Cyclic Mechanical Stretch Ameliorates the Degeneration of Nucleus Pulposus Cells through Promoting the ITGA2/PI3K/AKT Signaling Pathway

Background Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain and motor deficiency. Nucleus pulposus (NP) degeneration plays a key role in the process of IVDD. The mechanical and biological interactions involved in NP degeneration have not been elucidated. The present study is aimed at investigating the effect and mechanism of cyclic mechanical stretch in regulating the function and degeneration of NP cells. Methods NP cells were subjected to cyclic tensile stress (10% deformation) of 0.1 Hz for 8640 cycles. Cell proliferation was conducted through the MTT assay. The cell cycle and apoptosis were detected by flow cytometry. A gene expression profile chip was used to analyze the differentially expressed genes between the tensile stress group and the control group. Enrichment analysis of Gene Ontology (GO) annotation and signaling pathways were analyzed. Western blot and RNA interference were carried out to investigate the role of the ITGA2/PI3K/AKT pathway in the effect of cyclic mechanical stretch on NP cells. Results NP cells exhibited a greater (P < 0.05) growth rate in the tensile stress group compared to the control group. Cyclic mechanical stress significantly promoted the cell cycle transition of NP cells from the S phase to the G2/M phase. A fewer proportion of apoptotic cells were found in the tensile stress group (P < 0.05), indicating that cyclic mechanical stretch inhibits NP cell apoptosis. Microarray analysis revealed 689 significant differentially expressed genes between the two groups (P < 0.05), of which 333 genes were upregulated and another 356 genes were downregulated. Cyclic mechanical stretch altered the expression of 31 genes involved in the ITGA2/PI3K/AKT pathway and remarkably promoted this pathway in NP cells. Downregulation of ITGA2 and AKT further demonstrated that the PI3K/AKT pathway was responsible for the proliferation and COL2A1 expression of NP cells upon cyclic mechanical stretch. Conclusions Cyclic mechanical stretch promoted the proliferation and cell cycle and reversely inhibited the apoptosis of NP cells. Cyclic mechanical stretch promoted COL2A1 expression and ameliorated the degeneration of NP cells via regulation of the ITGA2/PI3K/AKT signaling pathway. Our results may provide a potential target and a possibility of IVDD disease treatment by ameliorating the degenerative changes.


Introduction
Intervertebral disc degeneration (IVDD) is frequently associated with low back pain (LBP), which is one of the most prev-alent musculoskeletal problems and the leading cause of disability [1,2]. IVDD causes instability, stenosis, and deformity of the spine and is considered an aberrant, pathological, and cell-mediated response leading to progressive structural failure and pain [3]. Despite the global prevalence and high economic burden associated with IVDD diseases [4], the pathogenesis and molecular mechanisms leading to IVDD have not been fully elucidated.
The mature healthy intervertebral disc (IVD), a connective tissue located between the vertebrate bodies of the vertebrae, is composed of three components: nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplates (CEPs) that anchor onto the vertebrae [5]. The main functions of IVD include transmission and absorption of mechanical loadings onto the vertebral column in order to maintain the motional function that allows extension, flexion, lateral bending, and axial rotation [6,7]. The NP is a highly hydrated gelatinous mass that comprises the chondrocytes and proteoglycans incorporating an irregular network of elastin fibers and type II collagen [8]. Overall, the healthy NP generates intradiscal pressure that separates the vertebrae above and below, maintains the tension of the AF, and distributes the pressure evenly across two adjacent CEPs [8]. Degeneration of NP has been considered a vital step in IVDD. A degenerated NP becomes an unorganized mass of fibrous tissue with altered extracellular matrix (ECM) components that has mainly lost its capacity of binding water [9]. The pressure within the degenerated NP dwindles, and the disc height decreases, resulting in IVDD and spinal biomechanical instability [10]. The NP undergoes the highest degree of remodeling during IVDD.
A wealth of complex factors give rise to disc degeneration and commonly involve synergistic interactions between biomechanics and biological mechanisms [8]. During their interactions, mechanical factors, cell biological metabolism response, and the water-binding ECM play important roles in degeneration. Some changes occur in degenerating NP cells; for example, the predominant cell type changes from notochordal cells to chondrocyte-like cells, the ECM changes from anabolism to catabolism, and inflammatory mediators increased [11][12][13][14]. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway governs many fundamental cellular functions and participates in regulating transcription, translation, cell proliferation, differentiation, cell cycle, and apoptosis [15,16]. The PI3K/AKT pathway has been reported to participate in regulating NP cell proliferation, apoptosis, senescence, and ECM metabolism, and it has also been shown to be significantly associated with NP degeneration [17][18][19]. However, the mechanics and biological interaction mechanism in regulating the degeneration of NP have not yet been explored in depth.
In this research, we investigated the effect of cyclic mechanical stretch on the biological function of NP cells and the role of the ITGA2/PI3K/AKT pathway in response to the effect of cyclic mechanical stretch on NP cells. The results from our study may provide potential targets and the possibility of reversing the degenerative changes in IVDD. 2.3. Isolation and Culture of NP Cells. NP tissues were washed thrice with phosphate-buffered saline (PBS) solution and cut into 1 to 2 mm 3 pieces under sterile conditions. The tissues were digested with 0.25% trypsin (Gibco, USA) for 30 min in a water bath at 37°C. The tissue samples were centrifuged at 1200 rpm for 5 min. The obtained precipitates were then digested by incubation with 0.1% type II collagenase for 3 h in a water bath at 37°C with intermittent shaking every 10 minutes. After digestion, the NP cells were harvested through a 70 μm cell strainer and centrifuged (1200 rpm) for 5 min. The NP cells were incubated with a complete DMEM culture medium (Gibco, USA) containing 12% heat-inactivated fetal bovine serum (FBS, Gibco, USA), streptomycin (100 mg/mL), and penicillin (100 U/mL). The NP cells were then incubated in a humidified 5% CO 2 atmosphere. When cell growth reached 80-90% confluence, the NP cells were dispersed using 0.25% trypsin (Gibco, USA) and resuspended in the appropriate culture plates. NP cells from passages 2-5 were used for all subsequent experiments.

Cyclic Mechanical Stretch.
The cyclic mechanical stretch model of NP cells in vitro was established using a cell stretching instrument (Mechano Culture FX2, Cell Scale, Canada) that was controlled by a computer. The NP cells were seeded into a sixteen-well flexible-bottom Silicone Plate (Cell Scale, Canada) at a density of 2 × 10 5 cells/200 μL in the culture medium and incubated in a humidified 5% CO 2 atmosphere for 12 h. The cells were then subjected to cyclic tensile stress (10% deformation) of 8640 cycles at 0.1 Hz (2 s of stretch, 5 s of hold, 2 s of recover, and 1 s of rest for 1 cycle; Figure 1). As the control group, NP cells were cultured under the same conditions without any mechanical stretch. After the stretch was completed, the cells were harvested for the subsequent experiments.
2.5. Cell Proliferation Assay. The proliferation of NP cells was detected through MTT assay. The cells were seeded in a 96well plate (2:5 × 10 3 cells per well). An MTS kit was used to detect the cell growth rate based on the manufacturer's protocol (Promega, USA). The experiment was performed three times.
2.6. Detection of the Cell Cycle. Cells in the tensile stress and control groups were harvested, washed twice with the icecold PBS, and fixed with 75% ethyl alcohol at -20°C for 1 hour. The cells were then resuspended in 400 mL PBS and incubated with RNase at 37°C for 30 minutes. The cells were then incubated with propidium iodide in the dark at 4°C for 2 Oxidative Medicine and Cellular Longevity 60 minutes and analyzed using a flow cytometer (Beckman, USA). The experiment was performed three times.

Detection of Cell
Apoptosis. NP cells were washed twice in ice-cold PBS, resuspended in 400 mL binding buffer, and incubated with Annexin V-fluorescein isothiocyanate (Solarbio, China) in the dark for 15 minutes at 4°C. The NP cells were then incubated with propidium iodide in the dark at 4°C for 5 minutes and visualized in a flow cytometer (Beckman, USA). The experiment was performed three times.

Gene Expression Profile Microarray and Bioinformatics
Analysis. The GeneChip® PrimeView™ Human Gene Expression Array (Affymetrix, Santa Clara, CA, USA) was used to analyze the gene expression profile microarray. The experiments and bioinformatics analysis were conducted by Gene Co., Ltd. (Shanghai, China). The quality of the original chip data was evaluated, including signal strength distribution, Pearson's correlation analysis, and principal component analysis. The probe signal strength that ranged in the lowest 20% was taken as background noise, and probes with a coefficient of variation greater than 25% were filtered out. After data cleaning, the remaining data were conducted to analyze the differentially expressed genes (DEGs) and function analysis. The log 2 | fold change | ≥1:5 and a P value cutoff of 0.05 were used as filters to identify the DEGs. The significance enrichment analysis of Gene Ontology (GO) annotation was analyzed by Fisher's exact test. A gene set enrichment analysis of all DEGs was conducted based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database to identify significantly enriched signaling pathways.
2.9. Total RNA Extraction and qRT-PCR. Total RNA was extracted using TRIzol solution (Invitrogen, California, USA) based on the manufacturer's instructions. The qRT-PCR was carried out as previously described [21] using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control. The primers are listed in Table 1. The Ct (threshold cycle) value was analyzed based on the threshold cycles via SDS 2.3 software. Relative expression levels of  3 Oxidative Medicine and Cellular Longevity target genes were normalized to the geometric mean of GAPDH and analyzed via the comparative threshold cycle (2 -ΔCT ) method.
2.10. siRNA (Small Interfering RNA) Transfection. siRNAs targeting ITGA2 (siRNA-ITGA2) and AKT (siRNA-AKT) (GenePharma Company, Shanghai, China) were used to knock down the expression of ITGA2 and AKT proteins in NP cells. Briefly, NP cells were seeded in a 6-well plate (1 × 10 6 cells per well). When the NP cell growth reached about 50% confluence, the cells were transfected with a 500 μL mixed siRNA transfecting reagent at 37°C for 48 hours. The siRNA transfecting reagent was prepared according to the manufacturer's protocol. Briefly, 200 pmol siRNA-ITGA2 and 200 pmol siRNA-AKT were diluted with 250 μL Opti-MEM I (Invitrogen, California, USA) dilution and placed at room temperature for 5 min and then mixed with 5 μL Lipofectamine RNAiMAX (Invitrogen, California, USA) diluted with 250 μL Opti-MEM I. The siRNA-NC was used as a negative control. The ITGA2 and AKT levels were measured 48 hours after transfection. The siRNA sequences used in this study were as follows: GAACGG GACUUUCGCAUCA for siRNA-ITGA2, ACAAGGACG GGCACATTAA for siRNA-AKT, and UUCUCCGAACG UGUCACGUTT for siRNA-NC.
2.11. Protein Extraction and Western Blot. NP cells were lysed using RIPA (Beyotime, Shanghai, China). The lysates were harvested through centrifugation (12,000 rpm) at 4°C for 30 min. Western blot was performed to determine the protein expression of target genes as previously described [21]. Densitometry was used to determine the band intensity using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, USA). The protein expression levels were normalized against 2.12. Statistical Analysis. Statistical analyses were conducted through SPSS 22.0 software (IBM, Armonk, USA), and a P value < 0.05 was considered statistically significant. Student's t-test was used for the comparison of continuous variables between groups. All data were shown as the mean ± standard deviation ðSDÞ.

Morphological Observation of NP Cells
Responding to the Cyclic Mechanical Stimuli. Biomechanical factors have been reported to be remarkably associated with NP degeneration [8,9]. Biomechanics influence the multiple biological functions of NP cells, such as regulating the balance of ECM anabolism and catabolism, the abnormal expression of growth factors and inflammatory molecules, and the nutrition and metabolism of NP cells [8,9]. As an important component of IVDs, the NP region is subjected to a different tension, pressure, and shear force under different physiological states. These different biomechanics lead to different effects on the function and degeneration of NP cells [7].

Oxidative Medicine and Cellular Longevity
This study investigated the effect of cyclic mechanical stretch on the biological function of NP cells. As observed under a microscope, NP cells were spindle-shaped or irregular in shape and appeared singly or as 2-to 4-cell colonies scattered at the bottom of the bottle on day 5 of culture ( Figure 2(a)). On day 9 of culture, the number of NP cells increased gradually, and the cells from each colony were closely connected (Figure 2(b)). On day 15 of culture, the culture reached 40-60% confluence with good morphology and refraction (Figure 2(c)). On culture day 25, cell growth reached 80-90% confluence (Figure 2(d)). NP cells from passages 2-5 were used to load the cyclic mechanical stretch. Results showed that NP cells in the tensile stress group were irregularly shaped with a full growth state and good refraction (Figure 2(e)), whereas the condition of NP cells in the control group was not good, with longer secondary processes and appeared aging (Figure 2(f)).

Cyclic Mechanical Stretch Promotes NP Cell Proliferation
and Cell Cycle Progression. The effect of cyclic mechanical stretch on the proliferation of NP cells was then evaluated. As shown in Figure 3(a), NP cells exhibited a significantly higher growth rate in the tensile stress group compared to the control group (P < 0:05). We then analyzed the effects of cyclic mechanical stretch on the cell cycle by flow cytometry. The percentages of NP cells in the G1, S, and G2/M phases in the tensile stress and control groups were 41:22 ± 5:17% vs. 41:71 ± 2:45%, 20:74 ± 2:32% vs. 32:47 ± 1:95%, and 22:27 ± 2:91% vs. 15:53 ± 0:78%, respectively (Figures 3(c) and 3(d)). The percentage of NP cells in the S phase was remarkably lower in the tensile stress group compared to the control group (P < 0:01, Figure 3(b)). The percentage of NP cells in the G2/M phase was significantly higher in the tensile stress group than in the control group (P < 0:05, Figure 3(b)). The results revealed that cyclic mechanical stretch significantly promoted the cell cycle transition of NP cells from the S phase to the G2/M phase.

Cyclic Mechanical Stretch Inhibits NP Cell Apoptosis.
We analyzed the effects of cyclic mechanical stretch on the NP cell apoptosis. The apoptosis percentages of NP cells in the tension and control groups were 8:99 ± 0:88% and 10:75 ± 0:40%, respectively (Figures 4(a) and 4(b)). A lower percentage of apoptotic NP cells was found in the tensile stress group  Oxidative Medicine and Cellular Longevity compared with the control group (P < 0:05, Figure 4(c)), indicating that cyclic mechanical stretch inhibited apoptosis in NP cells. These results suggested that cyclic mechanical stretch may affect the degeneration of the NP by regulating the proliferation, cell cycle, and apoptosis of NP cells. Matsumoto et al. reported that cyclic mechanical stretch increases the growth rate of NP cells in vitro [22]. On the contrary, Yang et al. and Wang et al. reported that abnormal mechanical stretch stress led to excessive apoptosis and degeneration of NP cells [23,24]. Cyclic mechanical tension with 20% elongation induced NP cell apoptosis [25]. When comparing the mechanical loading parameters of high amplitude (>10%), low frequency (<0.1 Hz), and long duration (>24 7 Oxidative Medicine and Cellular Longevity hours) in the previous study, the tensile stress used in the current study was applied at 0.1 Hz frequency for 24 h to produce 10% deformation. Together with these reported studies, our findings indicate that it is possible that high mechanical stretch would promote NP apoptosis and degeneration, while moderate mechanical stretch would inhibit NP cell apoptosis, promote NP cell proliferation, and potentially ameliorate NP degeneration.
We then performed GO annotation analysis, which consists of three main branches, namely, biological process (BP), molecular function (MF), and cellular component (CC). Figure 5(c) presents the top ten significantly enriched BP, MF, and CC associated with cyclic mechanical stretch stimulation. GO annotation analysis showed that the main genes from the BP category were positive regulation of gene expression, cell proliferation and division, and apoptotic processes; the genes from the MF category were mainly ECM structural constituents; and those from the CC category included focal adhesion and ECM. Abnormal NP cell proliferation, apoptosis, and ECM metabolism have been shown to result in NP degeneration [8]. The microarray results revealed that the genes with significant differential expression that were involved in these biological functions and processes are closely related to NP degeneration.
In addition, we analyzed the involvement of all DEGs associated with signaling pathways based on the KEGG database. Figure 5(d) shows the top ten enriched signaling pathways associated with cyclic mechanical stretch stimulation, and these signaling pathways included proteoglycans in cancer, PI3K/AKT, thyroid hormone, MAPK, FoxO, Rap1, Ras, biosynthesis of amino acids, AGE-RAGE signaling pathway in diabetic complications, and ECM-receptor interaction. Of these, the PI3K/AKT pathway has previously been reported to regulate cellular functions such as cell proliferation, growth, cell cycle, and apoptosis [15,16]. The PI3K/AKT pathway was reported to participate in the regulation of NP cell apoptosis, senescence, and ECM degradation and is significantly associated with NP degeneration [19,26,27]. Based on the above experimental results, it is reasonable that the PI3K/AKT signaling process might play important roles in the cyclic mechanical stretch-mediated effects on NP cells.
A total of 31 genes involved in the PI3K/AKT signaling pathway were uncovered to show significant differential expression between the two groups. Of these, 18 genes, including ITGA2, ITGA10 (integrin subunit alpha 10), BCL2L11 (BCL2-like 11), COL2A1 (collagen type II alpha 1), and FGF1 (fibroblast growth factor 1), which are positive effectors in this pathway, were markedly upregulated. And conversely, 13 genes that negatively regulate the pathway, such as MYC (MYC proto-oncogene), IL7R (interleukin 7 receptor), and THBS1 (thrombospondin 1), were markedly downregulated. We further conducted qRT-PCR to confirm the microarray results. Results showed that the mRNA levels of ITGA2, FGF1, COL2A1, and BCL2L11 were significantly increased, and the MYC and IL7R mRNA levels were markedly decreased in NP cells in the tension group compared with the control group (log 2 | fold change | ≥1:5, P < 0:05, Table 2). These results further supported our findings from the microarray data. Integrins are widely considered the predominant mechanoreceptors during the process of mechanical transduction in IVD cells [28]. Mechanosensing in NP cell loading with dynamic mechanics occurs via integrin signaling [28]. The integrin pathways play an important role in the susceptibility of notochordal cells to mechanical loading-induced IVDD [29]. ITGA2, also known as integrin α2, encodes the alpha subunit of a transmembrane receptor for collagens and other related proteins. ITGA2 forms a heterodimer with an integrin beta subunit, mediating the adhesion of multiple cell types  Oxidative Medicine and Cellular Longevity to the ECM. Integrin α2β1 participates in the estrogenintervened PI3K/AKT pathway regulating the antiapoptotic effect of intervertebral disc aging and degeneration and participates in FAK/PI3K signaling regulating adherence capacity and anoikis of NP cells in IVD degeneration [30]. The FAK/-PI3K/AKT pathway has been reported to acts as integrindependent signaling in regulating apoptosis of NP cells during the process of disc degeneration [31]. Integrins function synergistically with SYND4, and FAK/PI3K signaling participates in regulating adherence capacity and anoikis of NP cells in IVD degeneration [32]. Yan et al. reported that static compression loading upregulated integrin α2β1 expression and downregulated α10β1 expression in NP cells [33]. Compression loading altered the expression of collagen, MMP, and integrin and promoted integrin α2β1 signaling in IVD [33]. However, the role of ITGA2 in the effect of cyclic mechanical stretch on NP cell degeneration has not been elucidated.
In this study, Western blot was performed to further corroborate the effects of cyclic mechanical stretch on the ITGA2/PI3K/AKT signaling process in NP cells. The results of the Western blot revealed that the expression levels of the ITGA2 and p-(phosphorylated-) AKT proteins were remarkably higher in the tensile stress group compared to the control group (Figure 6(a)), indicating that cyclic mechanical stretch remarkably promotes the ITGA2/PI3K/AKT signaling pathway in NP cells.
To further substantiate the functional effect of cyclic mechanical stretch on the ITGA2/PI3K/AKT signaling pathway, we used siRNAs to knock down the protein expression of the key molecules in this pathway in NP cells, i.e., ITGA2 and AKT. Results of the Western blot analysis showed that ITGA2 and AKT proteins were markedly decreased (Figure 6(b)), indicating that the ITGA2/PI3K/AKT signaling pathway was inhibited. And  Oxidative Medicine and Cellular Longevity the ITGA2 and AKT expression showed no obvious change when the NP cells were loaded with cyclic mechanical stretch. We then determined the NP cell proliferation. Inhibition of this pathway significantly inhibited the proliferation of NP cells (Figure 6(c)), whereas cyclic mechanical stretch did not significantly affect the proliferation of NP cells transfected with siRNA-ITGA2/AKT. These results indicate that the ITGA2/PI3K/AKT signaling pathway plays a key role in the effect of cyclic mechanical stretch on NP cell proliferation.
Interestingly, we found that the cyclic mechanical stretch elevated COL2A1 protein expression in NP cells (Figure 6(a)). And COL2A1 protein expression was remarkably decreased after the knockdown of the ITGA2/PI3-K/AKT signaling pathway (Figure 6(b)). The ECM of NP consists of collagens and proteoglycans, of which aggrecan is one of the most abundant proteoglycans in NP tissues [34,35]. COL2A1 is a main component of the ECM in NP tissues and has been considered a key molecule in IVD degeneration [12]. Many ECM molecules are altered in degenerated NP, and collagen type II to collagen type I is one such predominant shift in the NP [36]. These results indicated that cyclic mechanical stretch may regulate COL2A1 expression through the ITGA2/PI3K/AKT signaling pathway and ameliorate the degeneration of NP cells. Further studies will investigate the molecular mechanism of cyclic mechanical stretch in regulating the ECM and inflammation of NP cells in the process of NP degeneration.

Conclusion
In conclusion, we provided evidence that cyclic mechanical stretch promoted NP cell proliferation and cell cycle transition from the S phase to the G2 phase and inhibited apoptosis of NP cells. Results of the microarray analysis revealed that 689 genes had significant differential expression. Enrichment analysis of all the DEGs in regulatory signaling pathways found the PI3K/AKT pathway. The results further revealed that cyclic mechanical stretch promoted cell proliferation and COL2A1 expression through regulation of the ITGA2/-PI3K/AKT signaling pathway and ameliorates the degeneration of NP cells. These results of our study may provide potential targets and the possibility of treating IVDD disease by reversing degenerative changes.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.