Rapamycin prevents the intervertebral disc degeneration via inhibiting differentiation and senescence of annulus fibrosus cells

The effects of bleomycin and rapamycin on cellular senescence and differentiation of rabbit annulus fibrosus stem cells (AFSCs) were investigated using a cell culture model. The results showed that bleomycin induced cellular senescence in AFSCs as evidenced by senescence-associated secretory phenotype. The morphology of AFSCs was changed from cobblestone-like cells to pancake-like cells. The senescence-associated β-galactosidase activity, the protein expression of P16 and P21, and inflammatory-related marker gene levels IL-1β, IL-6, and TNF-α were increased in bleomycin-treated AFSCs in a dose-dependent manner. Rapamycin treatment decreased the gene expression of MMP-3, MMP-13, IL-1β, IL-6, TNF-α, and protein levels of P16 and P21 in bleomycin-treated AFSCs. Furthermore, neither bleomycin nor rapamycin changed the ribosomal S6 protein level in AFSCs. However, the phosphorylation of the ribosomal S6 protein was increased in bleomycin-treated AFSCs and decreased in rapamycin-treated AFSCs. AFSCs differentiated into adipocytes, osteocytes, and chondrocytes when they were cultured with respective differentiation media. Rapamycin inhibited multi-differentiation potential of AFSCs in a concentration-dependent manner. Our findings demonstrated that mammalian target of rapamycin (mTOR) signaling affects cellular senescence, catabolic and inflammatory responses, and multi-differentiation potential, suggesting that potential treatment value of rapamycin for disc degenerative diseases, especially lower back pain.


INTRODUCTION
Lower back pain is a prevalent disc disorder that affects millions of Americans and costs billions of healthcare dollars every year. Current clinical treatments for lower back pain are largely palliative because the precise cellular and molecular mechanisms of the diseases are not clear. There is a variety of reasons for back pain with disk degeneration only being one possible cause. Advanced age is thought to be a primary risk factor for intervertebral disc disorders (IVDD) [1,2]. Organismal aging results from time-dependent accumulation of molecular and cellular damage that leads to impaired tissue homeostasis and eventual physiological and functional decline [3]. In humans, aging is associated with increased incidence of disc pathology, including abnormal collagen and proteoglycan expression, degeneration and calcification [4,5]. It has been reported that decreased extracellular matrix production, increased production of degrading enzymes, and increased expression of inflammatory cytokines contribute to the loss of structural integrity and accelerate IVDD [5].
Because matrix changes largely reflect alterations in the biology of the cells, during aging and degeneration, the abilities to replace damaged or atrophic tissue decline as a result of stem cell loss and consequent progressive loss of regenerative function [6]. It was suggested that stem cell population might be involved in tissue homeostasis and repair, by replacing lost mature cells, or in the pathogenesis of degenerative diseases [7]. It has been reported that stem cells exist in different parts of intervertebral disc (IVD), such as nucleus pulposus (NP) [8], annulus fibrosus (AF) [9], and cartilage endplate [10]. Recent studies indicated that there are significant differences in morphology of stem cells between young and aging groups [11][12][13]. More studies showed that the proliferation and multi-differentiation potentials of stem cells decline with age [14][15][16]. Stem cells are progressively lost over time through a variety of mechanisms including apoptosis, replicative or cellular senescence and trans-differentiation [17].
It has been reported that aging is associated with decreased maximal life span and accelerated senescence of stem cells [18]. Cellular senescence is a state where cells can no longer divide, despite the abundance of appropriate growth factors. The stem cells isolated from old human bone marrow exhibited accelerated senescence than young stem cells [18]. Similarly, the involvement of cellular senescence has been linked to osteoarthritis and disc degeneration. However, the cellular and molecular pathway on disc cell senescence and degeneration is largely unknown.
The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that participates in the regulation of cell growth and proliferation [19]. MTOR pathway is also involved in cellular and organismal aging. As a specific inhibitor of mTOR, rapamycin increases lifespan and inhibits spontaneous tumorigenesis in inbred female mice [20]. Recent studies showed that rapamycin retards multiple aspects of aging in mice, including alterations in heart, liver, and tendon, and rapamycin also attenuates age-associated changes in tibialis anterior tendon viscoelastic properties [21]. However, whether mTOR pathway links cellular senescence and aging disc degenerative changes is largely unknown. Bleomycin is a cytotoxic antibiotic that inhibits DNA metabolism and causes DNA damage. In this study, we used a novel aging study model to investigate mTOR pathway in cellular senescence and degeneration of annulus fibrosus stem cells using bleomycin and rapamycin.

The effect of bleomycin and rapamycin on morphology and proliferation of AFSCs
In order to study the cellular and molecular pathway on aging disc degeneration, the AFSCs isolated from rabbit AF tissues were treated with bleomycin and rapamycin. The morphology analysis has shown that the AFSCs have changed their morphology from cobblestone-like shape (Fig. 1A) to pancake-like cells with bleomycin treatment (Fig. 1B, 1C, 1G, 1H). The proliferation of AFSCs was decreased by bleomycin (Fig. 1L) in a concentration-dependent manner. Although the proliferation of AFSCs was not enhanced by adding rapamycin into bleomycin-containing medium (Fig.  1L), the pancake-like cell numbers were decreased and the spindle-like cell numbers were increased (Fig. 1D, 1E, 1I, 1J) in rapamycin-treated AFSCs.
The effect of bleomycin and rapamycin on senescence-associated β-galactosidase activity of AFSCs

AGING
The senescence-associated β-galactosidase (SA-β-gal) staining indicated that 10 μg/ml of bleomycin induced more than 40% of AFSCs to senescent cells (Fig. 2B, 2G) and 50 μg/ml of bleomycin induced more than 68% of AFSCs to senescent cells (Fig. 2C, 2H). These results were further demonstrated by immunostaining (Fig. 3) as evidenced by pancake-like cells found in AFSCs cultured with bleomycin-containing medium and positively stained with red fluorescence (white arrows in Fig. 3G, 3H). The positive stained cell numbers were decreased with adding rapamycin in bleomycincontaining medium ( Fig. 2 and Fig. 3).

The effect of bleomycin and rapamycin on gene expression of AFSCs
The expression of catabolic and inflammatory genes in AFSCs was also studied.

The effect of bleomycin and rapamycin on protein expression of AFSCs
Western blot results indicated that the protein levels of P16 and P21 were enhanced by bleomycin treatment and decreased by rapamycin (Fig. 6). Furthermore, the phosphorylation of S6 protein (PS6) was increased with bleomycin treatment and decreased with rapamycin treatment (Fig. 7A, 7B). There were no significant differences on S6 protein expression in all groups (Fig.  7A, 7C).

The effect of rapamycin on differentiation of AFSCs
Finally, rapamycin effect on multi-differentiation potential of AFSCs was also studied. AFSCs were differentiated into adipocytes (

DISCUSSION
Low back pain is prevalent worldwide and causes huge socio-economic burdens. Intervertebral disc degeneration (IVDD) is a widely accepted cause of low back pain. However, the mechanism of IVDD is still not well established [22]. It has been found that cell apoptosis, pro-inflammatory cytokine storm and increased matrix catabolism involve in the pathogenesis of IVDD, but truly effective treatment options are few.
Aging is the main risk factor for most chronic diseases, disabilities and declining health [17]. Cellular senescence has been hypothesized to contribute to ageassociated tissue dysfunction, reduced regenerative ca- AGING pacity and disease [23]. Senescent cells have been shown to increase with aging in various organs and tissues. Therefore, cellular senescence has become a novel therapeutic target for aging and age-related diseases. The disc cell senescence has been determined in degenerative discs and the cells from degenerate discs exhibited increased expression of P16, and matrixdegrading enzyme gene expression. [6].
A novel aging study in vitro model has been established by using bleomycin and rapamycin to treat AFSCs. Our results showed that exposure to bleomycin, a DNA damaging agent, induced cellular senescence in rabbit AFSCs. The senescence was characterized by irreversible cell-cycle arrest, which is mediated predominantly by P21 and/or P16 Ink4a , increased cell size, altered morphology, resistance to apoptosis, and an up-regulation of senescence-associated β-galactosidase (SA-β-gal) activity. The senescent phenotype induced by bleomycin also supports similar previous findings in alveolar epithelial cells [24]. It has been reported that persistent DNA damage induces the secretion of various factors including inflammatory cytokines, growth factors and proteases [25]. In this study, we found that bleomycin up-regulated the expression of proinflammatory cytokine IL-1β, IL-6, and TNF-α, and catabolic enzymes MMP-3, and MMP-13, which was in correlation with previous findings that senescent cells had an excessive increase in the levels of MMPs, ADAMTS, and pro-inflammatory cytokines such as TNF-α [26,27].
Rapamycin has been found to extend lifespan in yeast, fruit flies and mice, with mechanisms as to decelerate DNA damage accumulation and cellular senescence [28,29]. Rapamycin is a prospect of pharmacological rejuvenation of aging stem cells [30]. Our study also demonstrates that rapamycin partially decreases SA-β- AGING gal activity and senescent morphological change, indicating that rapamycin affects senescence at both molecular and cellular levels in rabbit AFSCs. In addition, rapamycin dramatically decreased the expression of TNF-α, MMP-3, and MMP-13 induced by bleomycin in AFSCs.
It is believed that stem cells play a key role in tissue regeneration and degeneration. Disc stem/progenitor cells have been isolated from human and animal spinal disc tissues [31,32]. AF stem/progenitor cells differ from AF fibroblasts in their ability to proliferate and self-renew, as well as in their multi-differentiation potential, which allows them to differentiate into various cell types such as adipocytes, chondrocytes and osteocytes, in addition to differentiating into AF fibro-blasts. The discs from patients with spinal deformities have ectopic calcification in the cartilage end plate and in the disc itself [33]. It has been reported that lumbar disc degeneration is associated with modic change and high paraspinal fat content [34]. Our results have shown that the AFSCs have multi-differentiation potential to differentiate into adipocytes, osteocytes, and chondrocytes when they were cultured in various differentiation media. Rapamycin inhibited the differentiation of AFSCs.
Remarkably, rapamycin is a clinically approved drug that has been used for a decade in renal transplant patients. It was suggested that rapamycin could be used for extension of healthy lifespan and prevention of agerelated diseases by slowing down the aging process AGING [20]. Therefore, the use of rapamycin may represent a novel approach to slow the aging-associated IVDD. Further studies are clearly needed to confirm the poten-tial mechanisms of mTOR signaling involvement in the prevention of aging induced IVDD in vitro and in vivo.  (B) semi-quantification of PS6; (C) semi-quantification of S6. To ensure that equal amount of total protein was loaded, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control for protein normalization. The results indicated that bleomycin did not cause the significant changes in S6 protein levels in AF cells (A, C), however, bleomycin increased PS6 protein expression in AF cells with a concentration-dependent manner (A, B). Adding rapamycin in bleomycin treated AF cells decreased PS6 levels in AF cells (A, B), but not S6 protein levels (A, C). *p<0.05 compared to control, # P<0.05 compared to bleomycin 50 μg/ml.

AGING
Our findings demonstrated that mTOR signaling pathway affects AF cell senescence, catabolic and inflammatory responses, and stem cell differentiation, suggesting that potential treatment value of rapamycin for disc degenerative diseases, especially lower back pain.

AF stem cell isolation and culture
The stem cells were isolated from annulus fibrosus of lumbar spine of five New Zealand white rabbits (female, 5 months old) based on a previously published protocol [35]. The protocol for use of the animals was approved by the IACUC of Shandong University. Briefly, after euthanasia, the AF tissues were harvested from the L2-L4 lumbar IVD and cut into small pieces. After 5 hours digestion with 0.04% collagenase P, the resulting cell suspensions were passed through a 70 μm cell sieve and centrifuged at 500g for 10 min. The cell pellets were re-suspended in culture medium consisting of F-12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin and cultured at 37°C with 95% air and 5% CO 2 . The medium was changed every three days and the stemness of the cells was tested by stem cell markers according to the previous publication [31]. The stem cells at passage 2-3 were used for the later experiments.

Proliferation of AFSCs
The proliferation of AFSCs grown in five different conditions was assessed with population doubling time (PDT), defined as the total culture time divided by the number of generations [36]. The number of generations was expressed as log 2 N c /N 0 , where N 0 is the population of the cells seeded initially, and N c is the population at confluence.

SA-β-gal staining by histochemical staining kit
The cell senescence were detected with β-galactosidase positively stained cells using a senescent cell histochemical staining kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol. Briefly, the cells treated with various conditions were washed twice with PBS and then fixed with fixation buffer for 10 min. The cells were then washed three times with PBS. After the last wash, staining solution was added and the cells were incubated in a dry incubator at 37°C overnight. The senescent cells were positively stained in blue and photographed with an inverted microscope.

Immune staining for senescent marker protein analysis
The cell senescence was further detected with immune staining on AFSCs treated with different conditions. Briefly, the cells treated with various conditions were washed twice with PBS and then fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min. The cells were then washed with PBS for another 3 times and incubated either with mouse anti-p16 (1:500, Cell Signaling Technology, Danvers, MA) or mouse anti-SA-β-gal (1:500, Cell Signaling Technology, Danvers, MA) antibodies at 4°C overnight. After washing the cells with PBS 3 times, the cells were incubated with Cy3-conjugated goat anti-mouse IgG second antibody (1:1000, Abcam, Cambridge, MA) at room temperature for 2 hours. The cells were also counterstained with H33342 staining (1:500, Sigma, St. Louis, MO). The positively stained cells were examined using fluorescence microscopy (Nikon Eclipse, TE2000-U).

Quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted from the AFSCs with an RNeasy Mini Kit (Qiagen, Valencia, CA) and used for first-strand cDNA synthesis by reverse transcription with SuperScript II (Invitrogen, Carlsbad, CA). The gene expression was tested by qRT-PCR using QIAGEN QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA). Rabbit-specific primers including collagen type I for AF cell-related gene, MMP-3 and MMP-13 for catabolic genes, and IL-1β, IL-6, and TNF-α for inflammatory genes were tested. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. All primers were designed according to the published papers (Table 1) [37][38][39][40]. After an initial denaturation for 10 min at 95°C, PCR was performed for 50 cycles, and each cycle consisted of denaturation for 50 seconds at 95°C, followed by annealing for 30 seconds at 58°C for all the genes. At least three independent experiments were performed to obtain relative expression levels of each gene. Data were analyzed by the 2 −ΔΔCt method.

Western blot analysis
Cell lysates were prepared using mammalian cell lysis buffer according to standard procedures provided by the manufacturer (Sigma, St. Louis, MO

Semi-quantification of positive stained cells
The stained cells were examined under a microscope and three random images in each well were taken for the semi-quantification. Three wells were used for each group. The positively stained areas were determined by SPOT™ imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI). The total area viewed under the microscope was divided by the positively stained area to calculate the proportion of positive staining. These values were averaged to represent the percentage positive staining in each group.

Statistical analysis
One-way analysis of variance (ANOVA) was used and all statistical tests were conducted using GraphPad Prism 7 (GraphPad Software, San Diego, CA). Differences with a p< 0.05 were determined as statistically significant.

AUTHOR CONTRIBUTIONS
Changhong Gao performed experiments, evaluated data, and drafted the manuscript. Bin Ning performed experiments and analyzed data. Chenglin Sang provided experimental help and advice and revised manuscript. Ying Zhang conceived the study, supervised the project, and revised the manuscript. All authors discussed the results and commented on the manuscript.