Mechanism of ADSC-Exos promoting macrophage migration via Itgav/Fak/Src signaling pathway

Mechanism of ADSC-Exos promoting macrophage migration via Itgav/Fak/Src signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanism of ADSC-Exos promoting macrophage migration via Itgav/Fak/Src signaling pathway bo yuan, Jiayi Xie, Qinghua Zhu, Miao Ren, Yilun Wang, Ying Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7248636/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Macrophages play an important role in peripheral nerve regeneration, but the specific mechanisms remain unclear. Our previous studies demonstrated that adipose stem cell-derived exosomes (ADSC-Exos) promote peripheral nerve regeneration, correlating with their effects on macrophages. To test this, ADSC-Exos were isolated from primary ADSCs and co-cultured with RAW264.7 macrophages. We examined ADSC-Exos' impact on macrophage migration by assessing RAW264.7 cell viability, migration capability, and adhesion ability. Transcriptome sequencing identified differentially expressed genes (DEGs) and examined their interactions. Key gene expressions were verified by qRT-PCR, while pathway-related proteins were detected using Western blot and qRT-PCR. Results showed ADSC-Exos enhanced RAW264.7 cell viability, adhesion and migration. Transcriptomic analysis revealed 233 DEGs. GO analysis showed DEGs were enriched in biological processes including negative regulation of damage response and cell migration, cellular constituents like cytoskeleton and adhesion complexes, and molecular functions including redox activity and ubiquitination. KEGG analysis indicated DEGs were abundant in pathways of cell adhesion molecules, MAPK, folate-dependent one-carbon metabolism, and NF-κB. ADSC-Exos promoted Itgav expression and Fak, Src, and Bcar1 phosphorylation. The Itgav inhibitor cilengitide decreased Itgav expression and phosphorylation of Fak, Src, and Bcar1. These findings suggest ADSC-Exos enhance macrophage migration and adhesion through the Itgav/Fak/Src signaling pathway. Biological sciences/Cell biology Biological sciences/Molecular biology exosomes adipose-derived stem cells macrophages migration transcriptome sequencing peripheral nerve injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Peripheral nerve injury (PNI) is a prevalent clinical traumatic condition that often causes sensory or motor impairment, muscle atrophy, and other issues that impose a huge economic burden on patients and society 1 . A significant quantity of degraded myelin or axonal fragments accumulate at the site of wounds when the peripheral nerve trunk or its branches are subjected to external stimuli, obstructing axonal regeneration 2 . Macrophages and Schwann cells (SCs) travel to the periphery of the injured nerve, clear fragmented tissue, and stimulate nerve regeneration 3 . As such, macrophage migration is essential for peripheral nerve regeneration and repair 4 . As one important component of innate immunity, macrophages play an important role in maintaining internal environment homeostasis, tissue repair, and immune defense, due to their physiological functions such as polarization, phagocytosis, and migration 5 . Nerve repair and regeneration are delayed because molecules such as silent regulator protein 6 revent macrophages from performing phagocytosis, migration, and other physiological tasks 6 . Myelin degradation, Wallerian degeneration, and axon regeneration at the site of damage are delayed in mice with peripheral neuropathy when silica is applied in vivo to prevent macrophage infiltration 7 . Macrophages are crucial components of neuronal regeneration and repair, and are essential for the rapid formation of synaptic connections with target organs 8,9 . Adipose-derived stem cells (ADSCs) are frequently used as seed cells in neural tissue engineering because of their capacity to stimulate cell division, prevent apoptosis, and improve neuronal function 10 . However, the primary factor responsible for the therapeutic function of ADSCs in tissue repair, autoimmune disorders, and neuroregeneration, is their exosome-derived products 11,5 . Adipose stem cell-derived exosomes (ADSC-Exos) have the advantages of minimal immunogenicity and high stability, and function to partially mediate the therapeutic effect of ADSCs 12 . As such, they play increasingly important roles in the repair and regeneration of damaged nerve tissues 11,13 . A recent study demonstrated that ADSC-Exos, in conjunction with the immunosuppressive medication tacrolimus (FK506), accelerated nerve regeneration with neuroprotective effects by reducing macrophages in a mouse model of sciatic nerve crush injury 4 . ADSC-Exos reduced inflammation by encouraging macrophage polarization to an M2 (anti-inflammatory) phenotype by upregulating Nrf2 and HO-1 expression levels 14,15 . ADSC-Exos have further been shown to reduce neuronal death, promote neurogenesis, and regulate neuroinflammation after absorption by macrophages 16 . It has further become obvious that ADSC-Exos regulate biological functions, including the migration, polarization, and phagocytosis of macrophages, suggesting them as a possible therapeutic target for PNI. This study aimed to investigate the mechanisms by which ADSC-Exos modulate macrophage migration, providing an experimental and theoretical basis for the promotion of macrophage migration by ADSC-Exos. Results Acquisition and characterization of adipose-derived stem cells Primary ADSCs were generated by isolating rat parietal adipose tissue from the epididymis, and cultured in vitro. The cells tightly adhered to the wall, while some formed the shape of a long shuttle after 24-hour culture. After incubation for 7 days, the ADSCs exhibited a uniform shape, and were arranged in an orderly swirling pattern (Fig. 1 A-B, E). When third-generation ADSCs were lipogenically formed for 14 days, a considerable number of red lipid droplets were observed by Oil Red O staining (Fig. 1 C-D). Alizarin red staining further revealed red calcified nodules in ADSCs after 32 days of osteogenic induction (Fig. 1 F-G). Adipose-derived stem cells were immunofluorescently stained to determine the presence of CD29 and CD44 in the cytoplasm. There were (97.70 ± 11.21)% of the cells positive for CD29, and (97.73 ± 0.51)% positive for CD44(Figure 1 H-I). Western blot results also demonstrated that CD29 and CD44 were positively expressed (Fig. 1 J). Characterization of ADSC-Exos Under transmission electron microscopy, the ADSC-Exos appeared cup-shaped. The primary peak distribution of the ADSC-Exos particle size was approximately 132 nm, as assessed using a nanoparticle tracking analyzer. Western blot revealed that ADSC-Exos expressed the exosome markers CD9 and TSG101. A summary of this process was shown in Fig. 2 . Analysis of macrophage vitality following uptake of ADSC-Exos After co-culturing 10 µg/ml PKH26-labeled ADSC-Exos with RAW264.7 cells for 24 hours, the macrophage nucleus was encircled by the red fluorescence indicative of PKH26 positivity (Fig. 3 A). Compared with the control, the number of Edu-positive cells increased considerably ( P < 0.05) after macrophage absorption of ADSC-Exos in a dose-dependent manner, although there was no significance between the 20 µg/ml group and the 40 µg/ml group (Fig. 3 B-C). Impact of ADSC-Exos on RAW264.7 migration and adhesion capacity Compared to the control group, there was a substantial increase in adherent cells, scratch healing area, and migratory cells in the Transwell chambers of the ADSC-Exos-treated group ( P < 0.05). Moreover, the 20 µg/ml ADSC-Exos-treated group had a greater effect than the 10 µg/ml ADSC-Exos-treated group ( P 0.05). (Fig. 4 ). Transcriptomics sequencing analysis By using transcriptome sequencing to compare the gene sequences of RAW264.7 in the Exos group with those of RAW264.7 in the control group, 233 genes were found to be differentially expressed between the two groups of samples, including 151 up-regulated genes and 82 down-regulated genes. The clustering heatmap further illustrated the differences in DEG expression levels between the two groups of samples (Fig. 5 A-B). The GO classification and annotation of DEGs from the two sets of samples revealed that molecular functions were primarily enriched in functional clusters, such as regulation of ubiquitination and regulation of redox activity; biological processes were improved mainly in available clusters, such as negative regulation of damage response and cell migration; and cellular components were primarily enriched in functional clusters, such as protein complexes involved in adhesion and cytoskeleton. The results of KEGG enrichment analysis showed that DEGs were mainly enriched in cell adhesion molecules, MAPK signaling pathway, one carbon pool by folate, and NF-κB signaling pathway (Fig. 5 C-D)( https://www.kegg.jp/kegg/kegg1.html ). Using the String database, the interaction relationship of DEGs was extracted, the PPI network interaction graphwas obtained by CytoScape software, and a total of 5 functional modules were obtained by BC value, among which the most significant module included 15 nodes and 41 edges. (Fig. 5 E-F). qRT-PCR method for detecting the expression level of key genes qRT-PCR was performed to assess the mRNA expression levels of the 12 genes involved in this pathway. Compared to the control group, the mRNA expression of Dusp6, Itga5, Itgav, Nr4a1, Pdgfb, Plau, Ptgs2, CD276, Pdgfa, and Pdcd1 was considerably higher in the treated ( P < 0.05); however, the mRNA expression of Cldn11 and CD28 were lower ( P < 0.05) (Fig. 6 ). The influence of Cilengitide on RAW264.7 viability Following a 24-hour treatment of RAW264.7 cells with varying concentrations of Cilengitide, the CCK8 assay demonstrated that, in contrast to the control group, the various concentrations of Cilengitide exhibited more pronounced inhibitory effects on the proliferation of RAW264.7 cells, along with a clear concentration-dependent effect relationship. The inhibitor's concentration at a 50% clearance rate, or IC50 value, served as an evaluation measure, and the ideal action concentration was 0.5 µM (Fig. 7 A). Identification of factors involved in the Itgav/Fak/Src signaling pathway Compared with the control group, the Exo group had higher levels of Itgav protein expression and higher levels of phosphorylation of Fak, Src, and Bcar1 proteins ( P < 0.05). In contrast, the inhibitor group showed lower levels of Itgav expression and lower levels of phosphorylation of Fak, Src, and Bcar1 than the Exo group ( P < 0.05) (Fig. 7 B-M). Effect of ADSC-Exos on RAW264.7 migration and adhesion capacity after inhibiting the Itgav/Fak/Src pathway Compared with the control group, the addition of ADSC-Exos significantly increased the number of migrating cells, the scratch healing area, adherent cells, and migration distance of RAW264.7 ( P < 0.05). However, the number of migrating cells, scratch healing area, adherent cells, and migration distance of RAW264.7 were significantly reduced ( P < 0.05) in the inhibitor group (Figs. 8 and 9 ). Discussion Tissue engineering treatment is a new field of study which integrates molecular biology, cell biology, and other sciences 18 . After nerve damage, ADSCs, which are one of the most promising seed cells for tissue engineering therapy, are essential for regenerative repair as they reduce inflammation and preserve physiological homeostasis 19 . In this study, an enzymatic approach, which is quick, easy to operate, and has a short in vitro culture cycle, was used to isolate a significant number of ADSCs from rat parietal adipose tissue of the epididymis 20 . The collected cells exhibited a homogenous morphology, stable proliferation, multidirectional differentiation, and stem cell capabilities. Additionally, they demonstrated strong expression of CD29 and CD44, indicating successful extraction of ADSCs. ADSCs have been shown to release physiologically active molecules such as exosomes, which exert therapeutic effects in a variety of sectors 21,22 . The results of this study demonstrated that ADSC-Exos were successfully isolated as they were consistent in size, had a cup-like appearance under an electron microscope, and positively expressed the exosome-specific markers CD9 and TSG101 21 . Macrophages constitute a crucial constituent of an organism's intrinsic immunity, as they can enhance neural repair, phagocytose fragments of necrotic tissue debris, secrete growth factors, and stimulate angiogenesis 23,24 . Consequently, they play pivotal roles in preserving internal environmental homeostasis, tissue repair, and immunomodulation of the immune system 25 . Following phagocytosis of ADSC-Exos, macrophages interact with receptor cells by attaching proteins, nucleic acids, and other bioactive materials transported by ADSC-Exos to appropriate receptors 26 . ADSC-Exos were also found to enhance neuroregeneration by promoting macrophage M2 polarization in a rat model after PNI via the TSG-6/NF-κB/NLRP3 pathway 27 . A crucial component of nerve regeneration and repair is the timely migration of macrophages to the injury site, where they phagocytose tissue debris and remove obstacles to regeneration. The accumulation of myelin sheaths and axon fragments around damaged nerves further prevents axonal regeneration 28 . Identifying the factors that affect the migration ability of macrophages may be key to the treatment of PNI. In this study, we used the fluorescent dye PKH26 to mark ADSC-Exos for assays which showed that macrophages could specifically absorb ADSC-Exos. The migratory and adhesion abilities of macrophages enveloped in ADSC-Exos were enhanced, and transwell, scratch, and adhesion assays revealed a considerable increase in the viability of these cells. Therefore, cell migration is essential. Measuring parameters such as cell displacement, steering angle, and blocking coefficient in vitro using live-cell imaging can provide important information for studying cell migration properties 29 . In this study, laser confocal microscopy was performed to capture real-time images of macrophage migration. These findings revealed that ADSC-Exos could extend the migration distance of macrophages, supporting the notion that ADSC-Exos enhance macrophage migration ability. Cell adhesion serves as the foundation for cell migration, a dynamic process involving pseudopod protrusion, cytosolic contraction, and adhesion dissociation 28,30 . Wibbe et al. suggested that cell adhesion structures mediate the connections between individual cells and attach intercellular contact sites to the underlying actin cytoskeleton, thus facilitating the transfer of mechanical forces and phase separation 31 . Cell adhesion is also essential for maintaining cell morphology, activating signaling pathways, and a variety of other developmental functions. Shinde et al. stated that illnesses and developmental abnormalities result from mutations in adhesion receptors 32 . Thus, using an adhesion assay, we further investigated the impact of ADSC-Exos on macrophage adhesion. The results of the adhesion assay demonstrated that the macrophage adhesion ability was significantly increased following ADSC-Exos phagocytosis, and the effect's trend was consistent with the findings of the transwell assay, which indicated that ADSC-Exos could promote macrophage migration by increasing macrophage adhesion ability. Moreover, this effect was found to be dose-dependent. Nevertheless, no significant difference was observed between the 20 µg/ml and 40 µg/ml ADSC-Exos treatment groups. Holly et al. further demonstrated an asymmetric bell curve for cell migration, extracellular matrix, and the density and affinity of the receptor 33 . Specifically, when all three factors were in lower concentration, the forward gravitational force formed by adhesion was smaller, resulting in a lower rate of cell migration. When the three factors were gradually increased, the number of migrations increased in proportion to the strength of the cell's adhesion. However, after surpassing a certain value, the adhesion became too strong, ultimately influencing the subsequent dissociation, which in turn caused a decrease in the rate of cell migration. This indicates that the ideal dose of ADSC-Exos to encourage macrophage migration may be 20 µg/ml. We further conducted a transcriptome sequencing analysis to better understand the mechanism of action of ADSC-Exos in promoting macrophage migration. By comparing the gene expression profiles of samples from both ADSC-Exos and control groups, we were able to identify 233 DEGs, of which 151 were up-regulated and 82 were down-regulated. The clustering heatmap showed the differential expression levels of DEGs between the two sets of samples with good clustering effects. We used a STRING database to visualize and analyze the DEGs, and to determine how their component factors interacted. By utilizing the MCODE plug-in in the Cytoscape software to analyze the DEGs, we were able to identify five major functional modules, the most significant of which might be the essential nodes influencing the entire signal transduction cascade. The trend in the data, which was confirmed using qRT-PCR for 12 of these correlators, was consistent with the transcriptome sequencing findings. The DEGs were then subjected to GO and KEGG enrichment analyses. The findings of the KEGG study indicated that the DEGs were primarily enriched in signaling pathways that include NF-κB, MAPK, and cell adhesion molecules. Additionally, cellular composition in the GO classification annotation was primarily enriched in functional clusters, including adhesion-related protein complexes. According to Chen et al., ADSC-Exos were picked up by macrophages and transported to the lesion sites 16 . By preventing macrophage activation and polarization through mediation of the NF-κB and MAPK signaling pathways, nerve injury-related side effects may be reduced. ADSC-Exos are also able to reduce the production of inflammatory cytokines in macrophages and improve neuroinflammation by inhibiting the NF-κB and MAPK signaling pathways 34 . As transmembrane glycoproteins on the cell membrane, cell adhesion molecules are crucial for axonal growth and regeneration, myelin synthesis stimulation, and neuronal differentiation 35 . Integrins are an essential class of cell adhesion molecules that exhibit specificity towards distinct ligands present in the extracellular matrix. Upon engagement with these ligands, the conformation of integrins is modified, triggering downstream kinases. Focal adhesion kinase (Fak) is one such kinase that plays a crucial role in the integrins' downstream pathway 36 . Fak's N-terminal functional domain binds to the intracellular portion of the β-subunit of integrins when integrins are activated. Simultaneously, Fak autophosphorylation at the Tyr397 site provides a binding site for the Src kinase SH2 structural domain, forming the Fak/Src signaling complex that initiates downstream pathways, including the RAS-MAPK and PI3K-AKT pathways, and completes signaling from outside to inside 9 . When Fak and Src binding is mutually activated, Bcar1 is tyrosine-phosphorylated, creating the SH2 binding site of Crk, which facilitates cytoskeletal changes, motility, and migration 37 . Cycloheximide inhibits macrophage migration by downregulating proteins involved in the SRC/FAK/P130Cas cascade 38 . Another study suggested that hydrogen sulfide could stimulate the Itgb1-Src-FAK/Pyk2-Rac pathway, which in turn promoted macrophage migration 39 . Thus, the integrin pathway plays an important role in regulating cell migration and adhesion. This study demonstrated that the addition of ADSC-Exos increased Itgav expression while enhancing Fak, Src, and Bcar1 phosphorylation levels in RAW264.7, which was accompanied by an increase in the ability of the protein to migrate and adhere. Similarly, the addition of Cilengitide, an Itgav inhibitor, decreased Itgav expression and the phosphorylation levels of Fak, Src, and Bcar1 in RAW264.7, which triggered a significant decline in the migration and adhesion ability of RAW264.7 after the addition of Cilengitide. RAW264.7's ability to migrate and adhere was significantly diminished, along with decreased phosphorylation of Fak, Src, and Bcar1. This study indicated that ADSC-Exos could be taken up by macrophages, thus affecting their migration and adhesion abilities, and that the mechanism might be closely related to the Itgav/Fak/Src signaling pathway. Methods Experimental animals and cells culture Twelve 8-week-old male Sprague-Dawley rats (weighing 80–120 g) under specific pathogen-free (SPF) conditions were purchased from Beijing Huafukang Biotechnology Co., Ltd. (License No. SCXK (Jing) 2019-0008). The animals were housed under standard conditions (22–26°C) with ad libitum access to food and water before and after surgical procedures. All rats were used for the isolation and culture of adipose-derived stem cells (ADSCs). Surgical anesthesia was induced with 3% isoflurane inhalation and maintained with 1.5% isoflurane. The depth of anesthesia was monitored by pedal reflex. Euthanasia was performed via CO₂ asphyxiation, and death was confirmed by the absence of corneal reflexes. This study was conducted in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ) and approved by the Animal Care and Use Committee of Chengde Medical University (Approval No. CDMULAC-20230331-009; Date: March 31, 2023). All experimental protocols complied with the NIH Guide for the Care and Use of Laboratory Animals. The mouse monocyte-macrophage cell line RAW264.7 was generously provided by the Department of Medicine, Peking University. Isolation and culture of ADSCs After aseptic cutting of the rat adipose tissue next to the epididymis, the fascia and blood vessels were suitably removed to extract ADSCs, as previously described. Briefly, after the tissue was subjected to 40–60 minutes of shaking at 37°C with 0.1% type I collagenase (Solarbio, Beijing, China), the digestion process was stopped by adding an equal volume of DMEM/F12 complete media supplemented with 10% FBS (Procell, Wuhan, China). Next, the cell suspension was filtered through a 200 mesh filter, and centrifuged for 10 min at 1000 rpm to extract the sediment. The reconstituted cells were then plated on Petri dishes for incubation. The initial fluid exchange occurred after 48 h. After culturing for 8 or 10 days in vitro (div), ADSCs were collected for osteogenic and lipogenic differentiation induction, immunofluorescence staining, and Western blot assays. Multidirectional differentiation potential assay of ADSCs The in vitro differentiation of osteogenic and lipogenic cells was induced as previously described 4 . Briefly, third-generation ADSCs were inoculated into 6-well plates at a density of 5x10 4 /ml. Once cell confluency reached 80%, the lipogenesis and osteogenesis induction culture medium (ProCell) was switched. The media was regularly modified every two to three days. When the lipid droplets were clear and complete, 12–14 days after lipogenic induction, Oil Red O staining (Procell) was performed. Alizarin red (Procell) was used to stain the cells, indicating the shape or growth of the osteogenic material, which was induced for 28–32 days. An inverted microscope was used to observe the formation of the lipid droplets and calcium nodules. Immunofluorescence staining of ADSCs First, the cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, MO, USA) for 10 minutes and then blocked with 5% bovine serum albumin (Solarbio) for 30 minutes at room temperature. Next, the cells were incubated at 4°C overnight with primary antibodies (Rabbit anti-CD29, 1:100, Cat# A-11001, ABclonal, MA, USA; Rabbit anti-CD44, 1:200, Cat# A12410, ABclonal), and then incubated away from light at room temperature for 1 hour with secondary antibodies (goat anti-rabbit secondary antibody conjugated to ABflo® 488, 1:400, Cat# AS053, ABclonal; goat anti-rabbit secondary antibody conjugated to ABflo® 594, 1:800, Cat# AS039, ABclonal). The nuclei were then counterstained with DAPI (Solarbio) for 5 minutes, and examined under a fluorescence microscope. Western blot assay Proteins were obtained by adding the lysate (RIPA: PMSF = 100:1, Solarbio) and quantified using a BCA protein kit (Solarbio). Proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels (10%) and transferred on to polyvinylidene difluoride membranes (Millipore, MA, USA). After being blocked with Ready-to-use blocking solution for 5 minutes at room temperature, the membranes were incubated with the following primary antibodies at 4°C overnight: CD29 (1:1000, Cat# A-11001, Abclonal), CD44 (1:1000, Cat# A12410, Abclonal), CD9 (1:500, Cat# 20597-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Cat# 28283-1-AP, Proteintech), Itgav (1:1000, Cat# A19071, Abclonal), Fak (1:400, Cat# ab40794, Abcam, Cambridge, USA), Src (1:300, Cat# ab133283, Abcam), Bcar1 (1:1000, Cat# 16815-1-AP, Proteintech), P-Fak (1:1000, Cat# ab81298, Abcam), P-Src (1:1000, Cat# 6943T, CST, Danvers, USA), P-Bcar1 (1:1000, Cat# 4011S, CST). Next, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:10000; Cat# AS080, Abclonal) at room temperature for 1 hour, after which an ECL chemiluminescence analyzer was used to analyze the protein bands. Finally, the relative expression of the target protein was determined by comparing the gray value of the target protein band with that of the standard protein band. Isolation and extraction of ADSC-Exos When the wall-adherent cells third-generation ADSCs reached 60% confluency in standard culture, they were transferred to the exosome extraction-specific medium and cultured for another 48–72 hours. After the exosome isolation reagent (250µl/ml, KeyGEN, Jiangsu, China) was added to the supernatant, the mixture was incubated at 4°C for 2 hours, and then centrifuged at 4°C, 12000 g for 20 minutes. ADSC-Exos precipitate was resuspended with 100 µl sterile PBS for later use. Transmission electron microscopy After dropping 20 µl of ADSC-Exos on the copper mesh and allowing it to adsorb for 5 minutes spontaneously, 20 µl of a 2% phosphotungstic acid solution was added. Following a 5-minute drying period under an incandescent lamp, the sample was examined using a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan), and images were recorded. Particle size detection After a 35-fold dilution with sterile PBS, ADSC-Exos were filtered using a 0.22 µm filter. Next, the particle size distribution of ADSC-Exos was determined using a nanoparticle- tracking analyzer (NS300, Malvern, Malvern, UK). PKH26 labeling of ADSC-Exos We used PKH26-labeled ADSC-Exos to analyze the distribution of ADSC-Exos in macrophages. Cells were incubated with PKH26 (100 µM, Cat# UR52302, Umibio, Shanghai, China) at room temperature for 10 minutes, after which ADSC-Exos were extracted after fixing the volume to 10 ml with sterile PBS. The pellet (PKH26- ADSC -Exos) was then resuspended in PBS and co-cultured with macrophages for 24 h. Finally, ADSC-Exos within the macrophages were observed and photographed under a fluorescence microscope. Cell viability assessment To study the impact of ADSC-Exos on the vitality of macrophages, RAW264.7 cells were seeded at a density of 1×10 4 cells per well in 24-well culture chambers. The cells were cultured in DMEM supplemented with 10% FBS for 24 hours. Subsequently, different concentrations of ADSC-Exos (0 µg/ml (control), 10 µg/ml, 20 µg/ml, and 40 µg/ml) were added. 5-ethynyl-2′-deoxyuridine (EdU, 10 µM; Cat# C0071S, Beyotime, Shanghai, China) was then added to the culture medium for incorporation into proliferating cells. The cells were fixed in 4% paraformaldehyde for 30 minutes on ice 24 hours after EdU treatment. Then, the EdU-incorporated proliferating cells were visualized using a BeyoClick™EdU-488 Kit (Beyotime), in accordance with the manufacturer’s instructions. The cell nuclei were then stained with the nuclear marker Hoechst 33342(1:1000; Beyotime). Finally, the cells were observed under a fluorescence microscope, and the percentage of EdU-positive cells was calculated. Cell migration assay The migratory ability of RAW264.7 was determined using 6.5 mm Transwell chambers with 8 µm pores (Corning NY, USA). RAW264.7 were treated with 0, 10, 20, or 40 µg/ml ADSC-Exos, and then seeded in the upper chamber at a density of 1000 cells/µL, while 600 µL complete medium containing DMEM and 10% fetal bovine serum was added in the bottom chamber. RAW264.7 cells were cultured in Transwell chambers for an additional 24 hours. Cells that migrated to the lower chamber were stained with 0.1% crystal violet for 20 minutes, whereas those left in the upper chamber were wiped away with a cotton swab. Finally, a randomly selected field of view was obtained and studied under a microscope. Image J software was subsequently used to count the number of moving cells. Wound healing assay The migratory ability of RAW264.7 cells was further determined using the wound healing assay at a density of 1×10 5 cells per well in 12-well culture chambers. A linear scratch was then made on the cultured cells, after which they were treated with 0, 10, 20, and 40 µg/ml ADSC-Exos for an additional 48 hours. Images were then captured using an inverted microscope at 0, 24, and 48 hours after wounding. The relative healing area was calculated using the ImageJ software ( https://imagej.nih.gov/ij/ ). Adhesion assay To determine the impact of ADSC-Exos on macrophage adhesion capacity, RAW264.7 cells were seeded at a density of 5×10 4 cells per well in 24-well culture chambers for 4. After removing non-adherent cells with PBS, they were stored in 4% paraformaldehyde for 30 minutes at room temperature. After the nuclei were stained with DAPI, the membranes were permeabilized with a solution containing 3% bovine serum albumin (BSA) and 1% Triton-X100 for 15 minutes. After capturing images with a fluorescence microscope, the number of adherent cells was calculated using Image J software. RNA-sequencing analysis RAW264.7 cells were consistently cultivated for 24 hours and the cells then treated with 0 and 20µg/ml ADSC-Exos for an additional 24 hours. RNA extraction and library construction were carried out using Trizol reagent (Invitrogen, CA, USA) and Illumina's NEBNext® UltraTM RNA Library Prep Kit (Gene Biotechnology International Trade Co., Ltd), respectively, following the manufacturer’s protocols for reverse transcriptional synthesis of double-stranded DNA, terminal repair, sequencing linker linking, PCR amplification, and library quality control. Relative expression of genes were measured using the 2 -ΔΔCT method. The primer sequences used in this study are listed in Table 1 . Library quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and sequenced on an Illumina Novaseq 6000 platform (Novaseq Technology Co., Ltd, Beijing, China). To achieve high-quality clean data, multiple steps were implemented to screen the raw data, including filtering of reads using FASTP (ver. 0.19.7) 40 , and reference genomic mapping was performed using HISAT2 (2.0.5) 41 . Table 1 Primer sequences Gene name Primer sequences Dusp6 Forward:5’-CGGCTTCTGCTGATGGACT-3’ Reverse:5’-TCGCTGCTATTCTCGTCGTA-3’ Itga5 Forward:5’-CCTCTGCTGCTGCTACTGT-3’ Reverse:5’-ACCTGGCTAGTGTTAG-3’ Itgav Forward:5’-GAACAAGGAGAACCAGAACCATT-3’ Reverse:5’-ATACCAACACAGCCAGTAGCA-3’ Nr4a1 Forward:5’-CAATATGGAACACCAGCAACGA-3’ Reverse:5’-GGAGGAGGCAGAGGAACAAG-3’ Pdgfb Forward:5’-ATCGCACCAACGCCAACT-3’ Reverse:5’-GGTCACTACTGTCTCACACTTG-3’ Plau Forward:5’-CACAGCCATCCAGGTCCATA-3’ Reverse:5’-AAGCGGTCCTCCAGAATCG-3’ Ptgs2 Forward:5’-CTGGTGCCTGGTCTGATGAT-3’ Reverse:5’-CAATGCGGTTCTGATACTGGAA-3’ CD276 Forward:5’-GAGGTGTTCTGGAAGGATGGA-3’ Reverse:5’-ATGGTGACTGAGCCGTGAG-3’ Pdgfa Forward:5’-GTAACACCAGCAGCGTCAAG-3’ Reverse:5’-ACCTCACATCTGTCTCCTCCT-3’ Pdcd1 Forward:5’-TGAACTGGAACCGCCTGAG-3’ Reverse:5’-GGTAGATGCCACTGTCATTGC-3’ Cldn11 Forward:5’-TGGATTGGCATCATCGTCACA-3’ Reverse:5’-CCAGAACGGAGGCAGCAAT-3’ CD28 Forward:5’-GCTGCTGTTCTTGGCTCTC-3’ Reverse:5’-GCTGACCTCGTTGCTATCTAC-3’ Src Forward: 5’-GCCTATGTGGAGCGGATGA-3’ Reverse: 5’-CTGTGTATTCGTTGTCTTCTATGAG-3’ Ptk2 Forward: 5’-ACTTGGACGCTGTATTGGAGA-3’ Reverse: 5’-CTGACGCATTGTTAAGGCTTCT-3’ GAPDH Forward: 5’-GGTGAAGGTCGGTGTGAACG-3’ Reverse: 5’-CTCGCTCCTGGAAGATGGTG-3’ Gene function and pathway enrichment analysis Differentially expressed genes (DEGs) were analyzed using DESeq2 (1.16.1). Genes with a |log 2 Fold change|>0 42 and P-value < 0.05 were considered as DEGs. These DEGs were subjected to KEGG pathway and GO enrichment analyses using ClusterProfiler (4.0) software 43 . After Protein-protein interaction network analysis of the DEGs was performed using the String database ( https://www.string-db.org/ ), gene connecting networks were visualized using Cytoscape 3.9.1 software. Cell proliferation assay To determine the optimum concentration of Cilengitide, RAW264.7, cells were seeded at a density of 1x10 5 /ml. cells in a 96-well culture chamber for 24 hours. The cultures were divided into three groups: blank (no drugs), control (no cells), and inhibitor (concentrations of 0.1, 0.3, 0.5, 0.7, and 0.9uM, respectively). Each group included five replicates. Cell Counting Kit-8 reagent (Apexbio, Houston, TX, USA) was added to each well (10 µL/well), followed by a 2-hour incubation at 37°C. Absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA) to determine the optical density (OD) of the different groups. Cell proliferation was expressed as cell inhibition as follows: cell inhibition (%) = [A1(cilengitide)-A3 (blank)]/[A2 (control)-A3(blank)] × 100%. Confocal Laser Scanning Microscope RAW264.7 cells were routinely cultured for 24 hours at a density of 3×10 4 /ml. A linear scratch was made on the cultured cells prior to treatment with 0 µg/ml ADSC-Exos, 20 µg/ml ADSC-Exos, and 20 µg/ml ADSC-Exos + 5 µM cilengitide for an additional 4 hours. The real-time migration of RAW264.7 was determined using a Confocal Laser Scanning Microscope (FV3000, Olympus, Tokyo, Japan) 44 . Statistical Analysis Data are presented as the mean ± standard deviation (SD). GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA; www.graphpad.com ) was used for statistical analyses. Data were evaluated using Student’s t-test between two groups or one-way analysis of variance followed by Tukey’s post hoc test in more than two groups, depending on whether normality was indicated, with P < 0.05 considered statistically significant 45–46 . Declarations Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Author contributions Conceptualization, X.F.; data curation, B.Y. and J.X.; methodology, B.Y., J.X., and M.R.; validation, B.Y., J.X., and Y.W.; resources, Q.Z. and M.R.; formal analysis, B.Y. and X.F.; investigation, J.X., Y.W., and Q.Z.; writing—original draft, B.Y. and J.X.; writing—review and editing, X.F. and B.Y.; supervision, Y.W. and X.F.; project administration, X.F.; funding acquisition, X.F., B.Y., and Y.W. All authors have read and agreed to the published version of the manuscript. Additional information Competing interests: The authors declare no competing interests. Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 82371385); the Natural Science Foundation of Hebei Provincial Department of Science and Technology (Grant No. H2021406056); the Key Project of Hebei Provincial Department of Education (Grant No. ZD2020178); Heilongjiang Natural Science Foundation of China (Grant No. JQ2021H004); the Advantageous Discipline of Human Anatomy and Histoembryology at Chengde Medical University (Grant No. [2023]22); Hebei Provincial Key Laboratory of Nerve Injury and Repair Open Project Fund (Grant No. NJKF202403); and the School-level Research Project of Chengde Nursing Vocational College (Grant No. Y202501). Institutional Review Board Statement: All animal experiments were approved by the Animal Ethics Committee of Chengde Medical University (Approval No. CDMULAC-20230331-009) and were conducted in strict accordance with the institutional guidelines for the care and use of laboratory animals. Correspondence and requests for materials should be addressed to X.F. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G>T mutation in MtDNA (mt-Atp8). Sci. Rep. 15, 18717. https://doi.org/10.1038/s41598-025-03671-8 (2025). Xue, Y, et al. Exosomes derived from M2 macrophage promote HUVECs proliferation, migration and tube formation in vitro. Sci. Rep. 15, 17876. https://doi.org/10.1038/s41598-025-03113-5 (2025). Additional Declarations No competing interests reported. Supplementary Files WBTSG101CD9.pdf WBCD29CD44.pdf WBIntegrinpathwayrelatedfactors.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7248636","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":506106733,"identity":"06381065-35ae-4060-9b07-198c8095fc54","order_by":0,"name":"bo yuan","email":"","orcid":"","institution":"Chengde Nursing Vocational College","correspondingAuthor":false,"prefix":"","firstName":"bo","middleName":"","lastName":"yuan","suffix":""},{"id":506106734,"identity":"bb0ed17a-ffa1-4007-ba73-ea51e727cc7b","order_by":1,"name":"Jiayi Xie","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Xie","suffix":""},{"id":506106735,"identity":"d4e5aa7f-90e2-4079-819d-cc2006e1ce59","order_by":2,"name":"Qinghua Zhu","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Zhu","suffix":""},{"id":506106736,"identity":"f36be83f-4484-4d67-9ce9-70f94de4e3d5","order_by":3,"name":"Miao Ren","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Ren","suffix":""},{"id":506106737,"identity":"62b2d53a-24a2-417b-9c33-6690cdceace5","order_by":4,"name":"Yilun Wang","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yilun","middleName":"","lastName":"Wang","suffix":""},{"id":506106738,"identity":"ba615941-7b6f-49c7-a516-b8ced5fb8bde","order_by":5,"name":"Ying Wang","email":"","orcid":"","institution":"Mudanjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Wang","suffix":""},{"id":506106739,"identity":"32666853-f586-49bb-bd2c-76c2533718f5","order_by":6,"name":"Xiumei Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIie3PMQrCMBSA4RcKzfLUNaKHiAjiUNqrWAKZMniElIIuHsDFOzh1jgZ18QAODk46CR0dBK3iprS6OeRfXob38QiAy/WHUQ1giokNjySHnAdhJUHzIs1xmnamQym+Is/4djNqYb4kupIw1bE1FbRhF+tWwI0H1K7m5UQObC2TSKax7iq+rwNKuSsjEROmIBY9Fmuh+NEDhr1SgsVmQW7oPx59bomuJs8rBhEXSQpfETwOFrNMIKNJSiZcCr/qL0hVNz9nYRRZerpcrkHYoHZdSt7zf1t3uVwu16fuXttKeWBO7eUAAAAASUVORK5CYII=","orcid":"","institution":"Chengde Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiumei","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2025-07-30 05:23:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7248636/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7248636/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90116001,"identity":"96c75220-7115-4125-a59a-6e073549d477","added_by":"auto","created_at":"2025-08-28 16:13:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":429049,"visible":true,"origin":"","legend":"\u003cp\u003eExtraction and identification of adipose-derived stem cells (A) Isolation and extraction of adipose-derived stem cells. (B) Incubation for 24 hours cell morphology. Bar = 100 µm. (C) Adipogenic induction for 14 days. (D) Oil red O staining. (E) Cell morphology after 7 days of culture. (F) Osteogenic induction for 32 days. (G) Alizarin red staining. (H) Immunofluorescence staining, positive expression of CD29 was labeled with green fluorescence, positive expression of CD44 was labeled with red fluorescence, and DAPI staining nucleus were blue. Bar = 20 µm. (I) CD29 and CD44 positive cell rate. (J) Western blot bands of CD29 and CD44.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/3f1185c7b9e8d70d57cc181b.png"},{"id":90115106,"identity":"9b03c72e-b09d-46cf-b2a5-c5219134dbf5","added_by":"auto","created_at":"2025-08-28 16:05:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121101,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of ADSC-Exos. Bar = 100μm. (A) Morphology of ADSC-Exos under transmission electron microscopy. (B) Particle tracking analyzer to determine the particle size distribution range of ADSC-Exos. (C) Western blot bands of CD9 and TSG101.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/e65ad0456145db5105e4a320.png"},{"id":90115111,"identity":"df7af846-3da4-4200-8127-1185b165d658","added_by":"auto","created_at":"2025-08-28 16:05:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174986,"visible":true,"origin":"","legend":"\u003cp\u003eTracing of ADSC-Exos in macrophages and analysis of macrophage viability. (A) Immunofluorescence was used to detect the in vivo tracing of ADSC-Exos in macrophages, PKH26-labeled ADSC-Exos showed red fluorescence, and the nuclei were blue fluorescence after DAPI staining. Bar = 20 µm. (B) RAW264.7 activity was detected by Edu method, Edu positive showed green fluorescence, and the nucleus was blue fluorescence after Hoechst33342 staining. Bar = 50μm. (C) Quantitative analysis of immunopositivity for EdU (normalized by levels in the control group). Data were expressed as mean ± SD (n = 3). ***\u003cem\u003eP\u003c/em\u003e＜0.001, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ns: \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05 (one-way analysis of variance followed by the Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/44eed1365526aac28dcce900.png"},{"id":90116005,"identity":"5033cf28-6e4a-48dc-bf0a-2ce05db36776","added_by":"auto","created_at":"2025-08-28 16:13:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191492,"visible":true,"origin":"","legend":"\u003cp\u003eADSC-Exos facilitated RAW264.7 migration. (A) RAW264.7 migration in the Transwell assay: RAW264.7 was treated with 0, 10, 20, or 40μg/ml ADSC-Exos. Cells that migrated to the lower chamber were stained with 0.1% crystal violet, which indicatedmigrated cells. Bar = 50 µm. (B) Representative image of the scratch experiment. Bar = 100 µm. (C) Representative image of RAW264.7 nucleation staining by DAPI in adhesion experiment. Bar = 50 µm. (D, E, F) The number of RAW264.7 migrating cells, relative healing area and the number of adherent cells was calculated by Image J software. Data were expressed as the mean ± SD (n = 3 ); ***\u003cem\u003eP\u003c/em\u003e＜0.001, **\u003cem\u003eP\u003c/em\u003e＜0.01, *\u003cem\u003eP\u003c/em\u003e＜0.05, ns:\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05(one-way analysis of variance followed by the Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/70e296be3ad8fe1b6f4410ad.png"},{"id":90115114,"identity":"5c0726b4-1e8b-4a25-81f3-a5efbd22eaea","added_by":"auto","created_at":"2025-08-28 16:05:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":309115,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome analysis results. (A) Clustering heatmap of DEGs (CK represents the control group, and Exo denotes the Exo group; red represents up-regulated genes and green represents down-regulated genes). (B) Volcano plot of DEGs (\u003cem\u003eP\u003c/em\u003e-value\u0026lt; 0.05 and |log\u003csub\u003e2\u003c/sub\u003efold change|\u0026gt; 0; red represents up-regulated genes and blue represents down-regulated genes). (C, D) GO analysis (C) and KEGG pathway analysis (D) of DEGs. Data source: KEGG database (Kanehisa et al., 2025)\u003csup\u003e17\u003c/sup\u003e. ©2025 Kanehisa Laboratories. Used with permission. (E) Interaction mapping of the PPI network of DEGs. (F) The most significant modules consist of 15 nodes and 41 edges.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/61a3fdc6c4b2a6e6cec70045.png"},{"id":90116002,"identity":"7f41e01f-7729-4aaf-b49e-9ef3a217965d","added_by":"auto","created_at":"2025-08-28 16:13:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":146832,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative reverse transcription-polymerase chain reaction showed the mRNA expression of Itga5、CD276、Dusp6、Itgav、Pdcd1、Nr4a1、Pdgfa、Pdgfb、Plau、Ptgs2、Cldn11 and CD28 in each group (n = 3 ). ***\u003cem\u003eP\u003c/em\u003e＜0.001, **\u003cem\u003eP\u003c/em\u003e＜0.01, *\u003cem\u003eP\u003c/em\u003e＜0.05 (Student’s t-test).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/e08590687d52ef1099967600.png"},{"id":90115122,"identity":"0794203e-f4b9-4d61-a28b-c2fdbc909d13","added_by":"auto","created_at":"2025-08-28 16:05:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":227084,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of Itgav/Fak/Src pathway-related factors. (A) CCK8 method was used to detect the growth inhibition rate of Cilengitide on RAW264.7 cells, The abscissa represents the concentration of action of Cilengitide, and the ordinate represents the inhibition rate. (B) Western blot was used to detect the expressions of Itgav, T-Fak/P-Fak, T-Src/P-Src, and T-Bcar1/P-Bcar1 proteins. (C-I) Western blot results were quantified using Image J. (J-M) Quantitative reverse transcription-polymerase chain reaction showed the mRNA expression of Itgav, Src, Ptk2 and Bcar1 in each group (n = 3 ). ***\u003cem\u003eP\u003c/em\u003e＜0.001, *\u003cem\u003eP\u003c/em\u003e＜0.05, ns:\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05 (one-way analysis of variance followed by the Tukey’s post hoc test)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/7f1443acfe503f8c87b983e9.png"},{"id":90116006,"identity":"08f20ed6-92da-45b9-ac16-9e46e7aef9ee","added_by":"auto","created_at":"2025-08-28 16:13:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":558341,"visible":true,"origin":"","legend":"\u003cp\u003eADSC-Exos facilitated RAW264.7 migration. (A) RAW264.7 migration in the Transwell assay. Bar = 50 µm. (B) Representative image of the scratch experiment. Bar = 100 µm. (C) Representative image of RAW264.7 nucleation staining by DAPI in adhesion experiment. Bar = 50 µm. (D, E, F) The number of RAW264.7 migrating cells, relative healing area or the number of adherent cells was calculated by Image J software. Data are expressed as the mean ± SD (n = 3); ***\u003cem\u003eP\u003c/em\u003e＜0.001, **\u003cem\u003eP\u003c/em\u003e＜0.01, *\u003cem\u003eP\u003c/em\u003e＜0.05, ns:\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05(one-way analysis of variance followed by the Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/4e697c2842b7ae424863d65f.png"},{"id":90115117,"identity":"a62cb0d9-8927-4e7a-a303-33083da426db","added_by":"auto","created_at":"2025-08-28 16:05:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":181710,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time imaging of RAW264.7 using laser confocal. (A) Real-time imaging typical image of RAW264.7. Bar = 50 µm. (B) Quantification of migration distance . Data are expressed as the mean ± SD (n = 3 ), ***\u003cem\u003eP\u003c/em\u003e＜0.001 (one-way analysis of variance followed by the Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/48d3d4a0b8a51bb90e5ebd33.png"},{"id":96364892,"identity":"1e5e0a44-f123-47c4-bbdc-c7e0c6872055","added_by":"auto","created_at":"2025-11-20 10:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3418280,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/7bce1a33-19d8-487d-ad41-54f1a42f43b3.pdf"},{"id":90115108,"identity":"044bc7a4-5ca6-44e8-a903-5c5a4d0b949d","added_by":"auto","created_at":"2025-08-28 16:05:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":127774,"visible":true,"origin":"","legend":"","description":"","filename":"WBTSG101CD9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/f6f8ea4437622d3b13fe6ac6.pdf"},{"id":90115110,"identity":"a1095a37-4d3c-4e82-a4af-9bf7b8c524a5","added_by":"auto","created_at":"2025-08-28 16:05:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":150656,"visible":true,"origin":"","legend":"","description":"","filename":"WBCD29CD44.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/6f38835afe32c3c777715427.pdf"},{"id":90116329,"identity":"494a0575-029c-4b39-a876-a676476b2c11","added_by":"auto","created_at":"2025-08-28 16:21:31","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":688878,"visible":true,"origin":"","legend":"","description":"","filename":"WBIntegrinpathwayrelatedfactors.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7248636/v1/20845c87326c42afe516358e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism of ADSC-Exos promoting macrophage migration via Itgav/Fak/Src signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePeripheral nerve injury (PNI) is a prevalent clinical traumatic condition that often causes sensory or motor impairment, muscle atrophy, and other issues that impose a huge economic burden on patients and society\u003csup\u003e1\u003c/sup\u003e. A significant quantity of degraded myelin or axonal fragments accumulate at the site of wounds when the peripheral nerve trunk or its branches are subjected to external stimuli, obstructing axonal regeneration\u003csup\u003e2\u003c/sup\u003e. Macrophages and Schwann cells (SCs) travel to the periphery of the injured nerve, clear fragmented tissue, and stimulate nerve regeneration\u003csup\u003e3\u003c/sup\u003e. As such, macrophage migration is essential for peripheral nerve regeneration and repair\u003csup\u003e4\u003c/sup\u003e. As one important component of innate immunity, macrophages play an important role in maintaining internal environment homeostasis, tissue repair, and immune defense, due to their physiological functions such as polarization, phagocytosis, and migration\u003csup\u003e5\u003c/sup\u003e. Nerve repair and regeneration are delayed because molecules such as silent regulator protein 6 revent macrophages from performing phagocytosis, migration, and other physiological tasks\u003csup\u003e6\u003c/sup\u003e. Myelin degradation, Wallerian degeneration, and axon regeneration at the site of damage are delayed in mice with peripheral neuropathy when silica is applied in vivo to prevent macrophage infiltration\u003csup\u003e7\u003c/sup\u003e. Macrophages are crucial components of neuronal regeneration and repair, and are essential for the rapid formation of synaptic connections with target organs\u003csup\u003e8,9\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdipose-derived stem cells (ADSCs) are frequently used as seed cells in neural tissue engineering because of their capacity to stimulate cell division, prevent apoptosis, and improve neuronal function\u003csup\u003e10\u003c/sup\u003e. However, the primary factor responsible for the therapeutic function of ADSCs in tissue repair, autoimmune disorders, and neuroregeneration, is their exosome-derived products\u003csup\u003e11,5\u003c/sup\u003e. Adipose stem cell-derived exosomes (ADSC-Exos) have the advantages of minimal immunogenicity and high stability, and function to partially mediate the therapeutic effect of ADSCs\u003csup\u003e12\u003c/sup\u003e. As such, they play increasingly important roles in the repair and regeneration of damaged nerve tissues\u003csup\u003e11,13\u003c/sup\u003e. A recent study demonstrated that ADSC-Exos, in conjunction with the immunosuppressive medication tacrolimus (FK506), accelerated nerve regeneration with neuroprotective effects by reducing macrophages in a mouse model of sciatic nerve crush injury\u003csup\u003e4\u003c/sup\u003e. ADSC-Exos reduced inflammation by encouraging macrophage polarization to an M2 (anti-inflammatory) phenotype by upregulating Nrf2 and HO-1 expression levels\u003csup\u003e14,15\u003c/sup\u003e. ADSC-Exos have further been shown to reduce neuronal death, promote neurogenesis, and regulate neuroinflammation after absorption by macrophages\u003csup\u003e16\u003c/sup\u003e. It has further become obvious that ADSC-Exos regulate biological functions, including the migration, polarization, and phagocytosis of macrophages, suggesting them as a possible therapeutic target for PNI. This study aimed to investigate the mechanisms by which ADSC-Exos modulate macrophage migration, providing an experimental and theoretical basis for the promotion of macrophage migration by ADSC-Exos.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAcquisition and characterization of adipose-derived stem cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePrimary ADSCs were generated by isolating rat parietal adipose tissue from the epididymis, and cultured in vitro. The cells tightly adhered to the wall, while some formed the shape of a long shuttle after 24-hour culture. After incubation for 7 days, the ADSCs exhibited a uniform shape, and were arranged in an orderly swirling pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, E). When third-generation ADSCs were lipogenically formed for 14 days, a considerable number of red lipid droplets were observed by Oil Red O staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Alizarin red staining further revealed red calcified nodules in ADSCs after 32 days of osteogenic induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G). Adipose-derived stem cells were immunofluorescently stained to determine the presence of CD29 and CD44 in the cytoplasm. There were (97.70\u0026thinsp;\u0026plusmn;\u0026thinsp;11.21)% of the cells positive for CD29, and (97.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51)% positive for CD44(Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). Western blot results also demonstrated that CD29 and CD44 were positively expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacterization of ADSC-Exos\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eUnder transmission electron microscopy, the ADSC-Exos appeared cup-shaped. The primary peak distribution of the ADSC-Exos particle size was approximately 132 nm, as assessed using a nanoparticle tracking analyzer. Western blot revealed that ADSC-Exos expressed the exosome markers CD9 and TSG101. A summary of this process was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAnalysis of macrophage vitality following uptake of ADSC-Exos\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter co-culturing 10 \u0026micro;g/ml PKH26-labeled ADSC-Exos with RAW264.7 cells for 24 hours, the macrophage nucleus was encircled by the red fluorescence indicative of PKH26 positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Compared with the control, the number of Edu-positive cells increased considerably (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after macrophage absorption of ADSC-Exos in a dose-dependent manner, although there was no significance between the 20 \u0026micro;g/ml group and the 40 \u0026micro;g/ml group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eImpact of ADSC-Exos on RAW264.7 migration and adhesion capacity\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCompared to the control group, there was a substantial increase in adherent cells, scratch healing area, and migratory cells in the Transwell chambers of the ADSC-Exos-treated group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the 20 \u0026micro;g/ml ADSC-Exos-treated group had a greater effect than the 10 \u0026micro;g/ml ADSC-Exos-treated group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Nevertheless, there was no statistically significant difference between the 20 \u0026micro;g/ml and 40 \u0026micro;g/ml ADSC-Exos-treated groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eTranscriptomics sequencing analysis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBy using transcriptome sequencing to compare the gene sequences of RAW264.7 in the Exos group with those of RAW264.7 in the control group, 233 genes were found to be differentially expressed between the two groups of samples, including 151 up-regulated genes and 82 down-regulated genes. The clustering heatmap further illustrated the differences in DEG expression levels between the two groups of samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). The GO classification and annotation of DEGs from the two sets of samples revealed that molecular functions were primarily enriched in functional clusters, such as regulation of ubiquitination and regulation of redox activity; biological processes were improved mainly in available clusters, such as negative regulation of damage response and cell migration; and cellular components were primarily enriched in functional clusters, such as protein complexes involved in adhesion and cytoskeleton. The results of KEGG enrichment analysis showed that DEGs were mainly enriched in cell adhesion molecules, MAPK signaling pathway, one carbon pool by folate, and NF-κB signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D)(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/kegg/kegg1.html\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/kegg/kegg1.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using the String database, the interaction relationship of DEGs was extracted, the PPI network interaction graphwas obtained by CytoScape software, and a total of 5 functional modules were obtained by BC value, among which the most significant module included 15 nodes and 41 edges. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eqRT-PCR method for detecting the expression level of key genes\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eqRT-PCR was performed to assess the mRNA expression levels of the 12 genes involved in this pathway. Compared to the control group, the mRNA expression of Dusp6, Itga5, Itgav, Nr4a1, Pdgfb, Plau, Ptgs2, CD276, Pdgfa, and Pdcd1 was considerably higher in the treated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); however, the mRNA expression of Cldn11 and CD28 were lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe influence of Cilengitide on RAW264.7 viability\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFollowing a 24-hour treatment of RAW264.7 cells with varying concentrations of Cilengitide, the CCK8 assay demonstrated that, in contrast to the control group, the various concentrations of Cilengitide exhibited more pronounced inhibitory effects on the proliferation of RAW264.7 cells, along with a clear concentration-dependent effect relationship. The inhibitor's concentration at a 50% clearance rate, or IC50 value, served as an evaluation measure, and the ideal action concentration was 0.5 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eIdentification of factors involved in the Itgav/Fak/Src signaling pathway\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCompared with the control group, the Exo group had higher levels of Itgav protein expression and higher levels of phosphorylation of Fak, Src, and Bcar1 proteins (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the inhibitor group showed lower levels of Itgav expression and lower levels of phosphorylation of Fak, Src, and Bcar1 than the Exo group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-M).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEffect of ADSC-Exos on RAW264.7 migration and adhesion capacity after inhibiting the Itgav/Fak/Src pathway\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCompared with the control group, the addition of ADSC-Exos significantly increased the number of migrating cells, the scratch healing area, adherent cells, and migration distance of RAW264.7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the number of migrating cells, scratch healing area, adherent cells, and migration distance of RAW264.7 were significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the inhibitor group (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTissue engineering treatment is a new field of study which integrates molecular biology, cell biology, and other sciences\u003csup\u003e18\u003c/sup\u003e. After nerve damage, ADSCs, which are one of the most promising seed cells for tissue engineering therapy, are essential for regenerative repair as they reduce inflammation and preserve physiological homeostasis\u003csup\u003e19\u003c/sup\u003e. In this study, an enzymatic approach, which is quick, easy to operate, and has a short in vitro culture cycle, was used to isolate a significant number of ADSCs from rat parietal adipose tissue of the epididymis\u003csup\u003e20\u003c/sup\u003e. The collected cells exhibited a homogenous morphology, stable proliferation, multidirectional differentiation, and stem cell capabilities. Additionally, they demonstrated strong expression of CD29 and CD44, indicating successful extraction of ADSCs. ADSCs have been shown to release physiologically active molecules such as exosomes, which exert therapeutic effects in a variety of sectors\u003csup\u003e21,22\u003c/sup\u003e. The results of this study demonstrated that ADSC-Exos were successfully isolated as they were consistent in size, had a cup-like appearance under an electron microscope, and positively expressed the exosome-specific markers CD9 and TSG101\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMacrophages constitute a crucial constituent of an organism's intrinsic immunity, as they can enhance neural repair, phagocytose fragments of necrotic tissue debris, secrete growth factors, and stimulate angiogenesis\u003csup\u003e23,24\u003c/sup\u003e. Consequently, they play pivotal roles in preserving internal environmental homeostasis, tissue repair, and immunomodulation of the immune system\u003csup\u003e25\u003c/sup\u003e. Following phagocytosis of ADSC-Exos, macrophages interact with receptor cells by attaching proteins, nucleic acids, and other bioactive materials transported by ADSC-Exos to appropriate receptors\u003csup\u003e26\u003c/sup\u003e. ADSC-Exos were also found to enhance neuroregeneration by promoting macrophage M2 polarization in a rat model after PNI via the TSG-6/NF-κB/NLRP3 pathway\u003csup\u003e27\u003c/sup\u003e. A crucial component of nerve regeneration and repair is the timely migration of macrophages to the injury site, where they phagocytose tissue debris and remove obstacles to regeneration. The accumulation of myelin sheaths and axon fragments around damaged nerves further prevents axonal regeneration\u003csup\u003e28\u003c/sup\u003e. Identifying the factors that affect the migration ability of macrophages may be key to the treatment of PNI. In this study, we used the fluorescent dye PKH26 to mark ADSC-Exos for assays which showed that macrophages could specifically absorb ADSC-Exos. The migratory and adhesion abilities of macrophages enveloped in ADSC-Exos were enhanced, and transwell, scratch, and adhesion assays revealed a considerable increase in the viability of these cells. Therefore, cell migration is essential. Measuring parameters such as cell displacement, steering angle, and blocking coefficient in vitro using live-cell imaging can provide important information for studying cell migration properties\u003csup\u003e29\u003c/sup\u003e. In this study, laser confocal microscopy was performed to capture real-time images of macrophage migration. These findings revealed that ADSC-Exos could extend the migration distance of macrophages, supporting the notion that ADSC-Exos enhance macrophage migration ability.\u003c/p\u003e\u003cp\u003eCell adhesion serves as the foundation for cell migration, a dynamic process involving pseudopod protrusion, cytosolic contraction, and adhesion dissociation\u003csup\u003e28,30\u003c/sup\u003e. Wibbe et al. suggested that cell adhesion structures mediate the connections between individual cells and attach intercellular contact sites to the underlying actin cytoskeleton, thus facilitating the transfer of mechanical forces and phase separation\u003csup\u003e31\u003c/sup\u003e. Cell adhesion is also essential for maintaining cell morphology, activating signaling pathways, and a variety of other developmental functions. Shinde et al. stated that illnesses and developmental abnormalities result from mutations in adhesion receptors\u003csup\u003e32\u003c/sup\u003e. Thus, using an adhesion assay, we further investigated the impact of ADSC-Exos on macrophage adhesion. The results of the adhesion assay demonstrated that the macrophage adhesion ability was significantly increased following ADSC-Exos phagocytosis, and the effect's trend was consistent with the findings of the transwell assay, which indicated that ADSC-Exos could promote macrophage migration by increasing macrophage adhesion ability. Moreover, this effect was found to be dose-dependent. Nevertheless, no significant difference was observed between the 20 \u0026micro;g/ml and 40 \u0026micro;g/ml ADSC-Exos treatment groups. Holly et al. further demonstrated an asymmetric bell curve for cell migration, extracellular matrix, and the density and affinity of the receptor\u003csup\u003e33\u003c/sup\u003e. Specifically, when all three factors were in lower concentration, the forward gravitational force formed by adhesion was smaller, resulting in a lower rate of cell migration. When the three factors were gradually increased, the number of migrations increased in proportion to the strength of the cell's adhesion. However, after surpassing a certain value, the adhesion became too strong, ultimately influencing the subsequent dissociation, which in turn caused a decrease in the rate of cell migration. This indicates that the ideal dose of ADSC-Exos to encourage macrophage migration may be 20 \u0026micro;g/ml.\u003c/p\u003e\u003cp\u003eWe further conducted a transcriptome sequencing analysis to better understand the mechanism of action of ADSC-Exos in promoting macrophage migration. By comparing the gene expression profiles of samples from both ADSC-Exos and control groups, we were able to identify 233 DEGs, of which 151 were up-regulated and 82 were down-regulated. The clustering heatmap showed the differential expression levels of DEGs between the two sets of samples with good clustering effects. We used a STRING database to visualize and analyze the DEGs, and to determine how their component factors interacted. By utilizing the MCODE plug-in in the Cytoscape software to analyze the DEGs, we were able to identify five major functional modules, the most significant of which might be the essential nodes influencing the entire signal transduction cascade. The trend in the data, which was confirmed using qRT-PCR for 12 of these correlators, was consistent with the transcriptome sequencing findings. The DEGs were then subjected to GO and KEGG enrichment analyses. The findings of the KEGG study indicated that the DEGs were primarily enriched in signaling pathways that include NF-κB, MAPK, and cell adhesion molecules. Additionally, cellular composition in the GO classification annotation was primarily enriched in functional clusters, including adhesion-related protein complexes. According to Chen et al., ADSC-Exos were picked up by macrophages and transported to the lesion sites\u003csup\u003e16\u003c/sup\u003e. By preventing macrophage activation and polarization through mediation of the NF-κB and MAPK signaling pathways, nerve injury-related side effects may be reduced. ADSC-Exos are also able to reduce the production of inflammatory cytokines in macrophages and improve neuroinflammation by inhibiting the NF-κB and MAPK signaling pathways\u003csup\u003e34\u003c/sup\u003e. As transmembrane glycoproteins on the cell membrane, cell adhesion molecules are crucial for axonal growth and regeneration, myelin synthesis stimulation, and neuronal differentiation\u003csup\u003e35\u003c/sup\u003e. Integrins are an essential class of cell adhesion molecules that exhibit specificity towards distinct ligands present in the extracellular matrix. Upon engagement with these ligands, the conformation of integrins is modified, triggering downstream kinases. Focal adhesion kinase (Fak) is one such kinase that plays a crucial role in the integrins' downstream pathway\u003csup\u003e36\u003c/sup\u003e. Fak's N-terminal functional domain binds to the intracellular portion of the β-subunit of integrins when integrins are activated. Simultaneously, Fak autophosphorylation at the Tyr397 site provides a binding site for the Src kinase SH2 structural domain, forming the Fak/Src signaling complex that initiates downstream pathways, including the RAS-MAPK and PI3K-AKT pathways, and completes signaling from outside to inside\u003csup\u003e9\u003c/sup\u003e. When Fak and Src binding is mutually activated, Bcar1 is tyrosine-phosphorylated, creating the SH2 binding site of Crk, which facilitates cytoskeletal changes, motility, and migration\u003csup\u003e37\u003c/sup\u003e. Cycloheximide inhibits macrophage migration by downregulating proteins involved in the SRC/FAK/P130Cas cascade\u003csup\u003e38\u003c/sup\u003e. Another study suggested that hydrogen sulfide could stimulate the Itgb1-Src-FAK/Pyk2-Rac pathway, which in turn promoted macrophage migration\u003csup\u003e39\u003c/sup\u003e. Thus, the integrin pathway plays an important role in regulating cell migration and adhesion. This study demonstrated that the addition of ADSC-Exos increased Itgav expression while enhancing Fak, Src, and Bcar1 phosphorylation levels in RAW264.7, which was accompanied by an increase in the ability of the protein to migrate and adhere. Similarly, the addition of Cilengitide, an Itgav inhibitor, decreased Itgav expression and the phosphorylation levels of Fak, Src, and Bcar1 in RAW264.7, which triggered a significant decline in the migration and adhesion ability of RAW264.7 after the addition of Cilengitide. RAW264.7's ability to migrate and adhere was significantly diminished, along with decreased phosphorylation of Fak, Src, and Bcar1. This study indicated that ADSC-Exos could be taken up by macrophages, thus affecting their migration and adhesion abilities, and that the mechanism might be closely related to the Itgav/Fak/Src signaling pathway.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003eExperimental animals and cells culture\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTwelve 8-week-old male Sprague-Dawley rats (weighing 80\u0026ndash;120 g) under specific pathogen-free (SPF) conditions were purchased from Beijing Huafukang Biotechnology Co., Ltd. (License No. SCXK (Jing) 2019-0008). The animals were housed under standard conditions (22\u0026ndash;26\u0026deg;C) with ad libitum access to food and water before and after surgical procedures. All rats were used for the isolation and culture of adipose-derived stem cells (ADSCs). Surgical anesthesia was induced with 3% isoflurane inhalation and maintained with 1.5% isoflurane. The depth of anesthesia was monitored by pedal reflex. Euthanasia was performed via CO₂ asphyxiation, and death was confirmed by the absence of corneal reflexes. This study was conducted in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and approved by the Animal Care and Use Committee of Chengde Medical University (Approval No. CDMULAC-20230331-009; Date: March 31, 2023). All experimental protocols complied with the NIH Guide for the Care and Use of Laboratory Animals. The mouse monocyte-macrophage cell line RAW264.7 was generously provided by the Department of Medicine, Peking University.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and culture of ADSCs\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter aseptic cutting of the rat adipose tissue next to the epididymis, the fascia and blood vessels were suitably removed to extract ADSCs, as previously described. Briefly, after the tissue was subjected to 40\u0026ndash;60 minutes of shaking at 37\u0026deg;C with 0.1% type I collagenase (Solarbio, Beijing, China), the digestion process was stopped by adding an equal volume of DMEM/F12 complete media supplemented with 10% FBS (Procell, Wuhan, China). Next, the cell suspension was filtered through a 200 mesh filter, and centrifuged for 10 min at 1000 rpm to extract the sediment. The reconstituted cells were then plated on Petri dishes for incubation. The initial fluid exchange occurred after 48 h. After culturing for 8 or 10 days in vitro (div), ADSCs were collected for osteogenic and lipogenic differentiation induction, immunofluorescence staining, and Western blot assays.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMultidirectional differentiation potential assay of ADSCs\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe in vitro differentiation of osteogenic and lipogenic cells was induced as previously described\u003csup\u003e4\u003c/sup\u003e. Briefly, third-generation ADSCs were inoculated into 6-well plates at a density of 5x10\u003csup\u003e4\u003c/sup\u003e/ml. Once cell confluency reached 80%, the lipogenesis and osteogenesis induction culture medium (ProCell) was switched. The media was regularly modified every two to three days. When the lipid droplets were clear and complete, 12\u0026ndash;14 days after lipogenic induction, Oil Red O staining (Procell) was performed. Alizarin red (Procell) was used to stain the cells, indicating the shape or growth of the osteogenic material, which was induced for 28\u0026ndash;32 days. An inverted microscope was used to observe the formation of the lipid droplets and calcium nodules.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining of ADSCs\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFirst, the cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, MO, USA) for 10 minutes and then blocked with 5% bovine serum albumin (Solarbio) for 30 minutes at room temperature. Next, the cells were incubated at 4\u0026deg;C overnight with primary antibodies (Rabbit anti-CD29, 1:100, Cat# A-11001, ABclonal, MA, USA; Rabbit anti-CD44, 1:200, Cat# A12410, ABclonal), and then incubated away from light at room temperature for 1 hour with secondary antibodies (goat anti-rabbit secondary antibody conjugated to ABflo\u0026reg; 488, 1:400, Cat# AS053, ABclonal; goat anti-rabbit secondary antibody conjugated to ABflo\u0026reg; 594, 1:800, Cat# AS039, ABclonal). The nuclei were then counterstained with DAPI (Solarbio) for 5 minutes, and examined under a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eProteins were obtained by adding the lysate (RIPA: PMSF\u0026thinsp;=\u0026thinsp;100:1, Solarbio) and quantified using a BCA protein kit (Solarbio). Proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels (10%) and transferred on to polyvinylidene difluoride membranes (Millipore, MA, USA). After being blocked with Ready-to-use blocking solution for 5 minutes at room temperature, the membranes were incubated with the following primary antibodies at 4\u0026deg;C overnight: CD29 (1:1000, Cat# A-11001, Abclonal), CD44 (1:1000, Cat# A12410, Abclonal), CD9 (1:500, Cat# 20597-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Cat# 28283-1-AP, Proteintech), Itgav (1:1000, Cat# A19071, Abclonal), Fak (1:400, Cat# ab40794, Abcam, Cambridge, USA), Src (1:300, Cat# ab133283, Abcam), Bcar1 (1:1000, Cat# 16815-1-AP, Proteintech), P-Fak (1:1000, Cat# ab81298, Abcam), P-Src (1:1000, Cat# 6943T, CST, Danvers, USA), P-Bcar1 (1:1000, Cat# 4011S, CST). Next, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:10000; Cat# AS080, Abclonal) at room temperature for 1 hour, after which an ECL chemiluminescence analyzer was used to analyze the protein bands. Finally, the relative expression of the target protein was determined by comparing the gray value of the target protein band with that of the standard protein band.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and extraction of ADSC-Exos\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWhen the wall-adherent cells third-generation ADSCs reached 60% confluency in standard culture, they were transferred to the exosome extraction-specific medium and cultured for another 48\u0026ndash;72 hours. After the exosome isolation reagent (250\u0026micro;l/ml, KeyGEN, Jiangsu, China) was added to the supernatant, the mixture was incubated at 4\u0026deg;C for 2 hours, and then centrifuged at 4\u0026deg;C, 12000 g for 20 minutes. ADSC-Exos precipitate was resuspended with 100 \u0026micro;l sterile PBS for later use.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter dropping 20 \u0026micro;l of ADSC-Exos on the copper mesh and allowing it to adsorb for 5 minutes spontaneously, 20 \u0026micro;l of a 2% phosphotungstic acid solution was added. Following a 5-minute drying period under an incandescent lamp, the sample was examined using a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan), and images were recorded.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eParticle size detection\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter a 35-fold dilution with sterile PBS, ADSC-Exos were filtered using a 0.22 \u0026micro;m filter. Next, the particle size distribution of ADSC-Exos was determined using a nanoparticle- tracking analyzer (NS300, Malvern, Malvern, UK).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ePKH26 labeling of ADSC-Exos\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe used PKH26-labeled ADSC-Exos to analyze the distribution of ADSC-Exos in macrophages. Cells were incubated with PKH26 (100 \u0026micro;M, Cat# UR52302, Umibio, Shanghai, China) at room temperature for 10 minutes, after which ADSC-Exos were extracted after fixing the volume to 10 ml with sterile PBS. The pellet (PKH26- ADSC -Exos) was then resuspended in PBS and co-cultured with macrophages for 24 h. Finally, ADSC-Exos within the macrophages were observed and photographed under a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eCell viability assessment\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo study the impact of ADSC-Exos on the vitality of macrophages, RAW264.7 cells were seeded at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well in 24-well culture chambers. The cells were cultured in DMEM supplemented with 10% FBS for 24 hours. Subsequently, different concentrations of ADSC-Exos (0 \u0026micro;g/ml (control), 10 \u0026micro;g/ml, 20 \u0026micro;g/ml, and 40 \u0026micro;g/ml) were added. 5-ethynyl-2\u0026prime;-deoxyuridine (EdU, 10 \u0026micro;M; Cat# C0071S, Beyotime, Shanghai, China) was then added to the culture medium for incorporation into proliferating cells. The cells were fixed in 4% paraformaldehyde for 30 minutes on ice 24 hours after EdU treatment. Then, the EdU-incorporated proliferating cells were visualized using a BeyoClick\u0026trade;EdU-488 Kit (Beyotime), in accordance with the manufacturer\u0026rsquo;s instructions. The cell nuclei were then stained with the nuclear marker Hoechst 33342(1:1000; Beyotime). Finally, the cells were observed under a fluorescence microscope, and the percentage of EdU-positive cells was calculated.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eCell migration assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe migratory ability of RAW264.7 was determined using 6.5 mm Transwell chambers with 8 \u0026micro;m pores (Corning NY, USA). RAW264.7 were treated with 0, 10, 20, or 40 \u0026micro;g/ml ADSC-Exos, and then seeded in the upper chamber at a density of 1000 cells/\u0026micro;L, while 600 \u0026micro;L complete medium containing DMEM and 10% fetal bovine serum was added in the bottom chamber. RAW264.7 cells were cultured in Transwell chambers for an additional 24 hours. Cells that migrated to the lower chamber were stained with 0.1% crystal violet for 20 minutes, whereas those left in the upper chamber were wiped away with a cotton swab. Finally, a randomly selected field of view was obtained and studied under a microscope. Image J software was subsequently used to count the number of moving cells.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eWound healing assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe migratory ability of RAW264.7 cells was further determined using the wound healing assay at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well in 12-well culture chambers. A linear scratch was then made on the cultured cells, after which they were treated with 0, 10, 20, and 40 \u0026micro;g/ml ADSC-Exos for an additional 48 hours. Images were then captured using an inverted microscope at 0, 24, and 48 hours after wounding. The relative healing area was calculated using the ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eAdhesion assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo determine the impact of ADSC-Exos on macrophage adhesion capacity, RAW264.7 cells were seeded at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well in 24-well culture chambers for 4. After removing non-adherent cells with PBS, they were stored in 4% paraformaldehyde for 30 minutes at room temperature. After the nuclei were stained with DAPI, the membranes were permeabilized with a solution containing 3% bovine serum albumin (BSA) and 1% Triton-X100 for 15 minutes. After capturing images with a fluorescence microscope, the number of adherent cells was calculated using Image J software.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eRNA-sequencing analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRAW264.7 cells were consistently cultivated for 24 hours and the cells then treated with 0 and 20\u0026micro;g/ml ADSC-Exos for an additional 24 hours. RNA extraction and library construction were carried out using Trizol reagent (Invitrogen, CA, USA) and Illumina's NEBNext\u0026reg; UltraTM RNA Library Prep Kit (Gene Biotechnology International Trade Co., Ltd), respectively, following the manufacturer\u0026rsquo;s protocols for reverse transcriptional synthesis of double-stranded DNA, terminal repair, sequencing linker linking, PCR amplification, and library quality control. Relative expression of genes were measured using the 2\u003csup\u003e-ΔΔCT\u003c/sup\u003e method. The primer sequences used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Library quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and sequenced on an Illumina Novaseq 6000 platform (Novaseq Technology Co., Ltd, Beijing, China). To achieve high-quality clean data, multiple steps were implemented to screen the raw data, including filtering of reads using FASTP (ver. 0.19.7)\u003csup\u003e40\u003c/sup\u003e, and reference genomic mapping was performed using HISAT2 (2.0.5)\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer sequences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDusp6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-CGGCTTCTGCTGATGGACT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-TCGCTGCTATTCTCGTCGTA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItga5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-CCTCTGCTGCTGCTACTGT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-ACCTGGCTAGTGTTAG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItgav\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-GAACAAGGAGAACCAGAACCATT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-ATACCAACACAGCCAGTAGCA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNr4a1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-CAATATGGAACACCAGCAACGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-GGAGGAGGCAGAGGAACAAG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePdgfb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-ATCGCACCAACGCCAACT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-GGTCACTACTGTCTCACACTTG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlau\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-CACAGCCATCCAGGTCCATA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-AAGCGGTCCTCCAGAATCG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePtgs2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-CTGGTGCCTGGTCTGATGAT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-CAATGCGGTTCTGATACTGGAA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-GAGGTGTTCTGGAAGGATGGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-ATGGTGACTGAGCCGTGAG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePdgfa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-GTAACACCAGCAGCGTCAAG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-ACCTCACATCTGTCTCCTCCT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePdcd1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-TGAACTGGAACCGCCTGAG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-GGTAGATGCCACTGTCATTGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCldn11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-TGGATTGGCATCATCGTCACA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-CCAGAACGGAGGCAGCAAT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward:5\u0026rsquo;-GCTGCTGTTCTTGGCTCTC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse:5\u0026rsquo;-GCTGACCTCGTTGCTATCTAC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSrc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: 5\u0026rsquo;-GCCTATGTGGAGCGGATGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse: 5\u0026rsquo;-CTGTGTATTCGTTGTCTTCTATGAG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePtk2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: 5\u0026rsquo;-ACTTGGACGCTGTATTGGAGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse: 5\u0026rsquo;-CTGACGCATTGTTAAGGCTTCT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: 5\u0026rsquo;-GGTGAAGGTCGGTGTGAACG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse: 5\u0026rsquo;-CTCGCTCCTGGAAGATGGTG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eGene function and pathway enrichment analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eDifferentially expressed genes (DEGs) were analyzed using DESeq2 (1.16.1). Genes with a |log\u003csub\u003e2\u003c/sub\u003eFold change|\u0026gt;0\u003csup\u003e42\u003c/sup\u003e and P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as DEGs. These DEGs were subjected to KEGG pathway and GO enrichment analyses using ClusterProfiler (4.0) software\u003csup\u003e43\u003c/sup\u003e. After Protein-protein interaction network analysis of the DEGs was performed using the String database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.string-db.org/\u003c/span\u003e\u003cspan address=\"https://www.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), gene connecting networks were visualized using Cytoscape 3.9.1 software.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eCell proliferation assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo determine the optimum concentration of Cilengitide, RAW264.7, cells were seeded at a density of 1x10\u003csup\u003e5\u003c/sup\u003e/ml. cells in a 96-well culture chamber for 24 hours. The cultures were divided into three groups: blank (no drugs), control (no cells), and inhibitor (concentrations of 0.1, 0.3, 0.5, 0.7, and 0.9uM, respectively). Each group included five replicates. Cell Counting Kit-8 reagent (Apexbio, Houston, TX, USA) was added to each well (10 \u0026micro;L/well), followed by a 2-hour incubation at 37\u0026deg;C. Absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA) to determine the optical density (OD) of the different groups. Cell proliferation was expressed as cell inhibition as follows: cell inhibition (%) = [A1(cilengitide)-A3 (blank)]/[A2 (control)-A3(blank)] \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eConfocal Laser Scanning Microscope\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRAW264.7 cells were routinely cultured for 24 hours at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e/ml. A linear scratch was made on the cultured cells prior to treatment with 0 \u0026micro;g/ml ADSC-Exos, 20 \u0026micro;g/ml ADSC-Exos, and 20 \u0026micro;g/ml ADSC-Exos\u0026thinsp;+\u0026thinsp;5 \u0026micro;M cilengitide for an additional 4 hours. The real-time migration of RAW264.7 was determined using a Confocal Laser Scanning Microscope (FV3000, Olympus, Tokyo, Japan)\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003cspan address=\"http://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for statistical analyses. Data were evaluated using Student\u0026rsquo;s t-test between two groups or one-way analysis of variance followed by Tukey\u0026rsquo;s post hoc test in more than two groups, depending on whether normality was indicated, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant\u003csup\u003e45\u0026ndash;46\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eConceptualization, X.F.; data curation, B.Y. and J.X.; methodology, B.Y., J.X., and M.R.; validation, B.Y., J.X., and Y.W.; resources, Q.Z. and M.R.; formal analysis, B.Y. and X.F.; investigation, J.X., Y.W., and Q.Z.; writing\u0026mdash;original draft, B.Y. and J.X.; writing\u0026mdash;review and editing, X.F. and B.Y.; supervision, Y.W. and X.F.; project administration, X.F.; funding acquisition, X.F., B.Y., and Y.W. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the National Natural Science Foundation of China (Grant No. 82371385); the Natural Science Foundation of Hebei Provincial Department of Science and Technology (Grant No. H2021406056); the Key Project of Hebei Provincial Department of Education (Grant No. ZD2020178); Heilongjiang Natural Science Foundation of China (Grant No. JQ2021H004); the Advantageous Discipline of Human Anatomy and Histoembryology at Chengde Medical University (Grant No. [2023]22); Hebei Provincial Key Laboratory of Nerve Injury and Repair Open Project Fund (Grant No. NJKF202403); and the School-level Research Project of Chengde Nursing Vocational College (Grant No. Y202501).\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003eAll animal experiments were approved by the Animal Ethics Committee of Chengde Medical University (Approval No. CDMULAC-20230331-009) and were conducted in strict accordance with the institutional guidelines for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to X.F.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit https://creativecommons.org/licenses/by-nc-nd/4.0/ .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNocera, G. \u0026amp; Jacob, C. Mechanisms of Schwann cell plasticity involved in peripheral nerve repair after injury. Cell. Mol. Life. Sci. 77, 3977-3989. https://doi.org/10.1007/s00018-020-03516-9 (2020).\u003c/li\u003e\n\u003cli\u003eQian, T., Fan, C., Liu, Q. \u0026amp; Yi, S. 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Rep. 15, 17876. https://doi.org/10.1038/s41598-025-03113-5 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exosomes, adipose-derived stem cells, macrophages, migration, transcriptome sequencing, peripheral nerve injury","lastPublishedDoi":"10.21203/rs.3.rs-7248636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7248636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMacrophages play an important role in peripheral nerve regeneration, but the specific mechanisms remain unclear. Our previous studies demonstrated that adipose stem cell-derived exosomes (ADSC-Exos) promote peripheral nerve regeneration, correlating with their effects on macrophages. To test this, ADSC-Exos were isolated from primary ADSCs and co-cultured with RAW264.7 macrophages. We examined ADSC-Exos' impact on macrophage migration by assessing RAW264.7 cell viability, migration capability, and adhesion ability. Transcriptome sequencing identified differentially expressed genes (DEGs) and examined their interactions. Key gene expressions were verified by qRT-PCR, while pathway-related proteins were detected using Western blot and qRT-PCR. Results showed ADSC-Exos enhanced RAW264.7 cell viability, adhesion and migration. Transcriptomic analysis revealed 233 DEGs. GO analysis showed DEGs were enriched in biological processes including negative regulation of damage response and cell migration, cellular constituents like cytoskeleton and adhesion complexes, and molecular functions including redox activity and ubiquitination. KEGG analysis indicated DEGs were abundant in pathways of cell adhesion molecules, MAPK, folate-dependent one-carbon metabolism, and NF-κB. ADSC-Exos promoted Itgav expression and Fak, Src, and Bcar1 phosphorylation. The Itgav inhibitor cilengitide decreased Itgav expression and phosphorylation of Fak, Src, and Bcar1. These findings suggest ADSC-Exos enhance macrophage migration and adhesion through the Itgav/Fak/Src signaling pathway.\u003c/p\u003e","manuscriptTitle":"Mechanism of ADSC-Exos promoting macrophage migration via Itgav/Fak/Src signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-28 16:05:26","doi":"10.21203/rs.3.rs-7248636/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d343a87-d375-444a-85b7-cb810a73e753","owner":[],"postedDate":"August 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53758105,"name":"Biological sciences/Cell biology"},{"id":53758106,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-11-19T10:23:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-28 16:05:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7248636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7248636","identity":"rs-7248636","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}