Overexpression of microRNA-126 in Adipose-Derived Mesenchymal Stem Cells Alleviate the Alcoholic Liver Injury

doi:10.21203/rs.3.rs-4713143/v1

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Overexpression of microRNA-126 in Adipose-Derived Mesenchymal Stem Cells Alleviate the Alcoholic Liver Injury

https://doi.org/10.21203/rs.3.rs-4713143/v1

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Alcoholic liver disease (ALD) is a major global health issue. This study explores the therapeutic efficacy of microRNA-126 (miR-126)-engineered adipose-derived mesenchymal stem cells (ADMSCs) in ALD, particularly focusing on their impact on the intestinal barrier. Male mice was used to establish an ALD model. Subsequently, both unmodified and miR-126-engineered ADMSCs were transplanted into these ALD models. A variety of techniques were then employed to assess liver injury, the integrity of the intestinal epithelial barrier (IEB), and the gut vascular barrier (GVB) across different intervention groups. ADMSCs effectively mitigated liver injury in the ALD model, as evidenced by improving liver function (serum ALT and AST levels) and injury, and reversing the serum LPS translocation. Furthermore, miR-126-overexpressing engineered ADMSCs demonstrated the most potent effects compared to other groups. ALD-induced damage to the intestinal epithelium and vascular barriers was not only ameliorated by ADMSCs but also further enhanced by ADMSCs^miR−126(+) treatment. Additionally, the expression levels of PV-1 (a positive marker for GVB injury) and ZO-1 (a negative marker for IEB injury) were most markedly reduced and elevated following treatment with ADMSCs^miR−126(+), respectively. Further analyses revealed that ADMSCs treatment actives PI3K/Akt/eNOS pathway and subsequently repressing the expression of caspase-3, thereby repairing IEB and GVB, in which miR-126 can improve the above effect. ADMSCs can alleviate ALD by regulating the IEB and GVB, and miR-126-engineered ADMSCs offer enhanced therapeutic benefits. These findings unveil a novel therapeutic mechanism for ALD that involves protection against damage to the IEB and GVB.

Acholic liver diseases

Adipose-derived mesenchymal stem cells

microRNA-126

Intestinal epithelial barrier

Gut vascular barrier

Alcoholic Liver Disease (ALD) is characterized by liver damage resulting from prolonged, excessive alcohol consumption [1–3]. The disease initiates as fatty degeneration or fatty liver and progresses through stages of alcoholic liver injury, fatty hepatitis, chronic hepatitis with liver fibrosis, eventually culminating in cirrhosis and potentially hepatocellular carcinoma. Despite extensive research, the underlying mechanisms of ALD remain largely elusive [3, 4].

Conversely, disruption of the intestinal barrier due to long-term alcohol consumption is increasingly recognized as a contributing factor to ALD [5–13]. Serving as the body's frontline defense between the external environment and the intestinal lumen, the intestinal barrier comprises both the gut-vascular barrier (GVB) and the intestinal epithelial barrier (IEB), which work in tandem to maintain the integrity of the intestinal barrier. Disruption of this barrier leads to increased intestinal permeability, a well-known pathogenic factor in both intestinal and extra-intestinal diseases [14]. In particular, this disruption allows for the translocation of bacteria and associated products like LPS into the liver, thereby exacerbating liver inflammation and damage. Despite its importance, there is a notable lack of research exploring the specific roles of the IEB and GVB in ALD. These barriers are layers of closely packed cells; the IEB covers external or internal surfaces, while the GVB comprises a structured endothelium situated below the epithelial layer. Recent studies on the GVB and IEB have provided new insights into the etiology and management of gut-liver axis diseases, implicating them in various inflammatory processes of gastrointestinal diseases [15–19].

The primary curative intervention for alcohol-induced cirrhosis is orthotopic liver transplantation. However, the scarcity of organ donors remains an unresolved issue [20–22]. As a result, stem cell-based therapies have emerged as a promising alternative. Mesenchymal stem cells (MSCs), a subtype of adult stem cells mainly isolated from bone marrow, have garnered attention for their self-renewal and differentiation capabilities, making them viable candidates for cell-based therapies [23]. Moreover, studies investigating MSCs from alternative sources like umbilical cord tissue, blood, liver, dental pulp, and adipose tissue have shown that their functional characteristics can vary based on factors such as collection method, cell count, maturity, and cellular composition. MSCs from different origins have been shown to facilitate tissue repair via cytokine secretion.

Adipose-derived mesenchymal stem cells (ADMSCs) are isolated from adipose tissue and are approximately 500 times more abundant than their bone marrow counterparts. They have proven effective in a range of applications, including hepatobiliary diseases [24]. MicroRNA (miRNA) is a class of small RNA molecules that regulate various biological processes, including metabolism and differentiation [25, 26]. Recent studies indicate that miR-126 plays a promoting role in angiogenesis and intestinal barrier formation [27, 28]. Furthermore, miR-126 has been shown to enhance MSC survival and promote the secretion of angiogenic factors, thereby improving the therapeutic efficacy of MSCs in treating vascular defects [29, 30].

In light of these findings, the aim of this study is to investigate whether ADMSCs can mitigate liver damage in ALD through the regulation of the GVB and IEB and whether miR-126-engineered ADMSCs can further enhance these protective effects.

Animals

Male C57BL/6 mice weighing 18-22g and aged 4-6 weeks were sourced from the Shanghai Laboratory Animal Center (Shanghai, China). They were kept in a temperature and humidity-controlled environment with a 12-h light/dark cycle and fed a standard diet. All animal protocols were approved by the Animal Ethics Committee of Wenzhou Medical University. All work was carried out in compliance with the National or Institutional Guide for the Care and Use of Laboratory Animals.

ADMSCs preparation

ADMSCs of C57BL/6 mice were obtained from Cyagen Biosciences Inc (Cyagen Biosciences, Guangzhou, China). ADMSCs phenotypic properties were examined by flow cytometry analysis and Cyagen Biosciences Inc provided the result: CD29⁺，CD44⁺，Sca-1⁺ (>70%) and CS117^- (<5%). They also evaluated the cells’ multipotency of ADMSCs in vitro for osteogenic and adipogenic differentiations. ADMSCs were cultured in the complete C57BL/6 mouse mesenchymal stem cell medium (Cyagen Biosciences, Guangzhou, China). For all experiments, the sixth generation of ADMSCs were used to keep experimentally consistent.

Vector construction and transduction

Three plasmids were utilized, pLV-THM as a transfer vector, pMD2.G as an envelope plasmid, and psPAX2 as a packaging plasmid. Initially, target sequences were integrated into the pLV-THM vector following the Addgene website protocol. The recombinant lentiviral vector was then introduced into Escherichia coli DH5α using the calcium chloride method, and positive clones were identified on LB plates containing ampicillin. Post-clone identification and growth, the plasmid DNA was extracted using a Takara plasmid isolation kit (Shiga, Japan). Subsequently, these plasmids were introduced into HEK-293T cells. For the production of miR-126, HEK-293T cells were co-transfected with miR-126 shuttle plasmids along with the packaging plasmids psPAX2 and pMD2G using Lipofectamine2000 (Invitrogen, Carlsbad, CA). Following the established protocol, the supernatant was harvested and passed through a 0.45-µm filter for miR-126 purification, yielding LV-miR-126. Similarly, LV-miR-126 sponge and LV-miR-CON were generated as miR-126 inhibitors and controls, respectively.

Then, ADMSCs were transduced according to the instructions after they were at 80% confluence. ADMSCs were maintained in DMEM medium adding LV-miR-CON, LV-miR-126, and LV- miR-126 sponge to generate the ADMSCs^null, ADMSCs^miR-126(+), and ADMSCs^{miR-126 (-)}, respectively. Medium was changed 24 h after transduction. After 72 h, immuno-fluorescence was performed for the identification of transfection efficiency.

Establishment of ALD model and treatment options

C57BL/6 mice were allocated into 6 distinct groups with 6 mice each, namely normal, ALD model, ADMSCs, ADMSCs^null, ADMSCs^{miR-126 (+)}, and ADMSCs^{miR-126 (-)}. For the initial 4 weeks, the control group was fed the Lieber-DeCali diet (sourced from Trophic Animal Feed High-tech Co., Jiangsu, China). Subsequently, the mice were transitioned to a diet combining Lieber-Decarli and ethanol (5% ethanol concentration), following a phased approach over one week: the ratio changed from 2:1 to 1:1, and finally to 1:2 on days 2, 4, and 6, respectively. This was followed by a consistent Lieber-DeCali ethanol diet for another 4 weeks to establish an alcoholic liver injury model. Intraperitoneal injections of PBS, ADMSCs (>1×10⁵ cells), ADMSCs^null (>1×10⁵ cells), ADMSCs^{miR-126 (+)} (>1×10⁵ cells), and ADMSCs^{miR-126 (-)} (>1×10⁵ cells) were administered to ALD mice on days 14, 21, and 28. At the end of the 4th week, both the ALD model and control groups were given either ethanol (5 g/kg body weight, 40%) or an equivalent volume of isothermal dextrin (8.9 g/kg body weight). Ten h post-gavage, all mice were euthanized for blood and liver tissue collection.

Histopathological examination and immuno-histochemical staining

Tissue samples from the liver or ileum were initially preserved in 4% paraformaldehyde for a 24-h period before being encased in paraffin. These samples were then sectioned into slices 4 micrometers thick. For histological examination, the sections underwent H&E staining, involving a 4-min immersion in hematoxylin followed by a 1-min eosin application, before being analyzed using a light microscope. To evaluate lipid accumulation in liver tissues, a Modified Oil Red O Staining Kit (Beyotime, Shanghai, China) was employed. Liver tissue cryosections, each 8 micrometers thick, were treated with a 0.5% Oil Red O solution for 10 mins, and subsequently examined under a light microscope.

As for immuno-histochemical (IHC), ZO-1 antibody (1:1000 dilution, Abcam, UK) was used in the gut staining. The first step was the same as the H&E staining. Then we put the paraffin sections in citric buffer (pH 6.0) and heated in a microwave oven for 20 mins at 100°C. Subsequently, the sections were blocked with 10% FBS for 30 mins followed by primary antibodies at 4°C overnight. Secondary antibody (DakoCytomation) was then applied for 30 mins at room temperature. Finally, the sections were stained by hematoxylin solution. Above sections per mice were blindly examined by three experienced liver pathologist.

Examination of serum ALT, AST and endotoxin levels

We collected blood from mice in all groups and obtained serum by standard centrifugation method. Serum levels of ALT and AST were assessed using the Stanbio ALT kit (Stanbio, USA), per the manufacturer’s instructions. Additionally, serum endotoxin levels were measured with the aid of a Limulus Amebocyte Lysate Chromogenic Kit acquired commercially, featuring a detection range from 0.015 to 1.2 endotoxin units per milliliter (EU/mL, Thermo Fisher Scientific, Waltham, MA, USA).

Reverse transcription-quantitative polymerase chain reaction

Total RNA was extracted from 50 mg liver tissue by Trizol reagent (Invitrogen, Shanghai, China). One microgram of total RNA was used to synthesize the first strand of cDNA by the RevertAid First Strand cDNA kit (Thermo Fisher, Shanghai, China) according to the manufacturer’s instructions. The primers for ZO-1 and PV-1 gene were designed and synthesized by Invitrogen Biotechnology Ltd. (Invitrogen, Shanghai, China). The sequences of ZO-1 and PV-1 primers were as follows: ZO-1 forward: 5’-TGCTATTACACGGTCCTC-3’ and reverse: 5’-TGGTGCTCCTAAACAATC-3’; PV-1 forward: 5’-CAGGCTGGACTCAGTGGTG-3’ and reverse: 5’-TCGCTCATTCAGTTCCTCATC-3’. The gene expression was quantitated by real time PCR with the following protocol: pre-denaturation: 95°C for 30 seconds; PCR reaction: 40 Cycles, 95°C, 5 seconds, 60°C for 34 seconds. The concentration of mRNA was calculated using the 2^-ΔΔCT method.

Detection of protein expression by western blot assay

The proteins expression was assessed by Western blotting with standard protocols. The total protein was extracted from about 40 mg of liver tissues in protein lysis buffer. 40 g protein of each sample was mixed with electrophoresis buffer at a ratio of 1 to 4, and then heated at 100℃ for 5 mins. The denatured protein was separated by running on 10% SDS-PAGE. After electrophoresis, the protein was transferred onto PVDF membrane, and non-specific binding of the membrane was blocked by incubation of 5%BSA in TBS at room temperature for 1 h. The membrane was then incubated with primary antibodies against different proteins overnight at 4°C, respectively: PV-1 (Abcam), PI3K and p-PI3K (Thermo Fisher Scientific), Akt and p-Akt (Cell Signaling Technology), eNOS and p-eNOS (Thermo Fisher Scientific), and capase-3 (Abcam). After washing in TBST, the membrane was incubated in different secondary antibodies. After washing by TBST, the bands were visualized by incubation with chemiluminescent substance, and the bands were quantitated and analyzed with the Quantity One image analysis system.

Immunofluorescence staining

Immunofluorescence staining was conducted on paraffin-embedded tissue sections, which were first fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) and then permeabilized using 0.05% TritonX-100 (Servicebio). Post-blocking, these sections were treated overnight with primary antibodies (PV-1: Abcam, UK; CD31: BD Pharmingen, UK) at 4°C within a moisture-controlled environment. This was followed by a 1-h room temperature incubation with FITC-labeled secondary antibodies in an antibody buffer. Subsequent to three PBA washes, the slices were stained with DAPI for nuclear visualization. Fluorescent images were then captured using an EVOS M7000 Cell Imaging System (Thermo Fisher Scientific).

Statistical analysis

Data were analyzed using SPSS19.0 and GraphPad Prism 5. One-way ANOVA and SNK-q tests were used for comparisons between groups. Results were considered statistically significant at P<0.05.

Establishment of Engineered ADMSCs with miR-126

ADMSCs were transfected with LV-miR-126, LV-miR-126 sponge, or LV-miR-CON. The transfection efficiency was confirmed through immunofluorescence staining (≥ 90%, data not shown). Subsequent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed a significant increase in the levels of miR-126 and miR-126 sponge, confirming the successful engineering of miR-126 into ADMSCs (Figure 1A). A CCK-8 assay was conducted to assess whether miR-126 or miR-126 sponge had any impact on cell viability, and the results indicated no reduction in ADMSC viability post-transfection (Figure 1B).

Efficacy of ADMSCs in mitigating Liver damage in ALD

Liver tissues from the ALD group exhibited extensive hepatocyte steatosis, significant inflammatory cell infiltration, disrupted hepatic cell arrangement, and the presence of variously-sized fat vacuoles, along with condensed and dissolved hepatocyte nuclei, compared to control tissues (Figure 2 A and B). Additionally, serum ALT and AST levels were markedly elevated in the ALD group (Figure 2 G and H). However, mice treated with unmodified ADMSCs or ADMSCs^null showed less severe liver lesions and reduced ALT and AST levels (Figure 2 C, D, G, and H). Notably, the ALD mice treated with ADMSCs^miR-126(+) displayed the most significant improvement in liver pathology and transaminases levels (Figure 2 E, G, and H). Conversely, the therapeutic effects were largely negated when treated with ADMSCs^miR-126(-) (Figure 2 F, G, and H).

Impact of ADMSCs on serum LPS level

Given the observed liver improvements, we hypothesized that ADMSCs might also reduce the translocation of bacterial products, such as LPS, thereby attenuating liver injury through various mechanisms, such as the suppression of inflammatory agents like cytokines and reactive nitrogen species. To test this, we measured the typical serum LPS level. Our findings showed elevated LPS levels in the ALD and ADMSCs^miR-126(-) groups, but not in the unmodified ADMSCs, ADMSCs^null, or ADMSCs^miR-126(+) groups (Figure 2 I). Furthermore, the most significant reduction in LPS level was observed in ALD mice treated with ADMSCs^miR-126(+).

ADMSCs protect the IEB and GVB of small intestinal in ALD

Given the observed liver improvements and reduced serum LPS level, we further investigated the effects on the small intestine (ileum). There were significant vascular dilation and congestion, as well as increased gaps between the mucosa and villi in the ALD group (Figure 3 A and B). Consistent with previous results, these injuries were mitigated by treatment with unmodified ADMSCs and ADMSCs^null (Figure 3 C and D). Moreover, the ileum of the ALD group treated with ADMSCs^miR-126(+) showed most significant improvement compared to other groups (Figure 3 E). In contrast, no significant improvement was observed in the ALD group treated with ADMSCs^miR-126(-) (Figure 3 F).

Intestinal epithelial injury was assessed through the expression of ZO-1 (Figure 4). The ALD model group displayed disrupted and absent ileal mucosa compared to the control group, characterized by epithelial cell swelling and reduced ZO-1 protein and mRNA expression (Figure 4 A, B, and G). Similar to previous results, the ALD group treated with ADMSCs^miR-126(+) showed most significant improvement in epithelial cells and increasing of ZO-1 levels, while the ADMSCs^miR-126(-) group exhibited even worse outcomes (Figure 4 F and G).

Podocalyxin-related protein 1 (PV1), a marker for endothelial cell permeability and GVB injury as well, was significantly upregulated in the ALD model at both the mRNA and protein levels (Figure 5 and 6). ADMSC treatment reduced PV-1 mRNA and protein expression in the ALD group, with the most significant effect observed in the ADMSCs^miR-126(+) group. This effect was partially negated by ADMSCs^miR-126(-).

ADMSCs repairing small intestinal in ALD via PI3K/Akt/eNOs pathway

Furthermore, we observed a significant decrease in p-PI3K, p-Akt and p-eNOS levels in the intestinal tissues of the ALD model. In contrast, ALD models treated with ADMSCs^miR-126(+) showed the highest levels of p-Akt (Figure 7). Subsequently, we also observed the decrease of caspase-3 according to the activation of PI3K/Akt/eNOS, which is most significantly decreased with ADMSCs^miR-126(+)treatment.

These above findings suggest that ADMSCs play therapeutic efficacy in protecting the IEB and GVB via activation PI3K/Akt/eNOS pathway, and miR-126 may enhance this effect.

The therapeutic potential of MSC transplantation has been extensively investigated and validated in a variety of diseases, including ALD [20-22]. However, the use of unmodified MSCs may be insufficient for effectively treating ALD [31]. Consequently, several strategies have been employed to augment the therapeutic efficacy of MSCs, such as gene engineering [31]. Additionally, various miRNAs have been identified that enhance the therapeutic effectiveness of MSCs in ALD [32]. However, the majority of these studies have primarily focused on the liver, neglecting other relevant organs affected during ALD progression. Previous research has demonstrated that alcohol consumption can compromise intestinal integrity by altering gut microbiota and disrupting the mucosal barrier, thereby facilitating the absorption of toxins that exacerbate liver damage and fibrosis [33]. One study revealed that alcohol upregulates miR-212 expression in intestinal epithelial cells, which in turn inhibits the key tight junction protein, ZO-1 [34]. To date, there is a significant gap in the literature concerning the role of miRNAs engineered ADMSCs in mitigating intestinal barrier damage in ALD.

Our study centered on miR-126, recognized as a key regulator in various aspects of vascular development [35-37]. Specifically, miR-126 stabilizes vascular integrity by modulating VCAM-1 expression, a critical component of the GVB, and mitigates blood-brain barrier disruption caused by cerebral hemorrhage, suggesting its potential protective effects against microvascular leakage [38]. Moreover, miR-126 enhances the activity of endothelial progenitor cells, promoting their homing and sustaining late-stage outward growth, endothelial-to-endothelial transformation, and neovascularization, thereby preventing vascular injury [39]. Furthermore, a study using a mouse model of cholestasis found that miR-126a-5p could be a potential therapeutic strategy for enhancing hepatic repair and metabolic function post liver damage [40]. Another study identified miR-126 knockdown may induce the incomplete mucosal epithelium in inflammatory bowel disease mice model [28]. Based on these findings, we engineered ADMSCs with miR-126 to assess its potential in enhancing the therapeutic effects of ADMSCs on ALD. To our knowledge, this is the first study to demonstrate the protective effects of miRNA-engineered ADMSCs on both GVB and IEB in ALD. We hypothesize that the observed ADMSC-mediated alleviation of ALD liver injury is linked to reduced intestinal permeability.

The current study revealed that ADMSCs significantly mitigate ALD-induced liver injury, as evidenced by reduced serum ALT and AST levels and improved liver histology. Notably, these improvements were most pronounced in mice treated with ADMSCs^miR-126(+), and less so with ADMSCs^miR-126(-). Consistent with these findings, we observed elevated serum LPS level in ALD mice, which were reduced following ADMSC treatment and most significantly decreased with ADMSCs^miR-126(+). The compromised integrity of the gut barrier is characterized by elevated levels of various markers, including LPS [41]. Therefore, we further evaluated the gut barrier, encompassing both IEB and GVB.

We assessed the pathological changes in the gut barrier and measured indicators PV1 and ZO-1 for IEB and GVB, respectively. Our results indicated that ALD mice treated with ADMSCs^miR-126(+) exhibited the most significant reductions in PV1 and increases in ZO-1 expression, suggesting a therapeutic effect on the repairing GVB and IEB. In contrast, ADMSCs^miR-126(-), which inhibits miR-126, yielded less favorable outcomes. This suggests that miR-126-overexpressing ADMSCs may alleviate liver injury by maintaining intestinal barrier.

In our study, we observed that ADMSC treatment directly increased the levels of p-PI3K in the ileum of mice with ALD, which correspondingly led to an increase in p-Akt and p-eNOS levels. This sequence of events resulted in a decrease of caspase-3, indicating the activation of the PI3K/Akt/eNOS pathway and the suppression of caspase-3 through ADMSC treatment. Previous studies have demonstrated that exosomes derived from MSCs enhance the survival and angiogenic functions of epithelial cells (ECs) damaged by hypoxia/reoxygenation, by delivering miR-126. This delivery activates the PI3K/Akt/eNOS pathway, subsequently reducing the expression of cleaved caspase-3 and increasing the expression of angiogenic and growth factors [30]. Moreover, the PI3K/Akt pathway has been shown to play a crucial role in alcohol-induced gastric mucosal damage [42]. Thus, we hypothesized that ADMSCs might activate the PI3K/Akt/eNOS pathway and consequently reduce the expression of caspase-3, thereby promoting the repair of the intestinal barrier. Specifically, treatment with ADMSCs^miR-126(+) demonstrated the most significant effect on the activation of PI3K/Akt/eNOS pathway and repress of caspase-3.

In summary, our study provides compelling evidence that ADMSCs can mitigate liver injury in ALD, potentially by repairing the intestinal barrier in the ileum through the activation of the PI3K/Akt/eNOS pathway. Additionally, miR-126 appears to enhance the therapeutic efficacy of ADMSCs. Nevertheless, there are several limitations to our study that warrant attention. For example, we did not directly assess the changes in growth factors in the ileum, such as VEGF, EGF, PDGF, and bFGF, which would provide more robust evidence to support our hypothesis. To provide a more comprehensive understanding, other indicators like gut microbiota should also be investigated to assess the impact of an impaired gut barrier more fully. In light of these limitations, future studies are warranted to comprehensively evaluate the therapeutic efficacy of ADMSCs in gut-mediated ALD.

This study elucidates the therapeutic potential of human ADMSCs in mitigating liver damage in an ALD model. ADMSCs ameliorate hepatic injury by repairing the compromised gut barrier and reducing leakiness induced by excessive ethanol consumption. Overexpression of miR-126 further enhances the therapeutic efficacy of ADMSCs. These findings may pave the way for a novel ADMSC-based treatment strategy for ALD patients.

Ethical approval

All mice were treated according to the protocols reviewed and approved by the institutional ethics committee of The First Affiliated Hospital of Wenzhou Medical University.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This research was supported by the National Natural Science Foundation of China (Youth Program, 82200632) and Basic Scientific Research Funds of Department of Education of Zhejiang Province (KYZD202201)

Author Contribution

Conceptualization, Xiaodong Wang, Yongping Chen and Dazhi Chen; methodology, Qianjing Du, TongTong Pan, Yuanhang Xia, Xiaodong Wang, Yongping Chen and Dazhi Chen; formal analysis, Qianjing Du, Weijian Yang, Xiaozhi Jin, Shiyi Zeng and Dazhi Chen; investigation, Ru Jin and Rongrong Shao; data curation, Rongrong Shao, Xiaozhi Jin, and Dazhi Chen; writing—original draft preparation, review and editing, Qianjing Du and Dazhi Chen.

Data Availability

Data is provided with request.

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No competing interests reported.

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Overexpression of microRNA-126 in Adipose-Derived Mesenchymal Stem Cells Alleviate the Alcoholic Liver Injury

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Abstract

Figures

Introduction

Materials and methods

Animals

ADMSCs preparation

Vector construction and transduction

Establishment of ALD model and treatment options

Histopathological examination and immuno-histochemical staining

Examination of serum ALT, AST and endotoxin levels

Reverse transcription-quantitative polymerase chain reaction

Detection of protein expression by western blot assay

Immunofluorescence staining

Statistical analysis

Results

Establishment of Engineered ADMSCs with miR-126

Efficacy of ADMSCs in mitigating Liver damage in ALD

Impact of ADMSCs on serum LPS level

ADMSCs protect the IEB and GVB of small intestinal in ALD

ADMSCs repairing small intestinal in ALD via PI3K/Akt/eNOs pathway

Discussion

Declarations

Ethical approval

Conflict of Interest

Funding

Author Contribution

Data Availability

References

Additional Declarations

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