Small extracellular vesicles from iPSC-derived mesenchymal stem cells ameliorate tendinopathy pain by inhibiting mast cell activation

Pain is defined as an unpleasant sensory and emotional experience of actual or potential tissue damage or an experience expressed in such terms [1], and can be acute or chronic. Persistent stimulation by acute pain would cause neural plasticity remodeling in pain-coding pathways and develop into chronic pain [2]. Thus the key to preventing chronic pain is to control the progression of pain during the acute phase. Tendinopathy is a prevalent musculoskeletal disease characterized by pain, swelling and limited joint movement [3]. It has been reported that the prevalence and incidence rates of lower extremity tendinopathy are 11.83 and 10.52 per 1000 person-years [4]. Chronic pain derived from tendinopathy would limit the movements of joints, even resulting in disability. To date, most studies related to tendinopathy have focused on promoting tendon regeneration without considering relieving acute pain. Clinically, the effectiveness of current treatments for acute pain in tendinopathy remains ambiguous. Though corticosteroid injection has been a mainstay treatment for tendon-related disorders for many years, its effectiveness remains controversial [5]. In conclusion, it is essential to explore a new therapeutic strategy to relieve acute pain derived from tendinopathy.

Mesenchymal stem cells (MSCs) have been well investigated in regulating the immune response and tissue regeneration [6,7]. Recently, we have generated MSCs from induced pluripotent stem cells (iPSCs) [8]. The MSCs derived from iPSCs (iMSCs) have a more robust proliferation and differentiation potential than adult bone marrow-derived MSCs [9]. Accumulating studies have indicated that the efficacy of MSCs is attributed to the paracrine small extracellular vesicles (sEVs), lipid bilayer nanoparticles containing proteins, lipids, nucleic acids and other biomolecules [10,11]. Our previous research has shown that sEVs derived from iMSCs (iMSC-sEVs) could attenuate osteoarthritis by alleviating inflammation and promoting chondrocyte proliferation [12,13]. Currently emerging studies have found that modification of sEVs can optimize anti-inflammatory and regeneration ability [14,15]. Previous studies have also demonstrated that bone marrow stem cell-derived EVs could promote tendon healing by suppressing inflammation and apoptotic cell accumulation and increasing the proportion of tendon-resident stem/progenitor cells [16,17]. Nevertheless, the therapeutic potential of iMSC-sEVs for alleviating acute pain in tendinopathy has barely been reported so far.

As proinflammatory cells and the immune system’s first responders [18], mast cells are localized in proximity to afferent fibers [19]. The proximity of mast cells to afferent nerve fibers potentiates critical molecular crosstalk, contributing to initiating and developing pain responses [20]. They are a critical link between the nervous system and the immune system [21]. Studies have shown that mast cells play a vital role in pain transmission in tendinopathy [22–24], indicating that suppressing the infiltration and activation of mast cells may be a target for relieving acute pain in tendinopathy. In addition, Cho et al. reported that sEVs from human adipose tissue-derived MSCs could ameliorate atopic dermatitis and reduce the infiltration of mast cells in vivo [25]. Kuwabara et al. showed that intravenous administration of MSCs after aneurysm formation prevented aneurysmal rupture in mice, and the protective effect appears to be mediated in part by the stabilization of mast cells by MSCs [26]. Accordingly, we hypothesized that iMSC-sEVs could ameliorate pain by inhibiting mast cell activation in tendinopathy.

In the present study, we first found that iMSC-sEVs could relieve acute pain and inhibit inflammation in a rat tendinopathy model. Then we showed that iMSC-sEVs could inhibit mast cells’ activation and interaction with nerve fibers in vivo. Moreover, iMSC-sEVs inhibited substance P (SP)-induced activation of mast cells in vitro. Mechanically, iMSC-sEVs might regulate the HIF-1 signaling pathway in mast cells. Herein we demonstrate for the first time that iMSC-sEVs possess the therapeutic potential to ameliorate pain in tendinopathy by stabilizing mast cells, at least in part, via the HIF-1 signaling pathway.

Materials & methods

Derivation & characterization of induced MSCs

The human iPSC line (iPS-S-01) used in this study was provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences in agreement with Liao and Xiao [27]. The derivation of iMSCs was described previously in our studies [8,12,28]. Briefly, the medium was replaced with a serum-free MSC culture medium containing basal medium (Nuwacell™ Nova Missoin Basal Medium, Nuwacell Biotechnology, RP02010-01, Anhui, China) and supplement (Nuwacell Nova Missoin Supplement, Nuwacell Biotechnology, RP02010-02) after 14 days of culturing in iPSC culture medium (Nuwacell Biotechnology, RP01001). The morphology of cells was changed to fibroblast-like cells at passage four, and the cells were utilized to identify iMSC phenotypic characteristics. The cells were continually passaged after reaching 90% confluence, and cells from passages five to ten were used in the subsequent experiments.

Flow cytometry was used to detect phenotypic markers of iMSCs. The cells were incubated with 1% (w/v) bovine serum albumin (BSA) (Gibco, CA, USA) to block the nonspecific antigens. Then, 1 × 10⁶ cells were stained with the following conjugated mouse monoclonal antibodies: CD24-PE (1:100, 560991, BD Biosciences, NJ, USA), CD29-PE (1:100, 561795, BD Biosciences), CD44-FITC (1:100, 560977, BD Biosciences), CD146-PE (1:100, 561013, BD Biosciences), CD133-PE (1:100, 130080081, MACS, Colonge, Germany), CD105-FITC (1:100, 560943, BD Biosciences), CD73-PE (1: 100, 561014, BD Biosciences), CD90-PE (1:100, 328109, Biolegend, NJ, USA), CD34-APC (1: 100, 560940, BD Biosciences), CD45-FITC (1:100, 560976, BD Biosciences) and HLA-DR-PE (1:100, 560943, BD Biosciences). After being washed in 1% (w/v) BSA twice, the cells were resuspended in 1% BSA and analyzed by CytoFLEX flow cytometer (Beckman Coulter Life Science, CA, USA).

Isolation of iMSC-sEVs

According to the 2018 guideline on minimal information for studies of extracellular vesicles [29], iMSC-sEVs were isolated from the cell-culture medium of iMSCs by differential ultracentrifugation protocols. Briefly, the obtained medium was centrifuged at 300× g for 10 min and 2000× g for 10 min. After centrifugation at 10,000× g for 1 h, the supernatant was filtered through a 0.22-μm filter sterilizer Steritop™ (Millipore, MA, USA) to remove cellular debris and microvesicles. The collected medium was further ultracentrifuged at 100,000× g for 70 min twice. After removal of the supernatant, the pellet was resuspended in phosphate-buffered saline (PBS).

Size distribution & particle concentration of iMSC-sEVs

The size and concentration of the iMSC-sEVs were assessed using a nanoflow cytometer (N30 Nanoflow Analyzer, Nano FCM, Inc., Xiamen, China) as previously described [30]. Briefly, isolated iMSC-sEVs diluted 100-fold with PBS (for a nanoparticle concentration of ~10⁸/ml) were loaded to the nanoflow to measure the side scatter intensity. The concentration of iMSC-sEVs was calculated according to the ratio of side scatter intensity to particle concentration in the standard polystyrene nanoparticles. The size distribution of the iMSC-sEVs sample was calculated according to the standard cure generated by standard silica nanoparticles.

Western blot analysis

To identify sEV using western blot analysis, three positive markers of iMSC-sEV (CD9, TSG101 and CD63) and one negative marker (GM130) were evaluated. Cells or iMSC-sEV proteins were harvested using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China; P0013C) supplemented with protease inhibitor cocktail (Beyotime; ST505). Lysates were cleared by centrifugation at 12,000× g for 20 min. The supernatant fractions were used for western blot analysis. Protein extracts were resolved by 10% SDS-PAGE and probed with the indicated antibodies. The antibodies against the following proteins were used for western blot analysis: rabbit monoclonal anti-CD9 (1:1000, Cell Signaling Technology, MA, USA; 13174s), mouse monoclonal anti-TSG-101 (1:1000, Abcam, Cambridge, UK; ab83), rabbit monoclonal anti-CD63 (1:1000, Abcam; ab134045) and mouse polyclonal anti-GM130 (1:500, Abcam; ab169276). Anti-rabbit IgG or anti-mouse IgG, horseradish peroxidase-linked antibody (1:2000; Cell Signaling Technology) was used as the secondary antibody. Protein level was detected using the ECL™ detection system (Thermo Fisher Scientific, MA, USA).

Animal model & experimental design

Animal care and experimental procedures were approved by the Animal Research Committee of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (approval code: DWLL2021-0910). Previous studies have established a rat tendinopathy model by carrageenan [31]. Male Sprague–Dawley rats aged 34 weeks underwent sham surgery or tendinopathy model establishment and were randomly and averagely divided into three groups: sham group; tendinopathy + PBS group; tendinopathy + sEVs (1 × 10⁹ particles/ml) group; n = 3. For tendinopathy model establishment, the quadriceps tendon was injected with 100 μl 4% (w/v) carrageenan under ultrasound guidance while the sham group were injected with PBS. Then, 1 week later, sEVs or PBS was injected into the tendon once a week for 4 weeks to determine the analgesic effect of iMSC-sEVs. Pain-related behaviors were analyzed 1 week after the administration of iMSC-sEVs or PBS. The test order for the assessment of pain-related behaviors was firstly gait analysis, then static weight bearing (SWB) and lastly hind paw withdrawal threshold (PWT). Reversal (%) of pain-related behaviors was calculated as follows:

where ‘value’ represents the values for SWB or PWT. In addition, we used isoflurane to perform excessive gas anesthesia to euthanize rats in the end.

Pain assessment

Hind PWT

PWTs were measured as described previously [32]. The electronic von Frey instrument (model BIO-EVF4; Bioseb, Vitrolles, France) was used to vertically stimulate the center of the rat hind paw with increasing intensity. The probe tip was gently placed perpendicularly into the mid-plantar surface of the paw, and steadily increasing pressure was applied until the hind paw was first lifted. When the withdrawal reaction was positive, and there were three positive withdrawal reactions within the five consecutive stimuli, the value was defined as PWT and was expressed in grams.

Static weight bearing

The SWB distribution over the right and left knee was assessed by measuring the postural balance between the injected and non-injected leg [32]. Briefly, a rat was placed in the chamber of a weight bearing measuring device (model #BIO-SWB-TOUCH-M; Bioseb). The force applied through each hind limb to the paw resting on the floor of the chamber was measured in grams, and an SWB index was calculated as follows:

For each rat, the test was given at least three times in each assessment period.

Gait analysis

Dynamic pain-related behavior was measured by the gait of the rats [33]. The CatWalk system objectively quantifies behavioral gait adaptation after daily use of a painful limb, automatically documenting paw placements on a surface and related parameters of inter-limb co-ordination [34]. Briefly, rats were placed on a walkway apparatus (Shanghai Mobiledatum Information Technology, Shanghai, China). A camera below the walkway captures and digitally records footprint images. These paw print placements and gait parameters were collected and further analyzed by WalkAnalysator (Shanghai Mobiledatum Information Technology). The CatWalk gait test was administered at week 4 after treatment. Print area, swing speed, duty cycle and max contact mean intensity were recorded and analyzed as right/left.

Histology & immunohistochemistry

For histological analysis, the rat tendon samples were fixed en bloc in 4% paraformaldehyde for 24 h, then dehydrated with a graded ethanol series, embedded in paraffin and sectioned (5 μm thick) parallel to the long axis of the tendon. The sections were prepared for hematoxylin and eosin staining (H&E) and immunohistochemical analysis.

To assess the proinflammatory cytokine distribution in rat tendon samples, we performed immunohistochemistry (IHC) staining on paraffin-embedded sections. The following antibodies were used: anti-IL-1β (1:500, Abcam, ab283818), anti-TNF-α (1:500, Abcam, ab217706), anti-IL-6 (1:500, Abcam, ab9324) and anti-NGF (1:500, Abcam, ab52987). Horseradish peroxidase-conjugated antibodies were used with 3,3′-diaminobenzidine as the chromogen for visualization. In some cases, a hematoxylin counterstaining was done for nuclear counterstaining. Histological and immunohistochemical staining were evaluated and photo-documented digitally with the microscope (DM6B, Leica, Wetzlar, Germany). Interpretation of the slides was performed by the semiquantitative grading scale of Movin score for tendon abnormalities [35].

Immunofluorescence staining

The rats were sacrificed, and cardiac perfusion was performed with ice-cold saline, followed by 4% (w/v) paraformaldehyde perfusion. Lumbar dorsal root ganglion (DRG) at levels L3–L5 and tendon tissues were dissected from the surrounding tissue, fixed in 4% formaldehyde overnight at 4°C and dehydrated with gradient sucrose solutions (20, 30 and 35% w/v). After being embedded and frozen in an optimal cutting temperature compound, the tissues were sliced into 10-μm-thick coronal sections. The sections were then stained with specific markers, including CGRP (1:400, Cell Signaling Technology, 14959), iNOS (1:400, Cell Signaling Technology, 13120), tryptase (1:100, Abcam, ab2378) and PGP9.5 (1:100, Abcam, ab108986). Fluorescence images were acquired using a fluorescence microscope (Leica, DM6B). ImageJ software (NIH, MD, USA) was performed to quantify tryptase-positive cell number, staining area and relative expression of CGRP and iNOS.

Uptake of iMSC-sEVs by mast cells in vitro

To determine the uptake of iMSC-sEVs into RBL-2H3 cells in vitro, we labeled iMSC-sEVs with Dil fluorochrome (Thermo Fisher Scientific) under room temperature for 1 min, followed by ultracentrifugation at 100,000× g in PBS to get rid of the unlabeled dye. Next, Dil-labeled sEVs were incubated with RBL-2H3 cells for 12 h, then the culture medium was discarded, and the cells were rinsed twice with PBS before image capture under the fluorescence microscope.

SP-induced degranulation in RBL-2H3 cells

RBL-2H3 cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. The cells were grown in Eagle’s Modified Essential Medium supplemented with glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml) and 10% fetal bovine serum (Gibco) in a humidified 5% CO₂ atmosphere at 37°C, plated on six-well culture dishes at a cell density of 9 × 10⁵ cells per well. After 24 h, RBL-2H3 cells were stimulated with SP (10 μM) or vehicle (PBS) and incubated for 15 min at 37°C in a 5% CO₂ atmosphere.

β-hexosaminidase release assay

The RBL-2H3 cells were subjected to a β-hexosaminidase release assay as previously described, with a small modification to determine the degranulation activity [36]. Briefly, after being stimulated by SP, the RBL-2H3 cells were treated with iMSC-sEVs (10⁹/ml) or vehicle for different times (6, 9, 12 or 24 h) at 37°C in a 5% CO₂ atmosphere. The supernatants (15 μl) were incubated with 60 μl of the substrate (1 mM p-nitrophenyl-N-acetyl-β-d-glucosaminide in citrate 0.05 M, pH 4.5) for 1 h at 37°C. Furthermore, the cells were lysed with 0.1% Triton™ X-100 and incubated with the substrate to determine the degranulation activity in the supernatants to determine the total amount of released β-hexosaminidase. The reaction was stopped by 150 μl of 0.1 M sodium bicarbonate buffer (pH 10.0), and the reaction product was monitored by measuring the optical density (OD) at 405 nm by using a GENios Pro reader (Tecan, Männedorf, Switzerland). The results were calculated by using the following formula: % degranulation = (OD-supernatant/[OD-supernatant + OD-triton x-100]) × 100.

Toluidine blue staining

SP-stimulated RBL-2H3 cells were treated and incubated as described above for the β-hexosaminidase release assay. After 12 h incubation with iMSC-sEVs (10⁹/ml) or PBS, the cells were fixed with paraformaldehyde at 4% for 20 min and incubated for 3 min with toluidine blue at 0.01% in 3% acetic acid. Subsequently, the cells were washed for 5 min in distilled water. The cells were observed using a Leica DMI6000 digital microscope.

Enzyme-linked immunosorbent assay

The supernatants collected at 18 h after different treatments were evaluated for proinflammatory molecules and NGF by ELISA. IL-1β, TNF-α, IL-6, IL-10 and NGF concentrations were measured by using a rat ELISA kit (Shanghai Westang Bio-Tech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The absorbance was measured by a microplate reader (Thermo Fisher Scientific) at 450 nm.

RT-qPCR analysis

The expression of targeted genes was analyzed by RT-qPCR. Briefly, the total RNA of samples was extracted using the EZ-press RNA Purification Kit (EZ Bioscience, MN, USA). RNA quantity and purity were confirmed with a Nanodrop spectrophotometer (Thermo Fisher Scientific). A 4× Reverse Transcription Master Mix (EZ Bioscience) was used for the reverse transcription reaction. PCR reactions were run using the ABI Prism^® 7900HT Real-Time System (Applied Biosystems, CA, USA) with 2 × SYBR^® Green qPCR Master Mix (EZ Bioscience). The primer sequences used in this study are listed in Supplementary Table 1.

RNA sequencing analysis

RNA sequencing (RNA-seq) analysis was performed by Shanghai Biotechnology Corporation (Shanghai, China). Poly(A) RNA was purified from total RNA, then converted to double-stranded cDNA; the resulting cDNA samples were sequenced using the standard Solexa protocols. The sequencing reads were mapped to the human genome using TopHat (v1.0.13, MD, USA). Avadis NGS (v1.3) was used to calculate the number of reads per kilobase per million mapped reads. Differentially expressed genes were called at twofold changes of these mapped reads. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed with the Database for Annotation, Visualization and Integrated Discovery ( https://david.ncifcrf.gov/).

Statistical analysis

Data are presented as mean ± standard deviation. Student’s t-test was used to assess the difference between two groups, and one-way analysis of variance with the Bonferroni post hoc test was applied for comparisons among multiple groups. All experiments were independently performed at least three times. Statistical analysis was performed using GraphPad Prism software (v8.0; GraphPad, CA, USA). Significant differences were considered to be denoted by a p-value < 0.05.

Results

Characterization of iMSCs & iMSC-sEVs

First, flow cytometry was applied to evaluate the cell surface antigen profile of the iMSCs. The results showed that the iMSCs highly expressed antigen markers including CD73, CD105, CD90, CD29, CD146 and CD105, but not CD34, CD45, CD133 or HLA-DR (Figure 1A). iMSC-sEVs were isolated from the cell culture supernatant of iMSCs and identified using transmission electron microscopy, nanoflow cytometer and western blot analysis. Transmission electron microscopy showed that the iMSC-sEVs were typical cup-shaped vesicles (Figure 1B). Nanoflow cytometer analysis revealed that the average diameter was 50–200 nm, and the concentration of the iMSC-sEVs was approximately 1.6 × 10¹⁰ particles/ml (Figure 1C). Western blot analysis determined the presence of exosomal markers CD9, TSG101 and CD63, but the cis-Golgi matrix protein GM130 was not detected (Figure 1D). As shown in Figure 1E & F, the mean particle concentration was 0.62 × 10⁸ ± 0.09 × 10⁸ particles/ml conditioned medium, or 420.20 ± 61.01 particles per cell (Figure 1E). The mean protein concentration was 6102.39 ± 190.29 ng/ml conditioned medium, or 100.32 × 10^-6 ± 13.56 × 10^-6 ng per particle (Figure 1F).

iMSC-sEVs alleviated the tendinopathy-related pain in a rat model

Firstly, we established the tendinopathy model by injecting carrageenan into the quadriceps tendon, and then iMSC-sEVs (1 × 10⁹ particles) or PBS were administered (Supplementary Figure 1). Then we performed H&E and IHC staining on the quadriceps tendon. H&E staining revealed that tendons in the iMSC-sEVs group showed more continuous and regular arrangement than disorganized tendons in the vehicle group (Figure 2A). In addition, the Movin score in the iMSC-sEVs group was significantly lower than that in the vehicle group (Figure 2B). IHC staining showed that proinflammatory cytokine expression significantly decreased in the iMSC-sEVs group compared with the vehicle group at 2 and 4 weeks (Figure 2A & B). Our results suggested that iMSC-sEVs could reduce proinflammatory cytokine production and repair the injured tendon.

Pain is a dominant character of inflammation. Therefore, we evaluated whether iMSC-sEVs could relieve the pain in tendinopathy. We accessed SWB and PWT after iMSC-sEVs administration for 4 weeks. For the static state, the reversals (%) of PWT and SWB in the iMSC-sEVs group were significantly increased compared with the vehicle group (Figure 2C & D). CatWalk tests were applied to examine whether iMSC-sEVs improved gait and motor function at 4 weeks after treatment. The lower limbs of the vehicle group showed less co-ordination than those of the iMSC-sEVs treatment group during walking (Figure 2E). Specifically, iMSC-sEVs significantly elevated the right/left hind values ratio in the print area, swing speed and max contact mean intensity compared with the vehicle group (Figure 2F). In addition, He et al. reported that bone marrow stem cell-derived sEVs could reduce the expressions of CGRP (a neuropathic pain marker) and iNOS (an inflammatory marker) in osteoarthritic rats' DRG tissues [37]; therefore, we performed immunofluorescence staining to detect the expressions of CGRP and iNOS. The result showed that the expression of these two proteins was significantly downregulated in DRG of the iMSC-sEVs group compared with the vehicle group (Supplementary Figure 2A & B). These results indicated that iMSC-sEVs could mitigate tendinopathy-related pain.

iMSC-sEVs inhibited mast cells’ infiltration & interactions with nerve fibers in the quadriceps tendon

Previous reports have demonstrated that mast cells play a vital role in tendinopathy-related pain [22,23]. We conducted double immunofluorescence staining on tendon sections for tryptase (marker of mast cells) and PGP9.5 (marker of nerve fibers) to assess the number of activated mast cells and the anatomical interaction between mast cells and nerve fibers. The results showed that compared with the sham group, tryptase-positive mast cells increased markedly in the tendinopathy group, as evidenced by the mean gray value of the tryptase staining area (Figure 3A & B). Besides, the number of tryptase-positive mast cells located close (<5 μm) to PGP9.5-positive nerve fibers also significantly increased (Figure 3A & B).

It is well known that NGF plays a prominent role in mediating interactions among mast cells and nerve fibers [38]. As expected, IHC staining of NGF showed a significantly increased expression compared with the sham group (Figure 3C & D). Therefore, these data supported the assumption of enhanced activation of mast cells after model establishment.

We then investigated whether iMSC-sEVs could regulate the activated mast cells’s infiltration and interaction with nerve fibers. Double immunofluorescence staining for tryptase and PGP9.5 was applied as described above. Compared with vehicle treatment, iMSC-sEV treatment significantly reduced the infiltration and interaction, as reflected by the significantly decreased number of tryptase-positive mast cells and those located near to PGP9.5-positive nerve fibers (distance <5 μm) (Figure 3A & B). Additionally, iMSC-sEV treatment significantly decreased the positive area of NGF compared with vehicle treatment (Figure 3C & D). Altogether, these results suggested that iMSC-sEVs treatment could stabilize mast cells under inflammatory conditions and impede their crosstalk with nerve fibers in a tendinopathy model.

iMSC-sEVs restrained SP-induced activation of mast cells

To further investigate the effect of iMSC-sEVs on the function of mast cells, SP was applied to activate RBL-2H3, a widely used mast cell line [39], in vitro. First of all, we determined whether iMSC-sEVs could be internalized by RBL-2H3 cells. iMSC-sEVs were labeled with Dil fluorescent dye and added to the culture medium. After 12 h of incubation, Dil-labeled iMSC-sEVs were efficiently taken up by RBL-2H3 cells (Figure 4A). RT-qPCR results showed that iMSC-sEVs reduced the mRNA expression of IL-1β, IL-6, TNF-α and NGF from SP-stimulated RBL-2H3 cells in a dose-dependent manner (Figure 4B). Therefore, iMSC-sEVs at a dose of 1 × 10⁹ particles/ml were chosen for the following experiments. The β-hexosaminidase release assay showed that iMSC-sEVs significantly reduced the degranulation of SP-stimulated RBL-2H3 cells, especially after 12 h incubation (Figure 4C). Toluidine blue staining showed that iMSC-sEVs significantly reduced the percentage of degranulated mast cells compared with vehicle treatment (Figure 4D & E). In addition, the expression of proinflammatory cytokines in the supernatant was significantly decreased in the iMSC-sEVs group, as determined by ELISA (Figure 4F). Collectively, these data indicated that iMSC-sEVs restrained SP-induced activation of mast cells in vitro.

iMSC-sEVs modulated the gene expression pattern of mast cells

To elucidate the underlying molecular mechanism by which iMSC-sEVs restrained the degranulation of mast cells, we performed RNA-seq analysis to profile the gene expression patterns in SP-stimulated RBL-2H3 cells treated with vehicle and iMSC-sEVs. We identified 768 upregulated genes (>twofold, p < 0.05) and 530 downregulated genes (<0.5-fold, p < 0.05) after iMSC-sEVs treatment (Figure 5A & B). KEGG pathway analysis indicated that the downregulated genes in the iMSC-sEVs group were enriched for functional annotations related to the HIF-1 signaling pathway and other metabolic pathways (Figure 5C). Similarly, gene ontology analysis revealed that the downregulated genes in the iMSC-sEVs group were enriched for biological processes such as regulation of apoptotic cell clearance and positive regulation of IL-1β secretion and response to type I interferon (Figure 5D). Subsequently, the heat map verified the expression of specific genes in the HIF-1 signaling pathway analysis and biological processes such as positive regulation of IL-1β and response to interferon, which showed a significant difference between groups (fold change >2) (Figure 5E). Furthermore, RT-qPCR analysis confirmed that the expression of HIF-1 signaling pathway-related genes was downregulated after iMSC-sEVs treatment (Figure 5F). These results suggested that iMSC-sEVs restrained mast cell activation through regulating the HIF-1 signaling pathway.

On the whole, iMSC-sEVs decreased the infiltration of inflammatory cytokines and inhibited the interactions between mast cells and nerve fibers. Moreover, iMSC-sEVs could restrain the activation of mast cells, which was partly mediated by the HIF-1 signaling pathway. Collectively, iMSC-sEVs could relieve tendinopathy-related acute pain through inhibiting mast cell activation via the HIF-1 signaling pathway (Figure 6).

Discussion

In this study we demonstrated for the first time that the application of sEVs isolated from iPS-MSCs significantly alleviated acute pain in a rat tendinopathy model. We found that iMSC-sEVs could notably inhibit mast cell activation and suppress inflammation in vivo. Furthermore, in vitro study illustrated that iMSC-sEVs could restrain SP-induced mast cell activation, in part via the HIF-1 signaling pathway.

Tendinopathy describes a spectrum of changes in damaged and diseased tendons, resulting in pain and dysfunction [40]. The pathogenesis of tendinopathy is multifactorial and complex, but it is widely accepted that overuse and inflammation contribute to tendinopathy [40]. This study focused on the pathogenicity of inflammation. Pain is an essential manifestation of inflammation, but pain can generally be classified into nociceptive pain, inflammatory pain and neuropathic pain according to the pathogenesis. Nociceptive and inflammatory pain are adaptive and protective, while neuropathic pain occurs after damage to the nervous system. In tendinopathy, inflammatory mediators evoke pain via direct activation and sensitization of nociceptors [41]. In contrast, persistent nociceptive input results in the growth of central sensitization and neuropathic pain, characterized by the hyperactivity and hyperexcitability of neurons in the brain and spinal cord [42]. Previous studies suggest that tendinopathy-related pain is a combinational form of inflammatory and neuropathic pain [43,44]. CGRP is generally involved in transmitting nociceptive information and pain sensitization in the peripheral and spinal cords [37]. Consistent with these, we determined the increased expression of CGRP and iNOS in the DRG of tendinopathy rats. Meanwhile, iMSC-sEVs treatment relieved the pain associated with tendinopathy, reducing central sensitization and neuropathic pain, as reflected by decreased expression of CGRP and iNOS.

After injuries occur in local tissue, mast cells and other immune cells are activated and release inflammatory mediators such as bradykinin, prostaglandin, protease and histamine to stimulate adjacent nociceptor afferent fibers [45,46]. In turn, affected afferent fibers of nociceptors also release neuromodulators, such as SP, calcitonin-producing peptide and vasoactive intestinal protein to activate mast cells. Consequently, an inflammatory cascade reaction of mast cell activation and peripheral neurohypersensitivity is formed, further amplifying pain and inflammation [47–49]. Scott et al. revealed that the number of mast cells in patellar tendon specimens of patients with patellar tendinopathy was significantly increased [50]. Consistently, in this study, we found that the number of activated mast cells and those located close (<5 μm) to PGP9.5-positive nerve fibers significantly increased in the tendon after model establishment in rats. Therefore, mast cells may be a target for relieving acute pain in tendinopathy.

MSCs have been proven to possess anti-inflammatory, analgesic and regenerative capacities [51]. However, current methods for the large-scale preparation of MSCs face several limitations and challenges. iMSCs can avoid the ethical problems and immune rejection, and iMSC-sEV production offers several advantages for applications of MSCs [11]. The anti-inflammatory and analgesic effects of iMSCs are mediated by paracrine action, which is dominated by sEVs containing various nucleic acids, DNA and proteins. Previous studies have reported that MSC-sEVs could inhibit mast cells’ activation via a PGE₂-dependent mechanism [52]. Our present research found that iMSC-sEVs dose-dependently downregulated SP-induced release of proinflammatory cytokines and degranulation of RBL-2H3 mast cells in vitro. In addition, the in vivo study showed that iMSC-sEVs decreased the expression of proinflammatory cytokines, the activation of mast cells and the distance from nerve fibers, confirming the ability of iMSC-sEVs to suppress mast cell activation. As expected, our in vivo study showed that iMSC-sEV treatment increased reversals (%) of PWT and SWB and improved gait performance and motor function, revealing the analgesic effect of iMSC-sEVs on tendinopathy in vivo. Overall, these results demonstrate that iMSC-sEVs alleviate pain derived from tendinopathy partially through inhibiting the activation of mast cells.

RNA-seq and bioinformatic analysis of sequencing data identified differentially expressed genes involved in several signaling pathways. Interestingly, the expression of genes in metabolism-related signaling pathways like glycolysis/gluconeogenesis, biosynthesis of amino acids and carbon metabolism was upregulated significantly in the vehicle group. This might be attributed to the activation of mast cells caused by SP. So far, an increasing number of studies have reported that the HIF-1 signaling pathway figures prominently in regulating mast cells. Mast cell-derived HIF-1 significantly contributes to regulating mast cell function, which promotes the development of colorectal cancer [53]. Abebayehu et al. found that lactic acid could suppress IL-33-mediated mast cell inflammatory responses via HIF-1α-dependent miR-155 suppression [54], and Yan et al. discovered that SP could upregulate the level of HIF-1α in gingival fibroblasts and participate in periodontitis [55]. According to the KEGG analysis, 17 genes in the HIF-1 signaling pathway were significantly downregulated after iMSC-sEVs treatment, and RT-qPCR confirmed it. Thus our study suggests that iMSC-sEVs could modulate the activation and function of mast cells by regulating the HIF-1 signaling pathway.

Conclusion

In summary, our study reports for the first time that iMSC-sEV treatment relieves acute pain derived from carrageenan-induced rat tendinopathy by modulating neuroimmune interactions via the suppression of mast cells. In addition, iMSC-sEVs could suppress SP-stimulated activation in mast cells partly through regulating the HIF-1 signaling pathway. These findings unravel molecular mechanisms underlying the application of iMSC-sEVs on mast cells and provide a novel treatment strategy for pain derived from tendinopathy.