Antioksidativno ponašanje a2-makroglobulina kod degeneracije intervertebralnog diska
Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration
Chen Yuhonga, Wei Huaixiangb, Xu Fengc
aChun'an County Hospital of Traditional Chinese Medicine, Department of Orthopedics, Hangzhou, China
bXiangxi Tujia and Miao Autonomous Prefecture People's Hospital, Department of Pain, Jishou, China
cBoji Hospital Changxing Zhejiang Province, Huzhou, China
Ključne reči: antioksidans; nukleus pulposus ćelije; degeneracija intervertebralnog diska; a2-makroglobulin; ekstracelularni matriks
Keywords: antioxidant; nucleus pulposus cells; intervertebral disc degeneration; a2-macroglobulin; extracellular matrix
Uvod: Cilj rada je bio da razjasni da li a2-makroglobulin (a2M) ima antioksidativni efekat tokom progresije degeneracije intervertebralnog diska (IVDD). Metode: Izmeren je sadržaj a2M i reaktivnih vrsta kiseonika (ROS) da bi se uporedilo blago i teško degenerisano ljudsko nukleus pulposus (NP) tkivo imunohistohemijom, masenom spektrometrijom i enzimskim imunosorbentnim testom (ELISA). Pored toga, egzogeni a2M je korišćen za kultivaciju teško degenerisanog NP tkiva in vitro. Procenjeni su efekti a2M na NP ćelije tretirane hipohloritom (HOCl), koje sadrže antioksidativne enzime, nivo ROS, kolagen II i ekspresiju agrekana, MMP3/13 i ADAMTS4/5. Rezultati: Povećan nivo ROS kod teško degenerisanog NP, praćen je smanjenim sadržajem a2M. Dodatak a2M mogao bi smanjiti nivo ROS kultivisanog NP in vitro, u međuvremenu, ekspresija MMP13 i ADAMTS4 je takođe smanjena. Utvrđeno je da tretman HOCl dovodi do oksidativnog oštećenja NP ćelija i smanjuje ekspresiju a2M na način koji zavisi od doze i vremena. Štaviše, egzogena a2M stimulacija je preokrenula akumulaciju ROS-a izazvanu HOCl. Takođe je primećena promocija SOD1/2, CAT, GPKS1, kolagena II i agrekana i supresija ekspresije MMP3/13, ADAMTS4/5 izazvane a2M. Zaključak: Naša studija pokazuje da a2M ima antioksidativnu sposobnost u degenerisanim NP ćelijama promovišući proizvodnju antioksidativnih enzima.
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 enzyme-linked 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. Conclusions: Our study indicates that a2M has an antioxidative ability in degenerated NP cells by promoting the antioxidative enzyme production.
The intervertebral disc has no blood supply and consists of a peripheral annulus fibrosis, a central nucleus pulposus (NP), and upper and lower endplates [
α2-macroglobulin (α2M) 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 [
Our present study aimed to clarify the role of α2M in IVDD, especially its antioxidative behavior,using cultured NP tissue and NP cells
Source of patient samples
To clarify the difference of α2M 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 [
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
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, α2M 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 μm 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.
Sections were suffered as follows: dewaxed, hydrated, and blocked with 10% goat serum for 1 hour. Sections were then incubated with α2M (ab58703, Abcam, Cambridge, MA, USA) primaryantibody overnight at 4°C. After incubated with biotinylated IgG and Elite ABC reagent (Beyotime, Shanghai, China), sections finally were developed by 3, 3-diaminobenzidine and counterstained with hematoxylin.
Enzyme-linked immunosorbent assay (ELISA)
The levels of α2M, 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 content of NP tissue or NP cells was determined using
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from NP cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed to cDNA templates by PrimeScript™ RT Mix (RR036A, TaKaRa, Tokyo, Japan). RT-PCR assay was performed to assay relative gene expression containing superoxide dismutase1 (SOD1), SOD2, catalase (CAT), glutathione peroxidase1 (GPX1), MMP3, MMP13, ADAMTS4, and ADAMTS5 by normalization of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) according to 2−ΔΔCt methods.
Table 1. Primer sequences of the genes for RT-PCR. RT-PCR, quantitative reverse-transcription polymerase chain reaction
Gene name Forward (5’>3’) Reverse (5’>3’) SOD1 GGTGGGCCAAAGGATGAAGAG CCACAAGCCAAACGACTTCC SOD2 GGAAGCCATCAAACGTGACTT GCGTTGATGTGAGGTTCCAG CAT TGGAGCTGGTAACCCAGTAGG CCTTTGCCTTGGAGTATTTGGTA GPX1 CAGTCGGTGTATGCCTTCTCG GAGGGACGCCACATTCTCG MMP-3 AGTCTTCCAATCCTACTGTTGCT TCCCCGTCACCTCCAATCC MMP13 ACTGAGAGGCTCCGAGAAATG GAACCCCGCATCTTGGCTT ADAMTS4 GAGGAGGAGATCGTGTTTCCA CCAGCTCTAGTAGCAGCGTC ADAMTS5 GAACATCGACCAACTCTACTCCG CAATGCCCACCGAACCATCT GAPDH ACAACTTTGGTATCGTGGAAGG GCCATCACGCCACAGTTTC
Table 1. Primer sequences of the genes for RT-PCR.
RT-PCR, quantitative reverse-transcription polymerase chain reaction
The primers of the genes were listed in
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-2-phenylindole (DAPI), and the positive fluorescence was visualized by the fluorescence microscope.
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).
α2M level decreased in degenerated NP tissue
To clarify the relation of α2M 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 1 α2M level decreased in degenerated NP tissue. Representative images of (A) MRI, the yellow arrows indicated the operation section, (B) HE staining (magnification: 200×) (C) IHC targeting α2M of both mildly and severely degenerated NP tissue. (magnification: 200×) (D) Quantification analysis of IHC. NP tissue from the 16 patients was lysed to measure (E) α2M with ELISA methods and (F) total ROS level. The values are mean ± SD of three independent experiments. (***P<0.001)
Download as PPT slide
Download as image
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 (
α2M supplement suppressed ROS of degenerated NP tissue in vitro
To determine the antioxidative effect of α2M on the NP cells, we used α2M from human plasma to culture the severe degeneration NP tissue. After 3 days, the α2M expression in NP cells of the severe group was upregulated in a dose-dependent manner resulting from the exogenic α2M stimulation (
Figure 2 α2M stimulation decreased ROS, MMP13, and ADAMTS4 of severely degenerated NP tissue. We cultured severely degenerated NP tissue in α2M (50, 150, 200 nmol/L) growth medium for 3 days. The protein expression level of α2M was determined by (A) IHC (magnification: 200×) and (B) quantification analysis. (C) Total ROS level of NP tissue. (D) The content of MMP13 and ADAMTS4 was assayed by ELISA. The values are mean ± SD of three independent experiments. (*P<0.05, **P<0.01)
Download as PPT slide
Download as image
Surprisingly, α2M was efficient to suppress the accumulation of ROS along as the increased α2M expression (
α2M supplement suppressed HOCl-induced ROS of degenerated NP cells in vitro
To further elucidate the effect of abundant oxidative stress on the expression of α2M and the role of α2M 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 3 α2M stimulation reversed HOCl-induced oxidative stress of NP cells. NP cells of mildly degenerated NP tissue were treated with HOCl (from 10 to 50 μmol/L) for 6 h, or treated with 30 μmol/L from 3 h to 24 h; Besides, NP cells were pretreated with 30 μmol/L HOCH for 6 h and then cultured with α2M (from 50 to 200 nmol/L) for another 24 h, or cultured with 200 nmol/L α2M from 3 h to 24 h. (A, B) The protein expression level of α2M was determined by ELISA. (C, D) Total ROS level of NP cells. The values are mean ± SD of three independent experiments.
Download as PPT slide
Download as image
In addition to this, HOCl affected the α2M level in a time-dependent manner as well, which presented a sharp drop in the first 12 hours (
α2M 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 α2M 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 4 α2M level decreased in degenerated NP tissue. Representative images of (A) MRI, the yellow arrows indicated the operation section, (B) HE staining (magnification: 200×) (C) IHC targeting α2M of both mildly and severely degenerated NP tissue. (magnification: 200×) (D) Quantification analysis of IHC. NP tissue from the 16 patients was lysed to measure (E) α2M with ELISA methods and (F) total ROS level. The values are mean ± SD of three independent experiments. (***P<0.001)
Download as PPT slide
Download as image
However, the exogenic stimulation of α2M effectively promoted these antioxidative enzymes expression, suggesting the antioxidative effect of α2M 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 α2M treatments (
α2M was originally used as a protease inhibitor to clear exogenous and excessive endogenous proteases from tissues, which contains almost all types of proteases [
To uncover the relation of α2M in the IVDD, we tested the α2M expression in NP tissue of different degradation from 16 patients. The findings indicated α2M decreased along with the IVDD. To further clarify the antioxidative function of α2M in the IVDD, we cultured the NP tissue with α2M, 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 H2O2 [
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 α2M. 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 [
In conclusion, α2M 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. α2M as a therapeutic drug has good clinical application prospects, but obtaining α2M 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.