Extracellular vesicles‐derived miR‐150‐5p secreted by adipose‐derived mesenchymal stem cells inhibits CXCL1 expression to attenuate hepatic fibrosis

Hepatic fibrosis (HF) is involved in aggravated wound‐healing response as chronic liver injury. Extracellular vesicles (EVs) carrying microRNA (miR) have been reported as therapeutic targets for liver diseases. In this study, we set out to explore whether adipose‐derived mesenchymal stem cells (ADMSCs)‐derived EVs containing miR‐150‐5p affect the progression of HF. Carbon tetrachloride (CCl4) was firstly used to induce HF mouse models in C57BL/6J mice, and activation of hepatic stellate cells (HSCs) was achieved using transforming growth factor β (TGF‐β). EVs were then isolated from ADMSCs and co‐cultured with HSCs. The relationship between miR‐150‐5p and CXCL1 was identified using dual luciferase gene reporter assay. Following loss‐ and gain‐function experimentation, HSC proliferation was examined by MTT assay, and levels of fibrosis‐, HSC activation‐ and apoptosis‐related genes were determined in vitro. Additionally, pathological scores, collagen volume fraction (CVF) as well as levels of inflammation‐ and hepatic injury‐associated genes were determined in in vivo. Down‐regulated miR‐150‐5p and elevated CXCL1 expression levels were detected in HF tissues. ADMSCs‐derived EVs transferred miR‐150‐5p to HSCs. CXCL1 was further verified as the downstream target gene of miR‐150‐5p. Moreover, ADMSCs‐EVs containing miR‐150‐5p markedly inhibited HSC proliferation and activation in vitro. Meanwhile, in vivo experiments also concurred with the aforementioned results as demonstrated by inhibited CVF, reduced inflammatory factor levels and hepatic injury‐associated indicators. Both experiments results were could be reversed by CXCL1 over‐expression. Collectively, our findings indicate that ADMSCs‐derived EVs containing miR‐150‐5p attenuate HF by inhibiting the CXCL1 expression.


| INTRODUC TI ON
Hepatic fibrosis (HF), a resultant of chronic liver injury, is associated with an aggravated wound-healing response. 1,2 HF presents a rising tendency on a globe scale, because of drinking, obesity, as well as chronic hepatitis B and C. 3 HF can often lead to severe outcomes such as liver cirrhosis or even hepatocellular carcinoma which further burden the medical infrastructure. 4 Clinically, HF is characterized by hepatic stellate cells (HSCs) activation along with extracellular matrix accumulation. 5 Furthermore, a diagnosis of HF can only be made with invasive procedures like biopsy, which always remain unfavourable among patients. 6 Currently, liver transplantation is regarded as the most optimal treatment strategy for patients with end-stage HF, but faces many obstacles such as shortage of donor organs and complications during surgery. 7 Recently, HSCs-specific strategy has been highlighted to possess therapeutical potential for HF therapy, 8 while the therapeutic role of extracellular vesicles (EVs) in HF has also garnered the interest of numerous researchers. 9 Therefore, extensive research on the effect of EVs in HF aimed at HSC activation could prove beneficial in regard to HF treatment strategies.
EVs, including exosomes and microvesicles, are known as membrane-bound particles secreted by multiple cell types, which exert crucial functions in cell-to-cell communication. 10 Meanwhile, mesenchymal stem cells (MSCs) are also known to yield paracrine functions through the release of EVs containing microRNA, mRNA, as well as proteins. 11 Importantly, MSCs or MSC-derived microvesicles are capable of treating hepatic diseases. 12 More notably, adipose-derived mesenchymal stem cells (ADMSCs) were recently highlighted to possess the ability to suppress HSC activation and attenuate HF. 13 EVs are also capable of carrying biologically active cargos to target cells from donor cells. 14 Additionally, microRNAs (miRs) are regarded as the cargos which can be encapsulated in EVs and transferred between cells. 15 Recent studies have further reported that EVs secreted by ADMSCs can deliver miRs such as miR-181-5p and miR-122 to inhibit the development of HF. 16,17 Interestingly, researchers have also found that one of the miR members, miR-150, could inhibit HSC activation. 18 Moreover, hsa-miR-150-5p was previously suggested to play an important role in HF by regulating processes such as metabolism and extracellular matrix protein organization. 19 Therefore, we have suggested that ADMSCs-derived EVs containing miR-150-5p may be conducive for HF amelioration. Of note, with miR-150-50 as a focus of this study, our targeting binding prediction further verified CXC chemokine-ligand-1 (CXCL1) as the downstream target of miR-150-5p. In addition, a negative correlation was observed between miR-150 and CXCL1 expression in mice overexpressing or underexpressing Kruppel-like factor 2. 20 CXCL1, a ligand for CXC chemokine receptor 2, has also been documented to be expressed in HSCs. 21 In addition, CXCL1 was previously indicated as a profibrotic chemokine partially responsible for fibroinflammatory liver injuries. 22 Taken all the above into consideration, we set out to perform a series of experiments to verify our hypothesis that miR-150-5p-containing EVs derived from ADMSCs can influence the development of HF by targeting CXCL1.

| Ethical approval
All animal studies were approved by the Ethics Committee of Jingmen First People's Hospital and performed strictly following the Guide for Institutional Animal Care and Use Committee of Jingmen First People's Hospital. Extensive efforts were made to minimize the suffering of the included animals.

| Experimental animals and model establishment
C57BL/6J male mice (aged 6 weeks old; calculated mean weight of 20 ± 2 g) were reared in a specific pathogen-free grade animal laboratory. The mice were used for experimentation after two weeks of adaptive feeding, with the mice being 8 weeks old. As a result, HF mouse models were established by injecting 10 mL/kg 10% carbon tetrachloride (CCl 4 ; 48 604, Sigma-Aldrich, St. Louis, MO, USA) into the mice peritoneum twice a week, for continuous eight weeks.

| Culture of ADMSCs
The abdominal adipose tissues were extracted after the mice were euthanized by cervical dislocation and then rinsed repeatedly with pre-cooled phosphate-buffered saline (PBS) containing 2% penicillin/streptomycin (15140-122; Gibco, Carlsbad, California, USA). The isolated adipose tissues were then sliced into pieces (1 mm 3 ), treated with 0.1% (mg/mL) type I collagenase digestion solution (5135; Sigma-Aldrich, St. Louis, MO, USA) and placed in a 15-mL centrifuge tube for detachment at 37℃ for 0.5-1 hour. The detachment was stopped with the addition of equal volumes of basic medium (MEL08-500ML; AmyJet Scientific, Wuhan, China).
Centrifugation was subsequently conducted at room temperature at 1000 r/minute (radius of 8 cm) for 8 minutes, and the supernatant was removed. The obtained cells were then resuspended in the basic culture medium, filtered with a 200-mesh cell sieve (DE2007; Biodee, Beijing, China) and centrifuged another time.
The supernatant and suspended adipocytes were discarded before the cells were resuspended with the MSC culture medium (S1569; Sigma). The cells were seeded at a density of 5 × 10 5 cells per 3.5-cm culture dish and recorded as primary cells (P0); they were sub-cultured until 80-90% confluency in a humidified incubator with 5% CO 2 at 37°C. The ADMSCs were subjected to sorting analysis by fluorescence activation using a BD LSRII analytical device (BD Biosciences, Franklin Lakes, NJ, USA). No spontaneous differentiation was observed during the culture process. In accordance with the instructions of the three-line induction and differentiation kit (CHEM-200004/5/6, Linmeng Biotechnology Co., Ltd., Shanghai, China), Alizarin Red S, oil red O and Alcian blue staining were adopted to observe the osteogenic, adipogenic and chondrogenic differentiation abilities of ADMSCs, respectively.
Primary mouse HSCs were obtained using pronase/collagenase perfusion through gradient centrifugation. The purity of isolated HSCs was evaluated with the help of alpha smooth muscle actin (α-SMA) staining based on immunocytochemistry with purity over 95%. Mouse HSCs were obtained by collagenase and cultured in Dulbecco modified eagle's medium (DMEM) comprising of 10% foetal bovine serum (FBS), streptomycin (100 g/mL) and penicillin (100 U/mL). The obtained cells were placed in a humidified incubator with 5% CO 2 at 37°C.
Subsequently, the prepared lentiviral particles were used to infect the ADMSCs and their control cells for 24 hours. After 48 hours of culture, purinomycin (p8230, Solarbio Technology Co., Ltd., Beijing, China) was adopted to screen the stably infected cell lines.

| Isolation and identification of EVs
The supernatant of ADMSCs (500 g) was collected and centrifuged, filtered through a 0.22 μM filter and centrifuged at 110 000 × g for 70 minutes. The precipitate was collected, resuspended with PBS, centrifuged at 110 000 × g for 70 minutes and then resuspended with 100 μL sterile PBS. All the ultracentrifugation steps were carried out at 4℃, using a Beckman ultracentrifuger (TL-100, Beckman Coulter Inc, Chaska, MN, USA) and a TLS-55 swing bucket rotor.
Low-speed centrifugation was performed with a Beckman Allegra X-15R desktop centrifuge.
A total of 20 μL of EVs was dropped on a copper net and then soaked for 3 minutes. Filter paper was then used to absorb the liquid from the side, followed by the addition of 30 μL phosphotungstic acid solution (pH 6.8). EVs were subsequently counterstained at room temperature for 5 minutes, dried with an incandescent lamp and photographed under a transmission electron microscope. Particle size analysis 23 was performed with nanoparticle tracking analysis (NS300, Malvern Instruments Ltd., Worcestershire, UK). Western blot assay was also applied to identify the surface markers of EVs.
The EV suspensions were determined using a bicinchoninic acid kit (BCA; 23227, Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA). After the sulphate-polyacrylamide gel electrophoresis gel was prepared, the proteins were denatured and underwent electrophoresis. Afterwards, the proteins were transferred onto a membrane and the specific marker proteins of EVs, including tumour susceptibility gene 101 (TSG101) (ab30871, dilution ratio of 1:1000), CD63 (ab68418, dilution ratio of 1:1000) and CD81 (ab109201, dilution ratio of 1:2000) and negative control GRP94 (ab3674, dilution ratio of 1:3000) were determined. Ponceau red served as the loading control. All the aforementioned antibodies were obtained from Abcam Inc (Cambridge, MA, USA).

| Immunofluorescence
After conventional detachment and transfection, the cells were counted and cultured in an immunofluorescence chamber at a density of 2 × 10 5 cells per well. Next, 1 mL of 4% paraformaldehyde was added to fix the cells, followed by reaction with 0.3% Triton.
The PBS-prepared primary antibodies to vimentin (ab92547, dilution ratio of 1:300) and α-SMA (ab108424, dilution ratio of 1:500) were incubated with the cells, followed by further incubation with green fluorescence-labelled Annexin V-fluorescein isothiocyanate (FITC)-goat anti-rabbit secondary antibody (ab6717, dilution ratio of 1:2000) in the dark for 1 hour at room temperature. After the nuclei were stained with DAPI in the dark for 15 minutes, the cells were observed and photographed under the same exposure condition using a fluorescent microscope.
The volume of each well was set to 0.2 mL, with six duplicate wells.
The plates were taken out after 24 hours, 48 hours and 72 hours of incubation, respectively. The medium containing 10% MTT solution (5 g/L) (GD-Y1317; Guduo Biotechnology, Shanghai, China) was then adopted for further 4 hours of culture. The supernatant was subsequently removed, and 100 μL dimethyl sulfoxide (DMSO) was added to each well (D5879-100 mL; Sigma) to fully dissolve the crystal of methylzan produced by the living cells. Afterwards, the optical density (OD) value of each well was measured at a wavelength of 490 nm using a microplate reader (BS-1101; Detie Experimental Equipment Co., Ltd., Nanjing, China).

| Western blot assay
The radioimmunoprecipitation assay lysate pre-cooled at 4℃ con- (GAPDH) (ab8245, 1:5000) at 4°C. The following day, the membrane was rinsed with Tris-buffered saline containing Tween-20 and reprobed with the diluent of goat anti-rabbit to immunoglobulin G (IgG; ab6721, 1:5000) labelled with horseradish peroxidase for 1 hour at room temperature (all the above antibodies were purchased from Abcam, except CXCL1). The substrate enhanced chemiluminescence reagent of horseradish peroxidase was purchased from Lianshuo Biotechnology (WBKLS0050, Shanghai, China). Finally, with GAPDH serving as the internal reference, a gel imaging analysis system (GIS-500, Qian Ming Gene Technology Co., Ltd., Beijing, China) and the ImageJ software were applied to analyse the protein expression patterns, which were indicated by relative grey value of the corresponding protein bands and of the internal reference protein bands.

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
RNA extraction kits (AM1552, Thermo Fisher Scientific) were used to extract the total RNA content from the cells and tissues and RNA content from EVs following the instructions, and the RNA concen-  Table 1). The detection of target gene expression was performed with a fluorescence quantitative PCR instrument (ABI ViiA 7, Daan International Holdings Limited, Guangzhou, China). With GAPDH and U6 serving as the internal parameters, the relative expression of the target gene was calculated using the relative quantitative method (2 −△△ CT method).

| Liver injury induced by CCl 4 administration
A total of 64 mice were randomly classified into control (without were subsequently injected aseptically into the mice twice a week for 8 weeks. Forty-eight hours before CCl 4 injection, mice in each group were injected with related lentivirus via the tail vein using high-pressure, and the recombinant lentivirus (7.6 × 10 7 IFU per mouse) was injected into mice at a concentration of 10 mg/kg 24,25 After CCl 4 treatment, the mice were euthanized, and their livers and serums were collected for further analysis.

| Masson staining
Paraffin sections were obtained from the liver tissues of mice, and the degree of HF was detected following the protocols of the Masson staining kit (G1340, Solarbio). The results were identified as positive if basement membrane and collagen fibres were stained blue or green, immune complex was stained red, and nucleus was stained blue brown. Next, 5 visual fields were randomly observed in each section under a polarized light microscope, and the Image Pro Plus 5.1 image analysis software (Cybernets, USA) was adopted for image analysis. Collagen volume fraction (CVF) was calculated as follows: CVF (%) = collagen area/ full field area × 100%.

| miR-150-5p was poorly expressed and CXCL1 was highly expressed in HF
Initially, mice were injected with CCl 4 to establish HF mouse models. HE and Masson staining were subsequently used to observe the pathological changes and degree of HF, which revealed that the pathological score and CVF of HF mice were significantly higher than those of control mice ( Figure 1A,B). The protein expression patterns of vimentin and collagen I in liver tissue were were all notably higher than those in the control mice ( Figure 1D).
Moreover, ELISA results illustrated that the expression levels of TNF-α, IL-6 and IL-17 as well as ALT, AST and TB levels were markedly higher in HF mice relative to control mice ( Figure 1E,F).
These findings verified the successful establishment of HF mouse models.
Subsequently, the expression patterns of miR-150-5p and CXCL1 were determined with the help of RT-qPCR and Western blot assay, which revealed that HF mice presented with suppressed miR-150-5p and elevated CXCL1 levels compared with the control mice ( Figure 1G,H).

| Characterization of ADMSCs and EVs
The third generation of ADMSCs was used to prepare a suspension to be significantly up-regulated in the EVs compared with the control cells, while the GRP94 protein was almost not expressed ( Figure 2D), indicating that ADMSCs-EVs were successfully isolated.

| ADMSCs-EVs transferred miR-150-5p to HSCs
The miR-150-5p expression patterns in ADMSCs and ADMSCs-EVs separated from control and HF mice were assessed by RT-qPCR, which illustrated that ADMSCs and ADMSCs-EVs presented with a markedly increased levels of miR-150-5p in comparison with those in control-ADMSCs or control-ADMSCs-EVs ( Figure 3A). Subsequently, ADMSCs were infected with lentiviral plasmids oe-miR-150-5p and miR-NC, and their EVs (miR-150-5p-EVs and miR-NC-EVs) were isolated. RT-qPCR revealed that after treatment of miR-150-5p-mimic, the expression levels of miR-150-5p increased in ADMSCs and their secreted EVs ( Figure 3B). Furthermore, PBS or RNase was added to miR-150-5p-EVs or miR-NC-EVs, respectively, to verify the stability of miR-150-5p. Additional RT-qPCR displayed that the expression of miR-150-5p were significantly decreased when the EV membrane was damaged by lysate and affected by RNase ( Figure 3C).
Additionally, in order to investigate whether ADMSCs-EVs containing miR-150-5p could be delivered to HSCs, PKH-67 was adopted to label the ADMSCs-miR-150-5p-EVs in three treatment groups

| miR-150-5p down-regulated CXCL1
The TargetScan website (http://www.targe tscan.org/vert7 1/) was initially retrieved to predict the downstream target gene of miR-150-5p, which revealed the presence of a targeting binding site between miR-150-5p and CXCL1 ( Figure 6A). Subsequently, dual luciferase reporter gene assay was carried out to verify the targeting relationship between miR-150-5p and CXCL1. After site-directed mutation of 3'-UTR region of CXCL1 mRNA, the luciferase signal in response to CXCL1-WT/miR-150-5p mimic co-transfection was found to be markedly decreased, indicating that miR-150-5p could specifically bind to CXCL1 ( Figure 6B). In addition, HSCs were then infected with miR-150-5p mimic and its inhibitor. As shown in RT-qPCR and Western blot assay, miR-150-5p mimic inhibited the CXCL1 expression, while the miR-150-5p inhibitor brought about the opposite effect ( Figure 6C,D). After HSCs were co-cultured with ADMSCs-EVs, miR-NC-EVs and miR-150-5p-EVs, respectively, the related RNA and total protein contents were extracted. RT-qPCR and Western blot assay illustrated that the expression levels of

| ADMSCs-EVs containing miR-150-5p mediated CXCL1 inhibited TGF-β-induced HSC activation
To elucidate the effect of ADMSCs-EVs-derived miR-150-5p on the activation of HSCs by regulating the expression of CXCL1, TGF-β was used to induce HSC activation, followed by treatment with miR-NC-EVs, miR-150-5p-EVs + oe-NC and miR-150-5p-EVs + oe-CXCL1. RT-qPCR results revealed that when compared to miR-NC-EVs, miR-150-5p-EVs markedly increased the miR-150-5p expression, while oe-CXCL1 did not alter the miR-150-5p expression compared with oe-NC ( Figure 7A). In addition, MTT assay demonstrated that relative to miR-NC-EVs, miR-150-5p-EVs led to inhibited HSC proliferation, while CXCL1 over-expression reversed this trend ( Figure 7B). Western blot assay further displayed that miR-150-5p-EVs brought about a decline in the expression levels of CXCL1 and Bcl-2, and increased those of Bax and cleaved caspase-3, while oe-CXCL1 treatment could reverse these trends ( Figure 7C). Western blot assay on the protein expression patterns of fibrosis-related factors displayed that the expression levels of collagen I, collagen III and fibronectin were inhibited in response to miR-150-5p-EVs than that upon miR-NC-EVs, but the trend could be reversed by oe-CXCL1 treatment ( Figure 7D). Finally, immunofluorescence revealed that miR-150-5p-EVs inhibited the expression levels of vimentin and α-SMA compared with miR-NC-EVs, relative to miR-150-5p-EVs + oe-NC, and CXCL1 over-expression could abrogate this trend ( Figure 7E).
Collectively, these findings indicated that CXCL1 expression was inhibited by miR-150-5p in the ADMSCs, which prevented the activation of HSCs induced by TGF-β.

| ADMSCs-EVs containing miR-150-5p inhibited CXCL1 to alleviate HF in vivo
Lastly, in vivo experimentation was carried out to explore the regulatory role of ADMSCs-EVs containing miR-150-5p in HF in Subsequent HE and Masson staining illustrated that compared with oe-NC, miR-150-5p-EVs reduced the pathological scores and CVF in HF mice, which were reversed by oe-CXCL1 treatment ( Figure 8A,B). As expected, immunohistochemistry showed that in HF mice, versus oe-NC, miR-150-5p-EVs reduced the protein expression levels of vimentin and collagen I, and these could be reversed by CXCL1 over-expression ( Figure 8C). Moreover, RT-qPCR results displayed that in comparison with oe-NC, miR-150-5p-EVs up-regulated the expression levels of miR-150-5p in liver tissues, but relative to miR-150-5p-EVs + oe-NC, oe-CXCL1 injection did not alter the expression of miR-150-5p ( Figure 8D).

| D ISCUSS I ON
HF is an intense repair as well as cicatrization mechanism-related disorder, with its end-stage cirrhosis accounting for high global morbidity and mortality rates. 27 It has been highlighted that EVs have the potential for HF therapy through mediation of intercellular miR delivery between HSCs. 9 In the current study, we aimed to investigate the role of ADMSCs-derived EVs containing miR-150-5p in HF, which uncovered an alleviatory role.
Firstly, we uncovered that miR-150-5p was poorly expressed and CXCL1 was highly expressed in HF. Similarly, down-regulated expression levels of serum miR-150-5p have been previously documented in patients with higher HF grading. 28 More notably, a particular study demonstrated that miR-150 suppressed HSC activation by down-regulating HOXA transcript at the distal tip, thereby serving as a potential therapeutic target for HF. 29 On the other hand, diminished expressions of CXCL1 as a result of deficiency of IL-17RA F I G U R E 7 ADMSCs-EVs containing miR-150-5p mediates CXCL1 to inhibit TGF-β-induced HSC activation. A, The expression of miR-150-5p in HSCs in co-culture system as determined by RT-qPCR. B, The proliferation of HSCs in co-culture system as examined by MTT assay. C, The expression of CXCL1 and apoptosis-related factors (Bax, Bcl-2 and caspase-3) in HSCs in co-culture system as determined by RT-qPCR. D, The protein expression of fibrosis-related factors (collagen Ⅰ, collagen Ⅲ and fibronectin) in HSCs in co-culture system as determined by Western blot assay. E, The expression of HSC activation markers (vimentin and α-SMA) in HSCs in co-culture system as determined by immunofluorescence. vimentin shows green fluorescence, α-SMA shows red fluorescence (× 200, scale bar = 50 μm). *P < .  TGF-β + miR NC-EVs TGF-β + miR-150-5p-EVs + oe-NC TGF-β + miR-150-5p-EVs + oe-CXCL1 were previously found to be capable of mediating cholesterol synthesis, indicating its regulatory role in HF. 30 Up-regulation of CXCL1 by CD147 is also known to stimulate HSC activation via autocrine, which further implicates CXCL1 with HF development. 31 All these findings and reports highlight the involvement of miR-150-5p and CXCL1 in HF development.
Transplantation of ADMSCs was previously demonstrated to be beneficial for the amelioration of CCl 4 -induced HF in rat models. 32 Consistently, ADMSCs have also been found to be effective at alleviating HF through the suppression of HSC activation and proliferation, which is very much in line with our findings. 33  Tregs to dendritic cells, 35 whereas EVs have been documented to transport miR-150 from smooth muscle cells to endothelial cells in other instances. 36 Furthermore, MSCs-derived EVs could accelerate the intracellular transfer of miR-150-5p between cells, which was highlighted as potential therapeutic strategy against joint destruction in RA. 37 Further experimentation in our study revealed that ADMSCs-   well-known pro-inflammatory cytokines responsible for HF pathogenesis, 40 and further, the presence of IL-6 in human fibrotic livers also plays a role in favouring HF development. 41 Moreover, diminished levels of AST, ALT and TB have also been previously reported to aid in the attenuation of HF. 42 Previous study unfolded that miR-150 could target and decrease CXCL1 expression in mice overexpressing or underexpressing Kruppel-like factor 2. 20 All these evidences indicate that miR-150-5p delivered by ADMSCs-EVs could target CXCL1, and consequently suppress HF development both in vivo and in vitro.
To summarize, the current study revealed that EVs derived from ADMSCs deliver miR-150-5p to down-regulate the expression of CXCL1, which inhibits the development of HF. Our findings highlight the therapeutic potential of miR-150-5p-containing ADMSCsderived EVs for HF treatment, which still requires further validation to improve the quality of life of patients plagued by HF.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data not shared.