Exosomes Derived from liver failure patients' plasma stimulated Mesenchymal Stem Cells alleviate acute liver failure

doi:10.21203/rs.3.rs-4995574/v1

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Exosomes Derived from liver failure patients' plasma stimulated Mesenchymal Stem Cells alleviate acute liver failure

https://doi.org/10.21203/rs.3.rs-4995574/v1

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Background: Exosomes derived from pre-stimulated mesenchymal stem cells have been found have a different therapeutic effect in disease, and bio-artificial liver therapy has been shown to play an effective role in liver failure; Here, we proposed to investigate the therapeutic potential of liver failure patients’ plasma stimulated MSCs-derived exosomes (LF-Exos).

Methods: We extracted untreated exosomes(NC-Exos) and LF-Exos, identified them using NTA, TEM and Western blotting, followed by miRNA sequencing. CCK-8, flow cytometry, H&E staining, immunohistochemistry, and Western blot were used to detect the protective effects of LF-Exos on D-GalN/LPS induced acute injured hepatocytes and the ALF mouse model.

Results: Our study shows that after stimulated with liver failure patients’ plasma, the morphology of MSCs was significantly changed, and proliferation activity was weakened. A total of 31 differentially expressed miRNAs were screened using a gene chip, and further analysis showed that the differentially expressed miRNAs might affect the PI3K-AKT signaling pathway. In addition, LF-Exos could induce AKT phosphorylation and reduce activation of the NLRP3 inflammasome in hepatocytes or liver tissue, inhibit D-GalN/LPS induced apoptosis in hepatocytes, and reduce pathological liver injury of ALF mouse.

Conclusion: The biological effects of LF-Exos will be altered after stimulated with liver failure patients’ plasma (LF plasma), and LF-Exos may inhibit the activity of NLRP3 inflammasome and activating the PI3K-AKT signaling pathway to exert a better protective effect on acute injured hepatocytes and ALF mouse models than NC-Exos.

Exosomes

Mesenchymal stem cells

Liver failure

Liver function can be impaired by a variety of etiologies; when it develops to liver failure, there is high short-term mortality^{[1, 2]}. Currently, the most effective clinical treatment for liver failure is liver transplantation, but there are problems such as shortage of donors, high cost, and immune rejection^[3]. Therefore, there is urgent to find more treatment modalities.

Mesenchymal stem cells (MSCs) are a class of low immunogenic cells possess self-renewal and multi-lineage differentiation potentials^[4], previous studies have proved that MSCs transplantation is an effective treatment for liver failure, they have been found to play a role in the treatment of various diseases by regulating immune function, promoting cell regeneration or tissue repair^[5]. However, MSCs transplantation is still considered to have the risk of capillaries blockage, teratoma formation, and demanding transport and storage conditions^[6–8]. With the deepening of research, MSC-derived exosomes (MSC-Exos) have been found to play a similar therapeutic function as parent cells^[9], and have the advantages of have a smaller size and less immunogentic to reach the affected tissue, exhibit no risk of tumor formation, and easier to produce and store^[10–13], thereby cell-free therapy based on MSC-Exos has been widely researched in recent years.

Previous research has shown that exosomes are heterogeneous, exosomes derived from different parent cells or the parent cells cultured in different condition, will have differential expression of nucleic acids, lipids, proteins and other contents^[14]. The heterogeneity of exosomes will affect their functional impact on recipient cells and plays various roles in immunomodulation, cell proliferation and apoptosis^{[15, 16]}. It has been found that applying the microenvironment of the disease to train MSCs can enhance the therapeutic role of MSC-Exos^{[17, 18]}; therefore, based on the heterogeneity of exosomes, training MSCs and deriving “trained MSC-Exos” for targeted therapeutic effects is an issue that could be further addressed in the current study.

In this study, we stimulated MSCs with liver failure patients’ plasma (LF plasma) and extracted plasma-stimulated MSC-derived exosomes (LF-Exos), observed the differences between LF-Exos and untreated exosomes (NC-Exos), and assessed the therapeutic effects of LF-Exos in acute injured hepatocytes and ALF mice.

2.1 Cell culture

The umbilical cord mesenchymal stem cells (hUCMSCs) were generously provided by Tianjin Ansai Cell Bioengineering Co., Ltd, The MSCs used in the experiments were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. After 12h, replace the culture medium containing LF plasma and the cell culture medium without added plasma was used as a control. When the cells reached 70% confluence, the culture was continued with serum-free medium for 48h to collect the supernatant.

The mouse AML12 hepatocyte cell line was obtained from Wuhan Pricella Biotechnology Co. Ltd. AML12 cells were cultured in DMEM/F12 containing 10% FBS, 1% penicillin–streptomycin, insulin, transferrin, selenium (ITS) Liquid Media Supplement, and 40 ng/ml dexamethasone.

Murine macrophage RAW264.7 cells were provided from our central laboratory and cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin.

2.2 Proliferation analysis

MSCs were cultured overnight in 96-well plates (3, 000 cells/pore) and then treated with different concentrations of LF plasma. CCK-8 solution was added to the pores according to the manufacturer’s protocol to measure cell proliferation, and a multifunctional microplate reader (Thermo) was used to read the optical density at 450 nm (OD450).

2.3 Exosomes isolation and identification

The cell culture supernatant was centrifuged at 4°C, 300 × g for 10 min to remove cells, 3,000 × g for 15 min to remove dead cells, 10,000 × g for 30 min to remove subcellular components, and the supernatant was transferred to an ultracentrifuge tube and ultracentrifuged at 120,000 × g for 90 min to obtain the sediment that contained exosomes, then resuspended exosomes in pre-cooled PBS. The protein concentration of exosomes was determined by BCA, and exosomes were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot.

2.4 Exosome miRNA sequencing and sequence analysis

Total RNA was extracted from exosomes using Trizol reagent (Life Technologies, Carlsbad, CA, USA) and purified using an RNeasy mini kit (Qiagen, Valencia, CA, USA). Using GeneChip to detect the relative expression levels of exosomal miRNAs, biotinylated cDNA was prepared according to the standard Affymetrix protocol, using the Ambion® WT Expression Kit. Following labeling, the fragmented cDNA was hybridized, washed, and stained in the Affymetrix Fluidics Station 450. All arrays were scanned by using Affymetrix® GeneChip Command Console (AGCC)

2.5 Treatment of cells with exosomes

AML12 cells were cultured in cell culture plates (2.5×10⁵cells/ml) for 24h, then 4 mg/ml D-GalN, 40 µg/ml LPS, and 20 µg/ml exosomes were added to the pores co-cultured with AML12 for 24h. The CCK-8 Assay Kit and 7AAD Apoptosis Assay Kit were used to measure the effect of exosomes on AML12 cells according to the manufacturer’s instructions or to collect total cell proteins.

RAW264.7 macrophages were cultured in 6-well plates (5×10⁵ cells/ml) overnight and then co-cultured with exosomes (20 µg/ml) for 6h. Subsequently, 200 ng/ml LPS was added to the pores and incubated for 24 h to collect the total cell proteins.

2.6 ALF mice model treat with exosomes

To establish the ALF model, 8-week-old male C57BL/6 mice were injected with D-GalN (250 mg/kg) and LPS (2.5 mg/kg), and then NC-Exos, LF-Exos (100 µg), or an equal volume of PBS was injected into the tail vein immediately. 6h after the intervention, anesthetized the mice with 5% isoflurane by inhalation, blood from the eyeballs was drawn as serum samples and entire livers were collected from sacrificed mice for further analysis. The work has been reported in line with the ARRIVE guidelines 2.0, with additional supporting documents provided in the supplementary materials.

The blood samples were centrifuged at 3,000 rpm for 15 min to collect the serum, and the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to the manufacturer’s protocol of the ALT test kit and AST test kit.

A portion of liver tissue was fixed in 4% paraformaldehyde, then dehydrated with gradient concentration of ethanol and embedded in paraffin wax, sections cut in succession were subjected to HE staining or TUNEL staining to observe the histopathological changes. The remainder of the tissue samples was preserved at -80℃ for further Western blot analysis.

2.7 Western blot analysis

Proteins from cell or tissue samples were extracted using RIPA lysate buffer (containing 1% protein phosphatase inhibitor), quantified by BCA, diluted with the appropriate amount of sample buffer, and boiled at 105°C. The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes. After sealing with skim milk, the membrane was incubated overnight with primary antibody at 4°C on a shaker. Blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 1h at room temperature. The primary antibody anti-AKT (1:1000) was purchased from Abcam. Anti-phospho-AKT (1:2000), anti-Phospho-PI3K (1:1000), and anti-ASC (1:1000) were purchased from CST. Anti-PI3K (1:1000) was purchased from Zenbio. Anti-NLRP3 (1:1000) and anti-mouse caspase-1 (1:1000) were bought from Adipogen. Anti-mouse-cleaved IL-1β (1:2000) was from R&D Systems. Anti-GAPDH (1:2000) and anti-Lamin B (1:1000)was used as an internal control.

2.8 Statistical analysis

SPSS 19 software was used for statistical analysis, GraphPad prism 8.3 was applied for image drawing. Data were presented as the mean ± standard deviation (X̄± SD). Student’s t-test was used for comparisons between two samples, and one-way ANOVA was used for comparisons between multiple samples; P < 0.05 considered statistical significant.

3.1 LF plasma changed the morphology of MSCs and affected the proliferation

We observed the morphology of MSCs under a microscope; the morphology of normal cultured MSCs showed a spindle-like shape, grew in a spiral arrangement, packed tightly with uniform morphology and size, and with a clear ovoid nucleus in the center of the cells. While the shape of MSCs cultured with LF plasma was widened and the gaps between cells were enlarged, the arrangement of cells became disordered, and many coarse particles appeared around the cell nuclei (Fig. 1A).CCK-8 analyzed that the proliferation curves of normal cultured MSCs showed typical "S" curves, while LF plasma will inhibit the growth of MSCs in a higher plasma concentration (Fig. 1B).

3.2 Identification of MSC-Exos

Two groups of MSC-Exos were isolated from the cell culture supernatant as previously described. Transmission electron microscopy (TEM) showed that the particles extracted from the two groups of supernatants, both in the form of teacups or elliptical discs, had a double membrane structure, which is typical of exosomes (Fig. 2A). Nanoparticle tracking analysis (NTA) analyzed the size of NC-Exos and LF-Exos between 30–200 nm, which was in line with the size of exosomes (Fig. 2B), and Western Blot confirmed that the NC-Exos and LF-Exos both expressed specific exosomal surface markers CD63, TSG101, and Alix, and did not express calnexin, which is not expressed on exosomes (Fig. 2C). In addition, the protein content of LF-Exos was significantly higher than that of NC-Exos (Fig. 2D). These results confirmed that the extracted particles were exosomes.

3.3 LF Plasma induces changes in miRNAs in MSC-Exos

We sequenced the miRNAs extracted from NC-Exos and LF-Exos, screened a set of miRNAs that were differentially expressed between NC-Exos and LF-Exos. There were 31 differentially expressed miRNAs in LF-Exos compared with NC-Exos; of these, 11 were upregulated and 20 were downregulated (|Fold change|>1.2, P < 0.05) (Fig. 3A-B).

The miRanda and RNAhybrid algorithms were used to predict target gene interaction binding sites for miRNAs, to match the target relationship between the predicted miRNAs and mRNAs, took the intersection of the matched mRNAs from the two databases, and 5933 predicted mRNAs were obtained. The 5933 predicted target mRNAs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The results showed that these target genes were enriched in several signaling pathways, GO enrichment analysis showed that these genes could be significantly enriched in the annotations of the regulation of signaling, cell junction, and protein binding. KEGG signaling pathway enrichment analysis showed that these genes could be significantly enriched in the annotation of PI3K-AKT signaling pathway, Wnt signaling pathway, and other signaling pathways (Fig. 3C-D).

3.4 MSC-Exos inhibits apoptosis in D-GalN / LPS-induced hepatocytes

The CCK-8 assay was used to measure the viability of AML12 cells, and the results showed that MSC-Exos promoted the viability of AML12 cells under the inhibitory effect caused by D-GalN/LPS, and the viability of AML12 cells was significantly increased in the LF-Exos group (Fig. 4A).

The apoptosis assay revealed that D-GalN/LPS significantly increased the ratio of cells undergoing apoptosis, and both NC-Exos and LF-Exos reversed D-GalN/LPS-induced apoptosis in AML12 cells. The apoptosis rate of AML12 cells in the LF-Exos group was significantly lower than that in the NC-Exos group (Fig. 4B-C).

3.5 MSC-Exos alleviate liver injury in D-GalN/LPS-induced ALF mice model

After observing the survival time of ALF mice, we found that mice started to die at 3 h after D-GalN/LPS injection for modelling, and the mortality rate reached 100% within 8 h. A few mice survived in both groups injected with NC-Exos or LF-Exos via the tail vein, but there was no significant difference in the survival analysis (Fig. 5A).

The serum ALT and AST levels of mice in the model group were significantly elevated, indicating that D-GalN/LPS caused significant liver injury. After injection with NC-Exos or LF-Exos, the serum ALT and AST levels of mice were significantly decreased, but only the serum ALT level of the LF-Exos group was more significantly reduced compared with that of the NC-Exos group (Fig. 5B-C). This may be due to the fact that ALT is mainly distributed in the cytoplasm of hepatocytes and ALT is more sensitive to early liver injury, while AST is mainly distributed in the mitochondria of hepatocytes and AST in the mitochondria is only released when hepatocytes are severely damaged and necrotic^[19], therefore, we only observed a significant decrease in ALT in the ALF mice model.

The livers of mice in the model group were generally congested and enlarged, and the hepatic organ coefficient (LW/BW) was significantly increased (Fig. 5D). Observed the pathology of mouse liver, HE staining showed that the structure of liver in normal mice was clear and obvious, the hepatocytes were tightly arranged around the central vein. The liver of the model group was significantly damaged, with blurring of hepatic lobular structure, large areas of hepatocytes were denatured and necrotic, the structure of the liver lobules blurred, and inflammatory cell was infiltrated. In the NC-Exos or LF-Exos groups, the hepatic lobular structure was restored, the necrotic area and inflammatory cell infiltration were reduced, while in the LF-Exos group, the amelioration was improved compared to that in the NC-Exos group (Fig. 5E).

Apoptosis of mice hepatocytes was detected by TUNEL staining, there is a number of TUNEL-positive hepatocytes observed in liver tissue from the Model group, and the number of TUNEL-positive cells in the NC-Exos group and LF-Exos group was reduced (Fig. 5F).

3.6 Effect of MSC-Exos on PI3K-AKT signaling pathway and NLRP3 inflammasome in D-GalN/LPS-induced injured hepatocytes and ALF mouse

Based on miRNA sequencing analysis, we speculate that LF-Exos may ameliorate the progression of ALF through the PI3K-AKT signaling pathway. In addition, it has been reported that the PI3K-AKT signaling pathway might be associated with NLRP3 inflammasome^{[20, 21]} and that the activation of the NLRP3 inflammasome is also a factor in the exacerbation of liver failure^{[22, 23]}.

We analyzed the expression levels of major proteins in the PI3K-AKT signaling pathway and NLRP3 inflammasome and found that LF-Exos significantly increased the expression of PI3K and p-AKT in AML12 cells and ALF mice. Also significantly reduced the expression of NLRP3 in AML12 cells, RAW264.7 cells, or ALF mice.

Mesenchymal stem cell-derived exosomes (MSC-Exos) have been considered to have therapeutic effects similar those to of MSCs, and have been shown to have effective therapeutic prospects for ALF^[24–26]. With the deepening of the study, researchers found that "training" MSCs may improve the therapeutic effects of MSCs and MSC-Exos^[27–29], effective stimulation of MSCs might make MSC-Exos more targeted and efficient in treating diseases, and search for this effective stimulation has become a hot spots in current exosome research. In this study, we selected LF plasma to stimulate MSCs, observed the differences between NC-Exos and LF-Exos, and preliminarily verified the therapeutic effect of LF-Exos on ALF.

The heterogeneity of exosomes leads to different contents of MSC-Exos that have been stimulated in different ways^[30]. miRNA is one of the most abundant components of exosomes and is the main biologically active substance^[31], miRNA can inhibit the gene expression and protein synthesis by competitively binding mRNA^[32], which plays an important role in the physiopathological process of the organism. Shao et al.^[31] stimulated hUCMSCs with IL-6, obtained exosomes (Exos-IL6), and conducted sequencing of small RNAs extracted from Exos-IL6 and Exos-NC, identified 37 differentially expressed miRNAs, and found that these miRNAs are widely involved in the regulation of IL-6-related signaling pathways, including the PI3K-AKT signaling pathway. Ma et al.^[33] pretreated MSCs with TNF-α and obtained exosomes (T-Exos), screened 180 differentially expressed miRNAs between T-Exos and NC-Exos, and revealed that some of the differentially expressed miRNAs were closely related to immunoinflammation-related signaling pathways. Our study found that MSCs stimulated with LF plasma will affect their morphology and inhibited their proliferation. Meanwhile, sequencing results showed that 31 differentially expressed miRNAs existed between NC-Exos and LF-Exos, and KEGG analysis predicted that the differentially expressed miRNAs might be related to PI3K-AKT signaling pathway.

The mechanisms by which MSC-Exos ameliorates acute liver failure are complex and varied, but most of them are closely related to the regulation of immune homeostasis, inhibition of apoptosis, and promotion of regenerative functions of the remaining cells. Focus on our sequencing results we found that PI3K-AKT signaling pathway is a classical pathway that is closely related to a wide range of physiological and pathological processes, such as cell growth, proliferation, migration, metabolism, and immunomodulation^{[34, 35]}. There have many studies found that the PI3K-AKT signaling pathway can play an important role in the treatment of ALF^[36], and it has been reported that there is a relationship between the PI3K-AKT signaling pathway and NLRP3 inflammasome. Zhao et al.^[20] revealed that AKT directly acts on NLRP3 and will inhibits NLRP3 oligimerization during LPS priming and reduces cytokine secretion after NLRP3 activation. Wang et al.^[21] found that NLRP3 inflammasome activation attributable to the inhibition of the ROS-PI3K/AKT pathway. Tabaa et al.^[37] found that, suppress the activation of NLRP3 will regulate PI3K/AKT/mTOR cascade. Our results suggested that LF-Exos may activate the PI3K-AKT signaling pathway and inhibit the activation of NLRP3 inflammasome, and plays a role in promoting cell proliferation, inhibiting apoptosis and immunomodulation.

MSC-Exos as an emerging therapeutic technology has been widely noticed, and lots of the studies have confirmed that MSC-Exos has a good therapeutic effect on liver failure, but how to obtain more therapeutically useful exosomes and apply them in the clinic is still a big distance. Our team has carried out research on bio-artificial liver therapy, which consists of a combination of extracorporeal circulation and a bioreactor, in bio-artificial liver system, hepatocytes are usually inserted into the bioreactor, and the patient's plasma is exchanged with the cells in the bioreactor and then returned to the blood, thus playing a role in promoting the repair of liver damage. Our study results concluded that the modification of MSCs with LF plasma could alter the expression of miRNAs in LF-Exos, and might play a role in inhibiting apoptosis and regulating immune responses in injured cells and ALF mice by activating the PI3K-AKT signaling pathway and inhibiting the activation of NLRP3 inflammasome, suggesting that LF-Exos may play a better therapeutic role in acute liver failure. The therapeutic effect of LF-Exos will be fulfilled if MSCs are placed into the bioreactor to constitute a MSCs-based bio-artificial liver, and this study will provide a basis for the study of bioartificial liver based on MSCs.

In summary, our study revealed that stimulate of MSCs by LF plasma could regulate the biological function of MSC-Exos, and LF-Exos might play a protective role against acute liver injury or liver failure by activating PI3K-AKT signaling pathway and inhibiting the activation of NLRP3 inflammasome. This lays the foundation for obtaining more therapeutically useful MSC-Exos and for the development of novel bio-artificial liver systems.

Contributions

Zhuoran Wang and Shaoli You contributed to the conception and design of the study; Zhuoran Wang and Jun Ling contributed to the data collection and experimental procedures; and Zhuoran Wang and Bing Zhu contributed to drafting the article, critical revision of the article, and final approval of the version to be published.

Ethics declarations

Ethical approval

The study protocol was approved by the Ethics Committee of The Fifth Medical Center of Chinese PLA General Hospital (Title:Study of a new protocol for the diagnosis and treatment of severe hepatitis B (liver failure) with immunomodulatory therapy; Grant number: 2019086D; Date of approval: 09/25/2020). Written informed consent was obtained from the patients or their first relative if they could not provide informed consent themselves.

Consent for publication

All authors have read and approved the final version of the manuscript.

Competing interests

The authors declare no conflicts of financial interest.

Funding

This work was supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China [grant number 2017ZX10203201].

Acknowledgements

The authors thank the excellent technical assistance provided by Zhaofang Bai. The authors declare that they have not use AI-generated work in this manuscript.

Availability of data and materials

All experimental protocols and data obtained in this study were available upon request to the author. All the sequencing data supporting the conclusions of this study are included within the article and supplementary data that name “sequencing date”.

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Exosomes Derived from liver failure patients' plasma stimulated Mesenchymal Stem Cells alleviate acute liver failure

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Abstract

Figures

1. Introduction

2. Methods and Materials

2.1 Cell culture

2.2 Proliferation analysis

2.3 Exosomes isolation and identification

2.4 Exosome miRNA sequencing and sequence analysis

2.5 Treatment of cells with exosomes

2.6 ALF mice model treat with exosomes

2.7 Western blot analysis

2.8 Statistical analysis

3. Results

3.1 LF plasma changed the morphology of MSCs and affected the proliferation

3.2 Identification of MSC-Exos

3.3 LF Plasma induces changes in miRNAs in MSC-Exos

3.4 MSC-Exos inhibits apoptosis in D-GalN / LPS-induced hepatocytes

3.5 MSC-Exos alleviate liver injury in D-GalN/LPS-induced ALF mice model

3.6 Effect of MSC-Exos on PI3K-AKT signaling pathway and NLRP3 inflammasome in D-GalN/LPS-induced injured hepatocytes and ALF mouse

4. Discussion

5. Conclusion

Declarations

Contributions

Ethics declarations

Ethical approval

Consent for publication

Competing interests

Funding

Acknowledgements

Availability of data and materials

References

Supplementary Files

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