Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration

Background To clarify if a2-macroglobulin (α2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD). Methods The content of α2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NP) tissue by immunohistochemistry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Additionally, exogenic α2M was used to culture severely degenerated NP tissue in vitro. The effects of α2M on hypochlorite (HOCl)-treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5. Results ROS level increased in severely degenerated NP, accompanying with a decreased α2M content. Supplement of α2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCl resulted in oxidative damage to NP cells and decreased α2M expression in a dose and time-dependent manner. Furthermore, exogenic α2M stimulation reversed the HOCl-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by α2M were also observed. Conclusions Our study indicates that α2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.


Summary
Background: To clarify if a2-macroglobulin (a2M) has an antioxidative effect during the progression of the intervertebral disc degeneration (IVDD). Methods: The content of a2M and reactive oxygen species (ROS) were measured to compare mildly and severely degenerated human nucleus pulposus (NP) tissue by immunohistochemistry, mass spectrometry, and enzymelinked immunosorbent assay (ELISA). Additionally, exogenic a2M was used to culture severely degenerated NP tissue in vitro. The effects of a2M on hypochlorite (HOCl)treated NP cells were evaluated, containing antioxidative enzymes, ROS level, collagen II, and aggrecan expression, MMP3/13, and ADAMTS4/5. Results: ROS level increased in severely degenerated NP, accompanying with a decreased a2M content. Supplement of a2M could decrease the ROS level of cultured NP in vitro, meanwhile, the MMP13 and ADAMTS4 expression were also reduced. It was found that treatment of HOCl resulted in oxidative damage to NP cells and decreased a2M expression in a dose and time-dependent manner. Furthermore, exogenic a2M stimulation reversed the HOCl-triggered ROS accumulation. The promotion of SOD1/2, CAT, GPX1, collagen II, and aggrecan, and suppression of MMP3/13, ADAMTS4/5 expression caused by a2M were also observed.

Introduction
The intervertebral disc has no blood supply and consists of a peripheral annulus fibrosis, a central nucleus pulposus (NP), and upper and lower endplates (1). NP has fewer cells but abundant extracellular matrix (ECM), such as collagen II and aggrecan, which are normally secreted by NP cells (2). Inter vertebral disc degeneration (IVDD) is mainly manifested in the imbalance in the synthesis and degradation of cellular and ECM components. Some proteolytic enzymes can degrade the ECM of intervertebral discs, of particular interest, are matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), which promote abnormal apoptosis of NP cells and cause further degeneration of the intervertebral disc (3,4).
a2-macroglobulin (a2M) is an ancient, evolutionarily conserved polymer glycoprotein, which has a variety of active forms and complex functional effects involving in the regulation and transport of substances in the body (5). Especially when people realized the broad-spectrum anti-protease effect of a2M, it was more fully applied in motor system diseases such as acromion bursitis, tendinitis (6), osteoarthritis (7), and intervertebral disc disease (8). In addition to the ability of protease inhibitor in eliminating excessive endogenous and exogenous proteases, it has also been confirmed in anti-radiation and anti-tumor (9) effects. Recently, more and more reports mention a2M involving in the inhibition of reactive oxygen species (ROS) (10). However, whether a2M related to the mediation of oxidative stress of IVDD is not fully understood. Oxidative stress refers to the serious imbalance between the generation of oxygen radicals and the antioxidant defense, leading to the accumulation of ROS in the body or cells, which leads to cytotoxicity and tissue damage (11). ROS can cause degradation of ECM by inhibiting collagen II and aggrecan synthesis and destroying ECM structure. As people get older, ROS accumulates, and the level of oxidative stress in the body gradually increases, which leads to the disability of intervertebral disc cells, inflammatory infiltration, and ECM disorders that resulting in IVDD (12).
Our present study aimed to clarify the role of a2M in IVDD, especially its antioxidative behavior, using cultured NP tissue and NP cells in vitro. Our finding provides a new understanding of a2M in the therapeutic strategy of IVDD that containing oxidative stress balance.

Source of patient samples
To clarify the difference of a2M in NP tissues of different degenerated degree, 16 NP samples were collected from patients undergoing disc herniation operations, which were grouped into mildly degenerated group and severely degenerated group based on the Pfirrmann score 13 (Grade II or III belongs to the mild group; Grade IV or V belongs to the severe group). This research project was approved by the Ethics Committee of our hospital.

NP tissue treatment
We conserved the tissues in cold sterile dulbecco's modified eagle medium (DMEM) medium (Gibco, Rockville, MD, USA) immediately after cutting from the patients and transferred to the lab for tissue culture in vitro. We used different concentrations of a2M from human plasma (Sigma-Aldrich, St. Louis, MO, USA) to culture the severely degenerated NP tissue for 3 days and collected for the following experiment.

NP cells isolation and treatment
The tissues were minced and digested with type II collagenase (0.2%) and trypsin (0.15%) solution at 37 °C overnight. We filtrated the cell solution, centrifuged and resuspended in DMEM/F-12 medium (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA). We used different concentrations of hypochlorous acid (HOCl) resulting in direct oxidative damage to NP cells. Additionally, a2M from human plasma (Sigma-Aldrich, St. Louis, MO, USA) was used to reverse HOCl.

Hematein eosin staining (HE)
NP tissue was first treated as follows: fixed with 4% formaldehyde, dehydrated with a gradient of alcohol, embedded in paraffin, and cut into 5 mm thick slices. Sections were then dewaxed, hydrated, hematoxylin stained for 5 min, 0.7% hydrochloric acid ethanol differentiated for 5 s, and eosin-stained for 30 s.

Conclusions:
Our study indicates that a2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.

Enzyme-linked immunosorbent assay (ELISA)
The levels of a2M, MMP13, and ADAMTS4 in by NP tissue or NP cells were analyzed by ELISA kit (ab108888; ab100605; ab213753, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions.

ROS measurement
ROS content of NP tissue or NP cells was determined using in vitro ROS/RNS assay (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. The relative fluorescence units (RFU) at an excitation/emission wavelength of 488/525 nm was measured by a microplate reader.

Immunofluorescence (IF) staining
After treatment, NP cells were washed with phosphate buffered saline (PBS), fixed with 4% formaldehyde, and blocked with 5% bovine serum albumin (BSA). NP cells were following incubated with collagen II and aggrecan primary antibodies (Abcam, Cambridge, MA, USA) overnight at 4 °C. Followed incubated with goat anti-rabbit IgG antibody (Beyotime, Shanghai, China) for 1 h at room temperature. Nucleus was stained with 4',6-diamidino-2phenylindole (DAPI), and the positive fluorescence was visualized by the fluorescence microscope.

Statistical analysis
Statistical analysis was performed using Statistical Product and Service Solutions (SPSS) 22. 0 software (IBM, Armonk, NY, USA). Data were displayed as the means ± standard deviations (SD) with triplicated experiments. Differences between two groups were analyzed by using the Student's t-test. Comparison between multiple groups was done using One-way ANOVA test followed by Post Hoc Test (Least Significant Difference). P<0.05 was considered statistically significant.

a2M level decreased in degenerated NP tissue
To clarify the relation of a2M level with the NP tissue's degenerated degree, we collected 8 mildly and severely degenerated NP tissue based on the Pfirrmann score, separately. As shown in Figure 1A, the yellow arrows indicated the operation section, which was the source of our NP specimen. The height of the severely degenerated NP tissue was lower than the mild one, and the border between the NP and the annulus fibrosus was more blurred than the mild one. From the HE staining, we can see that the arrangement of ECM in the severe degeneration group is more chaotic, and the NP cells are more hypertrophic compared to the mild degeneration group ( Figure  1B). We used two methods to analyzed the a2M level in NP tissue of the two groups containing IHC staining ( Figure 1C and 1D) and ELISA ( Figure 1E), the results of which indicated that a2M expression significantly reduced in severely degenerated condition compared to the mild group. Additionally, we also measured the total ROS expression and found the severe group had a higher ROS level, as expected ( Figure 1F). Therefore, degenerated NP tissue normally expresses a lower a2M level but a higher ROS level as the degree of degeneration increases.

a2M supplement suppressed ROS of degenerated NP tissue in vitro
To determine the antioxidative effect of a2M on the NP cells, we used a2M from human plasma to culture the severe degeneration NP tissue. After 3 days, the a2M expression in NP cells of the severe group was upregulated in a dose-dependent manner resulting from the exogenic a2M stimulation (Figure  2A and 2B). Surprisingly, a2M was efficient to suppress the accumulation of ROS along as the increased a2M expression ( Figure 2C). As a widely used anti-protease, we also tested the content of MMP13 and ADAMTS4 after the application of a2M. The result of ELISA indicated that a2M significantly decreased the MMP13 and ADAMTS4 expression in dose-dependent ( Figure 2D). The data suggest that a2M acts not only an anti-protease but also an antioxidant in the degenerated NP tissue.

a2M supplement suppressed HOCl-induced ROS of degenerated NP cells in vitro
To further elucidate the effect of abundant oxidative stress on the expression of a2M and the role of a2M in the oxidative stress state, we isolated NP cells from the mild degeneration tissue and used HOCl to activate the reactive oxygen and caused NP cells oxidative stress. As shown in Figure 3A, HOCl gradually decreased the a2M expression with the increased concentration from 10 mmol/L to 50 mmol/L. In addition to this, HOCl affected the a2M level in a time-dependent manner as well, which presented a sharp drop in the first 12 hours ( Figure 3B). Furthermore, we cultured the HOCL-pretreated NP cells with exogenic a2M from 50 nmol/L to 200 nmol/L, and the total ROS gradually decreased caused by the stimulation of a2M ( Figure 3C). Besides, the suppression of ROS based on a timedependent manner was also observed ( Figure 3D).
These results indicate that the accumulation of ROS caused by HOCl truly affects the a2M expression of NP cells, however, the supplement of a2M also rejects the HOCl-induced oxidative stress of NP cells.

a2M supplement protected antioxidative enzymes expression in HOCl-treated NP cells
The accumulation of ROS results from the imbalance between the generation of oxygen radicals and the antioxidant. Therefore, we concerned whether a2M played a role in the regulation of antioxidative enzymes such as SOD, CAT, and GPX. We pretreated NP cells with HOCl to trigger the vast ROS, which obviously reduced the mRNA expression of SOD1, SOD2, CAT, and GPX1 ( Figure 4A). However, the exogenic stimulation of a2M effectively promoted these antioxidative enzymes expression, suggesting the antioxidative effect of a2M might be related to the promotion of antioxidative enzymes. The disorder of ECM is the main character of IVDD containing the loss of collagen II and aggrecan. MMPs and ADAMTS regulate the dynamic balance of ECM and can degrade different ECM components. We found the excessive ROS could increase the MMP3, MMP13, ADAMTS4, and ADAMTS5 mRNA level, which also was reversed by the a2M treatments ( Figure 4B). Finally, the protein expression of collagen II ( Figure 4C) and aggrecan ( Figure 4D) was determined by IF. HOCl significantly decreased the collagen II and aggrecan compared to the control. After using the a2M, the collagen II and aggrecan content were upregulated again compared to the HOCl group. Therefore, we think a2M plays a role in the promotion of antioxidative enzymes and inhibition of protease, which leads to the protection of collagen II and aggrecan synthesis of NP cells.

Discussion
a2M was originally used as a protease inhibitor to clear exogenous and excessive endogenous proteases from tissues, which contains almost all types of proteases (14). In addition to being a broad-spectrum protease inhibitor, a2M has many other functions: as a carrier of some small molecules such as cytokines and growth factors, including TGF-b, TNF, IFNg, PDGF (15), FGF, IL-6 (16), and NGF; regulate cell apoptosis (17); regulate thrombin activity and fibrin hydrolysis (18); regulate cell proliferation, adhesion, and migration (19); regulation of oxidative stress, which is one of the pathology of IVDD. The toxic effects of oxidative stress on the body are manifested in lipid peroxidation of biological membranes, denaturation of intracellular proteins and enzymes, and DNA damage, causing abnormal cell metabolism and eventually leading to cell death or apoptosis (20). ROS is a class of free radicals closely related to oxidative stress, mainly including superoxide anions (O 2 -), hydroxyl radicals (·OH), H 2 O 2 , NO, and HOCl, which can be produced by the reaction of mitochondria and catalase in cells and can also be induced under special physical and chemical environments (21).
To uncover the relation of a2M in the IVDD, we tested the a2M expression in NP tissue of different degradation from 16 patients. The findings indicated a2M decreased along with the IVDD. To further clarify the antioxidative function of a2M in the IVDD, we cultured the NP tissue with a2M, which presented a ROS inhibition compared to the control. Apart from this, we also isolated NP cells and established an oxidative stress model by HOCl. HOCl is a strong oxidant in ROS, that mainly produced by activated neutrophils and macrophages. It has a strong bactericidal effect and its bactericidal ability is about 50 times that of H 2 O 2 (22). The basic level of HOCl is positive for the body to resist the invasion of foreign pathogens, but when the level of HOCl is too high, it will induce oxidative stress to cause damage to the body (23). Hence, we used HOCl to cause the oxidative stress in NP cells as well as the reduction of a2M, which was consistent with the previous study (24). Hopefully, the additional supplement of a2M performed an excellent effect on the inhibition of ROS caused by HOCl with a dose and time-dependent.
Apart from the excessive ROS, the weak activity of antioxidative enzymes also contributes to the destruction of the oxidative balance in cells. Therefore, we also analyzed the gene expression of SOD1/2, CAT, GPX1 under the treatment of HOCl and a2M. Despite the continuous production of ROS, the body can still fight oxidative stress through an antioxidant system, mainly including enzymatic and non-enzymatic mechanisms to antagonize the oxidation response. The main endogenous enzymatic antioxidants are SOD, CAT, GPX, peroxidase, glutathione reductase, among which SOD and CAT are the main anti-ROS enzymes (25). They together form the body's antioxidant defense system. The balance between ROS and the antioxidant defense system is a necessary condition for maintaining the homeostasis of the disc (26). In our study, HOCl suppressed the SOD1/2, CAT, and GPX1 mRNA expression, but a2M showed a positive effect on their expression, which strengthened the antioxidative force in the degenerated NP cells. The antiprotease activity of a2M has been fully confirmed (27). Wang et al. (7) found a2M inhibited MMP13 expression in the prevention of posttraumatic osteoarthritis. Tortorella et al. (28) reported a2M was an endogenous inhibitor of ADAMTS4 and ADAMTS5 in osteoarthritis. In this present study, a2M was verified to suppress the MMP and ADAMTS both in the severely degenerated NP tissue and in the HOCl-treated NP cells. Therefore, the antioxidative and antiprotease effect of a2M contributes to the protection of collagen II and aggrecan expression, which is meaningful to the prevention of IVDD.

Conclusions
In conclusion, a2M participates in the development of IVDD, and it not only has anti-inflammatory effects but also has the antioxidative behavior relating to the upregulation of the antioxidative enzyme production. However, its specific mechanism still needs further research. a2M as a therapeutic drug has good clinical application prospects, but obtaining a2M with high purity, large yield, reasonable price, and more safety is also the direction that needs to be worked in the future.

Conflict of interest statement
All the authors declare that they have no conflict of interest in this work.