Novel 11β-HSD1 inhibitor ameliorates liver fibrosis by inhibiting the Notch signaling pathway and increasing the NK cell population.

doi:10.21203/rs.3.rs-4316599/v1

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Novel 11β-HSD1 inhibitor ameliorates liver fibrosis by inhibiting the Notch signaling pathway and increasing the NK cell population.

https://doi.org/10.21203/rs.3.rs-4316599/v1

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11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates hepatic glucose output and systemic glucose homeostasis. We aimed to investigate the anti-fibrotic effect of a novel 11β-HSD1 inhibitor in a liver fibrosis mouse model. Hepatic fibrosis animal model was induced by thioacetamide administration during 19 weeks. Bulk RNA sequencing was performed to evaluate mode of action. Changes of immune cell distribution was evaluated using mass cytometry in peripheral blood. 11β-HSD1 inhibitor treatment group showed a significant decrease in the fibrosis area and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels compared with the thioacetamide induced hepatic fibrosis animal model. Bulk RNA sequencing data showed Notch signal decreased and natural killer (NK) cell pathway increased after 11β-HSD1 inhibitor treatment. Changes of NK cell population was reconfirmed by mass cytometry in In Vivo animal models, and expression of Notch ligands (Jag2, Dll1, Dll3, and Dll4), Notch signals (Hes1 and Sox9), and Notch receptors (Notch3 and Notch4) decreased after 11β-HSD1 inhibitor treatment. Therefore, the novel 11β-HSD1 inhibitor ameliorates liver fibrosis by inhibiting the Notch signaling pathway and increasing the NK cell population.

Biological sciences/Drug discovery/Drug delivery

Health sciences/Diseases/Gastrointestinal diseases/Liver diseases/Liver fibrosis

Non-alcoholic fatty liver disease

Non-alcoholic steatohepatitis

Liver fibrosis

11β-Hydroxysteroid dehydrogenase type 1

Natural Killer cell

Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most common liver diseases worldwide, affecting more than 25% of adults and a significant number of children and adolescents, and its prevalence increases with obesity.(1, 2) Liver fibrosis plays a critical role in the progression of MASLD to chronic liver diseases.(3) However, no effective pharmacological treatment for liver fibrosis is currently available.(4) Liver transplantation is the only treatment possible for the advanced disease stages.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), which is highly expressed in the liver and adipose tissue, converts inactive cortisone into active cortisol in humans.(5–7) 11β-HSD1 regulates hepatic glucose output and can be linked to intrahepatic fat deposition. Thus, abnormally high expression of 11β-HSD1 in visceral adipose tissue can promote adipogenesis, which may lead to Cushing’s disease (5, 8). Several selective inhibitors of 11β-HSD1 have been investigated for the treatment of MASLD. Inhibition of 11β-HSD1 by RO5093151 has been studied in insulin-resistant MASLD patients (9). A significant reduction in liver fat content, as well as liver enzyme concentrations, was observed in the patients; however, there were considerable adverse effects, including nervous system disorders, gastrointestinal disorders, and infections (9). AZD6925, another selective 11β-HSD1 inhibitor, decreases triglyceride accumulation in the liver, prevents hepatic steatosis, and increases mitochondrial biogenesis in db/db mice.(10) BVT.2733 inhibits lipogenesis in the liver and lipolysis in fatty tissues of mice fed a high-fat diet, thereby decreasing the delivery of glucocorticoids and free fatty acids to the liver and attenuating hepatic steatosis.(11, 12) Thus, 11β-HSD1 may play a significant role in MASLD development.

However, comprehensive research on the anti-fibrotic role of 11β-HSD1 inhibitors as well as it’s mode of action is lacking. Therefore, the aim of the present study was to investigate the anti-fibrotic effect and mechanism of action of a 11β-HSD1 inhibitor in mice with liver fibrosis.

Animal experiments and ethics statement

C57BL/6 wild-type male mice (six-week-old) were purchased from Dae Han Bio Link (Eumseoung, Republic of Korea) and bred and managed at the Hanyang Laboratory Animal Research Center. Mice were randomly assigned into the following four groups (Fig. 1A; n = 10 per group, n = 40 in total): normal control, 0.03% TAA (Sigma Aldrich, St. Louis, MO, USA) in drinking water for 19 weeks, TAA + 10 mg/kg/day OCA (APExBIO, Boston, MA, USA) for nine weeks, and TAA + 10 mg/kg/day 11β-HSD1 inhibitor (J2H Biotech, Suwon, Republic of Korea) for nine weeks. (13, 14). After 10 weeks of TAA administration, the treatment groups were administered TAA combined with OCA or 11β-HSD1 inhibitor for the next nine weeks. OCA and the 11β-HSD1 inhibitor were orally administered by gavage once a day (15). The study was approved by the Hanyang University Institutional Animal Care and Use Committee (HY-IACUC-21-0001). The research protocol was prepared, and the study was performed in compliance with their ethical guidelines.

Human natural killer cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated form healthy donor with density gradient in CPT tube (BD Biosciences, Cas.No 362753), centrifuged 1800G, RT, 20min. Freshly isolated NK cells using the Human Natural Killer Cell Isolation kit (Miltenyl Biotec, Cas.No 130-092-657). NK cells were cultured in NK MACS Medium human (Miltenyl Biotec, Cas.No 130-114-429) supplemented with 1% NK MACS Supplement human (Miltenyl Biotec), 5% human AB serum (Sigma Aldrich, Cas.No H3667), 500IU/ML IL-2 IS, premium grade (Milteny Biotec, Cas.No 130-097-744). Cells were maintained in a humidified atmosphere containing 5% CO₂ and 95% air at 37°C incubator. Total RNA from NK cells obtained from healthy donor (n = 1) was isolated using Trizol Reagent (Invitrogen, Cas.NO 15596018) and cDNA was prepared with PrimeScript RT reagent Kit (TaKaRa; Otsu, Shiga, Japan), 2µg of total RNA was reverse transcribed. qRT-PCR amplification was performed using a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) with LightCycler 480 SYBR Green I Master mix (Roche Diagnostics). All tests were performed in three replicated with 96 well plates and normalized to β-actin.

Study materials

The 11β-HSD1 inhibitor (J2H Biotech, Suwon, Republic of Korea) was synthesized, OCA (APExBIO, Boston, MA, USA) was purchased. The LX-2 and HepG2 cell lines were used for in vitro experiments.

Biochemical and histopathological analysis

Levels of serum ALT, AST, total bilirubin, and albumin were measured by chemistry analyzer AU480 (Beckman Coulter, Tokyo, Japan). H&E and Sirius Red staining were performed to assess inflammation and fibrosis. Inflammation and ballooning scores were determined using the MASLD activity score system for non-alcoholic steatohepatitis (16). The detailed procedures are described in the Supplementary Materials.

Western blotting

Total protein from the liver tissues was extracted with RIPA lysis buffer (GenDEPOT, Hanam, Republic of Korea), Protease inhibitor (GenDEPOT, Hanam, Republic of Korea) and Phosphatase inhibitor (GenDEPOT, Hanam, Republic of Korea). RIPA lysis buffer and protease inhibitor, phosphatase inhibitor was used in a ratio of 100:1. For electrophoresis, the isolated proteins (11 µg) were placed onto a 10% sodium dodecyl sulfate-polyacrylamide gel. The separated proteins were transferred onto nitrocellulose (NC) membranes (0.45 µm-pore size; Bio-Rad, Hercules, CA, USA), blocked with 1× EzBlock Chemi solution (ATTO, Tokyo, Japan) for 30 min, and incubated overnight at 4°C with the primary antibodies: Col1a1 (1:1000; Abcam, Cambridge, UK), FN-1 (1:1000; Abcam, Cambridge, UK), Vimentin (1:1000; Abcam, Cambridge, UK), IL6 (1:1000; Abcam, Cambridge, UK), NLRP3 (1:1000, Adipogen, Liestal, Switzerland), Hes1 (1:1000; Cell Signaling Technology, Massachusetts, USA), Spp1 (1:1000; Cell Signaling Technology, Massachusetts, USA), Sox9 (1:1000; Cell Signaling Technology, Massachusetts, USA), β-actin (1:1000; Santa Cruz Biotechnology, California, USA). After that, the membranes were incubated for 1 h at 25°C with a secondary HRP-conjugated anti-rabbit or mouse antibody (1:3000; Jackson Immunoresearch, West Grove, PA, USA). As a protein loading control, β-actin was used. The Dyne ECL STAR western blotting Detection Kit (Dyne Bio, Seongnam, Republic of Korea) was used to visualize positive protein bands, and the results were quantified using an image analyzer (Image Lab 3.0, Bio-Rad, Hercules, CA, USA).

LX-2 culture

The human HSC line LX-2 cells were cultured in high glucose DMEM (Gibco, Grand Island, NY, USA) with 2% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Welgene, Gyeongsan, Republic of Korea) at 37°C incubator with 5% CO₂. After cultured for 24 hours, LX-2 cells were treated with 20 ng/ml TGF-β (R&D Systems, Minneapolis, MN, USA), and 11β-HSD1 inhibitor (1 µM).

HepG2 culture

The human hepatocellular carcinoma cell line HepG2 cells were cultured in low glucose DMEM (Gibco, Grand Island, NY, USA) with 10% FBS and 1% penicillin/streptomycin at 37°C incubator with 5% CO₂. After cultured for 24 hours, HepG2 cells were treated with 0.02 g/ml TAA and 11β-HSD1 inhibitor (1 µM).

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from liver tissues, LX-2 cells and HepG2 cells using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA). 1 mL of TRIzol and 0.2 mL of Chloroform were added to liver tissue, and after centrifugation at 12000rpm, 15 min, 4 ℃, only the supernatant was separated. After that, 0.5 mL of Isopropanol per 1 mL of Trizol was added, incubated at 4℃ for 10 minutes, and centrifuged at 12000 rpm, 10 min, 4 ℃ to obtain an RNA pellet. The absorbance of RNA was measured using a Nanodrop ND-2000 spectrophotometer (Thermo Scientific Inc., DE, USA). High-purity RNA having a purity of 1.8 to 2.0 was obtained and used at absorbance A260/A280 (nm). The extracted RNA (2 µg) was synthesized into cDNA using PrimeScript™ RT Reagent Kit. qRT-PCR amplification was performed using a LightCycler 480 with LightCycler 480 SYBR Green I Master mix. All tests were performed in three replicated with 96 well plates and normalized to GAPDH and β-actin. The sequences of all primers are listed in Table S5 and S6.

RNA sequencing analysis

RNA sequencing was performed by Macrogen, Inc. (Seoul, Republic of Korea) on liver tissues from the groups of mice whose efficacy was evaluated: normal control (n = 3), TAA-treated group (n = 3), TAA + OCA group (n = 3), TAA + 11β-HSD1 inhibitor responder group (n = 3), and TAA + 11β-HSD1 inhibitor non-responder group (n = 3). 11β-HSD1 inhibitor group, the subgroup that showed alleviation of fibrosis (depending on the fibrosis score [≤ 4] and expression of fibrosis-related markers) was defined as the responder group, whereas the group that did not show alleviation of fibrosis was defined as the non-responder group. The detailed procedures are described in the Supplementary Materials.

Data analysis for RNA sequencing

FastQC was used to check the quality of the raw sequencing data (17). FASTX_Trimmer (18) and BBMap (19) were used to eliminate adapters and low-quality readings (< Q20). TopHat was used to map the trimmed reads to the reference genome (20). Gene expression levels were estimated using fragments per kilobase per million read values using Cufflinks (21). These values were adjusted by the quantile normalization method using EdgeR within R (R Development Core Team, 2016).

Immune profiling of mouse peripheral blood mononuclear cells (PBMC) by mass cytometry

The immunological mechanism of action of the 11β-HSD1 inhibitor was investigated by determining the change in the population of immune cell types from mouse PMBC samples using mass cytometry.(22) The isolated PBMCs were stained with lanthanide metal-tagged surface antibodies. The detailed procedures are described in the Table S7. The analysis groups for mass cytometry were: normal control (n = 5), TAA-treated (n = 6), and TAA + 11β-HSD1 inhibitor (n = 8). Few samples were excluded owing to the limited number of live cells. For data processing and visualization, Cytobank (https://www.cytobank.org) was used. Subsequently, the primary immune cell types and subtypes were manually gated using the gating strategy (Table S8). The t-distributed stochastic neighbor embedding (t-SNE) approach was applied to minimize dimensionality and show data at single-cell resolution. Automated clustering methods, such as the t-SNE and citrus methods, were used in addition to the manual gating approach in this study. Samples with less than 1000 cells were excluded from the analysis. When mass cytometry analysis was performed, 20% of the cells were unassigned, and the remaining were assigned as leukocytes.

Statistical analysis

Statistical analyses were performed using Statistical Package for GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). All data are expressed as mean ± standard deviation. Data were analyzed using the Mann–Whitney U test (if the general assumptions were met, e.g., not normally distributed according to Kolmogorov–Smirnov test), one-way analysis of variance for multiple comparisons, and Tukey’s test for post-hoc multiple comparisons (equal variances assumed). Statistical significance was set at p < 0.05.

11β-HSD1 inhibition attenuated hepatic fibrosis and inflammation in cirrhotic animal model

11β-HSD1 inhibitor treatment decreased hepatocyte ballooning (as indicated by H&E staining) (Fig. 1, Table S1), fibrosis area (%) (as indicated by Sirius Red staining, P = 0.0015) (Table S2), ALT and AST levels (Table S3) compared with the TAA induced liver cirrhosis animal model (Table S4). The mRNA levels of various fibrosis markers (including α-Sma, Col1a1, Fn1, and Timp1) were measured by RT-PCR in the liver tissues of mice. 11β-HSD1 inhibitor treatment showed a significant decrease in the expression levels of fibrosis markers compared with the TAA-treated control group (Fig. 2A). Protein expression levels of fibrosis markers (including Col1a1, FN-1 and vimentin) were also decreased in the 11β-HSD1 inhibitor group in the liver tissue of mice compared with those in the TAA-treated group (Fig. 2C). The mRNA expressions of Il-6, Mcp1, and Nlrp3 significantly decreased after 11β-HSD1 inhibition (Fig. 2A). The protein level of IL6 and NLRP3 also significantly decreased in the 11β-HSD1 inhibitor group (Fig. 2C).

11β-HSD1 inhibition decreased inflammation and fibrosis in In vitro model.

To verify the potential role of 11β-HSD1 inhibitor in alleviating liver fibrosis and inflammation, LX-2 and HepG2 cells, in which fibrosis and inflammation was induced by TGF-β (20 ng/ml) and TAA (0.02 g/ml), respectively, were treated with 1 µM 11β-HSD1 inhibitor. The expression levels of fibrosis markers (α-Sma, Col1a1, and Timp1) significantly decreased in the 11β-HSD1 inhibitor-treated groups compared with those in the fibrosis-induced group (without 11β-HSD1 inhibitor treatment) in LX-2 cell (Fig. 2B). Also the expression levels of inflammation markers (IL6, MCP1 and NLRP3) decreased in the 11β-HSD1 inhibitor-treated groups compared with inflammation-induced group (without 11β-HSD1 inhibitor treatment) in HepG2 cell (Fig. 2B).

Bulk RNA data suggested 11β-HSD1 inhibition decreased Notch signaling and increase NK cell related pathway

To estimate suggesting mode of action after 11β-HSD1 inhibitor treatment, bulk RNA sequencing was performed. Bulk RNA sequencing data demonstrated that fibrosis- and inflammation-related genes significantly decreased in the 11β-HSD1 inhibitor group compared with those in the TAA-treated group (Fig. 3A). Additionally, gene set enrichment analysis (GSEA) showed that the Notch signaling pathway, ERBB signaling pathway and JAK-STAT signaling pathway were significantly downregulated after 11β-HSD1 inhibitor treatment (Fig. 3B and 3C, Table S7). Notably, the expression of most of the genes related to the Notch signaling pathway (Notch ligand, receptor, and signal) decreased in the 11β-HSD1 inhibitor group (Fig. 3D). In particular, the expression levels of Notch ligands (Jag1, Jag2, Dll1, Dll3, and Dll4), Notch signals (Hes1, Sox9 and Spp1), and Notch receptors (Notch3 and Notch4) were significantly decreased after 11β-HSD1 inhibitor treatment (Fig. 3D). Moreover, the expression levels of NK cell-related genes (NKG2D, GZMB and Perforin1) significantly increased in the 11β-HSD1 inhibitor group compared with those in the TAA-treated group (Fig. 3D). Volcano plot showed that NK cell activation-related genes and negative regulation of inflammatory response-related genes increase in 11β-HSD1 inhibitor group compared with TAA group.

11β-HSD1 inhibition decreased Notch signaling pathway

Several markers of the Notch signaling pathway decreased after 11β-HSD1 inhibitor treatment in mouse liver tissue, LX-2 cells, and HepG2 cells. In the liver tissue, 11β-HSD1 inhibition significantly decreased the mRNA expression levels of the following markers: Notch receptors (Notch1 and Notch4), Notch ligands (Jag1 and DLL1), and Notch signal pathway subtype genes (Hes1, Sox9, and Spp1) (Fig. 4A). Additionally, LX-2 and HepG2 cell lines were used to confirm the down-regulation of the Notch signaling pathway upon inhibiting 11β-HSD1; fibrosis in LX-2 and inflammation in HepG2 cells was induced with TGF-β (20 ng/ml) and TAA (0.02 g/ml), respectively, followed by treatment with 11β-HSD1 inhibitor (1 µM). Expression of most Notch ligand, receptor, and subtype genes significantly decreased after treatment with the 11β-HSD1 inhibitor; however, Notch 2 receptor expression consistently increased (Fig. 4B). The protein level of Hes1, Sox9 and Spp1 also decreased in the 11β-HSD1 inhibitor group compared with TAA group (Fig. 4C).

11β-HSD1 inhibitor increased NK cell populations in peripheral blood

A single-cell-based, high-dimensional mass cytometry analysis revealed that the NK cell population significantly decreased in the TAA-treated group compared with that in the normal control group. However, the population considerably recovered after treatment with the 11β-HSD1 inhibitor, which is depicted in the manual gating and tSNE analysis (Fig. 5A and 5B, Table S8). The expression of surface markers in NK cells in all clusters increased after treatment with 11β-HSD1 inhibitor. When cellular clusters were analyzed at high resolution by immune cell type using PhenoGraph, the NK cell population in two clusters (#8 and #10) decreased in the TAA-treated group. However, they substantially recovered after treatment with the 11β-HSD1 inhibitor. (Fig. 5C). In contrast, the population of B cells in two clusters (#3 and #12) and that of CD4⁺ T cells in two clusters (#4 and #17) increased due to liver fibrosis, which decreased after 11β-HSD1 inhibition (Fig. 5C, Table S9).

We isolated human NK cell from normal donor’s peripheral blood and treated 1 µM of 11β-HSD1 inhibitor. GZMB and Perforin1 mRNA levels increased compared with normal control after 11β-HSD1 inhibitor treatment (Fig. 5D). And we also re-confirmed the changes of NK cell markers in mouse liver tissue. Interferon gamma, NKG2D, GZMB and Perforin1 mRNA levels were decreased in TAA group compared with normal control. But they were increased in 11β-HSD1 inhibitor treated group (Fig. 5E).

Changes of other immune cell population after 11β-HSD1 inhibitor in cirrhotic animal model.

Additionally, the immune cell clusters were analyzed according to the expression levels of surface markers through Citrus analysis (Fig. 6A). Surface marker expression in CD4⁺ T cells in clusters 59986 and 59980 decreased, and that of cluster 59979 increased after treatment with the 11β-HSD1 inhibitor. Surface marker expression levels in all B cell clusters were higher in the TAA-treated group than in the normal control group; however, they decreased in the 11β-HSD1 inhibitor group (Fig. 6B). CD4 + T cell and B cell total cluster expression was increased in TAA treated group compared with normal control, but decreased in 11β-HSD1 inhibitor treated group (Fig. 6C).

In the present study, we investigated the anti-fibrotic effect and possible mode of action of a 11β-HSD1 inhibitor in a TAA-induced liver fibrosis mouse model. We observed that liver fibrosis was alleviated after the 11β-HSD1 inhibitor treatment, and 11β-HSD1 inhibition was associated with a downregulation of the Notch signaling pathway and an increase in the NK cell population. NK cells play a crucial role in innate immunity. They are abundant in the liver and express NKG2D receptors on their surface. NKG2D plays an important role in liver fibrosis by binding to the retinoic acid early inducible gene-1 (Rae-1) observed in active hepatic stellate cells (HSCs).(23, 24) When NK cells are activated in the liver, NKG2D of NK cells and Rae-1 expressed by HSCs bind to each other, and IFN-γ is secreted from NK cells, which is known to have an anti-fibrotic function.(23, 25) Therefore, the anti-fibrotic effect of the 11β-HSD1 inhibitor may be attributed to the cytotoxic activity of NK cells, which can kill HSCs or inhibit their differentiation. Considering that the secretory activity of B cells increases in inflamed livers compared with that in normal livers, the decrease in the B cell population can be attributed to the attenuation of liver fibrosis and inflammation due to 11β-HSD1 inhibition.

RNA sequencing was performed using liver tissue to compare the gene expression changes caused by TAA and 11β-HSD1 inhibition and explore the possible mechanism of 11β-HSD1 inhibition in treating liver fibrosis. GSEA analysis showed that the Notch signaling pathway most significantly decreased among various other pathways after 11β-HSD1 inhibitor treatment. Activation of the Notch signaling pathway can promote the differentiation of HSCs into myofibroblasts and the progression of liver fibrosis.(26) When the Notch signal is activated, hepatocytes promote the secretion of SPP1 (OPN), which induces myofibroblast differentiation.(26) When the Notch signal is suppressed, liver sinusoidal endothelial cells secrete nitric oxide, thereby inhibiting the differentiation of HSCs into myofibroblasts. Notch 3 receptor expression increases in liver fibrosis and decreases after regression of liver fibrosis (27).

Gene ontology (GO) analysis of liver transcriptomic data demonstrated changes in the expression of genes related to fibrosis and NK cell activation. Among the genes involved in NK cell activation, the expression of genes corresponding to interleukins (e.g., Il15ra, Il18, Lyst, and Tox) were altered in the TAA-induced liver fibrosis model, whereas 11β-HSD1 inhibition showed the opposite effect on the expression of these genes (Table S10). This suggests that 11β-HSD1 inhibition contributes to the regulation of immune responses in the NK cells. Meanwhile, the results of the mass cytometry analysis of the immune cell phenotype demonstrated that the NK cell population decreased in the TAA-induced liver fibrosis model compared with those in the normal control but increased after 11β-HSD1 inhibition. NK cells elicit potential anti-fibrotic effects in the liver by directly or indirectly inhibiting HSCs through the production of various cytokines; however, further studies are needed to confirm these results.

Meanwhile, controversial data have also been reported. For example, Xiao et al. reported a positive correlation between the expression of 11β-HSD1 and the typical markers of liver fibrosis. 11β-HSD1 inhibition with a 11β-HSD1 inhibitor (BVT.2733) significantly reduced the degree of fibrosis (28). In contrast, another study reported that inhibition of 11β-HSD1 by UE2316 activated the hepatic myofibroblasts and worsened liver fibrosis in a reversible carbon tetrachloride-induced liver fibrosis mouse model (29). This study also suggested a dichotomous function of 11β-HSD1 during hepatic injury and resolution in a context-dependent manner, indicating the importance of the timing of 11β-HSD1 inhibitor administration (29).

In conclusion, 11β-HSD1 inhibition demonstrated the potential to alleviate liver fibrosis in a mouse model of liver fibrosis. This is possibly mediated through the Notch signaling pathway. Furthermore, since the NK cell population increased due to 11β-HSD1 inhibition, immune responses by NK cells could be the mechanism underlying liver fibrosis alleviation upon treatment with 11β-HSD1 inhibitor.

11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1

ALT, alanine aminotransferase; AST, aspartate aminotransferase

GO, Gene ontology

GSEA, gene set enrichment analysis

HSC, hepatic stellate cell

NAFLD, non-alcoholic fatty liver disease

NK, natural killer

OCA, obeticholic acid

PMBC, peripheral blood mononuclear cells

t-SNE, t-distributed stochastic neighbor embedding

TAA, thioacetamide

Acknowledgement

This work was supported by research funds from J2H Biotech, Korea Drug Development Fund, provided by the Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN21C1149000023), and the National Research Foundation of Korea 2020R1A2C2009227.

All authors have seen and approved the manuscript, contributed significantly to the work

Supplemental material

There are staining scores, serum levels, mouse liver weight, primer sequences and information of antibodies in supplemental material.

Conflict of interest

The authors declare no potential conflicts of interest.

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Novel 11β-HSD1 inhibitor ameliorates liver fibrosis by inhibiting the Notch signaling pathway and increasing the NK cell population.

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Abstract

Figures

Introduction

Materials and Methods

Animal experiments and ethics statement

Human natural killer cell isolation

Study materials

Biochemical and histopathological analysis

Western blotting

LX-2 culture

HepG2 culture

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

RNA sequencing analysis

Data analysis for RNA sequencing

Immune profiling of mouse peripheral blood mononuclear cells (PBMC) by mass cytometry

Statistical analysis

Results

11β-HSD1 inhibition attenuated hepatic fibrosis and inflammation in cirrhotic animal model

Bulk RNA data suggested 11β-HSD1 inhibition decreased Notch signaling and increase NK cell related pathway

11β-HSD1 inhibition decreased Notch signaling pathway

11β-HSD1 inhibitor increased NK cell populations in peripheral blood

Discussion

Abbreviations

Declarations

Acknowledgement

References

Additional Declarations

Supplementary Files

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