Exosomes Derived from miR-183-Overexpressing Human Adipose-Derived Stem Cells Alleviate Hepatic Ischemia-Reperfusion (I/R) Injury through miR-183/ALOX5 axisExosomes Derived from miR-183-Overexpressing Human Adipose-Derived Stem Cells Alleviate Hepatic Ischemia-Reperfusion (I/R) Injury through miR-183/ALOX5 Axis

Background Ischemia-reperfusion (I/R) injury a crucial factor causing liver injury in clinic. Recent research con�rmed that human adipose-derived stem cells (ADSCs) can differentiate into functional hepatocytes. However, the mechanism of ADSCs in the treatment of liver injury remains unclear. Methods Characteristics of ADSCs were �rst identi�ed, and exosomes from miR-183-overexpressed ADSCs were isolated and identi�ed. And the function and mechanism of microRNA-183 (miR-183) and arachidonate 5-lipoxygenase (ALOX5) were monitored through biological experiments in HL-7702 cells with I/R injury or I/R rats. Results We proved that inhibition of exosomes from the identi�ed ADSCs prevented proliferation, induced apoptosis and downregulated miR-183 in HL-7702 cells with I/R injury, while exosomes derived from ADSCs played an opposite role on the proliferation and apoptosis of HL-7702 cells with I/R injury in comparison with exosome inhibitors by upregulating miR-183. Meanwhile, the effect of miR-183 alone was similar to that of exosomes derived from ADSCs. Besides, ALOX5, as a target gene of miR-183, was involved in the related functions of miR-183. Moreover, in vivo experiments further con�rmed miR-183 or exosomes from ADSCs also could improve liver injury of rats and downregulate MAPK and NF-κB pathways.


Introduction
The liver is the largest organ in the body of mammals, with multiple functions, such as protein synthesis, detoxi cation, glycogen storage, etc [1,2].Because the liver needs a rich oxygen supply to maintain normal physiological function, it is highly susceptible to ischemia reperfusion (I /R) injury [3].Liver I/R injury is mainly caused by liver transplantation, severe trauma, hemorrhagic shock, etc [4,5].And it can reduce the capacities of liver metabolism and detoxi cation, cause microcirculation disorders and even liver failure, reduce the success rate of surgery, and affect disease prognosis [6,7].Therefore, further exploration of safe and effective methods to enhance the resistance of cells and improve liver regeneration is the key to reduce liver I/R injury.
ADSCs are a type of pluripotent stem cells with high self-renewal ability with adipogenic, osteogenic, chondrogenic and myoblast differentiation [8].Researches have proved that ADSCs have the ability to differentiate into hepatocytes with hepatocyte functions, such as glycogen synthesis, urea formation, expression of hepatocellular speci c genes, and cytochrome P450 enzyme activity, etc [9,10].Current researches have testi ed that ADSCs can be applied to treat liver diseases such as hepatitis, liver brosis and liver failure [11][12][13].Among them, ADSCs transplantation is an effective method to promote the recovery of normal function and liver regeneration after large-scale injury [14].It was reported that ADSCs can secrete cytokines and growth factors associated with liver regeneration [15,16].In animal models, ADSCs transplantation showed a protective effect against liver injury and stable integration into the diseased liver tissue [14,17].However, the key mechanism of ADSCs in the treatment of liver injury is unclear.
Exosomes are lipid bilayer vesicles with diameters of 50-150 nm, that are formed in the cytoplasm and released into the extracellular environment [18].Exosomes have been recognized as a novel mechanism of cell-cell communication and have protective effects on acute renal tubular injury, liver brosis, and nerve growth [19,20].Researches also veri ed that ADSCs-derived exosomes play essential roles in tissue repair and immune regulation [21].Besides, exosomes have stable properties, convenient storage and transportation, and no risk of immune rejection and tumor formation [12].Therefore, it is expected to be a new therapeutic strategy.However, the mechanism of ADSCs-paracrine exosomes on liver injury has not been fully elucidated.In this study, we isolated and identi ed exosomes from the authenticated ADSCs to elucidate the latent role and mechanism of speci c ADSCs-derived exosomes in liver I/R injury treatment.

Cell culture
Human ADSCs were obtained from Cellular Engineering Technologies (Coralville, IA).HL-7702 cells were supplied by American Type Culture Collection (Manassas).ADSCs and HL-7702 cells were cultured in 10% fetal bovine serum (FBS)-containing DMEM medium at 37°C, 5% CO2.The grown ADSCs were observed under a microscope.

Animal
Male Sprague-Dawley rats (180-200g) were obtained from Laboratory Animal Center of Sun Yat-Sen University.The experiment was carried out after clinical examination of health under the same conditions (temperature (20 ± 2°C), 50% humidity, 12 h light/dark cycle).All animal experiments have been approved by the Committee of the Ethics Committee of Southwest Hospital, Third Military Medical University (Army Medical University).

Rat model of hepatic I/R
On account of the previous researches [22,23], Brie y, after anesthesia, a traumatic clip was applied to occlude liver arterial/portal venous blood for 60 min, then the clip was removed for reperfusion.
After washing with PBS, cells were resuspended with 500 μL PBS.Then the positive rate of cell surface antigen in each group was examined by ow cytometry.

Oil red staining
The harvested ADSCs (1×10 5 /mL) were incubated with adipogenic differentiation medium (Millipore) for 16 days.After washing using PBS, ADSCs were xed using 4% formaldehyde for 30 min and treated with Oil red dye for 30 min.The staining effect was observed under a microscope.

Alizarin Red Staining
The harvested ADSCs (1×10 5 /mL) were grown with osteogenic differentiation medium (Millipore) for 2 weeks.After washing and xing, ADSCs were stained with alizarin red dye for 5 min.The staining effect was observed under a microscope.

Exosome extraction
As described in previous study [24], ADSCs were transfected with miR-183 mimics for 48 h, and the medium was harvested and ltered using 0.22 μm lter (Merck-Millipore).ExoQuick-TCExosome Precipitation Solution (System Biosciences) was applied to extract exosomes from ADSCs.

RT-qPCR analysis
The extraction of total RNAs was completed using TRIzol reagent (Invitrogen).cDNAs were synthesized using SuperScript II (Vazyme, Nanjing, China) with total RNAs.Ampli cation reaction was conducted using SYBR Green Mix (Promega).Ampli cation data was obtained through ABI Prism 7900 (Applied Biosystems) and counted using 2 −ΔΔCt method.

Hoechest staining
The treated HL-7702 cells (3×10 4 cells) were seeded into a 24-well plate for 48 h incubation at 37°C with 5% CO2, and added with 1 ml diluent Hoechest 33342 (Beyotime Institute of Biotechnology) at 37°C for 30 min.The stained apoptotic cells were observed using a uorescence microscope (Nikon).

Transmission electron microscopy (TEM)
The extracted exosomes from ADSCs were suspended in 0. 2% paraformaldehyde (Sigma-Aldrich), and 3 μL suspension was added onto Electron Microscopy Sciences coated with Formvar-Carbon at room temperature for 5 min.Then the samples were dyed with 1.75% uranyl acetate.After drying, the exosomes were observed using TEM (HITACHI H-7500, Hitachi, Tokyo, Japan).

Dynamic light scattering (DLS)
Nanosizer™ technology (Malvern Instruments, Malvern, UK) and Zetasizer software (Malvern) were applied for the detection of exosome size.

Isolation of RNA and protein from exosomes
Based on the instructions, total Exosome RNA & Protein Isolation Kit (Invitrogen) was adopted to extract the RNA and protein from exosomes.

TUNEL staining
Liver tissue section of rats in each group was dewaxed, and Apoptosis was examined using DeadEnd™ Fluorometric TUNEL System kit (Promega, Cat.no.G3250) in line with the manufacturer's instructions.

Immunohistochemistry (IHC) assay
After routine dewaxing and hydration, liver tissue sections were added with 3% H202 at room temperature for 10 min.After thermal antigen repair, the sections were sealed using 5% BSA at 37°C for 30 min, incubated with ALOX5 antibody (1:200, Abcam) overnight at 4 °C, followed by secondary antibody for 30 min.After DAB coloring for 10 min, the samples were dehydrated, transparent and then sealed with neutral gum.The results were photographed using a microscope.
Dual-luciferase reporter assay ALOX5 luciferase reporter plasmid was obtained from VIPOTION Biotechnology (Guangzhou, China).HL-7702 cells were co-transfected with ALOX5 luciferase reporter plasmid and miR-183 mimics, and the experiment was conducted using the Dual-Luciferase ® Reporter Assay System (Promega) based on the speci cation.

Statistical analysis
Measurement data with normal distribution were expressed as mean ± SD.SPSS 21.0 statistical software was used for data processing and analysis using t-test or one-way analysis of variance (ANOVA) with Duncan's test.P<0.05 signi ed that the data was statistically signi cant.

Characteristics of ADSCs
To collect exosomes from ADSCs, we purchased and identi ed ADSCs.Firstly, the surface markers of ADSCs were analyzed by ow cytometry, and the data signi ed that ADSCs were negative for CD31, CD34 and CD45, and positive for CD90 and CD105 (Figure 1A).Under the microscope, we discovered that cells were grown in a spindle-like shape (Figure 1B).Oil red staining results proved that there were small cytoplasmic lipid droplets in ADSCs, suggesting the adipogenic potential of ADSCs (Figure 1C).And Alizarin Red Staining results manifested that calcium mineral deposits were present in large numbers in the ADSCs, indicating the osteogenic potential of ADSCs (Figure 1D).Thus, we successfully identi ed ADSCs with the speci c characteristics.
Inhibition of exosomes prominently downregulates miR-183, suppresses proliferation and induces apoptosis in HL-7702 cells after I/R injury Subsequently, we further con rmed whether exosomes can affect the function of liver injury, including proliferation and apoptosis.Exosome inhibitor (GW4869) was adopted to treat the co-cultured ADSCs and HL-7702 cells with I/R injury.RT-qPCR data testi ed that was greatly reduced in I/R injury group relative to control (blank) group, while the reduction of miR-183 level could be reversed by co-culture of ADSCs in HL-7702 cells with I/R injury; interestingly, GW4869 could also prominently downregulate miR-183 in HL-7702 cells with I/R injury after co-culture with ADSCs (Figure 2A).Next, through the detection of ow cytometry, we proved that the decrease of cell proliferation (Edu-positive cells) could be signally attenuated by co-culture of ADSCs in HL-7702 cells with I/R injury, while this increase of cell proliferation in HL-7702 cells with I/R injury after co-culture with ADSCs also could be markedly reversed by GW4869 (P<0.01, Figure 2B).Additionally, we indicated that the trend of apoptosis was just opposite to the trend of cell proliferation in each group, that is, co-culture of ADSCs observably suppressed the apoptosis of HL-7702 cells with I/R injury, which also could be memorably reversed by GW4869 (Figure 2C).Besides, as displayed in Figure 2D, co-culture of ADSCs dramatically downregulated caspase 3 and Bax, and upregulated Bcl2 in HL-7702 cells with I/R injury, while these protein changes mediated by co-culture of ADSCs also could be notably reversed by GW4869 (Figure 2D).In summary, these data testi ed that inhibition of exosomes in ADSCs could prominently reverse the proliferation induction and apoptosis inhibition mediated by co-culture of ADSCs in HL-7702 cells with I/R injury.

Extraction and identi cation of exosomes in ADSCs after miR-183 overexpression
To further verify the roles of exosomes in ADSCs on HL-7702 cells after I/R injury, we extracted and identi ed exosomes from ADSCs.The TEM images exhibited the morphology of the extracted vesicles (Figure 3A), and DLS data showed that these vesicles mainly ranged from 80 to 120 nm in size (Figure 3B).Besides, western blotting data certi ed that the expressions of exosome markers (TSG101, CD9 and CD63) were markedly elevated in the extracted exosomes (Figure 3C).Therefore, we proved that the exosomes were successfully extracted.Meanwhile, we also certi ed that after co-culture, DID-labeling exosomes have been transferred in to HL7702 cells (Figure 3D).
Exosomes derived from miR-183-overexpressed ADSCs dramatically enhances proliferation and inhibits apoptosis of HL-7702 cells after I/R injury by upregulating miR-183 Next, we explored the impacts of exosomes and miR-183 on the proliferation and apoptosis of HL-7702 cells after I/R injury.Exosomes or/and miR-183 mimics were applied to treat HL-7702 cells after I/R injury.RT-qPCR data indicated that miR-183 level was observably decreased in I/R injury group relative to blank group, while the decrease of miR-183 level could also be signally reversed by exosomes in HL-7702 cells with I/R injury; meanwhile, we discovered that miR-183 overexpression then prominently upregulated miR-183, which was mediated by exosomes, in HL-7702 cells with I/R injury (Figure 4A).Functionally, just like co-culture of ADSCs, exosomes alone could also signi cantly promote the proliferation of HL-7702 cells with I/R injury, which also could be memorably energized by miR-183 overexpression (P<0.01, Figure 4B).Hoechest staining results also veri ed that the apoptosis of HL-7702 cells with I/R injury could be dramatically inhibited by exosomes, and exosome-mediated inhibition of apoptosis also could be further enhanced by miR-183 overexpression (Figure 4C).Besides, the data from western blotting analysis uncovered that exosomes markedly downregulated caspase 3 and Bax, and upregulated Bcl2 in HL-7702 cells with I/R injury, and changes in the expression of these proteins also could be further strengthened by miR-183 overexpression (Figure 4D).Overall, we disclosed that exosomes derived from ADSCs could alleviate liver I/R injury by upregulating miR-183 in vitro.

Overexpression of miR-183 markedly accelerates proliferation and prevents apoptosis of HL-7702 cells after I/R injury
In addition, we further tested whether miR-183 could independently affect proliferation and apoptosis of HL-7702 cells after I/R injury.Firstly, miR-183 was notably overexpressed in HL-7702 cells after I/R injury through the transfection of miR-183 mimics (Figure 5A).Secondly, ow cytometry data also manifested that overexpression of miR-183 dramatically accelerated the proliferation of HL-7702 cells after I/R injury (Figure 5B).Hoechest staining data certi ed that overexpression of miR-183 observably restrained the apoptosis of HL-7702 cells after I/R injury (Figure 5C).And we also found that overexpression of miR-183 memorably reduced caspase 3 and Bax expressions, and elevated Bcl2 expression in HL-7702 cells with I/R injury (Figure 5D).Consequently, these data proved that overexpression of miR-183 also could markedly alleviate liver I/R injury in vitro.
ALOX5, as a target gene of miR-183, is related to miR-183-medaited proliferation and apoptosis of HL-7702 cells with I/R injury Moreover, we further investigated the latent target gene of miR-183.As displayed in Figure 6A, we found that overexpression of miR-183 could signally downregulate ALOX5 in HL-7702 cells with I/R injury.Bioinformatics analysis results also signi ed the presence of binding sites for miR-183 and ALOX5 (Figure 6B).And dual-luciferase reporter results disclosed that the elevated miR-183 prominently attenuated the luciferase activity of WT-ALOX5, but not that of Mut-ALOX5 (Figure 6C).More importantly, HL-7702 cells with I/R injury were transfected with ALOX5 or/and miR-183 overexpression, and the data showed that ALOX5 overexpression markedly upregulated ALOX5, which then downregulated by miR-183 in HL-7702 cells with I/R injury (Figure 6D).Then we discovered that overexpression of ALOX5 could dramatically inhibited proliferation and facilitated apoptosis of HL-7702 cells with I/R injury, which also could be partially reversed by miR-183 (P<0.01, Figure 6E-6F).And we also found that of ALOX5 overexpression memorably increased caspase 3 and Bax expressions, and decreased Bcl2 expression in HL-7702 cells with I/R injury (Figure 6H).Thus, we revealed that miR-183 could attenuate liver I/R injury by targeting ALOX5 in vitro.

MiR-183 or exosomes from ADSCs ameliorates the liver function of I/R rats and inhibits MAPK and NF-κB pathways
Furthermore, we also veri ed the potential of exosomes from ADSCs and miR-183 on the liver function of rats with I/R injury.I/R rats were established and treated with exosomes and miR-183.As presented in Figure 7A, AST, ALT and LDH levels were memorably elevated in the I/R rats relative to that in the control rats, while exosomes introduction or miR-183 overexpression observably reduced AST, ALT and LDH levels in I/R rats (P<0.01, Figure 7A).Additionally, TUNEL staining results revealed that I/R injury could lead to signi cant enhancement of apoptosis in rats, while this enhancement of apoptosis also could be signally weakened (Figure 7B).IHC results then uncovered that ALOX5 expression was prominently increased in the I/R rats with respect to that in the control rats, while this increase also could be partially reversed by exosomes or miR-183 overexpression in the liver tissues of I/R rats (Figure 7C).Moreover, our data certi ed that ALOX5 p-p65 p-JNK p-p38 and p-ERK were memorably upregulated in the liver tissues of the I/R rats compared to that the control rats, while exosomes or miR-183 overexpression also could markedly attenuate the upregulation of these proteins in the liver tissues of the I/R rats (Figure 7D).On the whole, we demonstrated that exosomes introduction or miR-183 overexpression also could signally improve the liver function of I/R rats.

Discussion
Liver I/R injury is a complex and dynamic pathophysiological process, which mainly includes ischemia injury of local tissue cells and in ammation-mediated reperfusion injury [3,27].Researchers discovered that ADSCs transplantation can play a therapeutic role in liver injury by inhibiting in ammation, apoptosis, oxidative stress and autophagy, and promoting liver cell regeneration [28][29][30].In order to further investigate the therapeutic mechanism of ADSCs for liver I/R injury, we obtained human ADSCs.In our study, we found that the obtained ADSCs were negative for CD31, CD34 and CD45, and positive for CD90 and CD105.Meanwhile, we proved that the obtained ADSCs have the adipogenic and osteogenic potentials.It was reported that ADSCs are spindle-shaped in vitro; ADSCs can express stem cell speci c surface markers (CD29, CD44, CD73, CD90, CDL05 and CDL66), but lack hematopoietic markers (CD45 and CD34) and surface marker of neovascular endothelial cells (CD31); meanwhile, ADSCs have a strong collagen-forming ability [31,32].So, we successfully identi ed the obtained ADSCs.
As a vital component of cells, exosomes are composed of mRNA, small RNA and various proteins [33].Previous literature has reported that ADSCs-derived exosomes can promote the repair of damaged tissues by delivering mRNA and miRNA to nearby target cells [34,35].Besides, the activated proteins can also be transferred by exosomes to target cells and produce corresponding biological effects [36].Exosomes have been reported to play a major role in a variety of diseases, such as cancer [37,38], Parkinson's disease [39], cardiovascular diseases [40,41], ocular diseases [42], rheumatoid arthritis [43], atherosclerosis [44] and so on.Besides, exosomes have also been proven to be closely associated with drug-induced liver injury [45], acute liver failure [46], even liver cancer [47].In the current study, we further revealed that exosome inhibitor (GW4869) could signi cantly suppress proliferation and induce apoptosis of HL-7702 cells with I/R injury.Moreover, we discovered that GW4869 also could prominently downregulate miR-183, suggesting miR-183 is related to exosomes derived from ADSCs.Subsequently, our results proved that the isolated exosomes derived from miR-183-overexpressed ADSCs were mainly ranged from 80 to 120 nm in size with highly expressed TSG101, CD9 and CD63, suggesting that we have successfully isolated exosomes derived from miR-183-overexpressed ADSCs.Additionally, we veri ed that exosomes could be transferred in to HL7702 cells through co-culture.At present, ADSCs-derived exosomes have been proven to play signi cant roles in alleviating acute renal tubular injury, inducing functional recovery after stroke, relieving acute lung injury, and reducing the size of myocardial infarction, improving skin wound healing [48][49][50][51].In our study, we further discovered that the presence of ADSCs-derived exosomes could prominently enhance proliferation and inhibit apoptosis of HL-7702 cells with I/R injury, which also is relevant to miR-183.Furthermore, we testi ed that exosomes from ADSCs also have signi cant relieving effects on the liver function of I/R rats.
MiRNAs belong to a class of small non-coding RNAs, which have signi cant effects in a variety of processes by regulating genes closely related to cell functions [52,53].According to the literatures, we discovered that miR-183 is closely associated with a variety of diseases including cancer [54,55], lupus nephritis[56], retinal dysfunction [57], etc.In our study, we further uncovered that miR-183 overexpression also could accelerate proliferation and prevent apoptosis of HL-7702 cells with I/R injury, and improve the liver function of I/R rats, indicating the therapeutic effect of miR-183 on liver I/R injury.Meanwhile, we proved that and ADSCs-derived exosomes and miR-183 also could prevent MAPK and NF-κB pathways.
More importantly, we rst found that ALOX5 is as a target gene of miR-183 through bioinformatics analysis and experimental veri cation.ALOX5 gene, which is located on chromosome 10q11.2,can encode 5-lipoxygenase (5-LO) containing 673 amino acids [58].After activation, ALOX5 can be transferred to the nuclear membrane and interact with ALOX5 activated protein (FLAP), then converts AA to leukotriene and 5-hydroperoxy-eicostetraeoic acid (5-HpETE), which is further metabolized into leukotriene A4 (LTA4), LTB4 and LTC4 [59].Research manifested that LTA4, as an active lipid, critically related to in ammation.Currently, ALOX5 has been reported to be involved in the pathogenesis of multiple diseases, such as asthma, cardiovascular diseases, multiple sclerosis, tumors and allergic diseases, etc [59][60][61][62][63].Our study further demonstrated that ALOX5 could dramatically reverse miR-183-medaited proliferation and apoptosis of HL-7702 cells with I/R injury, indicating that miR-183 could notably attenuate liver I/R injury by targeting ALOX5.
We disclosed that exosomal miR-183 derived from ADSCs functions as an effective regulator by downregulating ALOX5 in liver I/R injury.Therefore, we indicated that control of exosomal miR-183 expression might be as a novel approach for the treatment of liver I/R injury.

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