Introduction
Lower back pain (LBP) is a common musculoskeletal disorder that severely affects the quality of life of human beings and imposes a substantial economic burden on society (1). Intervertebral disc (IVD) degeneration (IDD) has been shown to be the major contributor to LBP (2). IVD, composed of a central nucleus pulposus (NP) surrounded by annulus fibrosus and cartilaginous endplates, is an avascular organ. In healthy IVD, the hydrated NP, which is mainly composed of NP cells and is rich in the extracellular matrix (ECM), including type II collagen and aggrecan, is important in the physiological function of the disc in distributing the mechanical load acting on the spine, and in multi-axial flexibility (3,4). During the progression of IDD, the increased levels of pro-inflammatory cytokines, elevated cell apoptosis, increased production of ECM-catabolic proteinases, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and decreased synthesis of type II collagen and aggrecan have been observed within the NP tissue (5-7). These cellular and molecular changes lead to disruption of the physiological structure and function of IVD and to spinal instability, which are the main triggers of LBP (8).
The excessive production of proinflammatory molecules secreted by NP cells, including tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-2 and IL-6, has been demonstrated to serve critical roles in the initiation and development of IDD (5,9). Among the above-mentioned inflammatory cytokines, IL-1β is widely considered to be the predominant cytokine that is expressed at high levels in degenerative IVD tissues and cells and has been shown to be involved in several pathological processes in NP cells, including inflammatory responses, oxidative stress, apoptosis, and an imbalance of ECM synthesis and degradation (10,11). Therefore, inhibiting the effect of IL-1β on NP cells may postpone the progression of IDD.
Berberine (BBR) is an isoquinoline alkaloid that is derived from several medicinal herbs, including Rhizoma Coptidis, Cortex Phellodendri, and Mahonia bealei, which have long been used in traditional Chinese medicine. BBR has been reported to possess multiple pharmacological effects, including anti-oxidative, anti-inflammatory and anti-apoptotic effects (12-14). Previous studies have found that BBR has therapeutic effects on musculoskeletal disorders, including rheumatoid arthritis and osteoarthritis, owing to its anti-inflammatory properties (15,16). Zhao et al showed that BBR treatment can protect articular cartilage from degeneration via activating the Akt-p70S6K-S6 signaling pathway in IL-1β-stimulated articular chondrocytes and in a rat osteoarthritis model (17). Hu et al reported that BBR decreases glycosaminoglycan release and nitric oxide production in IL-1β-stimulated chondrocytes (16). In addition, the administration of BBR was found by Zhou et al to prevent nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in a rat model of osteoarthritis (18). As the morphology and avascular supply of NP cells are similar to those of chondrocytes, and BBR has been reported to inhibit the effects of oxidative stress in rat NP cells (19), it was hypothesized that BBR may prevent the development of IDD by protecting NP cells from IL-1β-induced degenerative effects. Therefore, the purpose of the present study was to investigate the influence of BBR on IL-1β-induced apoptosis and ECM degradation in human NP cells and to elucidate the underlying molecular mechanism.
Materials and methods
Patient tissue samples
Between March and October 2017, human lumbar NP tissues were collected from 10 patients (six women and four men; mean age, 24.7 years; age range, 15-42 years) with idiopathic scoliosis who underwent deformity correction surgery with the approval of the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Written informed consent was obtained from all participants involved in the study. The degrees of degeneration of the discs of all participants were assessed using the modified Pfirrmann grading system (20) and were classified as grade II.
Human NP cell culture and treatment
Human NP cells were isolated using a method reported previously by Kang et al (21), and were then cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 15% of fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% of a penicillin-streptomycin solution at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged twice for use in the following experiments. The human NP cells were seeded in a six-well plate at a density of 105 cells/well. On reaching 80-90% confluence, the NP cells were incubated with 25 µM BBR for 2 h prior to IL-1β (10 ng/ml) treatment for 24 h at 37°C. The NP cells were harvested for subsequent experiments. All experiments were conducted in triplicate.
Cell viability analysis
Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, the human NP cells were seeded in a 96-well plate (5×103 cells per well) and cultured as described above, followed by treatment with various concentrations of BBR (5, 10, 15, 20 or 25 µM) or IL-1β (10 ng/ml) for 24 h at 37°C. Subsequently, 10 µl of the CCK-8 dye was added into each well, followed by incubation at 37°C for 2 h. The optical density was measured at 450 nm on a microplate reader (Leica Microsystems GmbH, Wetzlar, Germany).
RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was isolated from the human NP cells with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The mRNA expression levels of type II collagen, aggrecan, MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 were quantified by RT-qPCR analysis on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the cycling conditions recommended by the manufacturer. qPCR was performed using a SYBR Prime Script™ RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The reaction conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 30 sec and 60°C for 30 sec. The gene expression levels were normalized to that of β-actin. Relative expression levels were analyzed using the 2−ΔΔCq method (22). The primer sequences used for RT-qPCR analysis were as follows: Type II collagen, forward 5′-AGA ACT GGT GGA GCA GCA AGA-3′ and reverse 5′-AGC AGG CGT AGG AAG GTC AT-3′; aggrecan, forward 5′-TGA GCG GCA GCA CTT TGA C-3′ and reverse 5′-TGA GTA CAG GAG GCT TGA GG-3′; MMP-3, forward 5′-TTC CTT GGA TTG GAG GTG AC-3′ and reverse 5′-AGC CTG GAG AAT GTG AGT GG-3′; MMP-13, forward 5′-CCC AAC CCT AAA CAT CCA A-3′ and reverse 5′-AAA CAG CTC CGC ATC AAC C-3′; ADAMTS-4, forward 5′-ACC CAA GCA TCC GCA ATC-3′ and reverse 5′-TGC CCA CAT CAG CCA TAC-3′; ADAMTS-5, forward 5′-GAC AGT TCA AAG CCA AAG ACC-3′ and reverse 5′-TTT CCT TCG TGG CAG AGT-3′; β-actin, forward 5′-AGC GAG CAT CCC CCA AAG TT-3′ and reverse 5′-GGG CAC GAA GGC TCA TCA TT-3′.
Western blotting
The western blotting procedure was performed to analyze protein levels. The treated human NP cells were lysed with RIPA lysis buffer, and protein concentration was measured using the BCA Protein Assay kit (Beyotime Institute of Technology, Haimen, China). The nuclear and cytoplasmic proteins were isolated with the Nuclear/Cytosolic Fractionation kit (BioVision, Inc., Mountain View, CA, USA). The concentration was measured using a BCA protein assay. A total of 40 µg protein per lane was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis on a 12% gel and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk in TBST, the membranes were incubated with primary antibodies against type II collagen (sc-7764; 142 kDa; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:8,000), aggrecan (ab3778; 50-60 kDa; Abcam, Cambridge, UK; 1:100), MMP-3 (14351; 60 kDa; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:1,000), MMP-13 (ab39012; 54 kDa; Abcam; 1:4,000), ADAMTS-4 (ab185722; 90 kDa; Abcam; 1:1,000), ADAMTS-5 (ab41037; 73 kDa; Abcam; 1:200), B-cell lymphoma 1 (Bcl-2; ab32124; 26 kDa; Abcam; 1:1,000), Bcl-2-associated X protein (Bax; ab32503; 20 kDa; Abcam; 1:1,000), cleaved caspase3 (9664; 17 kDa; Cell Signaling Technology, Inc.; 1:1,000), nuclear factor (NF)-κB p65 (ab16502; 60 kDa; Abcam; 1:500), phosphorylated p65 (p-p65; ab86299; 60 kDa; Abcam; 1:1,000), inhibitor of κBα (IκBα; (9242; 39 kDa; Cell Signaling Technology, Inc.; 1:1,000), β-actin (ab8227; 42 kDa; Abcam; 1:2,000) and lamin B1 (ab16048; 66 kDa; Abcam; 1:1,000) overnight at 4°C, followed by incubation with the respective secondary antibodies (BA1054; Boster Biological Technology, Pleasanton, CA, USA, 1:5,000) at room temperature for 1 h. The protein bands were detected using an enhanced chemiluminescence system and analyzed quantitatively using BandScan software (version 4.30; BioMarin Pharmaceutical Inc., UK). β-actin and lamin B1 served as loading controls.
Flow cytometry
Following treatment with the different agents, the human NP cells were harvested, washed with cold PBS, and stained using the Annexin V-FITC Apoptosis Detection kit (Beyotime Institute of Biotechnology, Haimen, China). The fluorescence of the cells was determined immediately by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The apoptotic rate was calculated as the sum of the percentages of early (Annexin V+/PI−) and late (Annexin V+/PI+) apoptotic cells.
Statistical analysis
The results are expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS v.18.0 software (SPSS, Inc., Chicago, IL, USA). Differences between groups were evaluated by one-way analysis of variance followed by the Tukey test. P<0.05 was considered to indicate a statistically significant difference.
Results
Cell viability of human NP cells following treatment with BBR
The present study evaluated the effect of BBR on the viability of human NP cells at various concentrations (5, 10, 15, 20 or 25 µM) for 24 h using the CCK-8 assay. The results indicated that BBR was not significantly toxic to human NP cells at concentrations ranging between 5 and 25 µM (Fig. 1A). In addition, BBR was administered to IL-1β-treated human NP cells at various concentrations (15, 20 or 25 µM). Pretreatment with BBR had a significant protective effect against IL-1β-induced cell apoptosis, particularly at 25 µM (Fig. 1B). Therefore, 25 µM BBR was selected for subsequent experiments.
Inhibitory effects of BBR on IL-1β-induced ECM degradation by human NP cells
To determine whether BBR affects IL-1β-induced ECM degradation by human NP cells, the present study assessed the mRNA and protein expression levels of the main components of the NP ECM, including type II collagen and aggrecan, and major NP ECM catabolic proteinases MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 by RT-qPCR and western blot analyses, respectively. The results revealed that IL-1β treatment markedly decreased the mRNA expression levels of type II collagen and aggrecan, whereas pretreatment with BBR attenuated the downregulation induced by IL-1β (Fig. 2A and B). It was also found that the mRNA expression levels of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in human NP cells were significantly increased following stimulation with IL-1β (Fig. 2C-F). However, BBR pretreatment resulted in a statistically significant attenuation of IL-1β-induced upregulation of these ECM catabolic proteinases (Fig. 2C-F). The same results were observed for protein expression levels (Fig. 2G-M)
BBR protects human NP cells from IL-1β-induced cell apoptosis
As NP cell apoptosis is important in the progression of IDD, the present study investigated the influence of BBR on IL-1β-induced human NP cell apoptosis. Flow cytometric analysis revealed that IL-1β increased the rate of apoptosis compared with that in the untreated control (Fig. 3A and B). When the human NP cells were cotreated with BBR and IL-1β, the results showed a significant decrease in the rate of apoptosis (Fig. 3A and B). Furthermore, the expression levels of apoptosis-related proteins (Bcl-2, Bax and cleaved caspase3) were assessed. The protein levels of Bax (pro-apoptotic) and cleaved caspase3 (pro-apoptotic) were increased and the protein level of Bcl-2 (anti-apoptotic) was decreased in the IL-1β-treated human NP cells (Fig. 3C-G). These trends were reversed by BBR pretreatment (Fig. 3C-G).
Suppressive effects of BBR on the IL-1β-induced activation of NF-κB in human NP cells
The NF-κB pathway has been found to be aberrantly activated in IDD and has been demonstrated to be an important mediator of the IL-1β-induced degenerative effects in human NP cells. Therefore, to further examine the underlying mechanism of BBR-induced anti-degenerative effects, the present study determined whether BBR regulates the NF-κB pathway in IL-1β-treated human NP cells. The extent of NF-κB p65 phosphorylation and the intracellular distribution of p65 were first evaluated by western blotting. Compared with the untreated group, a higher ratio p-p65/p65 and nuclear translocation of p65 from the cytoplasm were observed following IL-1β stimulation (Fig. 4A-C), indicating activation of the NF-κB signaling pathway in the IL-1β-treated human NP cells. However, pretreatment with BBR markedly suppressed this IL-1β-induced phosphorylation and translocation of p65 (Fig. 4A-C). IκBα is an inhibitor protein for p65, the phosphorylation of IκBα and its subsequent degradation induce the translocation of p65 to the nucleus. Therefore, the level of cytoplasmic IκBα was analyzed by western blotting in human NP cells. IL-1β treatment induced the degradation of cytoplasmic IκBα, whereas this effect was attenuated by BBR pretreatment (Fig. 4D).
Discussion
LBP affects 80% of the global population, and IDD is widely recognized as the main cause of LBP (2). The prevention or reversal of IDD is a potential treatment of LBP; however, the pathological basis and mechanisms underlying IDD remain to be fully elucidated. Current evidence suggests that the excessive inflammatory response, an increase in the proportion of apoptotic NP cells, and the imbalance between anabolism and catabolism of NP ECM are closely associated with the development of IDD (7,8,23); therefore, targeting these pathological processes is a novel strategy for the treatment of IDD. Medical treatments of LBP caused by IDD mainly include conservative approaches and, rarely, surgical procedures. Conservative approaches, including non-steroidal anti-inflammatory drugs, are prescribed to inhibit inflammation and to relieve the symptom of back pain temporarily; however, the widespread and long-term use of non-steroidal anti-inflammatory drugs results in considerable adverse effects (24). Surgical procedures, including spine fusion and discectomy (25), which cannot preserve the function of IVD, imposes a financial burden on the patient and is not an optimal choice. Therefore, investigating novel drug treatments that can promote endogenous repair of degenerative IVD and prevent IDD development is crucial, particularly for early-stage IDD. In previous years, plant-derived compounds have attracted the attention of those investigating IDD treatment owing to their anti-inflammatory effects and few adverse effects (10,26). BBR, an isoquinoline alkaloid extracted from Coptidis Rhizoma and Cortex Phellodendri, has been shown to have potent therapeutic potential against inflammatory diseases (12,27). The present study provides the first evidence, to the best of our knowledge, that BBR has pharmacological inhibitory effects on ECM degradation and apoptosis in IL-1β-stimulated human NP cells. The results also showed that BBR inhibits the IL-1β-induced activation of the NF-κB pathway in human NP cells (Fig. 5).
IL-1β is a major risk factor of IDD, with potent proinflammatory activity involving stimulation of the production of multiple proinflammatory mediators (28). It has been demonstrated that IL-1β is involved in multiple pathological processes related to the development of IDD (5,10,29). The progressive degradation of the NP ECM, which mainly consists of type II collagen and aggrecan (performing a vital function in the maintenance of the physiological function of IVD), is the most prominent feature of IDD. MMPs and ADAMTSs are the major ECM-degrading enzymes in IDD, and their functions in IDD have been investigated extensively (30). MMP-3 and MMP-13 have been identified as the main collagenases that lead to degradation of type II collagen in NP, whereas ADAMTS-4 and ADAMTS-5 are the primary aggrecanases due to their potent and specific activity in cleaving aggrecan. The expression of all these enzymes, which are often regarded as the catabolic markers of IDD, has been found to be upregulated in degenerative NP (31). The inhibition of MMPs and ADAMTSs has a therapeutic effect on IDD in vitro and in vivo. There is substantial evidence that, in NP cells, IL-1β can promote the production of ECM-degrading enzymes that degrade type II collagen and aggrecan in NP ECM (5,10). In the present study, the results showed that IL-1β treatment resulted in the upregulation of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 and the downregulation of type II collagen and aggrecan in human NP cells, as expected, and that BBR reversed these changes induced by IL-1β. These results suggest that BBR has the potential to control the progression of IDD by restoring the balance between ECM anabolism and catabolism in human NP cells.
NP cells are important for maintaining the structural integrity of IVD by generating the molecular components of ECM. Recently, an apoptosis-associated decrease in the NP cell number was proposed as an important mechanism underlying the development of IDD (32,33). Therefore, treatment of IDD not only depends on the NP ECM homeostasis but also on inhibiting the apoptosis of NP cells. Several studies have demonstrated that IL-1β is an important regulator of the apoptosis of NP cells (34,35). The majority of rabbit NP cells were shown to undergo apoptosis following IL-1β stimulation and exhibit morphological changes related to apoptosis, whereas a combined treatment with insulin-like growth factor 1 and IL-1β significantly reduced IL-1β-mediated apoptosis (35). Shen et al (34) reported that IL-1β induces the mitochondrial pathway in NP cells by increasing the expression ratio Bax/Bcl-2 and by releasing cytochrome c from the mitochondria to the cytoplasm, subsequently activating downstream caspases 9 and 3 to complete the apoptotic process. In addition, Chen et al (19) found that BBR may mitigate oxidative-stress-induced apoptosis through the mitochondrial pathway. The results of flow cytometric analysis in the present study revealed that BBR effectively prevented IL-1β-induced apoptosis. The data also indicated that BBR attenuated the downregulation of Bcl-2 and the upregulation of Bax and cleaved caspase 3 at the protein level in IL-1β-treated human NP cells. Taken together, these results suggest that BBR protects human NP cells from IL-1β-induced apoptosis.
Various intracellular signaling pathways are activated in response to inflammatory stimulation associated with IDD, thereby mediating the increase in the production of a downstream effector that is closely involved in the progression of IDD (36). As one of the most critical intracellular signaling proteins, NF-κB can regulate the expression of genes associated with ECM degradation and cell apoptosis in IL-1β-treated human NP cells (21,37). Inhibiting the activation of NF-κB is regarded as a potential therapeutic strategy against IDD. Under normal conditions, NF-κB is located in the cytoplasm bound to an inhibitory protein, IκB, which prevents NF-κB from entering the nucleus. Upon stimulation by IL-1β, the IκB protein is phosphorylated and degraded, resulting in the translocation of NF-κB from the cytoplasm to the nucleus. Subsequently, NF-κB facilitates gene transcription by binding to specific sequences in the promoter region of NF-κB-responsive genes, which upregulate the production of catabolic enzymes, inflammatory mediators and cyto-kines (5,10). To further elucidate the molecular mechanism underlying the inhibitory effect of BBR on ECM degradation and apoptosis in IL-1β-treated NP cells, the present study assessed the influence of BBR on the IL-1β-induced activation of NF-κB in human NP cells. The results revealed that BBR significantly inhibited the IL-1β-induced upregulation of the phosphorylation of NF-κB p65 and its nuclear translocation in human NP cells. In addition, the IL-1β-induced decrease in the level of cytoplasmic IκBα was reversed by BBR, indicating that treatment with BBR inhibited the degradation of IκBα, thereby maintaining NF-κB in an inactive state. Taken together, these results suggest that the therapeutic action of BBR on IDD may be mediated by suppression of the NF-κB signaling pathway.
However, as human NP cells cultured in vitro and human NP cells in vivo may respond differently to BBR treatment, whether BBR is effective as a IDD treatment in vivo remains to be fully elucidated. Therefore, animal experiments that can truly mimic IDD pathogenesis are urgently required to confirm the protective effect of BBR against IDD and to advance current understanding of the molecular mechanisms underlying this effect.
In conclusion, the results of the present study suggest that BBR exerts potent anti-ECM catabolic and anti-apoptotic actions by inhibiting the IL-1β-induced activation of NF-κB in human NP cells, indicating that BBR may be validated as an effective therapeutic agent for IDD in the future.