Omilancor mitigates the senescence of nucleus pulposus cells induced by DDP through targeting MAP2K6

MAP2K6 exhibited heightened expression levels in IDD

A comprehensive assessment was undertaken by analysing a collective compilation of three mRNA microarray datasets (including 8 healthy controls and 8 IDD patients) sourced from the Gene Expression Omnibus (GEO) repository to unearth the differentially expressed genes (DEGs) in IDD. A mean fold change >5 or <0.2 and p-values <0.01 were taken as criteria. As a result, 627 DEGs including 398 upregulated and 229 downregulated DEGs were identified in IDD samples compared with healthy controls (Figure 1A, 1B). And then, to unravel the underlying roles of DEGs in cellular senescence, these DEGs were intersected with senescence-related genes cataloged in KEGG database (Figure 1C). To substantiate these findings, 2 upregulated genes (MAP2K6, BRCA2) and 1 downregulated gene (SERPINE1) were examined by qRT-PCR analysis employing an independent cohort encompassing 3 IDD patients and 3 controls. MAP2K6 was found to be significantly upregulated in nucleus pulposus (NP) tissues extracted from IDD patients in contrast to those obtained from control patients (Figure 1D). This observation was further corroborated by immunofluorescence (IF), western blot and quantitative analyses (Figure 1E–1H). Therefore, we selected MAP2K6 for further investigation. These data collectively suggest a potential pivotal role for MAP2K6 in the progression of IDD.

Figure 1. MAP2K6 exhibited heightened expression levels in IDD. (A) Volcano plot illustrating the differential gene expression in human NP samples Note: GSE186542, GSE185728, GSE167199, (Control group [CTR]: n = 8, Intervertebral Disc degeneration [IDD]: n = 8). (B) Heatmap depicting the top 20 differentially expressed genes (DEGs) in human NP. (C) Venn diagram illustrating the intersection of DEGs between the CTR and IDD groups and senescence-related genes. (D) RT-qPCR results presenting the expression levels of candidate genes (MAP2K6, BRCA2, and SERPINE1) in degenerative NP cells. (E, F) Immunofluorescence staining (IF) and corresponding quantitative analysis of MAP2K6 in CTR and IDD human NP samples. (Original magnification 100×, scale bar = 400 μm; Red arrow: positive cells). (G, H) Western Blot analysis and quantitative assessment of MAP2K6 expression in human NP samples. CTR, control group; IDD, intervertebral disc degeneration group. n = 3 each group. Data are represented as mean ± standard deviation. ***p < 0.001, as determined by t-test.

MAP2K6 was upregulated in DDP-induced cellular senescence model

IL-1β and TNF-α are cytokines intricately involved in the pathogenesis of IDD, and frequently harnessed to establish an in vitro cellular model simulating the degenerative process. Cisplatin (DDP), known to initiate the DNA damage signaling cascade and activate sensory proteins triggering cellular senescence, has been widely applied in the establishment of cellular senescence models. Therefore, we first detected SA-β-gal activity to evaluate abovementioned modeling methods and found out that DDP was the ideal treatment for the construction of a robust cellular senescence model (Figure 2A, 2B). Subsequently, to investigate the role of MAP2K6 in the pathogenesis of IDD, qRT-PCR was used to ascertain the expression level of MAP2K6 within our cellular senescence models. Remarkably, our findings showed an upsurge in MAP2K6 expression across all cellular models, especially in DDP-induced cellular senescence model (Figure 2C). The veracity of this finding was further buttressed through IF and quantitative analyses (Figure. 2D–2F).

Figure 2. MAP2K6 was upregulated in DDP-induced cellular senescence model. (A, B) Rat NP cells were exposed to DDP (mM, 24h), IL-1β (100ng/ml, 24h), and TNF-α (50ng/ml) to establish an in vitro senescence NP cell model, which was confirmed by SA-β-gal staining and the corresponding quantitative analysis. (Original magnification 200×, scale bar = 200 μm; Red arrow: positive cells). (C) RT-qPCR results depict the expression levels of MAP2K6 following different treatments. (D, E) Immunocytochemistry (ICC) staining and the accompanying quantitative analysis of MAP2K6 in control (CTR) or DDP-treated Rat NP cell models. (Original magnification 400×, scale bar = 100 μm). (F) Western Blot analysis illustrating the expression of MAP2K6 in control (CTR) or DDP-treated Rat NP cell models. (G, H) Immunofluorescence (IF) staining of IVDs tissue in rat model and quantitative assessment of MAP2K6 expression in control (CTR) or IDD rats after 8 weeks of operation. (original magnification 400×, scale bar = 100 μm). CTR, control group; DDP, cisplatin treatment. n = 3 each group. Data are represented as mean ± standard deviation. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, as determined by t-test.

To investigate the role of MAP2K6 in the progression of IDD in vivo, we resorted to annulus fibrosus (AF) puncture-induced IDD rat model and found out that the expression level of MAP2K6 was significantly increased in the IDD group (Figure 2G, 2H). In summary, these findings underscore the plausible centrality of MAP2K6 in cellular senescence and IDD.

Omilancor reduced cellular senescence by regulating MAP2K6

Subsequently, we embarked on an investigation for the potential of a drug or compound to alleviate IDD by regulating MAP2K6-mediated cellular senescence. AlphaFold2 software was used to predict the structure of MAP2K6 and assess its reliability. Then, we used a natural drug library of 4948 compounds to screen out the ones with good docking with MAP2K6, docking prediction analysis showed that several compounds from the ligand library exhibited a great capability to interact with MAP2K6 (Figure 3A). In alignment with the predicted free energy of binding results, qRT-PCR analysis demonstrated that Omilancor significantly downregulated the expression of MAP2K6 in NP cells treated with DDP (Figure 3B). Subsequently, we predicted and visualized the binding sites and druggable sites by AutoLigand and PyMOL (Figure 3C, 3D).

Figure 3. Omilancor reduced cellular senescence by regulating MAP2K6. (A) The top 5 drugs and their corresponding binding energies (kcal/mol) predicted as potential inhibitors for MAP2K6. (B) RT-qPCR results revealing that Omilancor exhibited the most significant inhibitory effect on MAP2K6 expression (5ug/ml, 12h, n=3 each group). (C, D) Ligand interaction diagrams depicting the top-scoring molecular docking complexes between OMLC and MAP2K6 proteins (RMSD = 2.206; estimated free energy of binding = -12.1 kcal/mol). (E) Western blot analysis illustrating the expression levels of MAP2K6 and P21 following treatments with DDP and OMLC. (F) Immunocytochemistry (ICC) staining demonstrating the expression of MAP2K6 in rat NP cells subjected to various treatments. (Original magnification 400×, scale bar = 100 μm). (G, H) SA-β-gal staining and the corresponding quantitative analysis of rat NP cells under different treatment conditions. (Original magnification 100×, scale bar = 400 μm). Red arrow: positive cells). CTR, control group; DDP, cisplatin treatment; OMLC, Omilancor treatment. n = 3 each group. Data are represented as mean ± standard deviation. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA with post-hoc Bonferroni correction or Kruskal-Wallis H test with a Dunn’s correction.

To validate its potential in attenuating cellular senescence in vitro, we employed Omilancor as a pre-treatment for NP cells followed by subsequent DDP treatment. Western blot analysis suggested that pre-treatment with Omilancor significantly reduced the expression levels of MAP2K6 and p21, a recognized inhibitor of the cell cycle, compared to NP cells treated with DDP alone (Figure 3E). This result was further substantiated by subsequent IF experiments (Figure 3F). The SA-β-gal analysis revealed that pre-treatment with Omilancor notably inhibited DDP-induced cellular senescence in vitro (Figure 3G, 3H). Taken together, these results indicated that Omilancor mitigated cellular senescence, thereby alleviating IDD by downregulating the expression of MAP2K6 in vitro.

Omilancor alleviated senescence mediated IDD in vivo

To elucidate whether Omilancor could act as an effective therapeutic agent for IDD in vivo, we introduced Omilancor to the AF puncture-induced IDD rat model. Omilancor or PBS was locally injected into the punctured intervertebral disc during the surgical procedure. Subsequent to an 8-week observation period, radiological examination results indicated that locally administered Omilancor significantly alleviated the degeneration of IVDs (Figure 4A–4D), this encouraging radiological findings were further substantiated by histological assessments using H&E staining (Figure 4E). Moreover, IF results demonstrated that the expression levels of both MAP2K6 and P21 were significantly decreased in the Omilancor-treated group compared with the control or the PBS-administered group (Figure 4F).

Figure 4. Omilancor alleviated senescence mediated IDD in vivo. (A, B) X-ray images and disc height analysis of rats from the CTR, AFP plus PBS, and AFP plus OMLC groups, taken 8 weeks post-operation. (C, D) MRI images and Pfirrmann grade analysis of rats from the CTR, AFP plus PBS, and AFP plus OMLC groups, obtained 8 weeks after surgery. (E, F) H&E and IF staining depicting MAP2K6 and P21 in rats from the CTR, AFP plus PBS, and AFP plus OMLC groups, conducted 8 weeks post-operation. (Original magnification 200×, scale bar = 200 μm). CTR, control group; AFP, annulus fibrosus puncture group; n = 3 each group. Data are represented as mean ± standard deviation. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA with post-hoc Bonferroni correction or Kruskal-Wallis H test with a Dunn’s correction.

These results collectively indicated that Omilancor could impede the progression of cellular senescence by suppressing the expression of MAP2K6. This nuanced modulation, in turn, exerts a mitigating influence on IDD. Consequently, these findings supported the hypothesis that regulating MAP2K6 is a promising strategy for biological therapy of IDD.

NP tissues collection

A total of 6 NP samples was obtained from 6 patients (male: female=3: 3) underwent spinal surgery at the Luoyang Central Hospital. The degree of disc degeneration was graded according to the Pfirrmann system, based on T2-weighted images assessed by three independent observers. We collected degenerative NP tissues from disc herniation cases, which were graded as III, IV, or V. Control NP tissues, graded as I or II, were obtained from fresh traumatic lumbar fracture or hemivertebra cases (Table 1 and Supplementary Figure 1A).

GEO expression datasets and bioinformatics analysis

The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) is an international public repository that distributes high throughput gene expression datasets. To investigate the differences in expression profiling and relevant biological processes in IDD patients, we searched for datasets in the GEO database using the keywords: (1) “intervertebral disc degeneration” [All Fields] AND (2) “Homo sapiens” [porgn] AND (3) “gse” [Filter] AND (4) “2015/01” [Update Date]. The inclusion and exclusion criteria were as follows: datasets should be the whole-genome expression data of mRNA; expression data should be obtained from human annulus disc tissue of IDD patients and healthy controls. Finally, we included a total of three mRNA expression datasets (including GSE186542, GSE185728, GSE167199) in our study. The detail information of above-mentioned datasets was shown in (Table 2 and Supplementary Table 1).

Identifying essential cell Senescence regulator genes in IDD. In brief, 114 cell senescence regulator genes were obtained from the CellAge database (https://genomics.senescence.info/cells/). Finally, we took an intersection of the differentially expressed genes in IDD and cell senescence regulator genes and got a total of 3 essential cell senescence regulator genes (MAP2K6, BRCA2, and SERPINE1) in IDD for further analysis. The expression datasets were analyzed by the R software version 4.0.0 (http://www.r-project.org). After quantile normalization of the raw data, we identified differentially expressed mRNAs with statistical significance between the two groups using Volcano Plot and Heat Map filtering.

qRT-PCR

Total RNA extraction from NP tissue or cultured NP cells was performed with TRIzol Reagent (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. A Nanodrop (Thermo Fisher Scientific, USA) was used to analyze the RNA quantity. Then, the mRNA was converted to complementary DNA (cDNA) using a reverse transcription kit (Takara, Cat. #RR047A, Japan). Relative expression levels were analyzed using the comparative Ct (ΔΔCT) method (2^−ΔΔCT logarithm transformed). GAPDH was used to normalize mRNA expression level. The following primers were used: human MAP2K6 (F: 5’-GCCACAGTTAATAGCCAGGAA-3’, R: 5’-GCCCACATATCCAGAGCTTAT-3’), human BRCA2 (F: 5’-CGGATGGAAGAGAGACTCAAA-3’, R: 5’-ACAGGAAATTGAACAGCGATT-3’); human SERPINE1 (F: 5’-CAGTGGCTACCTGGTCGACGAAGTTGCTAAAGAGATCGATGCCGGTTGCAAACCATAC-3’); human MAP2K6 (R: 5’-GTATGGTTTGCAACCGGCATCGATCTCTTTAGCAACTTCGTCGACCAGGTAGCCACTG-3’); and human GAPDH (F: 5’-GACAGTCAGCCGCATCTTCTT-3', R: 5'-AATCCGTTGACTCCGACCTTC-3').

Western blot analysis

Total protein from NP tissue or cultured NP cells was extracted using RIPA lysis buffer supplemented with protease inhibitors cocktail (Thermo Fisher Scientific, USA). The protein concentrations were detected using a BCA Protein Assay Reagent Kit (KeyGEN BioTECH, China) according to the manufacturer’s instructions. Then, the lysates were separated on 8-12% sodium dodecyl sulphate-polyacrylamide gels (Epizyme Biotech, China), and transferred to 0.22μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were blocked for 2 h in TBS-T with 5% bovine serum albumin (BSA), and incubated with primary antibodies (MAP2K6, diluted 1:5000, Cat. #12280, Cell Signaling Technology, USA; p21, diluted 1:5000, Cat. #ab109199, Abcam, USA; β-Tubulin, diluted 1:1000, Cat. #5666, Cell Signaling Technology, USA) at 4° C overnight. After being washed with TBS-T, the membranes were incubated with HRP-linked anti-rabbit immunoglobulin G (IgG; diluted 1:5000, Cat. #7074, Cell Signaling Technology, USA) or anti-mouse IgG (diluted 1:5000, Cat. #7076, Cell Signaling Technology, USA) for 2 h. Finally, the blots were visualized with enhanced chemiluminescence (ECL) reagents (Millipore, USA). The results of this experiment were quantified by gray analysis using Image J software.

Culture of NP cells

Primary NP cells were freshly isolated as described in our previous studies with minor modification [38]. The NP tissue was dissected and minced, and then digested with 0.75 mg/ml collagenase (Sigma-Aldrich, USA) for 30 min at 37° C. The suspension was filtered (pore size, 100 μm) and resuspended with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) and 20% fetal bovine serum (FBS; Biological Industries, Israel). Subsequently, the cells were plated into 75 cm2 cell culture flasks. After 6-7 days, the cultured primary NP cells were passage into 10 cm cell culture plates (Corning, USA) with DMEM, 10% FBS and antibiotics (Gibco, USA). DDP (Sigma Aldrich, USA) was dissolved in DMF at a concentration of 10 mM.

To simulate cellular senescence, the cells were treated with 2×10^-6M DDP for 48 hours. RT-qPCR were conducted to validate the optimal concentration and treatment duration (Supplementary Figure 1B, 1C). Subsequently, the cells were treated with 0, 1, 2, 5, 10 or 20 ug/mL Omilancor for 0, 3, 6, 12, 24 or 48 hours to determine the optimal concentration and treatment duration. (Supplementary Figure 1D, 1E).

Senescence-associated β-galactosidase (SA-β-gal) staining and quantification

Cells were washed to remove drugs and incubated for an additional 3 days in fresh media. In situ staining of SA-β-gal with cells or frozen sections were performed using a SA-β-gal staining kit (Beyotime Institute of Biotechnology, China) following the manufacturer’s instructions. Cells were considered positive when the cytoplasm was stained with SA-β-gal. All experiments were performed in triplicate.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde in PBS for 15 min, after washed gently by PBS, cells were blocked by 10% goat serum (Thermo Fisher) for 1 h. Subsequently, cells were then incubated with MAP2K6 (diluted 1:200, Cat. #12280, Cell Signaling Technology, USA) or p21 (diluted 1:200, Cat. #2947, Cell Signaling Technology, USA) antibody over night at 4° C. The next day, the cells were incubated with fluorescent secondary antibody for 2 h at room temperature, and then counterstained with DAPI. The digital images of cells were taken using a Leica TCS SP8 microscope (Wetzlar, Germany). Fluorescence intensity was measured using ImageJ, and the count data were analyzed using the chi-square test.

Molecular docking screening and visualization

AutoDock is a suite of free open–source software for the computational docking and virtual screening of small molecules to macromolecular receptors. The structure of MAP2K6 was predicted using AutoDockTools-1.5.6. The model quality was assessed using the predicted aligned error. The molecular dockings and virtual screening of the predicted MAP2K6 and potential ligands structures were simulated and analyzed with AutoDock Vina and AutoLigand, and visualized with PyMOL 2.3.2 (Supplementary Table 2).

Animal experiment

An IDD rat model was successfully established as described in previous studies [39, 40]. In short, adult Sprague Dawley Rats (SD rats) (Shanghai Laboratory Animal Center, Shanghai, China) weighing approximately 200-250 g at about 10 weeks were used in this experiment. The procedures were approved by the Medical Ethics Committee of Luoyang Central Hospital (Luoyang, China). The rats were randomly divided into three groups: for the AF puncture model, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) and placed in the supine position. A midline abdominal incision was made in the front to visualize the L3/4 intervertebral discs. A 27-G needle was inserted into the L3-L4 disc parallel to the endplates by 1.0 mm, rotated in the axial direction by 180° and held for 30 s, and a vessel clamp was used to grip on the marker on the needle to control the depth.

For the Omilancor treatment, Omilancor (BioLegend, USA) was intraperitoneally injected at 4 mg/kg every two days, for a total duration of two weeks before operation.

After 8 weeks, the rats were anesthetized with pentobarbital sodium. After lumbar MRI examinations, discs were harvested for further experiments.

Statistical analysis

Each experiment is repeated at least three times independently. Data are presented as mean ± standard deviations. Data from multiple groups were compared using One-way analysis of variance (ANOVA). Student’s t test was used to analyze the differences between two groups. Statistical analyses were performed using SPSS software version 24.0 for Windows (IBM, USA). p < 0.05 was considered to indicate statistically significant.

Data and materials availability

All data of this study are included within the article.