Exosomal miR-221-3p Derived from Bone Marrow Mesenchymal Stem Cells Alleviates Asthma Progression by Targeting FGF2 and Inhibiting the ERK1/2 Signaling Pathway

Exosomes derived from human bone marrow mesenchymal stem cells (BMSCs) play potential protective roles in asthma. However, the underlying mechanisms remain not fully elucidated. Herein, exosomes were isolated from BMSCs, and the morphology, particle size, and exosome marker proteins were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot, respectively. Then airway smooth muscle cells (ASMCs) were treated with transforming growth factor-β1 (TGF-β1) to construct a proliferation model and then incubated with BMSCs-derived exosomes. We found that exosome incubation increased miR-221-3p expression and inhibited proliferation, migration, and the levels of extracellular matrix (ECM) proteins including fibronectin and collagen III. Moreover, FGF2 was identified as a target gene of miR-221-3p. FGF2 overexpression reversed the inhibitory effects of exosomal miR-221-3p on ASMC progression. Besides, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) is inhibited by exosomal miR-221-3p, which was reversed by FGF2 overexpression. And ERK1/2 signaling activator reversed the effects of exosomal miR-221-3p on ASMC progression. Additionally, an ovalbumin (OVA)-induced asthmatic mice model was established, and exosome treatment alleviated airway hyper-responsiveness (AHR), histopathological damage, and ECM deposition in asthmatic mice. Taken together, our findings indicated that exosomal miR-221-3p derived from BMSCs inhibited FGF2 expression and the ERK1/2 signaling, thus attenuating proliferation, migration, and ECM deposition in ASMCs and alleviating asthma progression in OVA-induced asthmatic mice. Our findings may provide a novel therapeutic strategy for asthma.


Introduction
Asthma is a common chronic respiratory disease with high morbidity and mortality among children worldwide [1]. It is a complex syndrome characterized by airway hyper-responsiveness (AHR), airway inflammation, and airway remodeling [2]. Airway remodeling is involved in asthma progression through multifaceted processes including basement membrane thickening, extracellular matrix (ECM) deposition and abnormal airway smooth muscle cell (ASMC) growth [3,4]. It has been emphasized that the abnormal proliferation and migration of AMSCs leads to airway wall thickening and airway narrowing and obstruction, which contributes to airway remodeling and severely affects lung function in patients with asthma [5]. Transforming growth factor-β1 (TGF-β1) was found to be elevated in the airway of patients with asthma, which facilitated airway remodeling by inducing ECM protein production and promoting ASMC proliferation and migration [6]. Although asthma symptoms can be relieved in children with asthma through several therapeutic methods, there is no available complete cure for childhood asthma up to now. us, novel effective therapeutic strategies based on a better understanding of molecular mechanisms of childhood asthma progression are urgently needed.
Bone marrow mesenchymal stem cells (BMSCs) are selfreplicating multipotent stromal cells extracted from bone marrow mesenchymal tissues. Recently, accumulating evidence showed that BMSCs played a critical role in the treatment of autoimmune diseases including asthma due to their potent anti-inflammatory and immunomodulatory properties [7]. Importantly, it was found that the therapeutic function of BMSCs mainly depended on cell-to-cell communication through the secretion and transfer of exosomes [8]. Exosomes are small enclosed vesicles with a diameter of 50 to 150 nm, which are secreted by various types of cells and engaged in intercellular communication through transportation of functional signaling factors such as proteins, lipids, mRNAs, and microRNAs (miRNAs) [9]. e regulative role of BMSCs-derived exosomes in asthma has been identified in several types of research. For instance, BMSCsderived exosomes promoted immunosuppression of regulatory T cells in asthma, indicating the therapeutic potential of BMSCs-derived exosomes for asthma [10]. BMSCs-derived exosomes inhibited airway remodeling and epithelialmesenchymal transition of the airway epithelium in the lungs of asthmatic rats [11]. However, the regulatory mechanisms of BMSCs-derived exosomes in asthma progression have not been explored clearly.
MiRNAs are a class of small non-coding RNAs that regulate the post-transcriptional expression of target genes to mediate multiple biological and pathological processes. Increasing studies have demonstrated that exosomal miRNAs derived from BMSCs are involved in asthma development. A study suggested that there were different miRNA and mRNA profiles after BMSCs treatment in an asthma mouse model, and the miR-21/activin A receptor IIA axis is an important mechanism for the induction of asthmatic inflammation [12]. Moreover, BMSCs exosomal miR-1470 exerted an immunomodulatory effect by promoting the differentiation of CD4 + CD25 + FOXP 3+ Tregs in asthmatic patients [13]. Interestingly, it was found that epithelial, sputum, and plasma miR-221-3p expression was significantly decreased in patients with asthma. e decreased expression of miR-221-3p might protect against airway eosinophilic inflammation by upregulating anti-inflammatory chemokine (C-X-C motif ) ligand (CXCL) 17 (CXCL17) [14]. Likewise, miR-221-3p was found to have existed in the BMSCs-derived exosomes, and miR-221-3p delivered by BMSCs-derived exosomes was found to be involved in human physiological diseases such as acute myelocytic leukemia and ischemic stroke [15,16]. us, we explored the roles of exosomal miR-221-3p derived from BMSCs in asthma progression.
In the present study, we investigated the effects and regulatory mechanism of exosomal miR-221-3p derived from BMSCs on TGF-β1-induced proliferation, migration, and ECM deposition in ASMCs, and then verified its effect on asthma progression in ovalbumin (OVA)-induced asthmatic mice, aiming to provide a novel therapeutic target for asthma.

BMSC Isolation and Culture.
e Sprague-Dawley (SD) rats aged 6 weeks old were supplied by the Laboratory Animal Center of Shandong University. BMSCs were isolated according to the previously reported method [17]. e rats were euthanized through intraperitoneally anesthetizing with 1% pentobarbital sodium (100 mg/kg), and bone marrow was obtained from the femur and tibia of each rat under aseptic conditions. e bone marrow was centrifuged at 1000 g for 5 min, and the BMSCs precipitation was collected and suspended with Dulbecco's Modified Eagle Medium (DEME, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). BMSCs were cultured under humidified conditions with 5% CO 2 at 37°C. e medium was changed in half volume after incubation for 24 h and then the whole medium was changed every 2 days. Cells were passaged at a confluence level of 80-90% at a ratio of 1 : 2.

Extraction and Identification of Exosomes from BMSCs.
BMSCs in passage 3 with 80% confluence were selected for exosome extraction. BMSCs-derived exosomes were extracted by ultra-highspeed centrifugation [18]. In brief, the culture medium of BMSCs was successfully centrifuged at 300g for 10 min, 2000g for 10 min, and 10, 000g for 35 min, and the supernatant was collected each time. en the supernatant was filtered by using a 0.22 μm filter membrane (Merck Millipore, Tullagreen, Ireland), followed by ultracentrifugation and centrifugation both 100, 000g for 2 h, respectively. Finally, the exacted exosomes were collected, resuspended in phosphate-buffered saline (PBS), and stored at −80°C. e protein quality of exosomes was determined by the BCA Protein Assay Kit (Takara Biotechnology, Dalian, China). e levels of exosomal surface marker proteins including CD9, CD63, CD81, and TSG101 were detected by Western blot assay. e exosome morphology was identified by using Transmission electron microscopy (TEM, Hitachi, Tokyo, Japan), and the particle size of the extracted exosomes was measured by nanoparticle tracking analysis (NTA; Malvern Panalytical, Malvern, UK).

ASMC Culture and Treatment.
ASMCs were obtained from patients without asthma who underwent lung resection surgery (N � 6) at the Chengyang District People's hospital of Qingdao City. Informed consent was obtained from each patient, and this study was approved by the Ethics Committee of Chengyang District People's hospital of Qingdao City. ASMCs were obtained according to a reported method [19]. In brief, the smooth muscle layer was isolated from healthy segments of the lobar or main bronchus of subjects through dissecting microscope. e tissues were washed with ice-cold PBS solution containing penicillin-streptomycin three times, and then were cut into small pieces and digested in Hanks' balanced salt solution (HBSS) containing 0.1% collagenase solution (Sigma, St. Louis, MO, USA) at 37°C for 30 min. en the isolated cells were collected by centrifugation and cultured in DEME supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin under humidified conditions with 5% CO 2 at 37°C. Cells cultured from 3 to 5 passages were used for subsequent experiments. ASMCs were stimulated with TGF-β1 (10 ng/ mL; R & D Systems, Minneapolis, MN, USA) for 24 h to induce the proliferation model, and 100 μg/mL exosomes were used to incubate with ASMCs in each group.

CCK-8 Assay.
Cell proliferation was measured by using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. Briefly, ASMCs were seeded into 96-well plates at a density of 1 × 10 4 cells/well. After incubation for 0, 24, 48, and 72 h at 37°C, 10 μL of CCK-8 solution was added to each well and incubated for 2 h at 37°C. e absorbance at 450 nm of each well was measured by using a microplate reader (Molecular Devices, Shanghai, China).

Transwell Migration Assay.
A Transwell assay was performed to measure ASMC migration. Briefly, HTR8/ SVneo cells with the serum-free medium were seeded into the upper chamber of Transwell chambers (8.0 μm pore size; Millipore Corporation, USA). And the DMEM medium was added to the lower chamber. After culturing for 24 h at 37°C, cells on the upper chamber were removed by using a cotton swab, while the cells in the bottom chamber were fixed with 70% ethanol for 10 min and stained with 0.1% crystal violet for 15 min. e migrated cells were quantified by counting five random fields at ×200 magnification under a light microscope (Olympus, Tokyo, Japan).

Dual-Luciferase Reporter Gene Assay.
e potential targets of miR-221-3p were predicted by using the online bioinformatics tool Starbase (https://starbase.sysu.edu.cn/). e binding sites at FGF2 3′-UTR were mutated from AUGUACC to CCCCGAA and then cloned into the pGL3control luciferase reporter vectors (Promega, Madison, WI) to construct the wild-type (FGF2-WT) and mutant type (FGF2-MUT) reporter vector. en the reporter plasmids and miR-221-3p mimic or NC-mimic were co-transfected into ASMCs. Following transfection for 48 h, relative luciferase activity was measured by using the Dual Luciferase Reporter Assay System (Promega, Madison, Wisconsin, WI, USA).

Animal Experimental
Protocols. BALB/c mice (6-8 weeks old, 20 ± 2 g) were obtained from the Laboratory Animal Center of Shandong University, which were Evidence-Based Complementary and Alternative Medicine maintained in sterile cages under 22-25°C temperature, 55-60% humidity, and 12 h light/dark cycle conditions with free access to food and water. All animal experiments in this study were approved by the Animal Ethics Committee of Laboratory Animal Center of Shandong University.
Mice were randomly divided into three groups (n � 8 per group): control group, OVA group, and OVA + Exo group. e Asthma mouse model was established as previously described [20]. e mice in the OVA group were sensitized on days 0, 7, and 14 by intraperitoneal injection of 20 μg of ovalbumin (OVA), emulsified in 1 mg aluminum hydroxide in a total volume of 200 μL. en the mice were exposed to a 1% OVA aerosol for up to 1 h From day 21 to 23. e mice in the control group were treated with an equal volume of saline instead of OVA. For exosome treatment, 100 μg of exosomes in 100 μL PBS was intratracheally administered every day on days 21 to 23. All mice were euthanized 24 h for the subsequent procedures after the last OVA challenge.

Measurement of Airway Hyperresponsiveness.
Within 24 h after the last OVA challenge, mice were anesthetized with 1% pentobarbital sodium and subjected to mechanical ventilation, and the airway hyperresponsiveness (AHR) of mice was measured by using an animal pulmonary function instrument (Buxco, CT, USA). Subsequently, all mice were exposed to increasing doses of methacholine aerosol (Sigma-Aldrich, USA) at 0, 5, 10, 25, and 50 mg/mL for 3 min, and the enhanced pause (Penh) value of unrestrained mice within 5 min was recorded according to the protocols of the instrument.

Histopathological Evaluation.
e rats were euthanized through intraperitoneally anesthetizing with 1% pentobarbital sodium (100 mg/kg). Lung tissues were collected, fixed with paraformaldehyde, and embedded with paraffin. en the tissues were cut into 5 μm thick sections and stained with hematoxylin and eosin (HE) using a standard protocol and analyzed by light microscopy (magnification ×200; Olympus, Tokyo, Japan).
Lung tissues were subjected to Masson trichrome staining to determine collagen deposition. Tissue sections were stained with hematoxylin solution for 6 min, ponceau and acid fuchsin solution for 1 min, and phosphomolybdic acid solution for 5 min. And then restained with aniline blue solution for 5 min. e tissue sections were analyzed by light microscopy (magnification ×400; Olympus, Tokyo, Japan).

BALF Collection and Cell
Counting. After mice were anesthetized, the tracheas were cannulated and lavaged with 0.8 mL of cold PBS twice to collect bronchoalveolar lavage fluid (BALF). e BALF samples were immediately centrifuged at 300g for 15 min to collect the supernatant. e number of total cells in BALF was determined with a hemocytometer. e differences in cell number of eosinophils, neutrophils, lymphocytes, and macrophages in BALF were evaluated by using the Kwik-Diff staining set ( ermo, USA) according to the manufacturer's instructions.

Measurement of the Serum Level of OVA-Specific IgE.
On day 24 after the last OVA challenge, the blood samples of mice were collected and centrifuged at 1000g for 10 min to obtain serum samples. e serum level of OVA-Specific immunoglobulin E (IgE) was measured by ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's instructions.

Statistical Analysis.
Statistical analysis was performed by using SPSS version 22.0 software. All data from at least three times independent experiments were presented as mean ± standard deviation (SD). Student's t-test and analysis of variance (ANOVA) followed by Tukey-Kramer correction were performed for the comparison between two groups and comparison among groups, respectively. P < 0.05 was considered to be statistically significant.

Identification of Exosomes Isolated from BMSCs.
We first identified the isolated exosomes from BMSCs. e expression of exosome-specific markers, including CD9, CD63, CD81, and TSG101 was measured by Western blotting, which showed that all exosome marker levels were significantly higher in the isolated exosomes than that in BMSCs and supernatant (Figures 1(a) and 1(b)). Moreover, the exosome morphology was identified by TEM (Figure 1(c)) and NTA revealed that the mean particle size of exosomes was 134.2 nm (Figure 1(d)).
ese results indicated that BMSCs-derived exosomes were successfully obtained in our experiments.

Exosomal miR-221-3p Derived from BMSCs Inhibited Proliferation, Migration, and ECM Deposition in ASMCs.
To explore whether exosomal miR-221-3p derived from BMSCs is involved in childhood asthma progression, ASMCs were treated with TGF-β1 to induce a proliferation model and then incubated with BMSCs-derived exosomes alone or together with transfection with miR-221-3p inhibitor. RT-qPCR showed that the level of miR-221-3p was decreased in TGF-β1-treated ASMCs, and exosome incubation upregulated miR-221-3p level, while transfection of miR-221-3p inhibitor reduced miR-221-3p level (Figure 2(a)). Moreover, it was observed that TGF-β1 stimulation promoted cell proliferation (Figure 2(b)) and migration (Figures 2(c) and 2(d)) in ASMCs, and exosome incubation markedly attenuated proliferation and migration, while miR-221-3p inhibition reversed these effects. Additionally, ASMCs play an important role in airway remodeling by producing ECM proteins. We next determined the functional role of exosomal miR-221-3p in TGF-β1-induced ECM deposition. Western blotting demonstrated that TGF-β1 stimulation induced the expression of fibronectin and collagen III in ASMCs, and exosome incubation significantly decreased the expression of fibronectin and collagen III, which were then reversed by miR-221-3p inhibition (Figures 2(e)-2(g)).

FGF2 is a Target Gene of miR-221-3p.
e potential target mRNAs of miR-221-3p were predicted by using the StarBase tool (https://starbase.sysu.edu.cn/), which suggested that there were potential binding sites between miR-221-3p and FGF2 (Figure 3(a)). Dual luciferase reporter gene assay further confirmed that only the relative luciferase activity of FGF2-WT was significantly inhibited after transfection with miR-221-3p mimic, while the relative luciferase activity of FGF2-MUT was not affected by any treatment (Figure 3(b)). Furthermore, miR-221-3p mimic, miR-221-3p inhibitor, and their negative controls were transfected into ASMCs, respectively. As shown in Figure 3(c), the expression of miR-221-3p in ASMCs was increased in the miR-221-3p mimic group and decreased in the miR-221-3p inhibitor group. Moreover, miR-221-3p overexpression apparently inhibited the expression of FGF2 at both mRNA and protein levels, while miR-221-3p inhibition markedly promoted the mRNA and protein expression of FGF2 (Figures 3(d)-3(f )). ese findings indicated that FGF2 was a target gene of miR-221-3p.

Exosomal miR-221-3p Derived from BMSCs Alleviated Asthma Progression in Mice.
To further explore the role of exosomal miR-221-3p in asthma progression in vivo, the OVA-induced asthmatic mice model was constructed, and exosomal miR-221-3p was injected for treatment. We and 6(c)) in lung tissues of the OVA group compared with the control group, and this expression pattern was reversed by exosomal miR-221-3p treatment. en the airway hyperresponsiveness was detected by measuring the Penh value, which showed that aerosolized methacholine caused a dose-dependent increase in Penh value in all groups, and the Penh value in the OVA group was higher than that in the control group, while exosomal miR-221-3p treatment significantly reduced the Penh value in OVA-challenged mice ( Figure 6(d)). Next, histopathological evaluation of lung tissue sections from each group was detected by HE staining and Masson staining. HE staining showed that OVA-challenged mice showed obvious inflammatory cell infiltration and bronchial wall thickening, while exosomal miR-221-3p treatment attenuated these histopathological changes ( Figure 6(e)). Masson staining indicated that a large amount of collagen deposition around the bronchioles in the OVA group, while exosomal miR-221-3p exerted a substantial effect on reducing ECM deposition (Figure 6(f )). Additionally, the number of inflammatory cells including (i) e levels of TGF-β1, IL-4, and IL-13 in BALF were examined by using ELISA. Data from at least three independent experiments were presented as mean ± SD * P < 0.05. neutrophils, eosinophils, macrophages, and lymphocytes in BALF was increased significantly in the OVA group compared with that in the control group, while treatment of exosomal miR-221-3p reduced the number of inflammatory cells (Figure 6(g)). ELISA results showed that the levels of serum OVA-specific IgE (Figure 6(h)) and inflammatory cytokines including TGF-β1, IL-3, and IL-14 levels ( Figure 6(i)) were significantly elevated in lung tissues of OVA-challenged mice, while exosomal miR-221-3p treatment significantly reversed this effect. Our results revealed that exosomal miR-221-3p derived from BMSCs alleviated asthma progression in mice.

Discussion
Emerging evidence has revealed the potential protective role of BMSCs-derived exosomes in asthma progression due to their anti-inflammatory and immunomodulatory properties [7]. However, the possible underlying mechanisms of BMSCs-derived exosomes in the protection of asthma remain not fully understood. A large number of studies have confirmed that BMSCs-derived exosomes play crucial roles in the human pathological process through the transportation of functional miRNAs [7,21]. A previous study illustrated that miR-221-3p delivered by BMSCs-derived exosomes promoted the development of acute myelocytic leukemia [15]. Moreover, BMSCs-derived extracellular vesicles carrying miR-221-3p exerted a neuroprotective effect in ischemic stroke by targeting activating transcription factor 3 [16]. Interestingly, it was found that epithelial, sputum and plasma miR-221-3p expression was prominently decreased in patients with asthma, and the decreased expression of miR-221-3p might protect against airway eosinophilic inflammation by upregulating anti-inflammatory chemokine CXCL17, indicating that epithelial and sputum miR-221-3p are novel biomarkers for airway eosinophilic inflammation in asthma [14].
us, our study investigated the roles and underlying mechanism of exosomal miR-221-3p derived from BMSCs in asthma progression. We found that exosomal miR-221-3p derived from BMSCs inhibited TGF-β1-induced proliferation and migration, and reduced the levels of ECM proteins including fibronectin and collagen III in ASMCs. Additionally, exosomal miR-221-3p alleviated AHR, histopathological damage, and collagen deposition in OVA-induced asthmatic mice. Importantly, we suggested that exosomal miR-221-3p achieved its protective roles by targeting fibroblast growth factor 2 (FGF2) in ASMCs.
FGF2, also known as the basic fibroblast growth factor (bFGF), is a potent mitogenic factor belonging to the FGF family. It was reported that FGF2 was overexpressed in asthma and promoted airway inflammation in airway epithelial cells [22]. Recently, studies have focused on the immunomodulatory function of FGF2 in chronic inflammatory airway diseases including asthma and chronic obstructive pulmonary disease [23]. Notably, FGF2 inhibited TGF-β1-induced differentiation of ASMCs in vitro [24]. Moreover, FGF2 and TGF-β1 synergized in human bronchial smooth muscle cell (BSMC) proliferation, and this synergistic effect might contribute to the hyperplastic phenotype of BSMC in asthmatic airway remodeling [25]. ese studies indicated that FGF2 was involved in asthma progression and might be a therapeutic target for asthma diseases. In our study, we showed that FGF2 was a target gene of miR-221-3p, and FGF2 expression was inhibited by miR-221-3p in ASMCs, Furthermore, FGF2 overexpression reversed the inhibitory effect of exosomal miR-221-3p on TGF-β1-induced proliferation, migration, and ECM deposition in ASMCs, indicating that exosomal miR-221-3p exerted its protective role in asthma by inhibiting FGF2 expression. e ERK1/2 signaling pathway is a common pathway regulated by various proliferative factors, which is identified as a pivotal factor in asthma progression [26]. And the role of ERK1/2 signaling in promoting the proliferation and migration of ASMCs was previously reported. Biased Bitter taste receptor (TAS2R) bronchodilators inhibited ASMC proliferation by downregulating phosphorylated ERK1/2 [27]. Vasoactive intestinal peptides inhibited airway remodeling in asthmatic mice, and inhibited phosphorylation of the ERK1/2 signaling on ASMCs, thus inhibiting the proliferation of ASMCs [26]. More notably, FGF2 is closely related to wound repair and cell proliferation. Recent studies provided us the evidence that FGF2 participated in regulating the ERK1/2 signaling in cell proliferation. For instance, FGF2 significantly enhanced liver cell proliferation by increasing the phosphorylation level of ERK1/2 and c-Jun N-terminal kinase (JNK) [28]. FGF2 promoted proliferation and migration of uterine luminal epithelial cells during early pregnancy, which was reversed by the treatment of inhibitor U0126 [29]. erefore, we explored whether the ERK1/2 signaling is involved in the regulation of the miR-221-3p/ FGF2 axis in ASMC progression. Our results illustrated that exosomal miR-221-3p suppressed FGF2-mediated phosphorylation of the ERK1/2 signaling, and ERK1/2 activator TBHQ reversed the inhibitory effects of exosomal miR-221-3p on proliferation, migration, and ECM deposition in ASMCs, indicating that exosomal miR-221-3p attenuated ASMC progression through inhibiting the ERK1/2 signaling [30].
In conclusion, our findings demonstrated that exosomal miR-221-3p derived from BMSCs inhibited FGF2 expression and the ERK1/2 signaling, thus attenuating proliferation, migration, and ECM deposition in ASMCs. Additionally, exosomal miR-221-3p alleviated asthma progression in OVA-induced asthmatic mice. Our findings may provide a novel therapeutic strategy for asthma.

Data Availability
e datasets used during the present study are available from the corresponding author upon reasonable request.

Ethical Approval
is study was approved by the Ethnic Committee of Chengyang District People's hospital of Qingdao City, and Evidence-Based Complementary and Alternative Medicine all subjects had read and signed the informed consent. All animal care and experimental procedures were approved by the Ethics Committee of Laboratory Animal Center of Shandong University.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Weike Liu and Jieting Wan designed the experiments. Hui Lin and Wuhui Nie performed the experimental work. Qian Jiang provided statistical analysis and figures for the manuscript. Aimei Zhang wrote the manuscript. All authors read and approved the final manuscript.