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. 2020 Jun 17;19(14):1727–1739. doi: 10.1080/15384101.2020.1769301

Mesenchymal stem cells-derived exosomes ameliorate nucleus pulposus cells apoptosis via delivering miR-142-3p: therapeutic potential for intervertebral disc degenerative diseases

Lifan Zhu a, Yuhui Shi a,b, Ling Liu a, Huan Wang a, Pengcheng Shen a,b, Huilin Yang a,✉
PMCID: PMC7469501  PMID: 32635856

ABSTRACT

Intervertebral disc degeneration (IDD) is the main cause of lower back pain (LBP), and puzzles massive individuals worldwide. Mesenchymal stem cells (MSCs) transplantation has been demonstrated to potentially ameliorate IDD progression, while the underlying mechanism has not been fully explained. Interleukin-1β (IL-1β) was used to induce nucleus pulposus cells (NPCs) injury. Bone marrow MSCs-derived exosomes were isolated using the super centrifugation method, and characterized using Transmission electron microscopy (TEM) and western blot. Cell viability was determined by MTT, while apoptosis was measured by Annexin-V staining using flow cytometry. miR-142-3fp and gene expressions were measured by real-time PCR. The protein expressions were determined by western blot. Herein, we found exosomes from bone marrow MSCs are circular vesicles, about 80 nm in diameter, and with robust expression of TSG101 and CD63, but without of Calnexin. MSCs exosomes alleviated NPCs apoptosis by reducing IL-1β-induced inflammatory cytokines secretion and MAPK signaling activation. Additionally, MSCs exosomes inhibited NPCs apoptosis and MAPK signaling by delivering miR-142-3p that targets mixed lineage kinase 3 (MLK3). Overexpression of MLK3 abolished the effects of MSCs exosomes on the inflammatory condition, cell apoptosis, and MAPK signaling activation in NPCs. The results confirmed that bone marrow MSCs-derived exosomes-packaged miR-142-3p alleviates NPCs injury through suppressing MAPK signaling by targeting MLK3. The work highlights the therapeutic effect of MSCs on IDD progression, and bone marrow MSCs exosomes might be apromising therapeutic strategy for IDD.

KEYWORDS: Intervertebral disc degeneration, MSCs exosomes, miR-142-3p

Introduction

Lower back pain (LBP) is a prevalent orthopedic disease that puzzled 70–80% individuals worldwide [1]. Intervertebral disc degeneration (IDD) is attributed to the cause of LBP, and believed to contribute to the disability [2]. The process of IDD implies plenty of structurally disrupting events, such as cell nutrition declines, cell death, and matrix molecules degradation [3]. Nucleus pulpous cells (NPCs), annulus fibrosus (AF), and cartilaginous endplates (CEPs) compose of intervertebral disc. NPCs maintain the stability of intervertebral disc and mediate the external force of the spine. The degeneration of intervertebral disc mainly attributes to the degradation and death of NPCs, as well as the inflammatory condition [4,5]. With the progression of IDD, the level of IL-1β was significantly increased in nucleus pulpous (NP), moreover, IL-1β treated NPCs showed an increased level of inflammatory mediators, such as cyclooxygenase-2 (COX-2), nitric oxide (NO), and NO synthase (NOS). In addition, IL-1β treated NPCs manifest increased cell apoptosis [6]. Therefore, to find an effective method to inhibit cell apoptosis and inflammation in NPCs is necessary.

Recently, therapeutic strategies for IDD can partially relieve the symptoms. Novel strategies, such as gene-target therapy and cell engineering approach are been developed to alleviate IDD or enhance intervertebral disc regeneration. Mesenchymal stem cells (MSCs)-based therapy has been considered to offer huge potential for the treatment of IDD [7]. A recent study shown the vesicles of MSCs could ameliorate IDD by suppressing inflammatory mediators in NPCs [5]. Nevertheless, the potential mechanism underlying MSCs-induced repair on NPCs remains unclear.

Accumulating evidence showed that exosomes, the vesicles with a diameter of 30–200 nm, are the messengers between cell-cell communications [8]. It has been found that exosomes can be originated from all kind of cells and released to the extracellular environment, and then merged to the adjacent cells to influence the recipient cells by delivering various contents, such as cytokines, proteins, lipids, RNA, and DNA [9]. The bone marrow-derived mesenchymal stem cells (MSCs) belongs to the MSCs, and MSCs-secreted exosomes are likely to affect the death of NPCs [10]. In addition, MSCs transplantation has been shown to regenerate the IDD in the pig model [7], and exosomes from MSCs promote bone regeneration [11]. What’s more the interaction between MSCs and NPCs showed appropriate for IDD therapy [12]. However, the mechanism by which of MSC-derived exosomes on NPCs is currently unclear.

MicroRNA (miRNA) is a kind of non-coding RNA with the length of less than 22 nucleotides and modulates gene expression in a post-transcription manner by binding to the 3ʹUTR region of mRNA. Various reports demonstrated that miRNA level is associated with the NPCs apoptosis and proliferation, extracellular matrix regeneration, and inflammatory response [13–16]. It has been reported that exosomes delivering miRNAs are attractively in searching for the mechanism underlying alleviating diseases [17]. Small RNA sequencing revealed that miR-142-3p is involved in the exosomes of bone marrow MSCs, and can be transferred to the target cells for gene communication [18–20]. While whether miR-142-3p in MSCs-derived exosome-mediated NPCs physiological status still not yet known.

The mitogen-activated protein kinase (MAPK) family is comprised by ERK1/2, JNK, and p38 MAPK proteins. Studies elucidated that ERK is mediated to inflammatory reactions and cell proliferation [21,22]. p38 MAPKs serves as a proinflammatory mediator and modulates cell apoptosis and inflammatory [23–25]. JNK is a neurotrophic factor that mediates cell apoptosis [26]. Therefore, MAPK signaling is involved in the inflammatory response and cell functions, such as cell proliferation, differentiation, and apoptosis. Mixed lineage kinase 3 (MLK3) is a kind of serine/threonine MAPK kinase kinase (MAP3 K), and ubiquitously expressed in the mammal. Of note, MLK3 activates the ERK MAPK pathway and is required in the cell migration and invasion in various diseases [27–29]. However, whether MLK3 and MAPK pathway are involved in MSCs-ameliorated IDD still need further identification.

In the present study, as a possible mediator of MSCs-derived exosomes in IDD, miR-142-3p is studied as a therapeutic potential target for NPCs injury. We verified that miR-142-3p could alleviate NPCs injury in IDD by targeting MLK3 and further regulating the activation of MAPK pathway.

Materials and methods

Cell culture

The bone marrow was obtained from C57BL/6 mice. The MSCs were isolated from bone marrow using adherent culture methods. The NP tissues obtained from C57BL/6 mice were pretreated by 0.25% tyrosinase and 0.2% collagenase type II to separate the NPCs. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, Sigma Aldrich, MO, USA) containing 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA). The culture environment was maintained at 37°C with 5% CO2 at a humidified atmosphere. The medium was changed every 3 days, and the cells in the third passenger were used for the following experiments. The study was approved by the Ethic Committee of The First Affiliated Hospital of Soochow University, and all experiments were conducted following the standards of animal experiments.

Exosome isolation and determination

The Bone marrow MSCs-derived exosomes were isolated using the super centrifugation methods. Briefly, after BMSCs were grown to passage 3, the medium was centrifugated at 300 g for 10 min, and the supernatant was harvested for the centrifugation at 2000 g for 20 min to eliminate cells and debris. To obtain vesicles, the supernatant was centrifugated at 20,000 g for 30 min, followed by filtrating through a 0.22-μm filter to remove particles larger than 200 nm. The ultracentrifugation was performed using the Optima L-100xp ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 120,000 g for 2 h. All procedures were processed at 4°C, and the isolated exosomes that resuspended by PBS were stored at −80°C. Exosomes were visualized using Transmission electron microscopy (TEM), identified by measuring the expression of TSG101, CD63, and Calnexin using western blot. The exosome size was determined by nanoparticle tracking analysis [30] with dedicated mouse anti-human monoclonal antibodies (MoAbs).

TEM analysis

Exosomes were fixed by 2.5% glutaraldehyde at pH 7.2 at 4°C for 24 h, and then fixed in 1% osmium tetroxide for 60 min at room temperature. The samples were embedded into 10% gelatin and fixed in glutaraldehyde at 4°C. After that, the sample blocks were dehydrated by an increasing concentration of alcohol (30%, 50%, 70%, 90%, 95%, and 100%) for 10 min, followed by the increasing concentration of propylene oxide. Next, the samples were infiltrated by a series of Quetol-812 epoxy resin (25%, 50%, 75%, and 100%) mixed with propylene oxide for 3 h at each step, and then embedded in the Quetol-812 epoxy resin and polymerized at 35°C for 12 h, 45°C for 12 h, and 60°C for 24 h. The samples were stained by the uranyl acetate for 10 min and lead citrate for 5 min. Exosomes morphology was observed using a TEM.

The determination of exosomes size

For the determination of exosome diameter, Nanoparticle tracking analysis (NTA) was conducted. First, the purified exosomes were infiltrated to an aldehyde/sulfate latex bead. After that, the exosome diameter was visualized using an Nanosight NS500 according to the manufacturer’s instruction.

Fluorescent labeling of exosomes

Purified exosomes in PBS were labeled using PKH67 green membrane dye (Sigma-Aldrich, St. Louis, MO). Briefly, 20 μg exosomes were incubated with 5 μl of PKH67 within 1 ml of diluent C for 15 min at room temperature. Next, to stop the labeling, 1 ml of 1% bovine serum albumin (BSA) was added, followed by adding to 22 ml using PBS. The mixture was centrifuged at 110,000 g for 80 min at 4°C, and the supernatant was removed. Finally, the pellets of PKH67-labeled exosomes were resuspended in 100 μl of PBS and observed under a fluorescence confocal microscope.

Cell apoptosis

For determine apoptosis, cells received the treatments of IL-1β (10 ng/mL), or IL-1β+exosome (50 μg/mL) or IL-1β+miR-142-3p mimic, or IL-1β+exosome+pcDNA-MLK3 for 24 h. After that, cells were resuspended in the binding buffer and double-stained with 0.5 μg/mL Annexin-V-FITC for 10 min. Apoptosis was analyzed by a flow cytometry. The Annexin-V-FITC positive cells were quantified using CellQuest software.

Real-time PCR

Total RNA was isolated from cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The isolated RNA was purified using DNase and subjected to reverse transcription by a Reverse Transcription Kit (Takara, Shiga, Japan) following the manufacture’s instruction. The obtained cDNA was used to perform the real-time PCR. The SYBR Green I real-time PCR kit (Invitrogen, Carlsbad, CA, USA) was used on an ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) to amply the products. The mRNA was calculated by the 2−ΔΔCt method. The relative expression of mRNAs and miRNAs were normalized to β-actin and U6. The primers sequences are as follows:

IL-1β: Forward: 5ʹ-AAAAATGCCTCGTGCTGTCT-3ʹ; Reverse: 5ʹ-TCGTTGCTTGTCTCTCCTTG-3ʹ

TNF-α: Forward: 5ʹ-AAATGGGCTCCCTCTATCAGTTC-3ʹ; Reverse: 5ʹ-TCTGCTTGGTGGTTTGCTACGAC-3ʹ

IL-6: Forward: 5ʹ-CCGGGAACGAAAGAGAAGCT-3ʹ; Reverse: 5ʹ-CGCTTGTGGAGAAGGAGTTCA-3ʹ

IL-8: Forward: 5ʹ-TTGGAAGCCACTTCAGTCAGAC-3ʹ; Reverse: 5ʹ-GGAGCAGGAGGAATTACCAGTT-3ʹ

IL-12: Forward: 5ʹ-TGGGCAATGTGATTGAAAATTGCC-3ʹ; Reverse: 5ʹ-AAATGGGTGTGCTGTGCCCCGTT-3ʹ

IL-18: Forward: 5ʹ-AAATTGGGGCCTTCTTGGATAGGTGG-3ʹ; Reverse: 5ʹ-TTTCCGTGTGTAATTGGGTAAATCC-3ʹ

miR-142-3p: Forward: 5ʹ-GGAGAGATCCCGAAATGTGTG-3ʹ; Reverse: 5ʹ-GGTGTCCGAATGCTTGAAACCCTTTGG-3ʹ

MLK3: Forward: 5ʹ-AGTGAGTGCGCTGTTTAAATCCCGG-3ʹ; Reverse: CCGTAATAGAGGCTTAGGTTAAATTCTCTGG-3ʹ

β-actin: Forward: 5ʹ-ACATTGGCATGGCTTTGTTT-3ʹ; Reverse: 5ʹ-GTTTGCTCCAACCAACTGCT-3ʹ

U6: Forward: 5ʹ-CTCGCTTCGGGCAGCAC ATATACT-3ʹ; Reverse: 5ʹ-ACGCTTCACGAATTTGCGTGTC-3ʹ

Western blot

Cells were incubated with RIPA lysis buffer (Thermo Fisher Scientific, Shanghai, China) for 30 min. After centrifugation for 15 min, the protein was extracted and then quantified by BCA protein assay kit. The protein was separated by the 10% SDS-PAGE, followed by transferring onto PVDF and incubating with the primary antibodies of TSG101, CD63, Calnexin, p-p38, p38, p-JNK, JNK, p-ERK, ERK, and GAPDH (purchased from Abcam, Cambridge, UK) at 4°C for 24 h. After that, the PVDF was washed using tris buffered saline tween (TBST) and incubated with the second antibody of horseradish-peroxidase coupled goat anti-rabbit antibodies (Beyotine, Shanghai, China) at room temperature for 1 h. The protein bands were visualized using an Enhanced Chemiluminescence (ECL) (Thermo Scientific, US). GAPDH served as an internal control.

Cell transfection

The mimic, inhibitor of miR-142-3p, and their negative control (NC) were purchased from Shanghai Sangon (Shanghai, China). For MLK3 overexpression, the sequence of MLK3 was synthesized by Shanghai Sangon (Shanghai, China), and its NC was purchased from Invitrogen (Carlsbad, CA, USA). After confluent into 70%, cells were collected for transfection. The transfection was conducted using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction. After 24 h, transfection efficiency was determined using real-time PCR.

Cell viability

In the present study, cell viability was determined using MTT assay. Cells were seeded in a 96-well plate and received the treatment of IL-1β (10 ng/mL), or exosome and pcDNA-MLK3. After incubation for 24 h, cells were cultured by 5 μL of MTT solution for 2 h, followed by the treatment of 200 μL dimethyl sulfoxide (DMSO). Cell viability was determined by measuring the absorbance at 490 nm.

Intracellular glutathione (GSH) and reactive oxygen species (ROS) detection

To determine the oxidative stress in NPCs, both oxidative stress-related indexes GSH and ROS were measured using 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA, Beyotime, Shanghai, China) method. Briefly, after pretreated with different treatments, the NPs were collected into a 5 ml tubes and incubated with 10 μM DCFH-DA and then cultured in the DMEM without of FBS at dark for 1 h at 37°C. After washed by PBS, the cells were digested with trypsin, and centrifuged to collected the cells. A flow cytometer with the excitation wavelength at 488 nm and emission wavelength at 525 nm was used to detect the content of GSH and SOD.

Luciferase reporter assay

The online TargetScan was used to predict the potential binding region between miR-142-3p and MLK3 3ʹUTR. The miR-142-3p mimic, and the inhibitor, as well as their NC was purchased from Shanghai Sangon (Shanghai, China). The HEK293 T cells (purchased from ATCC) were prepared in a 24-well plate and cultured in the DMEM with 10% fetal bovine serum for 24 h. The sequence of 3ʹUTR in MLK3 was amplified and then cloned into the pGL3 plasmids. Then, pGL3-MLK-WT (wide type) or pGL3-MLK-Mut (mutant) combined with miR-142-3p mimic or their negative control was co-transfected into cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction. After 24 h, the luciferase activity was measured using dual-luciferase reporter assay system (Promega, Madison, WI).

Data analysis

All data were presented as means±SD and analyzed using SPSS 18.0 software. The statistically significant difference was analyzed using student’s-t test between two groups and ANOVA among multi-groups. p < 0.05 was considered a significant difference.

Results

Identification of exosomes derived from bone marrow MSCs

In order to investigate the therapeutic potential of MSCs on IDD, we first try to isolate the exosomes from bone marrow MSCs and identify its morphology and phenotypes. TEM showed the isolated exosomes were circular vesicles (Figure 1(a)). The diameter of the exosomes was about 80 nm (Figure 1(b)). It had a robust expression of exosomal markers CD63 and TSG101, but not the endoplasmic reticulum protein Calnexin (Figure 1(c)). Intervertebral disc degeneration occasionally exhibits nucleus pulposus cells (NPCs) loss. As exosomes can be uptake by cells during coculture, next, we verified whether the purified exosomes can be untaken by nucleus pulposus cells (NPCs). After coculture, NPCs showed PKH67 expression in its cytoplasm around nucleases (Figure 1(d)). The results demonstrate that exosomes derived from MSCs were successfully uptaken by NPCs.

Figure 1.

Figure 1.

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The characterization of Bone marrow MSCs-derived exosomes. (a) Representative images of the exosomes were visualized using TEM. (b) Exosome size distribution was measured using NTA. (c) The biomarkers of TSG101, CD63, and Calnexin for exosomes characterization were determined using western blot. (d) Exosomes were labeled with PKH26 by immunofluorescence. Each experiment was repeated for three individual times, and data were presented as means±SD.

MSCs-derived exosomes attenuate IL-1β-induced NPCs apoptosis and inflammation

It reported that IL-1β could induce cell apoptosis in NPCs by activating inflammation signaling [31]. Subsequently, we assessed the role of MSCs-derived exosomes on the biological behavior of IL-1β-induced NPCs. As presented in Figure 2(a-c), compared with the untreated control, IL-1β-treated NPCs exhibited a higher level of apoptotic cells and lower level of cell viability. However, MSCs exosomes reversed IL-1β-induced cell apoptosis and -repressed viability in NPCs. Furthermore, to investigate the underlying mechanism, we identified the expressions of inflammatory cytokines and found that MSCs exosomes could significantly eliminate IL-1β-elevated IL-1β, TNF-α, IL-6, IL-8, IL-12, and IL-18 (Figure 2(d-i)). It known that SOD and GSH are very important antioxidants and can protect cells from oxidative damage-induced cell apoptosis. Next, we investigated the levels of SOD and GSH in IL-1β-induced apoptosis. We found that IL-1β treatment caused a decreased level of SOD and GSH. MSCs exosomes significantly reversed IL-1β-induced decreased SOD and GSH (Figure 2(j,k)). The data indicates that MSCs exosomes attenuate IL-1β-induced NPCs apoptosis.

Figure 2.

Figure 2.

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MSCs-derived exosomes attenuate IL-1β-induced NPCs injury and MAPK signaling. (a) Effect of MSCs exosome on cell apoptosis was measured by MTT. (b, c) Effect of MSCs exosome on cell viability was measured by flow cytometry using Annexin V staining. (d-i) Relative mRNA expressions of IL-1β, TNF-α, IL-6, IL-8, IL-12, and IL-18 were determined using real-time qPCR. (j, k) GSH and ROS contents in NPCs were measured using DCFH-DA. *p < 0.05, **p < 0.01, ***p < 0.001; Exo: exosomes. Each experiment was repeated for three individual times, and data were presented as means±SD.

MSCs-derived exosomes suppress IL-1β-induced NPCs apoptosis by delivering miR-124-3p

Since exosomes were described to mediate cells behavior by miRNA transfer [20], and exosomal miR-142-3p from bone marrow MSCs participates in various cell processes, like promoting colon cancer stem cell-like traits or cell communication [18,19]. Here, we investigated whether miR-142-3p is involved in bone marrow MSCs exosomes-mediated NPCs apoptosis. The level of miR-142-3p in NPCs was significantly decreased by IL-1β treatment that was remarkably reversed by exosomes treatment, suggesting the important role of miR-142-3p in exosomes-mediated cell apoptosis (Figure 3(a)). When NPCs were directly treated by overexpressing miR-142-3p (miR-142-3p mimic), the level of miR-142-3p was significantly increased (Figure 3(b)), while it was decreased after NPCs treated by miR-142-3p knockdown (miR-142-3p inhibitor) (Figure 3(c)). The cell viability and apoptosis of NPCs were significantly alleviated by miR-142-3p overexpression (Figure 3(d,f)). Moreover, miR-142-3p knockdown significantly reduced IL-1β-suppressed cell viability, but enhanced IL-1β-induced cell apoptosis (3e, 3 g). Those data demonstrates that MSCs-derived exosomes attenuate NPCs cell apoptosis by delivering miR-142-3p.

Figure 3.

Figure 3.

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Exosomes-packaged miR-124-3p suppresses IL-1β-induced NPCs apoptosis.

(a) MiR-142-3p expression in NPCs cells with the treatment of IL-1β combined with or without exosomes was determined using real-time qPCR. (b, c) MSCs were treated with miR-142-3p mimic or inhibitor, and combined with/without IL-1β,,the expression of miR-142-3p was determined using real-time qPCR (d, e) MSCs were treated with miR-142-3p mimic or inhibitor, and combined with/without IL-1β,cell viability was measured by MTT, (f, g) MSCs were treated with miR-142-3p mimic or inhibitor, and combined with/without IL-1β, cell apoptosis was determined by flow cytometry using Annexin V staining. *p < 0.05. Exo: exosomes. Each experiment was repeated for three individual times, and data were presented as means±SD.

MLK3 is the target of miR-142-3p

To determine the mechanism by which exosomal miR-142-3p mediates the NPCs apoptosis, we attempted to predict its target gene using TargetScan. Figure 4(a) shows that miR-142-3p could potentially bind to the 3ʹUTR of Mixed lineage kinase 3 (MLK3). The luciferase reporter assay was performed to verify its interaction. MLK3 3ʹUTR in WT or Mut was established and co-transfected with miR-142-3p overexpression or NC into NPCs. The results showed that the luciferase activity of WT MLK3 was significantly decreased by miR-142-3p overexpression, while not changed in Mut MLK3 (Figure 4(b)). Real-time PCR and western blot confirmed that MLK3 expression was suppressed by miR-142-3p overexpression and enhanced by miR-142-3p knockdown (miR-142-3p inhibitor) (Figure 4(c,d)). These findings suggest that MLK3 is a direct target of miR-142-3p.

Figure 4.

Figure 4.

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MLK3 is a direct target of miR-142-3p. (a) Interaction of miR-142-3p and MLK3 3ʹUTR was predicted by TargetScan. (b) Luciferase reporter assay was conducted to verify the interaction between miR-142-3p and MLK3. (c) The levels of MLK3 in cells that pretreated with miR-142-3p mimic and inhibitor were determined using real-time PCR. (d) Western blot was performed to analyze MLK3 protein expression in cells with miR-142-3p mimic and inhibitor. *p < 0.05. Each experiment was repeated for three individual times, and data were presented as means±SD.

MSCs-derived exosomes attenuate IL-1β-induced MAPK signaling activation

It was previously shown that MLK3 can activate MAPK signaling [28]. Next, we determined whether miR-142-3p/MLK3 axis was involved in the MAPK signaling activation. Interestingly, IL-1β-treated NPCs also showed higher expressions of p-p38, p-JNK, and p-ERK, while MSCs exosomes treatment significantly inhibited IL-1β-enhanced expressions of p-p38, p-JNK, and p-ERK (Figure 5(a)). Pretreatment with MAPK inhibitor LY2228820 significantly attenuates IL-1β-induced NPCs apoptosis (Figure 5(b)) and inflammation response (Figure 5(c-h)). The results indicate MSCs-derived exosomes mitigated NPCs apoptosis through MAPK signaling.

Figure 5.

Figure 5.

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MSCs-derived exosomes-mediated MAPK signaling activation in NPCs.

(a) Effects of exosomes on IL-1β-activated MAPK signaling pathway exhibited by protein expressions of p-p38, p38, p-JNK, JNK, p-ERK, and ERK. The effects of MAPK inhibitor LY2228820 on IL-1β-induced cell apoptosis was determined by Annexin V staining (b), and inflammatory response was assessed by determining the mRNA expression of IL-1β (c), TNF-α (d), IL-6 (e), IL-8 (f), IL-12 (g), and IL-18 (h). *p < 0.05, **p < 0.01, ***p < 0.001. Exo: exosomes. Each experiment was repeated for three individual times, and data were presented as means±SD.

miR-142-3p inhibits IL-1β-induced MAPK signaling activation by targeting MLK3

miRNAs belong to a family of small non-coding RNAs that has great advantages to develop drugs [32]. Next, we explored the potential therapeutic effects of miR-142-3p on NPCs using artificially generated miR-142-3p overexpression. Firstly, the transfection efficiency of MLK3 overexpression was determined by both real-time PCR and western blot (Figure 6(a)). Then, NPCs were treated by IL-1β in the presence or absence of miR-142-3p mimic combined with MLK3 overexpression by transfecting pcDNA-MLK3. The expression levels of p-JNK, p-ERK, and p-p38 were decreased by overexpressed miR-142-3p, as compared with the IL-1β treatment group, and further abolished by MLK3 overexpression (Figure 6(b)). Accordingly, MLK3-pcDNA abolished the effects of exosome on IL-1β-induced decreased cell viability and increased apoptosis and inflammatory cytokines (Figure 6(c-e)). Taken together, MSCs-derived exosomes ameliorate NPCs apoptosis through inactivating MAPK signaling via delivering miR-142-3p, which targeting MLK3.

Figure 6.

Figure 6.

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MiR-142-3p inhibits MAPK signaling activation by targeting MLK3. (a) Transfection efficiency of MLK3 on NPCs was measured using real-time qPCR and western blot. (b) Western blot was conducted to determine the expressions of p-p38, p38, p-JNK, JNK, p-ERK and ERK in different groups cells. (c, d) Cell viability and apoptosis were determined using MTT and flow cytometry, respectively. (e) The relative mRNA expression of IL-1β and TNF-α in NPCs was determined using real-time PCR. *p < 0.05, **p < 0.01; Exo: exosomes. Each experiment was repeated for three individual times, and data were presented as means±SD.

Discussion

Plenty of evidence has shown that bone marrow MSCs transplantation could ameliorate IDD progression, and MSCs-secreted exosomes served as an alternative way for IDD therapy [10]. In addition, small vesicle of exosome, which was recognized as a “rising star,” has been considered to play an important role in intercellular communication and cellular processed modulation [33,34]. In the present study, we found that bone marrow MSCs-derived exosomes could protect NPCs against IL-1β-induced apoptosis and inflammation, which is partially medicated by suppressing the activation of MLK3/MAPK signaling through delivering miR-142-3p.

MSCs are ideal parent cells for clinical therapy due to their available and expansible in the diverse tissues and not immunologically reactive [35]. The mechanisms underlying MSCs that responsible for the therapeutic benefits have been widely reported, such as the paracrine and bone marrow MSCs-derived exosomes [36,37]. Exosome-based therapy has been applied not only in the same individual but also in the different ones, even between species [35,38]. Recently, MSCs-exosomes have shown to mediate immunomodulation, wound healing, inflammation, and apoptosis [39,40]. It has been reported that MSCs could protect resident Intervertebral disc cells against death [41], and the interaction between MSCs differentiation and NPCs repair has been implicated in the therapeutic benefits [42,43]. In our study, the inflammatory response and increased cell apoptosis induced by IL-1β in NPCs was abolished by the MSCs exosomes treatment, indicating the MSCs exosomes had ameliorating effects on NPCs.

Accumulating evidence revealed that RNAs-containing exosomes play a significant role in altering the cellular activities and functions of recipient cells [44]. MSCs-exosomes have been proven to be enriched with miRNAs, and MSCs exosomes can deliver extensive miRNAs to the NPCs. Research about miRNAs mediate the NPCs injury in the IDD progression has been widely reported [45,46]. Consequently, it seems reasonable that miRNAs serve as the candidates for deducing the therapeutic mechanism of MSCs exosomes on IDD. In the present study, we found that miR-142-3p was enriched in the bone marrow MSCs exosome, and modulated IL-1β-induced NPCs injury. Moreover, MLK3 was demonstrated as a directly target of miR-142-3p in this process. Consequently, it is reasonable to suggest that bone marrow MSCs-derived exosomes delivering miR-142-3p to modulate NPCs loss by targeting MLK3.

The activated p38 MAPK pathway contributes to the annulus fibrosus cell apoptosis [47]. MAPK signaling facilitates endoplasmic reticulum stress and IL-6 release in IDD [48]. Suppression of MAPK activity showed a protective role in IL-1β-induced inflammatory and catabolic in human intervertebral disc cells [49]. In addition, the MAPK pathway was activated in the IDD [50], while the mechanism of how to regulate the activation of MAPK remains unclear. Our findings showed that IL-1β induced the activation of MAPK pathway, while overexpression of miR-142-3p in NPCs suppressed MAPK activation. Taken together, we concluded that MSCs exosomes protect NPCs against injury by delivering miR-142-3p that targets MLK3 and further inhibits the activation of MAPK signaling.

Taken together, this study describes the effect of bone marrow MSCs-derived exosomes on NPCs injury, and found that MSCs exosomes can ameliorate the NPCs injury by delivering miR-142-3p. In addition, the exosome-packaged miR-142-3p could target MLK3 to suppress MAPK signaling. The study uncovers the potential therapeutic effects of MSCs-packaged miR-142-3p on NPCs loss, and offers new opportunities for the therapeutic potential for IDD.

Funding Statement

The present study was supported by Hospital Fund of the Ninth People's Hospital of Suzhou (YK202003 to LZ)

Disclosure statement

No potential conflict of interest was reported by the authors.

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