Effects of extracellular vesicles from adipose‐derived stem cells on human keloid fibroblasts via the SOCS1/JAK2/STAT3 pathway

Keloid represents a benign skin tumor with many cancer‐like features. Extracellular vesicles (EVs) derived from human adipose‐derived stem cells (hADSCs) play a role in cell migration of multiple diseases.


| INTRODUC TI ON
Keloid is a dermatological condition characterized by excessive fibroproliferation in the skin, resulting from aberrant wound healing. 1 Keloid is distinguished by the excessive collagen deposition and the abberant proliferation of human keloid fibroblasts (HKFs). 2 The adverse effects of keloid formation encompass pain, hyperesthesia, and pruritus, which further reduce the quality of life and compromise the emotional well-being of affected individuals. 3However, there is currently a lack of widely effective treatment options, and current therapies demonstrate limited efficacy, benefiting for only a few patients while exhibiting high recurrence rates. 4Therefore, it's imperative to study and devise efficacious treatment methods for keloid.
Human adipose-derived stem cell (hADSC) is one of mesenchymal stem cells that enable the differentiation of large numbers of multipotent stem cells into multiple cell lineages and to promote tissue regeneration through the paracrine function. 5terature shows that hADSCs expedite the wound healing process. 6Morever, hADSCs inhibit the bioactivity of keloid fibroblasts. 7Also, hADSCs possess the ability to produce small round vesicles called extracellular vesicles (EVs), 8 which are heterogeneous collection of membrane-limited vesicles carrying microR-NAs (miRNAs), proteins, and signaling lipids. 9,10EVs can regulate the migration of fibroblasts which release growth factors to induce other cell proliferation and collagen production. 11EVs and their miRNA cargoes have been identified as key regulators of human skin homeostasis. 12hADSC-EVs have significant implications in various biological processes, including angiogenesis, cell survival, tissue regeneration, and attenuation of disease pathology. 13However, there has been no report about the role of hADSC-EVs on proliferation, migration, and collagen synthesis of HKFs to date.
Autophagy is tightly bound up with keloid, and autophagy is observed to be enhanced in the margin area of the keloid. 14LC3 upregulation in keloids indicates that the repression of autophagy could potentially be a therapeutic approach. 15Notably, suppression of autophagy has been documented to decrease the invasive and migratory capabilities of tumor cells, which play a crucial role in the initial phases of metastasis, such as local invasion and endocytosis. 16portantly, repression of autophagy stimulates apoptosis in HKFs, subdues their migration, and effectively manages keloid recurrence. 14The involvement of hADSCs in the modulation of autophagy of HKFs remains elusive.
Suppressor of cytokine signaling 1 (SOCS1) is a member of the SOCS family and has emerged as a negative modulator of signal transduction of cellular factors. 17It is noteworthy that there is a reduction in SOCS1 expression in fibroblasts of patients with idiopathic pulmonary fibrosis. 18Furthermore, SOCS1 plays a nonredundant role in hepatocytes and macrophages, exerting regulatory effects on liver fibrosis by reducing hepatocyte injury and inflammatory responses of macrophages. 19A study conducted by Wang et al. has manifested that knockdown of SOCS1 is associated with increased proliferation and invasion of rheumatoid arthritis synovial fibroblasts. 20Besides, Platycodon grandiflorus polysaccharide induces M1 polarization of porcine alveolar macrophages by activating autophagy through downregulation of SOCS1/2 proteins. 21Interestingly, bone marrow mesenchymal stem cells-EVs shuttling miR-29b-3p impedes the SOCS1 pathway and facilitates osteogenic differentiation. 22However, the action of SOCS1 in hADSC-EVs on HKF autophagy, proliferation and migration is not well understood.
4][25] Plus, SOCS1 serves as a suppressor of the JAK2/STAT3 pathway. 26Zhou and his colleagues have found that the SOCS1/JAK2/STAT3 pathway is engaged in the pro-angiogenic transformation of cancer-related fibroblasts provoked by exosomal miR-155-5p. 27As previously mentioned, SOCS1 deficiency in fibroblasts leads to enhanced collagen production, but SOCS1overexpression restrains collagen production. 28But whether SOCS1 carried by hADSC-EVs regulates HKF autophagy and collagen synthesis via the JAK2/STAT3 pathway has not been investigated yet.Hence, the objective of this present study was to investigate the specific mechanism of hADSC-EVs on HKFs to offer theoretical references for clinical trials targeting keloid treatment.

| Oil red O staining
Oil red O staining was performed to assess the adipogenic capacity.The specific steps were as previously reported. 29Briefly, hADSCs were induced in adipogenic induction medium for 2 weeks.Subsequently, the cells underwent fixation in 10% formalin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 min, followed by phosphate buffer saline (PBS) (10 mM Na2HPO4, 137 mM NaCl, 1.8 mM KH2PO4, and 2.7 mM KCl, pH 7.4, Sigma-Aldrich) washes.Later, cells were subjected to staining using a 2% (wt/vol) fresh oil red O solution (Sigma-Aldrich) at room temperature for 5 min to determine lipid droplets in the induced cells.Next, the staining results were observed using an inverted microscope (Olympus, Tokyo, Japan).

| Alizarin red staining
Calcium deposition in osteoblasts was visualized by alizarin red staining to assess osteogenic ability, following the established protocol as described in a previous study. 30Briefly, hADSCs were induced in adipogenic induction medium for 3 weeks.After being fixed with 95% ethanol (Sigma-Aldrich) for 10 min, the cells were cultivated with 0.1% alizarin red-tris-hcl (PH 8.3) (Sigma-Aldrich) for 30 min at 37°C.Thereafter, the cells underwent a series of washes until they became clear.The staining results were observed using an inverted microscope (Olympus).

| Alcian blue staining
The hADSCs were induced in adipogenic induction medium for 3 weeks.Alcian blue staining was implemented using the standard Alcian blue kit (pH = 2.5) (Solarbio, Beijing, China) as per the manuals of manufacturer to evaluate chondrogenic capacity, followed by observation of the staining results using an inverted microscope (Olympus).The correct EVs were lysed using lysis buffer, and the protein concentration was quantified by bicinchoninic acid assay (BCA) kits (Beyotime, Shanghai, China).The protein content was subsequently utilized as the benchmark for EVs.
Cells were maintained in DMEM comprising 10% FBS and 1% P/S at 37°C in 5% CO 2 .Cells at the 3rd passage were used.

| Labeling of EVs
EVs labeling was performed as previously described. 34Briefly, EVs were labeled using a PKH67 fluorescence kit (Sigma-Aldrich), diluted with PBS, and centrifuged min at 150 000 g and 4°C for 70 to discard the unbound dye.Subsequently, the labeled EVs were subjected to incubation with HKFs at 37°C with 5% CO 2 for 12, 24, and 48 h, respectively.After three PBS washes, HKFs were fixed with 4% paraformaldehyde, followed by DAPI staining to observe the nucleus and photography via a fluorescence microscope (Cal Zeiss Meditec AG, Jena, Germany).PBS was utilized as a solvent control.Quantitative analysis was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).

| Wound healing assay
Wound healing assay was performed as previously described. 36Briefly, HKFs were seeded onto 6-well plates at a density of 1 × 10 5 cells per well.The bottom side of the plates was streaked with horizontal lines with a gap ranging from 0.5 to 1 cm.HKFs were grown for 24 h in the medium supplemented with 10% FBS.Next, a sterile pipette was utilized to create 1 mm scratch perpendicular to cell surface.HKFs were washed with PBS and maintained in the medium with 1% serum of low-concentration.The scratch width at 0 and 24 h was calculated by Image Pro-Plus (Media Cybernetics, Rockville, MD, USA).

| Transwell assay
Transwell assay was performed as previously described. 36Briefly, transwell chamber (Corning, Tewksbury, MA, USA) was positioned within the 24-well plates and suspended in the respective well.The Transwell chamber was designated as the apical chamber and 24-well plate became basolateral chamber.HKFs (1 × 10 5 cells/well) were seeded in the apical chamber, and the medium with 20% FBS was added to the basolateral chamber at 37°C.Chambers were taken out after 24 h.HKFs were fixed for 20 min with 4% paraformaldehyde, washed twice with PBS, stained for 20 min with 0.1% crystal violet and washed twice again with PBS before being allowed to dry.The numbers of migrated cells were counted and observed by microscopy.

| RT-qPCR
RT-qPCR was performed as reported. 27In short, the TRIzol reagent (Invitrogen) was adopted for the purpose of extracting total RNA from hADSC-EVs or cells.Complementary DNA was synthesized from RNA by RT using PrimeScript RT Reagent kits (Takara, Tokyo, Japan).RT-qPCR was implemented using SYBR® Premix Ex Taq™ II (Takara) with a total volume of 20 μL: 5 min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C.The 2 −ΔΔCT method was used to compute relative gene levels.Primer sequences are presented in Table 1.

| Western blot
Total protein of hADSC-EVs or cells was extracted by Protein Extraction kits (WLA019, Wanleibio, Shenyang, China).BCA kit (Beyotime) was adopted for quantifying protein concentration.Then, a total of 40 μg protein was separated by SDS-PAGE and transferred onto the PVDF membrane.After blockade with 5% skimmed milktris-buffered saline with Tween 20 (TBST) and 2-h incubation, the membrane was rinsed with TBST and probed overnight with primary antibodies at 4°C and 1-h incubation with secondary antibodies at 37°C.Following this, the protein band was detected using chemiluminescence assay kits (ECL Plus, Life Technology, Foster City, CA, USA).
Image J (NIH, Bethesda, MD, USA) was adopted for gray value analysis.The relative protein levels were calculated using Gel-Pro-Analyzer (Media Cybernetics).The antibodies used are illustrated in Table 2.

| Immunofluorescence
As light chain 3 (LC3)-II serves as a widely accepted marker for autophagosomes and relatively specifically related to autophagosomes and autolysosomes, the quantification of LC3-positive puncta is deemed gold-standard for determining autophagosome numbers in cells. 37Immunofluorescence assay was performed as reported. 38HKFs were fixed for 15 min with 4% paraformaldehyde, rinsed with NaCl/Pi, permeabilized with 0.2% Triton X-100, and incubated at 4°C with LC3B primary rabbit antibody (1:200, #3868, CST, Danvers, MA, USA) overnight.Next, cells were probed with Alexa 594-conjugated goat anti-rabbit secondary antibody (1:200, #8889, CST) at 37°C for 1 h.The images were taken captured using a fluorescence microscope (Carl Zeiss Meditec AG).The LC3-positive puncta were qiuantified using the Image J. data among groups were done using one-way analysis of variance (ANOVA), followed by Tukey's test.The p values were obtained from two-sided tests.The difference was regarded statistically significant at the threshold of p < 0.05.

| hADSC-EVs were isolated and identified
Firstly, we detected antigen expression on hADSC surfaces.The levels of CD105 and CD90 were increased with the percentages of 98.33% and 98.51% respectively, while the levels of CD45 and CD31 were decreased with their percentages being 0.05% and 0.61%, respectively (Figure 1A).Alizarin red, oil red O, and alcian blue staining demonstrated promising capabilities for adipogenic, osteogenic, and chondrogenic differentiation in hADSCs (Figure 1B).The aforementioned findings indicated the successful identification of hADSCs.Subsequently, EVs were extracted from hADSCs by ultracentrifugation. Results of electronic microscopy and NTA showed that EVs predominantly displayed as vesicular bodies with bilayer membrane in the circular or oval shape with diameters at 50-130 nm and particle diameter peak at around 100 nm at the concentration of 1.81 × 10 8 EVs/mL (Figure 1C, D).Western blot results analysis revealed an upregulation in the levels of EV-specific markers CD9, CD63, TSG101, and Alix, and not expressed Calnexin protein (Figure 1E).Briefly, the correct hADSC-EVs were obtained.

| hADSC-EVs inhibited HKF proliferation and migration and reduced collagen deposition
To explore the role of hADSC-EVs in HKFs, hADSC-EVs were labeled using PKH67 staining solution.The uptake experiment con-

| hADSC-EVs suppressed HKF autophagy by carrying SOCS1
RT-qPCR and Western blot showed that SOCS1 mRNA and protein levels were prominently up-regulated in hADSC-EVs compared to GW4869-treated hADSC supernatants (Figure 3A, all p < 0.01).SOCS1 mRNA and protein levels were weakly expressed in the HKF group and significantly elevated in HKFs treated with hADSC-EVs (Figure 3B, all p < 0.01).We herein assumed that hADSC-EVs might play a role in modulating fibroblast proliferation and migration by transporting SOCS1.To further investigate whether hADSC-EVs carry SOCS1 to suppress HKF autophagy, hADSC-EVs were isolated from hADSCs transfected with siR SOCS1, and subsequent observation unraveled decreased SOCS1 mRNA and protein levels in both the hADSC-siR SOCS1 group and EVs-siR SOCS1 group (Figure 3C, D, all p < 0.01).
Then, EVs-siR SOCS1 was introduced into HKFs and the SOCS1 mRNA and protein levels were both decreased (Figure 3E, all p < 0.01).

| Inhibition of SOCS1 in hADSC-EVs partially abolished the suppressive actions of hADSC-EVs on HKFs
To determine if hADSC-EVs affect HKFs by carrying SOCS1, hADSC-EVs from different groups were introduced to HKFs and cultivated for 24 h.After silencing SOCS1 in hADSC-EVs, the proliferative and migratory abilities of HKFs were enhanced (Figure 4A-C, all p < 0.05), and collagen I and III levels were remarkably raised (Figure 4D, all p < 0.01).

TA B L E 2
Antibodies in western blot.These results demonstrated that SOCS1 inhibition in hADSC-EVs partially abated the suppressive effects of hADSC-EVs on HKF proliferation and migration, indicating that hADSC-EVs suppressed the HKFs proliferation and collagen deposition by carrying SOCS1.

| SOCS1 carried by hADSC-EVs inhibited the JAK2/STAT3 pathway
To investigate the potential regulatory role of SOCS1 in hADSC-EVs on HKF proliferation and migration and collagen production through the JAK2/STAT3 pathway, the levels of JAK2 and STAT3 in HKFs

| Activation of the JAK2/STAT3 pathway partially averted the repressive actions of hADSC-EVs on HKFs
To verify whether SOCS1 carried by hADSC-EVs suppresses HKF proliferation and migration by repressing the JAK2/STAT3 pathway, the JAK2/STAT3 pathway was activated using its activator CA1 after SOCS1 downregulation in hADSC-EVs, with DMSO as solvent of the control group.Western blot exhibited no significant differences in the expression levels of p/t-JAK2 and p/t-STAT3 in the HKF + EVs-siR

| DISCUSS ION
Keloids are anomalous tissue scars that typically manifest on injured skin and are attributed to the excessive proliferation of granulation tissue or type III collagen during the process of wound healing. 39Keloids pose a significant challenge in terms of treatment due to their inherent propensity for recurrence following excision. 40Recent evidence has found that hADSCs have the ability to remodel the fibrotic matrix in a scar and reduce myofibroblasts proliferation. 41EVs exhibit similar functions to their originating cells and offer several advantages over stem cells. 42is study found that SOCS1 carried by hADSC-EVs inhibited HKF autophagy, proliferation, migration, and collagen deposition by suppressing the JAK2/STAT3 pathway.
As multipotent stem cells, hADSCs show a potential effect on anti-fibrosis. 7Noticeably, an increase in fibroblast proliferation is a primary factor contributing to keloid. 43In our study, hADSC-EVs The protein levels of collagen I and III were reduced after hADSC-EVs were endocytosed by HKFs.According to a previous report, hADSCs inhibit the activity of HKF and collagen synthesis by paracrine signaling. 45hADSCs also facilitate wound healing, suppress proliferation of hypertrophic scar fibroblasts, and alleviate wound inflammation. 7Corneal stromal/mesenchymal stem cellsderived EVs can reduce stromal scarring. 46It has been reported that hADSC-EVs promote cutaneous wound healing and prevent hypertrophic scar formation. 47,48Altogether, the present study validated that hADSC-EVs inhibited HKF proliferation, migration, and reduced collagen deposition.
Alveolar macrophages have been found to secrete SOCS1 in vesicles. 49Notably, several studies provide suggestive evidence that SOCS1 overexpression can hinder the collagen production in fibroblasts, while silencing SOCS1 can facilitate fibroblast proliferation and invasion. 20,28Our results unraveled that SOCS1 expression was firstly decreased in HKFs and then increased after hADSC-EV treatment.An increase in SOCS1 expression was also observed in hADSC-EVs, revealing consistency with the previous finding that total EVs enhance SOCS protein levels in dendritic cells. 50Besides, hADSC-EVs influence osteoblast functions through autophagy and expedite the healing of skin wound by regulating cell apoptosis and autophagy. 51,52SOCS1 has been found to be bound up with cell autophagy. 21Our results unveiled that hADSC-EVs could hinder HKF autophagy by delivering SOCS1.Additionally, mice with SOCS1 knockout exhibit acceleration in hepatocyte proliferation. 53nsistently, our foundings proved the promotional effects of SOCS1 downregulation in hADSC-EVs on the proliferation and migration of HKFs.Regarding collagen deposition, it has been observed that mice lacking SOCS1 in hepatocytes and macrophages display an increase in collagen deposition. 19Not surprisingly, hADSC-EVs carrying downregulated SOCS1 elevated collagen I and III levels in HKFs.Therefore, SOCS1 carried by hADSC-EVs inhibited HKF proliferation and collagen deposition.
[25] hADSC-EVs decreased the levels of the JAK2/STAT3 pathwayrelated proteins, while SOCS1 downregulation further raised their expression levels.Activation of the JAK2/STAT3 signaling pathway in human dermal fibroblasts enhances the expression of matrix metalloproteinase and autophagy. 54Suppression of JAK2/ STAT3 activation represses senescence, fibrosis, autophagy, and anti-apoptosis of fibroblasts. 55The JAK2/STAT3 pathway activation partially counteracted the inhibitory action of hADSC-EVs on HKF autophagy, proliferation, migration and collagen deposition, which provides strong support to the previous finding that JAK2/STAT3 inhibition strongly suppresses proliferation, migration, and collagen production in keloid fibroblasts. 23

2. 5 |
Extraction, identification, and grouping of hADSC-EVshADSCs at passage 3 were cultured in DMEM with 10% FBS until cell confluence reached 70%.The medium was refreshed with serum-free medium and hADSCs were cultured with 5% CO 2 at 37°C for 24 h.The supernatant collected and subjected to two rounds of ultracentrifugation at 500 × g and 2000 × g (10 min each time) to remove cell debris, followed by centrifugation for 60 min at 100 000 × g and resuspension in PBS, and preservation at −80°C until further analysis.hADSC-EVs were identified using the following methods: (1) the morphology was observed by transmission electron microscopy (TEM, Olympus); (2) size distribution was analyzed by nanoparticle tracking analysis (NTA, NanoSight NS300, Malvern Panalytical, Malvern, UK); (3) the levels of surface markers (positive: CD9, CD63, TSG101 and Alix; negative: Calnexin) were determined by Western blot.The hADSC supernatant was treated with 10 nM GW4869 (GW, Selleck, Houston, TX, USA) for 8 h as the control.32 hADSC-EVs were grouped as follows: (1) GW group (hADSC supernatant treated with 10 nM GW for 8 h), (2) EVs group, (3) EVs-short interfering RNA (siR) negative control (NC) group (EVs extracted from hADSCs transfected with siR NC for 24 h), and (4) EVs-siR SOCS1 (EVs extracted from hADSCs transfected with siR SOCS1 for 24 h).Lipofectamine 2000 (11668-019, Invitrogen, Carlsbad, CA, USA) was employed for hADSC transfection at a final concentration of 100 nM in strict compliance with the instructions.siR NC and siR SOCS1 were purchased from GenePharma (Shanghai, China).
firmed hADSC-EVs (green fluorescence) can be endocytosed by HKFs compared with PBS (Figure 2A, all p < 0.001).Subsequently, CCK-8 method showed that hADSC-EVs, relative to GW treatment, can substantially inhibit HKF proliferation (Figure 2B, all p < 0.05).Wound healing and Transwell assays demonstrated that hADSC-EVs can significantly inhibit the migratory ability of HKFs compared with GW treatment (Figure 2C, D, all p < 0.01).In comparison to GW treatment, hADSC-EVs can reduce the protein levels of collagen I and collagen III in HKFs (Figure 2E, all p < 0.01).The aforementioned results suggested that hADSC-EVs inhibited HKF proliferation and migration and reduced collagen I and III deposition.
1 hADSC-EVs were isolated and identified.The procured hADSCs were cultured and passaged to the 3rd generation.(A) expression levels of hADSC surface positive markers CD105 and CD90 and negative markers CD45 and CD31 were determined by flow cytometry; (B) alizarin red, oil red O, and alcian blue staining was performed; (C) the morphology of hADSC-EVs was observed under a transmission electron microscope; (D) particle size of EVs was measured by NTA; (E) expression levels of CD9, CD63, TSG101, Alix and Calnexin proteins were determined by Western blot.Cell experiment was repeated three times independently.F I G U R E 2 hADSC-EVs inhibited HKF proliferation and migration and reduced collagen deposition.The procured HKFs were cultured and passaged to the 3rd generation.hADSC-EVs were added into HKFs with hADSC supernatant added with GW4869 as a control.(A) endocytosis of hADSC-EVs by HKFs detected by immunofluorescence with PBS as solvent control; (B) HKF proliferation detected using CCK-8 method; (C,D) HKF migration detected by wound healing and Transwell assays; (E) collagen levels in HKFs determined by Western blot.Cell experiment was repeated three times independently.Data were expressed as the mean ± SD.Data comparisons among multiple groups were analyzed using one-way ANOVA and Tukey's multiple comparisons test.*p < 0.05, **p < 0.01, ***p < 0.001.SOCS1 group versus the HKF + EVs-siR SOCS1 + DMSO group (Figure 6A, all p > 0.05) and showed elevated levels of p/t-JAK2 and p/t-STAT3 in the HKF + EVs-siR SOCS1 + CA1 group relative to the HKF + EVs-siR SOCS1 group (Figure 6A, all p < 0.05).Meanwhile, the JAK2/STAT3 pathway activation diminished the suppressive effect of hADSC-EVs on HKF autophagy, proliferation, migration and F I G U R E 3 hADSC-EVs suppressed HKF autophagy by carrying SOCS1.(A-E) SOCS1 mRNA and protein levels in cells measured by RT-qPCR and Western blot; (F) levels of autophagy-related proteins determined by Western blot; (G) expression level of LC3 measured by immunofluorescence.Cell experiment was repeated three times independently.Data were expressed as the mean ± SD.Data comparisons among multiple groups in figures A and C-G were analyzed using one-way ANOVA and Tukey's multiple comparisons test, data comparisons in figure B were analyzed using t test.*p < 0.05, **p < 0.01.collagen deposition (Figure 6B-F, all p < 0.05).Overall, SOCS1 carried by hADSC-EVs suppressed HKF proliferation and migration by deactivating the JAK2/STAT3 pathway.
were isolated and identified.hADSC-EVs labeled with PKH67 were endocytosed by HKFs.hADSC-EVs inhibited HKF proliferation F I G U R E 4 Inhibition of SOCS1 in hADSC-EVs partially abolished the suppressive effect of hADSC-EVs on HKF proliferation and migration.(A) HKF proliferation after hADSC-EVs treatment detected using CCK-8 method; (B,C) HKF migration after hADSC-EVs treatment detected by wound healing and Transwell assays; (D) collagen levels in HKFs treated with hADSC-EVs determined by Western blot.Cell experiment was repeated three times independently.Data were expressed as the mean ± SD.Data comparisons among multiple groups were analyzed using one-way ANOVA and Tukey's multiple comparisons test.*p < 0.05, **p < 0.01.and migration.Moreover, elevated collagen I and III levels have been noted in keloid tissues according to previous research.44 Overall, hADSC-EVs carried SOCS1 into HKFs, resulting in the inhibition of HKF proliferation and migration through the deactivation of the JAK2/STAT3 pathway.hADSCs secrete anti-fibrotic cellular factors and growth factors and deliver cellular factors associated with differentiation and cell cycle regulation to the extracellular environment through paracrine function.hADSC-EVs exhibit a high abundance of proteins and other noncoding RNAs with various functions in cellular and biological processes.In a groundbreaking manner, this study revealed that hADSC-EV-carried SOCS1 effectively inhibited the JAK2/ STAT3 pathway to suppress autophagy, proliferation and migration of HKFs, and to reduce collagen deposition.The effect and regulatory mechanism of SOCS1 on keloid has not been fully elucidated.Furthermore, it is still uncertain whether miRNAs transported by hADSC-EVs can produce regulatory effects on HKF proliferation and migration remains unclear.In addition, the mechanism by which proteins carried by hADSC-EVs modulate F I G U R E 5 SOCS1 carried by hADSC-EVs inhibited the JAK2/STAT3 pathway.The expression levels of JAK2 and STAT3 in HNFs and HKFs were determined by Western blot.Cell experiment was repeated three times independently.Data were expressed as the mean ± SD.Data comparisons were analyzed using one-way ANOVA and Tukey's multiple comparisons test.p, the phosphorylated protein level, t, the total protein level.*p < 0.05, **p < 0.01.