uses ATP to phosphorylate PtdIns, PtdIns4P and PtdIns(4,5)P2. In terms of candidate drugs targeting genes, candicidin, idelalisib, oxazepam, phenmetrazine, yohimbine are special inhibitors of PIK3CA.
TNFSF10: The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. The binding of this protein to its receptors has been shown to trigger the activation of MAPK8/JNK, caspase 8, and caspase 3.
2. One significant and special signaling pathway is associated with autophagy in LDD.
ERK signaling pathway:
Three significant genes, FASLG, TP53，and BDNF, are involved in this pathway.
FASLG: This gene is a member of the tumor necrosis factor superfamily. The primary function of the encoded transmembrane protein is the induction of apoptosis triggered by binding to FAS.
TP53: This gene encodes a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The encoded protein responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. In terms of candidate drugs targeting genes, bortezomib is a special inhibitor of TP53.
BDNF: This gene encodes a member of the nerve growth factor family of proteins. Alternative splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is proteolytically processed to generate the mature protein. Binding of this protein to its cognate receptor promotes neuronal survival in the adult brain.
Many types of research have demonstrated that the ERK signaling pathway is closely associated with LDD. Li et al. showed that autophagy attenuates compression-Induced apoptosis of human nucleus pulposus cells via MEK/ERK/NRF1/Atg7 signaling pathways during intervertebral disc degeneration . However, Chen et al. showed H2O2 stimulated an early autophagy response through the ERK/m-TOR signaling pathway. Autophagy inhibition significantly decreased the apoptosis incidence in the cells insulted by H2O2 (400UM) . Besides, Ni et al. showed that TGF-β1 reduces the oxidative stress-induced autophagy and apoptosis in rat annulus fibrosus cells through the ERK signaling pathway .
In conclusion, oxidative stress (H2O2) could activate autophagy through different signaling pathways. In Chen’s research, 400-µM H2O2 was used and caused autophagy that promoted apoptosis. By contrast, Jiang and Gao used 100-µM H2O2, which caused autophagy that inhibited apoptosis. From the previous studies, we can conclude that different levels of oxidative stress could lead to autophagy that play different roles.
Therefore, we can speculate the relationships between autophagy, apoptosis, and necrosis. Autophagy is bidirectional in that it can both inhibit and induce apoptosis. However, in our opinion, autophagy-induced apoptosis is not necessarily unfavorable. For severely damaged cells that cannot be saved, autophagy can accelerate their programmed apoptosis to avoid greater damage response caused by cell necrosis while reserving more energy to save the less damaged cells. In general, autophagy is a positive physiological process.