Mesenchymal stem cell-derived exosomal miR-26a induces ferroptosis, suppresses hepatic stellate cell activation, and ameliorates liver fibrosis by modulating SLC7A11

Abstract Liver fibrosis is a key contributor to hepatic disease-related mortality. Exosomes derived from mesenchymal stem cells (MSCs) have been revealed to improve liver fibrosis. To explore the effect and mechanism of MSC-derived exosomal miR-26a on liver fibrosis, exosomes were separated from bone marrow-derived MSCs (BMSCs) and used to treat with LX2 cells. The miR-26a level was decreased in BMSC-derived exosomes. Treatment with exosomes isolated from human BMSCs transfected with miR-26a mimics (miR-26a mimic-Exo) decreased the 5-ethynyl-2'-deoxyuridine-positive cell rate, the protein level of α-SMA and collagen I, and the glutathione (GSH) level but enhanced the apoptosis rate and the reactive oxide species (ROS) level in LX2 cells, which were reversed by the treatment of deferoxamine. Mechanically, miR-26a directly bound SLC7A11 mRNA and negatively modulated the level of SLC7A11 in LX2 cells. Overexpression of SLC7A11 reversed the miR-26a mimic-Exo-induced alterations in the level of ROS, Fe2+, malonaldehyde, and GSH in LX2 cells. In vivo, miR-26a mimic-Exo decreased the level of SLC7A11 and attenuated CCL4-induced liver fibrosis. Collectively, miR-26a mimic-Exo induced ferroptosis to alleviate liver fibrosis by regulating SLC7A11, which may provide new strategies for the treatment of liver fibrosis, and even other relevant diseases.


Introduction
Liver fibrosis is a wound-healing progress in response to chronic hepatic damage, which can be caused by a variety of factors, such as alcohol, hepatitis viruses, diabetes, obesity, and hepatotoxic drugs [1].Liver fibrosis, characterized by an excessive increase in the extracellular matrix (ECM) proteins in hepatic stellate cells (HSCs), can progress to cirrhosis and even ultimately result in hepatic failure and hepatocellular carcinoma and is one of the crucial causes of incidence rate and death rate around the world [1][2][3].Experimental and clinical evidence indicate that the pathological conditions of liver fibrosis can be reversible [1,3].However, no effective drugs are currently approved to treat liver fibrosis.Some compounds have been reported to exhibit antifibrotic properties, but the inadequate efficacy with various side effects greatly impedes their clinical application [4].Thus, identifying the target and developing the strategies based on the pathogenesis is of great significance for the treatment of liver fibrosis.
Induction of programmed cell death of activated HSCs, such as apoptosis, ferroptosis, and necroptosis, can improve the pathological progression of liver fibrosis [5,6].Ferroptosis is an iron-dependent lipid peroxidation that differs from apoptosis, pyroptosis, or necrosis [7,8].Ferroptosis is closely related to the progression of multifarious liver disorders.For instance, Zhang et al. [9] report that BRD7-P53-SLC25A28 axis modulates ferroptosis in HSCs.The study from the same research group showed that the RNA-binding protein ZFP36/TTP regulates autophagy to prevent ferroptosis in HSCs [10].Artemether has been identified as a potential medicine for the therapy of liver fibrosis, in which artemether promotes the extensive production of reactive oxide species (ROS) and the occurrence of ferroptosis by inhibiting the ubiquitination of iron regulatory protein 2 to cause its deposition and the enhancement of iron in HSCs [11].Tsurusaki and colleagues revealed that hepatic ferroptosis triggers the initiation of inflammation in nonalcoholic steatohepatitis [12].Metallothionein-1G suppresses ferroptosis to accelerate sorafenib resistance in hepatocellular carcinoma cells [13].Therefore, treating liver fibrosis by inducing ferroptosis in activated HSCs may be a promising approach [10,14].
Exosomes with a diameter of 30-100 nm are bilayer lipid vesicles that have been proven to have no risk for tumor formation and have low immunogenicity [15].Exosomes can be actively secreted into the extracellular circumstances by diverse mammalian cells [16], and mesenchymal stem cell (MSC)-derived exosomes have been revealed to improve liver fibrosis [17,18].Exosomes carry a large number of bioactive molecules, such as lipids, proteins, nucleic acids, non-coding ribonucleic acid (RNA), which can be functionally exchanged between cell types and even across species [19].microRNA (miRNA), a type of noncoding RNAs with 21-25 nucleotides, can conjugate with RNA-elicited silencing complex to bind on the 3′-untranslated region of target mRNA, which then restrains the formation of the translation initiation complex, thereby leading to the translation inhibition [20].Therefore, miRNAs play a crucial role in various human diseases, including in liver fibrosis [21].miR-26 has exhibited the inhibitory effect on myocardial fibrosis [22,23], kidney fibrosis [24], and pulmonary fibrosis [25].It has been reported that miR-26a ameliorates ethanol-induced acute liver injury by enhancing autophagy [26].miR-26a is specifically induced by endoplasmic reticulum stress and is involved in the progression of nonalcoholic fatty liver disease [27].miR-26a also directly participates in the liver regeneration by targeting the mdm2/p53 loop [28].Notably, miR-26a is differentially expressed between HSCs and liver tissues, suggesting that miR-26a is related to liver fibrosis [29].However, the specific role and mechanism of miR-26a in liver fibrosis remains unclear.
This study aimed to explore the effect and potential mechanism of MSC-derived exosomal miR-26a on liver fibrosis.The findings in this study will help to further understand the role of MSC-derived exosomal miR-26a in the pathogenesis of liver fibrosis and may provide a new approach for the therapy of liver fibrosis, and other relevant disorders.

Isolation and identification BMSCderived exosomes
The exosomes were isolated from the culture medium of BMSCs using an exosome isolation reagent (Cat#: 4478359, Invitrogen).Briefly, the media of BMSCs were centrifuged at 2,000 × g for half an hour to harvest the supernatant.Then, the Total Exosome Isolation reagents were appended into the supernatant and vortexed for the sufficient mixture.The mixture was hatched at 4°C for 24 h and centrifuged at 10,000 × g for 60 min to collect the pellet.The pellet was re-suspended into phosphate buffer saline (PBS, Cat#: P1020, Solarbio, Beijing, China) to obtain the isolated BMSCderived exosomes (BMSC-Exo).The BMSC-Exo was imaged under transmission electron microscopy (TEM) (Cat#: 1200EX, Jeol, Japan).Besides, the exosomal markers, containing CD9, CD63, and calnexin, were examined via western blot after the treatment of the RIPA solution (Cat#: R0010, Solarbio).

LX2 cells' treatment
LX2 cells were plated into the six-well plates with a density of 5 × 10 5 cells/well and incubated in exosome-exhausted culture medium with 10% FBS and 1% P/S.Then, 50 μg/mL exosomes separated from transfected MSCs were used to incubate with the LX2 cells.Besides, to further verify the effect of BMSC-derived exosomal miR-26a on ferroptosis, LX2 cells were administrated with miR-26a mimic-Exo as well as the ferroptosis inhibitor, deferoxamine (DFO, 100 µM) for 24 h.

Flow cytometry
As the previous studies [30,31], cell apoptosis was analyzed by flow cytometry with an Annexin V-FITC Apoptosis Detection Kit (Cat#: CA1020, Solarbio).Cells were rinsed with pre-cold PBS and mixed into binding buffer.Cells were dyed with FITC-Annexin V and propidium iodide for 5 min in a dark.Cells were examined on a FACScan flow cytometry (BD Biosciences, NJ, USA) and the apoptotic rate was determined by BD CellQuest Pro software (version 5.1, BD Biosciences).In addition, the ROS level was measured using a reactive oxygen species assay kit (Cat#: CA1410, Solarbio) and analyzed via flow cytometry.

In vivo experiments
Four-week-old male C57BL/6J mice (13-15 g) were acquired from Junke biological Co., LTD.(Nanjing, China) and fed in the laboratory environment with 12 h/12 h light-dark cycle and 40-60% the relative humidity at 22℃.All animal experiments were abided by the Guide for the Care and Use of Laboratory Animals [32] and authorized by the Animal Research Ethics Committee of the Second Affiliated Hospital of Soochow University.The liver fibrosis model was constructed through the application of CCl4 (Cat#: 488488, Sigma-Aldrich, St. Louis, MO, USA) dissolved in olive oil with a ratio of 1:10 based on the previous description [33].Mice were randomized into four groups, including control, CCl4, CCl4 + NC mimic-Exo, and CCl4 + miR-26a mimic-Exo.Mice in CCl4, CCl4 + NC mimic-Exo, and CCl4 + miR-26a mimic-Exo were modeled by intraperitoneally injecting with 0.5 mg/kg CCl4 for 8 weeks twice a week.Twenty-four hours after the model, mice were administrated with PBS, exosomes separated from MSCs infected with NC mimic (100 μg/mL), and exosomes from MSCs with miR-26a mimic (100 μg/mL) through the tail vein, respectively.The dose of exosomes applied in the present study was based on the previous research [34].
Mice were sacrificed by inhaling the excess isoflurane (Cat#: R510-22, RWD, Guangdong, China), and the blood and liver tissues were yielded for the following experiments.

Measurement of aminotransferase (ALT) and aspartate aminotransferase (AST)
Blood was collected and stood for 2 h at room temperature, and at 4°C overnight.Subsequently, blood samples were centrifuged at 4,000 × g for 10 min at 4°C to isolate the serum samples.The serum contents of ALT and AST were detected by using Alanine Transaminase Activity Assay kit (Cat#: ab105134, Abcam) and Aspartate Aminotransferase Activity Assay kit (Cat#: ab105135, Abcam) according to the operation instruction.

Western blot
As the previous descriptions [36,37], cells and tissues were treated with RIPA lysis buffer to extract the total proteins and then quantified with a BCA kit (Cat#: PC0020, Solarbio).

Statistical analysis
Data were displayed as mean ± standard deviation.The statistical difference was determined via the Student's ttest and one-way analysis of variance for two groups and more than two groups followed by post hoc Bonferroni test by SPSS 26.0 software (IBM, Armonk, New York, USA).p < 0.05 was defined as the significant difference.
Ethics approval: Ethical approval was obtained from the Ethics Committee of the Second Affiliated Hospital of Soochow University.

miR-26a was lowly expressed in BMSCderived exosomes
To explore the effect of MSC-derived exosomal miR-26a on liver fibrosis, the exosomes were isolated from human BMSCs.Small round or oval vesicles with uneven morphology were observed under TEM, with a diameter of 30-100 nm (Figure 1a), morphologically confirming the successful isolation of the exosomes.In addition, both CD9 and CD63 were expressed in BMSC-Exo, while calnexin was not expressed in BMSC-Exo (Figure 1b), further verifying the exosomes in molecular level.These results indicated that exosomes were successfully isolated from human BMSCs.
Then, the expression level of miR-26a was examined in exosomes.As shown in Figure 1c, the miR-26a level was markedly decreased in BMSC-Exo relative to that in BMSC cellular matrix (BMSC-CM).Moreover, the miR-26a expression in LX2 cells administrated with BMSC-Exo was significantly reduced compared with LX2 cells treated with PBS (Figure 1d).Together, these results suggested that the miR-26a level was reduced in BMSC-derived exosomes.

Overexpression of BMSC-derived exosomal miR-26a inhibited the HSC activation
HSC activation is a key pathogenesis of liver fibrosis; thus, the role of BMSC-derived exosomal miR-26a was assessed in the HSC activation.Since the expression level of miR-26a was decreased in BMSC-derived exosomes, miR-26a mimics were transfected into human BMSCs to upregulate the level of miR-26a, and then, the exosomes were isolated from human BMSCs.The miR-26a level was notably increased in exosomes isolated from human BMSCs transfected with miR-26a mimics (miR-26a mimic-Exo) compared with exosomes isolated from human BMSCs transfected with NC mimics (NC mimic-Exo) (Figure 2a), demonstrating a viable transfection efficiency.Treatment of miR-26a mimic-Exo prominently diminished the EDU-positive rate but markedly enhanced the apoptosis rate of LX2 cells compared with LX2 cells treated with NC mimic-Exo (Figure 2b and c).ECM deposition that can be promoted by the activation of HSC is a key characteristic of liver fibrosis.The protein levels of α-SMA and collagen I, two important proteins in ECM deposition, were significantly reduced in LX2 cells treated with miR-26a mimic-Exo compared with LX2 cells treated with NC mimic-Exo (Figure 2d).Collectively, these results indicated that upregulation of BMSC-derived exosomal miR-26a repressed the HSC activation.

Upregulation of BMSC-derived exosomal miR-26a induced ferroptosis
Then, the effect of BMSC-derived exosomal miR-26a on ferroptosis was detected.A prominent enhancement in the level of ROS, Fe 2+ , and MDA and a conspicuous decrease in the GSH level were observed in LX2 cells treated with miR-26a mimic-Exo compared with LX2 cells treated with NC mimic-Exo (Figure 3a and b), suggesting that BMSC-derived exosomal miR-26a induced ferroptosis in LX2 cells.To further verify the effect of BMSC-derived exosomal miR-26a on ferroptosis, LX2 cells were transfected with miR-26a mimic-Exo and treated with the ferroptosis inhibitor, DFO.Enhanced EDUpositive rate in LX2 cells treated with DFO was significantly counteracted by the incubation of miR-26a mimic-Exo, while reduced EDU-positive rate in LX2 cells treated with miR-26a mimic-Exo was markedly restored with the further treatment of DFO (Figure 3c).However, the opposite results were found in the ROS level (Figure 3d).Moreover, increased protein level of α-SMA and collagen I in LX2 cells with DFO were significantly counteracted with the incubation of miR-26a mimic-Exo, while decreased protein levels of α-SMA and collagen I in LX2 cells with miR-26a mimic-Exo were markedly restored with the further treatment of DFO (Figure 3e).Collectively, these results demonstrated that overexpression of BMSC-derived exosomal miR-26a promoted ferroptosis in HSCs.

MiR-26a targeted SLC7A11
To further explore the mechanism of miR-26a in liver fibrosis, the downstream targets of miR-26a were predicted by targetScan and miRDB.Binding sites were discovered between miR-26a and SLC7A11 mRNA due to the complementary base pairing, and the target rank and target score demonstrated the high possibility of the interaction between miR-26a and SLC7A11 mRNA (Figure 4a).Moreover, the relative fluorescence expression was markedly reduced in LX2 cells co-transfected with miR-26a mimic and SLC7A11-WT compared with LX2 cells transfected with NC mimic and SLC7A11-WT, while no difference was found in the relative fluorescence level in LX2 cells co-transfected with miR-26a mimic and SLC7A11-MUT, and cells co-transfected with NC mimic and SLC7A11-MUT (Figure 4b), suggesting that miR-26a could directly bind to SLC7A11.Transfection of miR-26a mimic into LX2 cells prominently decreased the relative protein level of SLC7A11 (Figure 4c), indicating that miR-26a negatively regulated the expression level of SLC7A11 in LX2 cells.Moreover, the SLC7A11 protein level was notably declined in LX2 cells treated with miR-26a mimic-Exo (Figure 4d).Altogether, these results expounded that miR-26a directly bound to SLC7A11 and negatively modulated the level of SLC7A11 in HSCs.

BMSC-derived exosomal miR-26a evoked ferroptosis of HSC by regulating SLC7A11
To investigate the direct role of SLC7A11 in BMSC-derived exosomal miR-26a-mediated ferroptosis, LX2 cells were administrated with miR-26a mimic-Exo and transfected with pcDNA vector plasmids carrying the sequences of SLC7A11 (designated as SLC7A11).The relative protein expression of SLC7A11 was significantly decreased with the treatment of miR-26a mimic-Exo, which was rescued with the overexpression of SLC7A11 in LX2 cells (Figure 5a).The ROS level was notably increased in LX2 cells treated with miR-26a mimic-Exo, which was markedly attenuated with the further overexpression of SLC7A11 (Figure 5b).Additionally, enhanced level of Fe 2+ and MDA in LX2 cells treated with miR-26a mimic-Exo was prominently antagonized with the further overexpression of SLC7A11, while decreased level of GSH in LX2 cells treated with miR-26a mimic-Exo was observably rescued with the further overexpression of SLC7A11 (Figure 5c).Therefore, these results indicated that BMSC-derived exosomal miR-26a elicited ferroptosis of HSC by regulating SLC7A11.
3.6 BMSC-derived exosomal miR-26a alleviated CCL4-induced liver fibrosis in vivo Moreover, the effect of BMSC-derived exosomal miR-26a on liver fibrosis was evaluated in CCL4-induced mice, an in vivo model of liver fibrosis.Masson staining results showed that CCL4 evoked an obvious fibrosis in mice, which was profoundly improved by injecting of miR-26a mimic-Exo (Figure 6a).The serum contents of ALT and AST were significantly augmented in CCL4-induced mice, which were prominently declined with the injection of miR-26a mimic- Exo (Figure 6b).Low level of miR-26a in CCL4-induced mice was markedly restored with the injection of miR-26a mimic-Exo (Figure 6c).In addition, CCL4 triggered a prominent enhancement in the SLC7A11 protein level, which was observably neutralized by injecting of miR-26a mimic-Exo (Figure 6d).Together, these results indicated that BMSC-derived exosomal miR-26a reduced the level of SLC7A11 and relieved CCL4-induced liver fibrosis in mice.

Discussion
MSCs have attracted more and more attention for the treatment of liver diseases, and accumulating evidences have revealed that MSCs can alleviate liver fibrosis and improve liver function [38][39][40][41].Moreover, MSC transplantation has been demonstrated to be effective for the treatment of human liver fibrosis in clinical trials [42].Nevertheless, the fact of cellular rejection and the risk for iatrogenic tumor formation remain unresolved in MSC transplantation [43].Thus, MSC-derived exosomes that have no risk for tumor formation and less immunogenicity [15] may be a better choice.A large number of studies have revealed the ameliorative effect of MSCs-derived exosomes on liver fibrosis [17,44,45].In the present study, exosomes were successfully isolated from human BMSCs and used in the subsequent experiments.
Exosomes have been found to carry many contents, such as miRNAs, to mediate intercellular communication [46].miR- MSC-derived exosomal miR-26a improves liver fibrosis  9 148a from MSC-derived exosomes prevents liver fibrosis by targeting KLF6/STAT3 pathway [18].MSC-derived exosomal miR-618 dampens the development of liver fibrosis by targeting Smad4 [33].Here, the miR-26a expression was reduced in BMSCderived exosomes.Treatment of miR-26a mimic-Exo decreased the EDU-positive rate and the protein level of α-SMA and collagen I but enhanced the apoptosis rate of LX2 cells.The enhancement of ECM proteins is a major feature of liver fibrosis [1].HSCs, the main source of ECM proteins, are quiescent under normal physiological state, which can be activated and transdifferentiated upon pathological conditions resulting from liver injury [47].HSCs, activated by cytokines and fibrogenic regulators, cause the generation of the section of collagens and α-SMA, which contribute to the ECM deposition [47][48][49].Activated HSCs serve as the myofibroblasts, which impel the fibrotic responses and promote the progression of liver fibrosis [47,50].Thus, upregulation of BMSC-derived exosomal miR-26a suppressed the HSC activation.Moreover, injection of miR-26a mimic-Exo reduced liver fibrosis, and improved the liver function in CCL4induced mice.Taken together, MSC-derived exosomal miR-26a relieved liver fibrosis.
Moreover, MSC-derived exosomal miR-26a induced ferroptosis in LX2 cells, as evidenced by the increased level of ROS, Fe 2+ , and MDA and the decreased GHS level.Furthermore, the application of DFO further verified that MSC-derived exosomal miR-26a promoted ferroptosis to relieve liver fibrosis in LX2 cells.More importantly, miR-26a directly bound to SLC7A11 mRNA and negatively regulated the level of SLC7A11 in LX2 cells.SLC7A11 is a critical protein related to the progression of ferroptosis, and it is an ingredient of the cystine/glutamate antiporter to be responsible for providing the cysteine necessary for the synthesis of GSH [7].SLC7A11 has been demonstrated to have potentially therapeutic effects in preventing or treating liver fibrosis.For instance, TRIM26 induces HSC ferroptosis to inhibit liver fibrosis by the ubiquitinated degradation of SLC7A11 [51].Sorafenib evokes HSC ferroptosis to attenuate liver fibrosis by regulating the HIF-1α/SLC7A11 axis [52].Wogonoside dampens liver fibrosis by evoking HSC ferroptosis through the SOCS1/P53/SLC7A11 axis [53].In the present study, overexpression of SLC7A11 reversed the miR-26a mimic-Exo-induced alterations in the level of ROS, Fe 2+ , MDA, and GSH in LX2 cells, indicating that BMSC-derived exosomal miR-26a elicited ferroptosis of HSCs by regulating SLC7A11.
In summary, our results expounded that BMSC-derived exosomal miR-26a induced ferroptosis to alleviate liver fibrosis by regulating SLC7A11.However, there are still some limitations in this study that needed to be addressed in the future.The direct role of miR-26a/SLC7A11 axis in liver fibrosis can be investigated both in vitro and in vivo.Whether the treatment of miR-26a will not affect the expression of SLC7A11 mRNA but modulate the post-transcriptionally protein level should be confirmed by RT-qPCR experiment in the following experiment.A SLC7A11 knockdown together with miR-26a treatment can be conducted to further confirm that miR-26a control liver fibrosis through the SLC7A11 pathway.In addition, more preclinical and clinical trials are essential in the subsequent study for the clinical application of exosomes.Collectively, our results provide the pre-clinical evidence for the discovery of the possible target for the treatment of liver fibrosis.

Figure 1 :
Figure 1: miR-26a was lowly expressed in BMSC-derived exosomes.(a) The exosomes were isolated from human BMSCs and then observed by TEM.Scale bar = 100 nm.The exosomes were labeled with white arrows.(b) The relative protein expression of CD9, CD63, and calnexin was examined by western blot.Data were presented after being normalized with β-actin.(c) The relative expression of miR-26a in BMSC-Exo or BMSC-CM was detected by RT-qPCR.Data were presented after being normalized with U6. ** p < 0.05 vs BMSC-CM.(d) The relative expression of miR-26a in LX2 cells treated with BMSC-Exo or PBS was detected by RT-qPCR.Data were presented after being normalized with U6. ** p < 0.05 vs PBS.

Figure 2 :
Figure 2: Overexpression of BMSC-derived exosomal miR-26a inhibited the HSC activation.(a) miR-26a mimics were transfected into human BMSCs to upregulate the level of miR-26a, and then, the exosomes were isolated from human BMSCs.The relative expression of miR-26a in miR-26a mimic-Exo or NC mimic-Exo was examined by RT-qPCR.Data were presented after being normalized with U6.(b) The proliferation of LX2 cells treated with miR-26a mimic-Exo or NC mimic-Exo was assessed by EDU assays.Scale bar = 50 nm.(c) The apoptosis rate of LX2 cells treated with miR-26a mimic-Exo or NC mimic-Exo was measured by flow cytometry.(d) The relative protein expression of α-SMA and collagen I of LX2 cells treated with miR-26a mimic-Exo or NC mimic-Exo was detected by western blot.Data were presented after being normalized with β-actin.** p < 0.05 vs NC mimic-Exo.

Figure 3 :
Figure 3: Overexpression of BMSC-derived exosomal miR-26a accelerated ferroptosis in HSCs.(a) The ROS level of LX2 cells treated with miR-26a mimic-Exo or NC mimic-Exo was detected by flow cytometry.(b) Measurement of Fe 2+ , MDA, and GSH of LX2 cells treated with miR-26a mimic-Exo or NC mimic-Exo.(c) The proliferation was assessed by EDU assays after LX2 cells were treated with miR-26a mimic-Exo, NC mimic-Exo, and DFO.Scale bar = 50 nm.(d) The ROS level was detected by flow cytometry after LX2 cells were treated with miR-26a mimic-Exo, NC mimic-Exo, and DFO.(e) The relative protein expression of α-SMA and collagen I was determined by western blot after LX2 cells were treated with miR-26a mimic-Exo, NC mimic-Exo, and DFO.Data were presented after being normalized with β-actin.** p < 0.05 vs NC mimic-Exo; ## p < 0.05 vs miR-26a mimic-Exo; aa p < 0.05 vs NC mimic-Exo + DFO.

Figure 4 :
Figure 4: miR-26a directly targeted SLC7A11.(a) The binding sites between miR-26a and SLC7A11 mRNA were predicted, and the target rank and target score between miR-26a and SLC7A11 mRNA were shown.(b) The direct interaction between miR-26a and SLC7A11 was determined by a dual-luciferase reporter assay.(c) The relative protein expression of SLC7A11 was examined by western blot after LX2 cells were transfected with miR-26a mimic or NC mimic.Data were presented after being normalized with β-actin.(d) The relative protein expression of SLC7A11 was detected by western blot after LX2 cells were treated with miR-26a mimic-Exo.Data were presented after being normalized with β-actin.** p < 0.05 vs NC mimic-Exo.

Figure 5 :
Figure 5: BMSC-derived exosomal miR-26a induced ferroptosis of HSC by regulating SLC7A11.LX2 cells were treated with miR-26a mimic-Exo as well as transfected with pcDNA vector plasmids carrying the sequences of SLC7A11.(a) The relative protein expression level of SLC7A11 was detected by western blot.Data were presented after being normalized with β-actin.(b) The ROS level was detected by flow cytometry.(c) Measurement of Fe 2+ , MDA, and GSH.** p < 0.05 vs NC mimic-Exo; aa p < 0.05 vs miR-26a mimic-Exo + Vector.

Figure 6 :
Figure 6: BMSC-derived exosomal miR-26a alleviated CCL4-induced liver fibrosis in mice.Mice were modeled by intraperitoneally injecting with 0.5 mg/kg CCl4 for 8 weeks twice a week.Twenty-four hours after the model, mice were injected with 100 μg/mL exosomes isolated from MSCs through the tail vein.(a) The liver fibrosis was assessed by Masson staining.(b) Examination of ALT and AST in sera.(c) The relative expression of miR-26a was detected by RT-qPCR.Data were presented after being normalized with U6.(d) The relative protein expression of SLC7A11 was detected by western blot.Data were presented after being normalized with β-actin.** p < 0.05 vs Control; aa p < 0.05 vs CCL4 + NC mimic-Exo.