Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen

Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen

Journal Pre-proof Intervertebral disc ageing and degeneration: the antiapoptotic effect of oestrogen Sidong Yang, Feng Zhang, Jiangtao Ma, Wenyuan Din...

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Journal Pre-proof Intervertebral disc ageing and degeneration: the antiapoptotic effect of oestrogen Sidong Yang, Feng Zhang, Jiangtao Ma, Wenyuan Ding

PII:

S1568-1637(19)30119-9

DOI:

https://doi.org/10.1016/j.arr.2019.100978

Reference:

ARR 100978

To appear in:

Ageing Research Reviews

Received Date:

20 April 2019

Revised Date:

20 October 2019

Accepted Date:

22 October 2019

Please cite this article as: Yang S, Zhang F, Ma J, Ding W, Intervertebral disc ageing and degeneration: the antiapoptotic effect of oestrogen, Ageing Research Reviews (2019), doi: https://doi.org/10.1016/j.arr.2019.100978

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Intervertebral disc ageing and degeneration: the antiapoptotic effect of oestrogen

Short Title: antiapoptotic effect of oestrogen in IVDD

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Department of Spine Surgery, The Third Hospital of Hebei Medical University, 139Ziqiang Rd, Shijiazhuang 050051, PR China.

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Sidong Yang1, Feng Zhang2, Jiangtao Ma3, Wenyuan Ding1*

Department of Rehabilitation Medicine, The Third Hospital of Hebei Medical

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University, 139Ziqiang Rd, Shijiazhuang 050051, PR China.

Laboratory of Immunology, Hebei Provincial Institute of Orthopaedic Research,

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139Ziqiang Rd, Shijiazhuang 050051, PR China.

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Correspondence should be addressed to: Prof. Wenyuan Ding, M.D., Department of Spinal Surgery, The Third Hospital of Hebei Medical University, 139Ziqiang Rd,

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Shijiazhuang 050051, PR China.

Telephone number: +86 31188602317 Fax number: +86 311 87023626

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E-mail address: [email protected]

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E-mail addresses:

Sidong Yang, [email protected]; Feng Zhang, [email protected]; Jiangtao Ma, [email protected]; Wenyuan Ding, [email protected]

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Graphical abstract

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Highlights Oestrogen deficiency causes intervertebral disc ageing and degeneration. Oestrogen can effectively alleviate intervertebral disc degeneration. Oestrogen inhibits aberrant apoptosis of disc cells. Oestrogen decreases disc cell apoptosis in multiple ways.

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Abstract

As an important part of the spinal column, the intervertebral disc (IVD) plays an

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important role in the intervertebral juncture and spinal movement in general. IVD degeneration (IVDD), which mimics disc ageing but at an accelerated rate, is a common and chronic process that results in severe spinal symptoms, such as lower

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back pain. It is generally assumed that lower back pain caused by IVDD can also develop secondary conditions, including spinal canal stenosis, spinal segmental

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instability, osteophyte formation, disc herniation and spinal cord and nerve root compression. Over the past few years, many researchers around the world have widely studied the relevance between oestrogen and IVDD, indicating that oestrogen can effectively alleviate IVDD development by inhibiting the apoptosis of IVD cells. Oestrogen can decrease IVD cell apoptosis in multiple ways, including the inhibition of the inflammatory cytokines IL-1β and TNF-α, reducing catabolism because of inhibition of matrix metalloproteinases, upregulating integrin α2β1 and IVD anabolism, activating the PI3K/Akt pathway, decreasing oxidative damage and 2 / 27

promoting autophagy. In this article, we perform an overview of the literature regarding the antiapoptotic effect of oestrogen in IVDD.

Abbreviations IVD, intervertebral disc; IVDD, intervertebral disc degeneration; AF, annulus fibrosus; NP, nucleus pulposus; ECM, extracellular matrix; MMP, matrix metalloproteinase; ER, oestrogen receptor; OVX, ovariectomy; BMD, bone mineral density; TNF-α, tumour necrosis factor-α; IL, interleukin; HRT, hormone replacement therapy; ERT, oestrorgen replacement therapy; PGE2, prostaglandin E2;

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GM-CSF, granulocyte macrophage colony stimulation factor; TGF-β, transforming

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growth factor-β; FGF, fibroblast growth factor; COX-2, cyclooxygenase-2

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Key words: Ageing; spine; intervertebral disc degeneration; oestrogen; apoptosis

1.1 Introduction

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Intervertebral disc (IVD) degeneration (IVDD), an important cause of discogenic lower back pain (LBP) (Millecamps and Stone, 2018; Podichetty, 2007; Wiet et al.,

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2017; Wáng et al., 2016), has traditionally been thought to be an age-related process of the disc tissue caused by decreased proteoglycan content, eventually leading to decreased intervertebral height, endplate sclerosis and osteophyte formation

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(Hadjipavlou et al., 2008; Raj, 2008; Sharma, 2018). The normal human IVD is a fibrocartilaginous structure that consists of three parts: (i) the outer annulus fibrosus

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(AF) composed of fibroblast-like cells and type I collagen; (ii) the inner soft nucleus pulposus (NP) composed of chondrocyte-like cells (or notochordal cells in a foetus) (Bach et al., 2015; Rodrigues-Pinto et al., 2016), proteoglycan and water; and (iii) cartilage endplates (Liu et al., 2018a; Pimenta et al., 2018). LBP is a common, worldwide disease that causes an enormous social economic burden and leads to a low quality of life. Here, IVDD is considered a major cause of LBP (Bressler et al., 1999; Hoy et al., 2012; Liu et al., 2018a; Prince et al., 2015). 3 / 27

Disc degeneration, although asymptomatic in many cases, is also associated with sciatica and disc herniation or prolapse (Boden et al., 1990; Luoma et al., 2000). IVD height, as well as the biomechanics of the rest of the spinal column, is altered in cases of IVDD, which may adversely affect the behaviour of other spinal structures, including the muscles and ligaments (Pimenta et al., 2018). In the long term, this may lead to spinal stenosis (Deng et al., 2012), a major cause of pain and disability for the elderly; its incidence is likely to rise exponentially with the current demographic changes and a rapid increase of the aged population (Luoma et al., 2000). The process

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of IVDD is complicated. Although the mechanism of IVDD is not fully understood, altered mechanical loading, degeneration of the extracellular matrix (ECM), increased

secretion of inflammatory factors, excessive senescence, and aberrant apoptosis of NP cells have proved to play important roles in its development (Gui and Yu, 2015;

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Kadow et al., 2015; Kepler et al., 2013; Le et al., 2007; Stokes and Iatridis, 2004). In NP tissue, the resident NP cells control ECM metabolism; they produce

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proteoglycan and type II collagen, which are the main molecules needed for maintaining the gelatinous property of NP tissue (Chung et al., 2003; Roberts et al.,

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2006; Walker and Anderson, 2004). NP cell death resulting from aberrant apoptosis may lead to metabolic disorders in the ECM, which has been found in IVDD (Roberts

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et al., 2006; Zhao et al., 2007). Furthermore, accumulative studies revealed that interventions targeting NP cell apoptosis could alleviate the metabolic disorders of the ECM and even slow the progression of IVDD (Liu et al., 2018b; Sudo and Minami,

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2010, 2011; Wang et al., 2016; Yang et al., 2014, 2016, 2015a). Therefore, more investigations on NP cell apoptosis and the target interventions may not only increase

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the pathogenetic knowledge of IVDD, but also provide potential therapeutic strategies.

In 1995, Imada et al. (Imada et al., 1995) reported that oophorectomy was a risk

factor for degenerative spondylolisthesis, having a high odds ratio of 7.5 (95% confidence interval, 1.6 to 46). The incidence of degenerative spondylolisthesis in oophorectomised patients was shown to be about three times higher than that in non-oophorectomised-matched control subjects, which strongly indicates that there 4 / 27

was a significant sex difference in terms of the prevalence of IVD-related diseases. This suggests the potential involvement of sex hormones, especially oestrogen, in the pathogenesis of IVDD. Ten years later, Ha et al., after harvesting the articular cartilage of the facet joints in 17 degenerative spondylolisthesis and in 15 spinal stenosis patients, found that a higher expression of oestrogen receptors (ERs) might aggravate the degenerative changes of the facet articular cartilage and might also be a causative factor for degenerative spondylolisthesis in postmenopausal women (Ha et al., 2005).

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For the past few years, attention has been increasingly placed on the effect that oestrogen has on the motor system, and research on the correlation between oestrogen and IVD-associated diseases has shown a dramatic trend. Data show that

middle-to-young-age males have a higher incidence of IVDD than females, but a

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distinct result comes from the elderly population: females are more inclined to

develop IVDD than males (de Schepper EI et al., 2010; Deng et al., 2012; Takatalo et

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al., 2009; Wang and Griffith, 2010; Wang et al., 2011; Wáng et al., 2016). Taken

age-related IVDD.

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together, oestrogen withdrawal, a characteristic of female ageing, is associated with

2.1 Oestrogen and ERs in IVD

In both males and females, oestrogen plays an important role in the

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reproductive system and many other organ systems. To date, there are four major naturally occurring oestrogens identified in women: oestrone (E1), oestradiol (E2),

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oestriol (E3), and estetrol (E4). To our knowledge, oestradiol works as the predominant oestrogen during reproductive years in terms of absolute serum levels as well as its oestrogenic effects. The first oestrogen – oestrone – was purified and

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crystallised in 1929, and oestradiol was discovered later in 1940 (Simpson and Santen, 2015). It is clear that the biosynthesis of oestrogen is through the conversion from androgen under an aromatisation reaction. The reaction sequences of the conversion have been found to be testosterone/19-hydroxyandrostenedione/ 19-oxoandrostenedione/oestrogen, and the intermediates 19-hydroxyandrostenedione and 19-oxoandrostenedione are regarded as the precursors of oestrogen (Morato et al., 1961; Simpson and Santen, 2015). In detail, three moles of oxygen and three 5 / 27

moles of NADPH are consumed when one mole of androgen is converted into oestrogen through the human placental aromatase (Thompson and Siiteri, 1974). The function of oestrogen is mediated by ER, which belongs to the independent receptor superfamily (Pelletier, 2000); it consists of two classic nuclear receptors – ER-α and ER-β – and also the membrane-bound G-protein-coupled receptor 30 (Wei et al., 2016). ER-α is the target site that has been classically stated; ER-β has lately been discovered in prostate cancer cells, ovarian blood vessel cells, astrocytes, osteoblasts, osteoclasts and articular cartilage cells, among others (Pelletier, 2000).

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As reported, ERs are expressed in all IVD structures, including AF (Gruber et al., 2002), NP (Song et al., 2017, 2014) and cartilage endplates (Kato et al., 2010),

but no data regarding the levels of oestrogen in local IVD tissues have been reported yet. Song et al. recruited 36 elderly patients with lumbar disc degeneration and

graded the degenerated IVDs based on MRI images using the Pfirrmann grading

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system (Song et al., 2014). By immunostaining gender-specific ER-α and ER-β in

NP cells, they found that both ER-α and ER-β existed in the nucleus and cytoplasm

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of NP cells, and the expression of both ERs significantly decreased with the aggravation of IVDD in aged patients, particularly ER-β. In addition, the expression

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of ER-α and ER-β in NP cells is significantly higher in males than in females. By exploring the effect of oestrogen on AF cell apoptosis induced by interleukin-1beta (IL-1β), Wang et al. detected the cytotoxicity and morphological changes of AF cells,

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concluding that 17β-oestradiol can inhibit the apoptosis of AF cells and hence improve cell survival and proliferation, which also indirectly confirmed the existence of ER in AF (Wang et al., 2014a). Hence, ERs are distributed in all IVD structures,

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including AF, NP and endplate cartilage. 3.1 The pathological changes of IVD because of oestrogen withdrawal

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The degeneration of cartilage endplates is associated with decreased oestrogen

levels. Being the most common manifestation of endplate degeneration, Schmorl’s node may lead to osteosclerosis, osteonecrosis and osteofibrosis, all of which prevent nutrients from penetrating into the endplate and ultimately lead to IVDD because of malnutrition (Moore, 2006). Before the age of 50, the number of Schmorl’s nodes is one time more in males than in females; however, for the elderly population, it is more common in females than in males (Moore, 2006). After comparing the MRI changes of IVD between premenopausal and postmenopausal 6 / 27

females, Wang et al. (Wang and Griffith, 2011) reported that IVDD in postmenopausal females was more severe than that in premenopausal females, confirming, from another aspect, that oestrogen withdrawal impaired endplate intensity after menopause. Oestrogen withdrawal affects the nutrient supply to IVD. For adults, IVD is the largest avascular tissue in the body, particularly in the central nucleus, a long way from a source of nutrition or metabolite clearance (Holm et al., 1981); there is no effective blood supply in IVD except for a few marginal capillaries in the outermost annulus and the endplates (Nachemson et al., 1970; Roberts et al., 2006). Nutrition

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supply for most IVD tissues, including the inner layer of AF and NP, depends on the diffusion of blood in spondyloepiphyseal terminal vessels through superior and

inferior cartilage endplates (Buckwalter, 1995; Deng et al., 2012; Holm et al., 1981; Urban et al., 1978, 1977, 1982). After menopause, females suffer from rapid

oestrogen withdrawal, degeneration of cartilage endplates and increased Schmorl’s

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nodes, which inevitably impedes blood perfusion of cartilage endplates (Wang and Griffith, 2010). With the assistance of dynamically enhanced MRI, Deng et al.

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observed the changes when applying contrast agents to the IVD of female rats, finding that both the maximum blood perfusion amount and rate became lower in

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ovariectomy (OVX) rats than in the normal control group, which indirectly proved that oestrogen deficiency in rats had a negative effect on blood circulation in IVD (Deng et al., 2012). The reason oestrogen can increase the blood supply in IVD may

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be attributed to its effects, which include vasodilation, prevention of intimal thickening and improvement of atherosclerosis. In addition, Griffith et al. performed a similar study analysing bone mineral density (BMD) and the perfusion parameters

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generated from OVX rats and compared these results with those from a control group (Griffith et al., 2010). Here, a synchronised decline in BMD and blood

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perfusion was found, indicating that oestrogen withdrawal was also relevant to blood perfusion in the vertebral body. The height of the IVD relies on the proper composition of various components

in the IVD. In a healthy IVD, the types of collagen, elastin, hydrophilic glycosaminoglycan, and water contained in NP and AF are evenly distributed (Raj, 2008). A previous study clearly demonstrated that a decrease in the IVD space was strongly related to oestrogen withdrawal because of menopause (Calleja-Agius et al., 2009). Baron et al. divided 100 women into three groups: a postmenopausal group 7 / 27

treated by oestrogen, a postmenopausal group with no treatment and a premenopausal group (Baron et al., 2005). Then, the authors measured the height between T12 and L3. It was found that the postmenopausal group without treatment had a minimum height and premenopausal group showed a maximum height, indicating that the oestrogen level could influence the height of the IVD due to the significant impact it had on the ECM, in particular the glycosaminoglycans and water content of the IVD. 4.1 Antiapoptotic effect of oestrogen on IVDD The behavioural alterations of degenerated IVD cells assume that a decreased

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cell count and degraded ECM are highly related to the initiation of IVDD. Excessive disc cell apoptosis leading to decreased cell density and catabolism of the ECM

(Park et al., 2001) both play pivotal roles in IVDD. The most significant biochemical change to occur in disc degeneration is the loss of proteoglycan (Lyons et al., 1981).

In IVDD, large numbers of IVD cells undergo programmed cell death. Moreover, the

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components of ECM that are generated by the surviving cells change, failing to

perform their normal functions (Ding et al., 2013; Zhang et al., 2011; Zhao et al.,

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2006). Notably, aberrant apoptosis and accelerated ageing of NP cells are considered to be the two major cellular processes associated with IVDD (Le et al., 2007; Li et

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al., 2017; Wang et al., 2014b), which implies that NP cell apoptosis could be an important target for the treatment of IVDD. Over the last few years, studies on the relevance between oestrogen and IVDD have indicated that oestrogen can effectively

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alleviate the progression of IVDD by inhibiting the aberrant apoptosis of IVD cells. Here, we performed an overview of the related mechanisms, as shown in Table 1. 4.1.1 Oestrogen decreases IVD cell apoptosis by inhibiting inflammatory factors.

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As shown in Figure 1, IVD cells have been identified as producing varieties of cytokines (Ahn et al., 2002; Podichetty, 2007; Weiler et al., 2005), including IL-1α

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(Doita et al., 2001), IL-1β (Miyamoto et al., 2000; Yang et al., 2015b), IL-6 (Kang et al., 1996, 1995; Rand et al., 1997; Takahashi et al., 1996), IL-8 (Ahn et al., 2002; Burke et al., 2002a, b), IL-10 (Ahn et al., 2002; Rand et al., 1997), tumour necrosis factor-α (TNF-α) (Doita et al., 2001; Larsson et al., 2005; Murata et al., 2006; Olmarker and Larsson, 1998; Séguin et al., 2005, 2006; Wang et al., 2017), granulocyte macrophage colony stimulation factor (GM-CSF) (Rand et al., 1997; Takahashi et al., 1996), transforming growth factor-β (TGF-β) (Matsunaga et al., 2003; Tolonen et al., 2006, 2001) and fibroblast growth factor (FGF) (Doita et al., 8 / 27

1996; Nagano et al., 1995; Tolonen et al., 2006, 1995). Cyclooxygenase-2 (COX-2) (Ding et al., 2018) and prostaglandin E2 (PGE2) (Kang et al., 1996, 1995; Weiler et al., 2005) can also be secreted by IVD cells. As a proinflammatory factor, IL-1β is highly expressed in degenerated IVD and facet joint (James et al., 2018; Wang et al., 2018); it is characterised by three aspects: promotes greater synthesis of matrix-degrading enzymes, such as MMP-2, MMP-3 and MMP-13; results in less production of proteoglycan and collagen; and induces the expression of IL-6, COX-2 and PGE2 (Yang et al., 2015b). We previously studied the effect of oestrogen on AF cell apoptosis induced by

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IL-1β and found that oestrogen can attenuate the apoptosis of AF cells induced by IL-1β, along with intensifying the survival and proliferation of AF cells under

inflammatory stimuli, which indicates that oestrogen was capable of downregulating

the activity of IL-1β (Wang et al., 2014a). Wei et al. found that oestrogen can protect NP cells against IL-1β-induced apoptosis and improve cell proliferation by bonding

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with both GPR30 and classic ERs (Wei et al., 2016).

Oestrogen can inhibit IVD cell apoptosis induced by TNF-α. By performing

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immunohistochemistry and real-time quantitative PCR, Chen et al. measured raloxifene, ADAMTS-4 and ADAMTS-5 in the cartilage endplates of patients who

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had undergone spinal surgery, finding that the expression of TNF-α and ADAMTS-5 increased in degenerated and herniated IVD cells (Chen et al., 2014). Hattori et al. reported that raloxifene, as an ER agonist, effectively inhibited human IVD

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chondrocyte apoptosis induced by TNF-α (Hattori et al., 2012). Based on the findings above, we deduce that oestrogen can protect IVD and delay the onset of IVDD induced by TNF-α. Indeed, we designed and performed such an in-vitro study

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and found that oestrogen can effectively inhibit TNF-α-induced apoptosis in human NP cells (Liu et al., 2017).

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4.1.2 Oestrogen decreases NP cell apoptosis by upregulating the PI3K/Akt pathway

In our previous study (Wang et al., 2016), NP cells were divided into four

groups: control, TNF-α, TNF-α with pretreated 17β-estradiol, TNF-α with pretreated 17β-estradiol and MK2206 (inhibitor of the Akt pathway). The data revealed that oestrogen effectively protected NP cells against TNF-α-induced apoptosis. Meanwhile, p-Akt expression generally increased in a time-dependent manner (0–48 hours) after treatment with TNF-α and 17β-oestradiol. Thus, oestrogen decreases NP 9 / 27

cell apoptosis by upregulating the PI3K/AKT pathway. Likewise, our other study (Yang et al., 2016) also suggested the effect of 17β-estradiol against apoptosis induced by IL-1β in rat NP cells via the PI3K/Akt/caspase-3 pathway. 4.1.3 Oestrogen decreases NP cell apoptosis by downregulating oxidative damage It has been well documented that oxidative damage can result in aberrant IVD cell apoptosis, and high glucose levels can cause oxidative damage (Ming-Yan et al., 2019; Ni et al., 2019). Yang et al. applied a high-glucose condition to induce apoptosis in rat NP cells, and then, they tried to inhibit the apoptosis induced by high glucose using 17β-estradiol (Yang et al., 2018). Consequently, they found that

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oestrogen reduced the content of reactive oxygen species, decreased NP cell apoptosis incidence and increased the gene expression of some ECM

macromolecules (aggrecan and type II collagen) when compared with the control NP

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cells. This is in line with the results we obtained in our previous study, which showed that 17β-oestradiol can effectively protect rat NP cells against

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peroxide-induced apoptosis in a dose-dependent manner, implying that 17β-oestradiol has the potential to prevent IVDD onset or slow its progression (Ning

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et al., 2016). Taken together, oestrogen can alleviate NP cell apoptosis and enhance ECM biosynthesis by reducing oxidative damage (Yang et al., 2018). 4.1.4 Oestrogen decreases NP cell apoptosis by promoting autophagy.

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Recently, it was revealed that oestrogen efficiently suppressed serum-deprivation-induced apoptosis and the expression of MMP-3 and MMP-13 by promoting autophagy in rat NP cells (Ao et al., 2018). In the study, autophagy and

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apoptosis were detected in NP cells under serum-deprivation conditions. It was shown that apoptosis and MMP-3/13 expressions deceased to a minimum value

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when 10-7 mole of oestrogen was administered to elevate autophagy incidence to a peak value at 48 h. By contrast, the pretreatment of 3-methyladenine (an inhibitor of autophagy) led to a dramatic decrease in autophagy, and the simultaneous use of oestrogen had no effect on the apoptosis with the expression of MMP-3 and MMP-13 increasing along with upward apoptosis. These data indicated that serum deprivation-induced apoptosis was efficiently suppressed by oestrogen by promoting autophagy in rat NP cells. 4.1.5 Oestrogen decreases IVD cell apoptosis by upregulating integrin expression. 10 / 27

Oestrogen can inhibit NP cell apoptosis by upregulating α2β1 integrin. As a membrane receptor, integrin regulates the interaction between cells and the ECM, adjusting the intracellular signal transduction pathways such as ERK/MAPK to control cell functions, which includes growth and differentiation, migration, adhesion and survival (Harburger and Calderwood, 2009). In the integrin family, the β1 subfamily is expressed widely in nature and mediates cell interactions through the components of the ECM (such as fibronectin, laminin and collagen). In addition, in the β1 family, α1β1, α2β1, α10β1 and α11β1 are important collagen receptors (Johnson et al., 2009; Leitinger and Hohenester, 2007). Recently, our research team performed

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an in-vitro study on the effect of oestrogen on rat NP cells and found that oestrogen can markedly decrease the apoptosis of NP cells induced by levofloxacin; here, the mechanism was that oestrogen upregulated the activity of integrin α2β1, improving

the adhesion of NP cells to type Ⅱcollagen (Yang et al., 2014). In addition, Zhao et al. reported that 17β-estradiol protected rat AF cells against apoptosis via α1

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integrin-mediated adhesion to type I collagen (Zhao et al., 2016). 4.1.6 Other possible pathways involved.

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Oestrogen can alleviate the process of IVDD by inhibiting the effect of MMPs and controlling the production of cellular enzymes. Previously, in a rat IVD puncture

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model, we found that 17β-oestradiol can effectively alleviate the progression of IVDD by downregulating MMP-3 and MMP-13 and upregulating type II collagen (Liu et al., 2018b). Pertaining to proteolytic enzymes, which are Zn2+-dependent,

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MMPs belong to one of the most important matrix-degrading enzymes (MacColl and Khalil, 2015). The activity of MMPs is an important event because they can trigger the catabolism of ECM followed by the apoptosis of IVD cells (Melrose et al., 2012).

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By detecting MMPs, nitric oxide and PGE2, Kang et al. carried out a comparative study of the IVD between patients with lumbar disc herniation and those with

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idiopathic scoliosis or traumatic burst fractures; the authors found that all the research data of MMPs were significantly higher in the herniation group than in the control group (Kang et al., 1996). Using a TUNEL assay and Annexin-V/PI double staining, we studied the effect of oestrogen on NP cell apoptosis induced by IL-1β and proved that oestrogen had the ability to protect NP cells against aberrant apoptosis by downregulating the expression of MMP-3 and MMP-13 (Yang et al., 2015a). 5.1 Oestrogen and treatment of IVDD 11 / 27

To date, researchers have not focused enough on the treatment of IVDD by utilising oestrogen. There are only a few studies on hormone replacement therapy (HRT) for IVDD. Recently, we conducted an animal experiment using oestrogen replacement therapy (ERT) to treat IVDD. As shown in Figure 2, ERT successfully retarded the progression of IVDD, which was induced by the combined effect of OVX and puncture. As shown in Figure 3, the expression level of cleaved caspase-3 decreased greatly because of ERT use. Indeed, comparing the postmenopausal IVD height with ERT and those without any treatment, Baron et al. found that the IVD height in the ERT group was higher than that in the control group, and the difference

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was significant (Baron et al., 2005); they further deduced that the remarkable differences might be caused by the profound effects that oestrogen has on the

hydrophilic polysaccharide, water content, collagen and elastin in IVD. Muscat et al. compared the IVD height between postmenopausal females with osteoporotic fractures and postmenopausal females treated with ERT, and the results were

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consistent with what Baron et al. had found (Muscat et al., 2007). In the treatment guidance for climacteric patients, Studd pointed out that ERT was beneficial for

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collagen and was able to improve the IVD and bone quality (Studd, 2010). It is well-known that males have a relatively lower chance of experiencing

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IVDD than females; thus, testosterone might exert a protective effect on the IVD tissue in the progression of ageing-related IVDD. Bertolo et al. reported that testosterone was found to effectively enhance in-vitro chondrogenesis in male IVD

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cells, particularly in NP cells, which are characterised by increased expressions of aggrecan, type I collagen and, especially, type II collagen, but they did not affect the chondrogenic differentiation of female IVD cells and mesenchymal stem cells from

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both genders (Bertolo et al., 2014). Interestingly, ECM expression of IVD cells decreased when anastrazole (an aromatase inhibitor) was administered to block the

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conversion of testosterone to oestradiol, suggesting that the protective effect of testosterone on IVD would be relevant to or depend on oestradiol. Recently, Wen et al. found that β-ecdysterone can ameliorate IVDD by protecting NP cells against aberrant apoptosis via the upregulation of autophagy (Wen et al., 2019), which could indicate another hormone-based therapeutic strategy for IVDD-related diseases. However, we should not neglect the possible risks for oestrogen-based treatment strategies. Previous data have indicated that the use of menopausal treatments for women with pre-existing cardiovascular disease was harmful (Miller 12 / 27

and Harman, 2017; Nair and Herrington, 2000). In another study, consecutive cases of idiopathic venous thromboembolism were evaluated in postmenopausal women who were between 45 and 70 years of age. The odds ratios for developing a venous thromboembolism in current users of oral and transdermal oestrogen compared with nonusers were 4.2 (95% CI: 1.5−11.6) and 0.9 (95% CI: 0.4−2.1), respectively (Canonico et al., 2007), indicating that the intake routes of ERT made a difference in these risks. In addition, it was previously reported that oestrogen prescribed for the treatment of menopausal symptoms might cause or exacerbate noncyclic breast pain/mastalgia (Crandall et al., 2012). However, the administration of lower-dose

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oestrogen over a 4-year period did not cause increased breast pain in healthy, recently menopausal women (Files et al., 2014). More importantly, it is well-known that oestrogen has been closely related to cancer risk, especially breast cancer

(Fuhrman et al., 2012). However, a double-blind, placebo-controlled, randomised

clinical trial indicated that among postmenopausal women with a prior hysterectomy

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who were followed up for 10.7 years, oestrogen-alone use for a median of 5.9 years

decreased the risk of breast cancer (LaCroix et al., 2011). That seems like new hope

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for future studies in terms of ERT, although there have been no studies reporting the link to breast cancer regarding ERT for IVD diseases by far. Collectively, it still

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remains elusive whether the potential benefits of postmenopausal ERT outweigh the risks.

IVDD is a complex pathological process and includes various degenerative

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events such as cell ageing, cell death, autophagy and the related signal transduction pathways. The potential biological treatment strategies for IVDD – injection of protein, gene transduction and cell transplantation – have been gradually studied in

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vivo. However, the application of these therapies is still in its infancy. Further studies on the molecular biological mechanisms will be of great help in exploring effective

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biological therapies that can delay or reverse the progression of IVDD (Anderson and Tannoury, 2005; Kepler et al., 2011; Weiler et al., 2005). 6.1 Conclusions As shown in Figure 4, oestrogen counters IVD cell apoptosis in multiple ways, including the inhibition of inflammatory cytokines IL-1β and TNF-α; reducing catabolism because of MMPs’ inhibition; upregulating integrin α2β1 and ECM anabolism; the activation of the PI3K/Akt pathway; decreasing oxidative damage; and promoting autophagy. Previous research is scarce on the clinical practice of 13 / 27

oestrogen replacement therapy targeting degenerative IVD diseases. Thus, a small dose of oestrogen replacement therapy targeting degenerative IVD diseases is an area that requires further research.

8.1 Declarations of interest None.

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7.1 Acknowledgements This work was supported by Natural Science Foundation of China (Grant No. 81601917, 81572166, 81871800), and Natural Science Foundation of Hebei

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Province (Grant No. H2016206073, H2018206313).

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Figure captions

Figure 1 Main inflammatory factors produced by intervertebral disc cells. IL, interleukin; TNF-α, tumor necrosis factor-α; GM-CSF, granulocyte macrophage colony stimulation factor; TGF-β, transforming growth factor-β; FGF, fibroblast growth factor ; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; MMP, matrix

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metalloproteinase.

Figure 2 The effect of estrogen on IVDD indicated by X-ray image. IVDD was

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caused by combined effect of OVX and acupuncture. Based on the rat model above, estrogen was utilized for the purpose of alleviating the IVDD development.

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Noticeably, estrogen effectively alleviated the progression of IVDD. IVDD, intervertebral disc degeneration; OVX, ovariectomy; ERT, estrogen replacement therapy; ICI, ICI182780 (estrogen receptor antagonist).

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Figure 3 The expression level of cleaved Caspase-3 determined by

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immunohistochemistry. IVDD was caused by combined effect of OVX and acupuncture. Based on the rat model above, estrogen was utilized for the purpose of

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alleviating the IVDD development. Noticeably, estrogen effectively inhibited the apoptotic incidence indicated by the decrease of cleaved Caspase-3. IVDD, intervertebral disc degeneration; OVX, ovariectomy; ERT, estrogen replacement

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therapy; ICI, ICI182780 (estrogen receptor antagonist). Scale bar, 20 μm

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Figure 4 The molecular mechanism underlying the anti-apoptosis effect of

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estrogen on IVD cells. E2, estrogen; PG, proteoglycan; COL, collagen; MMP,

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matrix metalloproteinase; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.

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Tables Table 1 Researches related to anti-apoptotic effect of estrogen on IVD cells

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Cell type Mechanism References AF; NP inhibiting IL-1β Wang et al, 2014; Wei et al, 2016 CEP; NP inhibiting TNF-α Hattori et al, 2012; Liu et al, 2017 NP; NP activating PI3K/Akt Wang et al, 2016; Yang et al, 2016 NP decreasing ROS Yang et al, 2018 NP promoting autophagy Ao et al, 2018 NP upregulating integrin α2β1 Yang et al, 2014 AF upregulating integrin α1 Zhao et al, 2016 NP inhibition of MMPs Yang et al, 2015 CEP upregulating ECM Sheng et al, 2018 IVD, intervertebral disc; AF, annulus fibrosus; NP, nucleus pulposus; CEP, cartilaginous endplate; ROS, reactive oxygen species; MMP, matrix metalloproteinase; ECM, extracellular matrix

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