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Introduction

11β-hydroxysteroid dehydrogenases (11β-HSD) are a class of oxidoreductases that catalyze the reversible transformation between corticosterone and dehydrocorticosterone. 11β-HSD can be divided into two isoforms in humans: 11β-HSD1 and 11β-HSD2 (1). 11β-HSD1 is a crucial enzyme that converts inactive cortisone into active hydrocortisone, thus regulating multiple functions of glucocorticoids (GCs) (2). 11β-HSD1 is widely expressed in tissues (3) and is particularly abundant in the liver, muscle and fat tissue (4). In renal tissue, 11β-HSD1 exhibits oxidase activity, whereas in non-renal tissues, such as liver and fat tissues, 11β-HSD1 exhibits reductase activity. In particular, the role of 11β-HSD1 in the liver is associated with elevated levels of active GCs, which is consistent with increased levels of GCs in liver fibrosis (5). Therefore, it has been speculated that 11β-HSD1 is involved in the early activation and signal transduction of hepatic stellate cells (HSCs) and may regulate the expression of extracellular matrix (ECM) components (6–9).

Hepatic fibrosis is a common pathophysiological feature of all chronic liver diseases (10). HSCs are the final target cell type of various profibrotic factors (11), serving as key mediators in the occurrence and development of hepatic fibrosis (12–15). Once HSCs are activated by inflammatory factors, including transforming growth factor (TGF)-β1 (16), their phenotype changes from resting to activated and then into myofibroblasts (MFB), which secrete abundant ECM components (17), resulting in increased ECM deposition and reduced matrix decomposition (18). Furthermore, activated HSCs exhibit reduced cell cycle, accelerated proliferation rates (19), increased numbers (20), decreased amounts of fat and vitamin A storage (21), and secretion of profibrotic factors [α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF)], which further stimulate HSC activation (19,22), resulting in a feedback mechanism (23). Activated HSCs promote the synthesis and secretion of tissue inhibitor of metalloproteinase (24) and inhibit the degradation of ECM by matrix metalloproteinases, thus promoting liver fibrogenesis (25). ECM is primarily composed of three types of organic substances: Collagen, glycoprotein and proteoglycan. Among these, type IV collagen (Col IV), N-terminal pro-peptide of collagen type III (PIIINP) and hyaluronic acid (HA) are often used as serological indicators of the degree of hepatic fibrosis (26,27). In addition, liver hydroxyproline (Hyp) content, a unique amino acid component of collagen, can reflect alterations in collagen metabolism, parallel to the degree of hepatic fibrosis (28). As an antigen substance, xenogeneic serum induces an immunoresponse in the liver, leading to the formation of immune complexes. If the excessive immune complexes are not removed in a timely manner, they are deposited in the liver and the hepatic vascular wall, causing cell infiltration, release of inflammatory mediators, cell degeneration of parenchymal liver cells, fibrous hyperplasia and hepatic fibrosis (27,29,30).

The present study developed an in vivo model of hepatic fibrosis in rats using porcine serum. In addition, using in vitro cell culture, LX2-HSCs were stimulated with TGF-β1. The two experimental approaches were established to systematically investigate alterations in 11β-HSD1 expression and activity in liver tissue and HSCs during hepatic fibrogenesis. Furthermore, the effects of 11β-HSD1 knockdown and overexpression on the development and progression of hepatic fibrosis and its underlying mechanism of action were studied in LX2-HSCs.

Materials and methods

Experimental animals

A total of 108 male Sprague Dawley (SD) rats (age, 4–6 weeks; weight, 130–150 g) in quarantine were obtained from Hunan SJA Laboratory Animal Co. Ltd. The SD rats were housed in barrier rooms at 22±2°C with 65–70% humidity, a 12-h light/dark cycle and free access to water and food. Rats were randomly assigned to the following three groups (n=36 per group): i) Normal control group (N); ii) the model group (M); and iii) the 11β-HSD1 inhibitor group (M+I). Rats in the M and M+I groups received an intraperitoneal injection of 0.5 ml porcine serum (Beijing Solarbio Science & Technology Co., Ltd.) twice a week. The N group received an equal volume of 0.9% NaCl via intraperitoneal injection at the same frequency for the same duration. In addition to porcine serum administration, rats in the M+I group were treated with the 11β-HSD1 inhibitor BVT.2733 (100 mg/kg), which was orally administrated by gavage. At 5, 10 and 15 weeks following treatment, 12 rats in each group were anesthetized with an intraperitoneal injection of 2% pentobarbital sodium (30 mg/kg body weight) and deep anesthesia was verified by moderate respiratory and cardiovascular depression, and good muscle relaxation. The experimental rats were euthanized by cervical dislocation. The abdominal aorta and right lobe of the liver were collected for subsequent experiments. Prior to sacrifice, 2 ml blood was collected from the abdominal aorta of the rats, which was stored at 4°C.

All animal experiments were performed in accordance with the guidelines of The Institutional Animal Care and Use Committee of Xiang [license no. SCXK (Xiang) 2011-0003].

Experimental reagents

The 11β-HSD1 inhibitor, BVT.2733, was purchased from MedChem Express. Rat TGF-β1 was purchased from Abbkine Scientific Co., Ltd. (cat. no. PRP100618). The anti-11β-HSD1 antibody was purchased from AtaGenix (cat. no. ATA24000; 1:200). Human TGF-β1 was purchased from PeproTech, Inc. (cat. no. AF-100-21). Rat Col IV (cat. no. MBS704982) and PIIINP ELISA kits (cat. no. MBS704287), and the Annexin V-FITC/PI apoptosis detection kit (cat. no. MBS355226) were purchased from MyBioSource, Inc. The HA ELISA kit was purchased from R&D Systems (cat. no. DHYAL0). The ProCell collagen colorimetric assay kit was purchased from Genmed Scientifics, Inc. (cat. no. GMS10373.8). The rabbit-derived rat α-SMA (cat. no. ab124964; 1:1,500), CTGF (cat. no. ab6992; 1:2,000) and β-actin (cat. no. ab8227; 1:1,000) primary antibodies, and the goat anti-rabbit secondary IgG-HRP antibody (cat. no. ab97080; 1:5,000) were all purchased from Abcam. The cortisol kit (11β-HSD1 activity detection kit) was purchased from Cisbio (cat. no. 62CRTPEG). The adenovirus and adenovirus vector were purchased from Miao Ling Biotechnology Co., Ltd. (cat. nos. P0241 and P0653). The Cell Counting Kit-8 (CCK-8) cell proliferation test kit (cat. no. CK04) was purchased from Dojindo Molecular Technologies, Inc.

Pathological examination of livers

At weeks 5, 10 and 15 of treatment, 12 rats were randomly selected, anesthetized with 2% pentobarbital sodium and sacrificed. The abdominal aorta and right lobe of the liver were harvested for pathological examinations. In brief, the tissues were fixed for 7 days in 4% neural-buffered formalin at room temperature, dehydrated in increasing concentrations of ethanol, embedded with paraffin wax and sliced into 4 µm-sections, and comparative pathological alterations were observed via hematoxylin (5–20 min) and eosin staining (10–30 min), both at room temperature. The stages of fibrosis were examined by two independent experienced pathologists using the following pathological scoring criteria (31): 0, no liver fibrosis; I, degeneration of hepatocytes with ≤0.5 of the radius of hepatic lobules and infiltration of inflammatory cells surrounding the portal area; II, degeneration of hepatocyte >0.5 of the radius of hepatic lobules, with primarily balloon-like degeneration, a large number of infiltrating inflammatory cells and obvious fibrous tissue hyperplasia in the portal vein; and III, degeneration and necrosis of hepatocytes extended to 0.66 of the radius of hepatic lobules with fragmentation and bridging necrosis of hepatocytes, and lots of fibrosis in the parenchyma.

Measurement of serum Col IV, HA and PIIINP via ELISA

Blood samples were centrifuged at 1006.2 × g for 15 min at 4°C to obtain serum. The concentrations of Col IV, HA and NPIIINP in the serum of the rats were detected using ELISA kits, according to the manufacturer's protocol.

Western blotting

Total protein from the liver tissues was extracted with ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology), containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1mM protease inhibitor PMSF. Total protein was quantified using a bicinchoninic acid assay to correct the sampling volume and 30 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto nitrocellulose membranes (EMD Millipore) and blocked with 5% BSA (Beyotime Institute of Biotechnology) for 1 h at room temperature. The membranes were then incubated with the primary antibodies for 1 h at room temperature. Following the primary antibody incubation, the membranes were washed with 1X TBS-1% Tween-20 (TBST) three times for 15 min each and incubated with the secondary antibodies (1:2,000; Boster Biological Technology; cat. nos. BA1050 and BA1054) 1 h at room temperature. The membrane was subsequently washed with 1X TBST twice for 15 min and 1X TBS once for 15 min. Protein bands were visualized using a SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Inc.) and protein expression was quantified using ImageJ v1.8.0 software (National Institutes of Health).

Measurement of Hyp in liver tissues

Hyp content in the liver tissue was detected using an A030-2 kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. In brief, liver tissues (~200 mg) were homogenized and incubated overnight at 110°C. After filtering acid hydrolysates with a 0.45 µm filter, samples or Hyp standards (50 µl) were dried at 60°C, dissolved with methanol (50 µl) and treated with 1.2 ml 50% isopropanol and 200 µl chloramine-T solution at room temperature for 10 min. Ehrlich's solution (1.3 ml) was added and the samples were further incubated at 50°C for 90 min. The optical density was read at a wavelength of 558 nm using a spectrophotometer. A standard curve was constructed using serial two-fold dilutions of 1 mg hydroxyproline solution.

Detection of 11β-HSD1 activity in liver tissue and HSCs

Liver tissues (1.5-2.0 g) were homogenized in PBS, and the resulting homogenate was centrifuged at 1,000 × g at 4°C for 5 min. The supernatant was collected and centrifuged at 42,000 × g for 3 min at 4°C. The protein concentration of the supernatant was determined using the bicinchoninic acid method. The supernatant was placed in an ice bath for subsequent analysis. HSCs were lyzed and total protein was quantified using the bicinchoninic acid assay. 11β-HSD1 activity in the supernatant of rat liver tissue and total protein of HSCs was detected using the cortisol kit according to the manufacturer's instructions.

Preparation, culture and verification of rat primary HSCs

Primary HSCs were obtained from 20 additional SD rats (all details on rats and housing the same as above) by perfusion with Pronase E/Collagenase IV. Briefly, the rats were euthanized with 30 mg/kg body weight sodium pentobarbital and CO2 (20% container volume/min). After determining that the rats were immobile, not breathing and the appearance of pupil dilation, the abdomen of the rats was disinfected with 75% ethanol. The abdominal cavity was opened to expose the inferior vena cava and liver. The inferior vena cava was clipped, and the portal vein was cut, and a syringe pump was used to infuse EGTA solution (20 ml/ml at 40°C) from the inferior vena cava into the liver at a constant rate until the liver turned white. Subsequently, the liver was infused with 15 ml Pronase E solution and 20 ml Collagenase IV solution for 20 min each at 37°C. The liver was separated from the abdominal cavity and plated in a 100-mm Petri dish, then 5 ml Pronase E (0.2 g/l)/Collagenase IV (0.2 g/l) digestion solution was added to digest the liver tissue at 37°C for 15 min. The liver was crushed with ophthalmic scissors and a pipette was used to disperse the hepatocytes. The digested hepatocyte suspension was transferred to a 50 ml centrifuge tube and shook for 25 min on a plate shaker at 37°C. Subsequently, 10 ml pre-chilled rat HSC complete medium (4°C; cat. no. CM-R041; Procell Life Science & Technology Co., Ltd.) was added to stop the digestion. After filtering through a 200-mesh screen, the suspension was placed in a 50 ml centrifuge tube to undergo one-step density gradient centrifugation with 18% Nycodenz (W/V) at 1,450 × g for 22 min at room temperature. Trypan blue staining was conducted to evaluate the relative population of living HSCs at 37°C for 3 min. Quiescent HSCs were examined under a fluorescence microscope and appeared blue under UV light irradiation (328 nm wavelength). Desmin immunocytochemistry was conducted to determine the purity of the isolated primary HSCs. After culture in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 20% FBS (Biological Industries) at 37°C for 24 h, the majority of HSCs had attached to the dishes. Cell culture medium was replaced with DMEM containing 0.5% FBS and cultured at 37°C for another 24 h. Subsequently, 5×106 HSCs were treated with or without 5 ng/ml TGF-β1 at 37°C for another 48 h.

Immunocytochemistry

Rat HSCs were inoculated into a petri dish containing coverslips and treated with or without TGF-β1. After cells were grown to a monolayer, the coverslips were removed and washed twice with 1X PBS. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature, after which the slides were washed with 1X PBS for 5 min and the cells were permeabilized with 0.5% Triton for 15 min at room temperature. The slides were then washed twice with 1X PBS for 5 min each, blocked with 1% BSA (Beyotime Institute of Biotechnology) solution for 30 min at room temperature and incubated with the primary antibody diluted with 1% BSA solution at 37°C for 2 h. Subsequently, the slides were incubated with Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (cat. no. bs-0295G-AF488; BIOSS) diluted with 1% BSA solution at 37°C for 1 h, washed twice with 1X PBS for 5 min each time and stained with a 5% DAPI solution at room temperature for 2 min. Subsequently, the slides were mounted with an anti-quenching sealer and visualized under a fluorescence microscope (magnification, ×200).

11β -HSD1 gene knockdown and overexpression in LX2-HSCs

To overexpress 11β-HSD1, the expression plasmid pSuper-11β-HSD1 was constructed. Briefly, the full-length 11β-HSD1 cDNA was synthesized by Miao Ling Biotechnology Co., Ltd. and inserted into the parental vector pSuper (Addgene, Inc.). The pSuper-11β-HSD1 expression vector was confirmed by double enzymatic digestion with BamH1/XhoI and sequencing. The custom interference plasmids, including small interfering (si)RNA-11β-HSD1 and non-specific control (NC) siRNA-11β-HSD1-NC, were purchased from Miao Ling Biotechnology Co., Ltd. The sequences of the siRNAs were as follows: siRNA-11β-HSD1 sense, 5′-CAGAGAUGCUCCAAGGAAAGATT-3′ and antisense, 5′-UUUCCUUGGAGCAUCUCUGGUTT-3′; and siRNA-11β-HSD1-NC sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. A total of 5×106 LX2-HSCs cells were divided into the following groups: i) Normal control group (N); ii) the siRNA group, in which LX2-HSCs were transfected with 2 µg interference plasmid siRNA-11β-HSD1 and expression of 11β-HSD1 was silenced; iii) the negative control group (NC), in which LX2-HSCs were transfected with 50 nM plasmid siRNA-11β-HSD1-NC; iv) the pSuper group, in which LX2-HSCs were transfected with 1 µg pSuper-11β-HSD1 plasmid to overexpress 11β-HSD1; and v) the Vec group, in which LX2-HSCs were transfected with 1 µg pSuper-11β-HSD1 plasmid, which was used as a control. All cells were transfected using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). At 72 h post-transfection, total protein was extracted and the expression of 11β-HSD1 was measured in each experimental group by western blotting.

Cell cycle distribution and HSC proliferation

HSC proliferation was measured after 12, 24, 48, 72 and 96 h of culture using the CCK-8 cell proliferation kit, according to the manufacturer's instructions. HSC cell cycle distribution was detected using propidium iodide (PI) staining. Briefly, HSC cells were trypsinized, collected and then fixed and permeabilized using 70% (v/v) cold ethanol for 2 h at room temperature. Cells were subsequently incubated with 10 µg/ml PI/0.1% Triton X-100/0.1% RNase in PBS solution at 37°C for 30 min in the dark. Stained cells were analyzed on a BD FACS Calibur (BD Biosciences) using a fluorescence emission wavelength of 585 nm after an excitation wavelength of 488 nm. For each analysis, 10,000 events were evaluated, and DNA content was determined using ModFit LT v3.0 software (Verity Software House, Inc.).

Effect of changes in 11β-HSD1 expression on the release of collagen in HSCs

Cells from each group were collected and the concentration of collagen was measured using the ProCell collagen colorimetric assay kit, according to the manufacturer's protocol. Briefly, Sircol dye reagent was added to the cell culture supernatants (1,000 × g; 10 min; room temperature), stirred for 20 min at room temperature and centrifuged at 15,000 × g for 10 min at room temperature. The absorbance of the bound dye was measured at a wavelength of 555 nm on a spectrophotometer.

Statistical analysis

Statistical analyses were conducted using SPSS statistical software (version 17.0; SPSS, Inc.). At least three independent experiments were performed for each analysis. Data are expressed as the mean ± standard deviation. The normality of the data was analyzed using the Kolmogorov-Smirnov test. The homoscedasticity of data was analyzed using the Levene test. For normally distributed data with homogeneity, differences among groups were analyzed using one-way ANOVA followed by Fisher's LSD post hoc test. Data containing >3 groups were compared with the control group using one-way ANOVA followed by Dunnett's post hoc test. For comparisons within groups (TGF-β1− and TGF-β1+) and among groups analyzed using two-way ANOVA followed by the Bonferroni post hoc test. For non-normally distributed data, the Kruskal-Wallis test followed by the Bonferroni post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

Results

Role of 11β-HSD1 inhibition in liver fibrosis

As shown in Fig. 1A and B, excessive formation of fibrotic tissues was observed in the livers of rats treated with porcine serum (M group), whereas slight periportal inflammatory infiltration was observed in fibrotic rats treated with the 11β-HSD1 inhibitor (M+I group). Histopathological analysis demonstrated that the degree of liver fibrosis was significantly higher in the M group compared with the N group (P<0.01) and the M+I group (P<0.05). Together, the results suggested that 11β-HSD1 inhibition was effective against experimental liver fibrosis.

Figure 1. Effect of BVT.2733 on porcine serum-mediated liver fibrosis in rats. Liver tissues were obtained from the following three groups: i) N; ii) M; and iii) M+I. Pathological alterations were analyzed using H&E staining. (A) Representative images of H&E staining (magnification, ×40). Inflammatory cell infiltration was observed in the portal area of the liver in the positive group; no obvious indication of degeneration, necrosis and fibrotic tissue hyperplasia was observed. (B) Quantitative analysis of the histological images. ##P<0.01 vs. N; **P<0.01 and *P<0.05 vs. M. N, normal control group; M, model group; M+I, 11β-HSD1 inhibitor (BVT.2733) group; 11β-HSD1, 11β-hydroxysteroid dehydrogenase 1; H&E, hematoxylin and eosin.

Serum markers of fibrosis are ameliorated after treatment with an 11β-HSD1 inhibitor

As hepatic fibrosis developed in the experimental model, the levels of Col IV, PIIINP and HA significantly increased in a time-dependent manner in the M group (P<0.01), which was not observed in the N group (Fig. 2A-C). Although the markers of liver fibrosis increased as hepatic fibrosis developed in the presence of the 11β-HSD1 inhibitor (M+I group), the increase was significantly decreased compared with the M group (P<0.01).

Figure 2. Effect of porcine serum on liver fibrosis. As liver fibrosis progressed, the expression of (A) Col IV, (B) PIIINP and (C) HA in the serum was measured. #P<0.05, ##P<0.01 vs N; *P<0.05, **P<0.01 vs. M. The liver (D) Hyp content and (E) 11β-HSD1 enzyme activity. ##P<0.01 vs. N; **P<0.01 and *P<0.05 vs. M. Protein expression levels were (F) determined by western blotting and semi-quantified for (G) α-SMA, (H) 11β-HSD1 and (I) CTGF. #P<0.05, ##P<0.01 and ###P<0.001 vs. N; *P<0.05, **P<0.01 and ***P<0.001 vs. M. Col IV, type IV collagen; PIIINP, N-terminal pro-peptide of collagen type III; HA, hyaluronic acid; N, normal control group; M, model group; M+I, 11β-HSD1 inhibitor (BVT.2733) group; Hyp, hydroxyproline; 11β-HSD1, 11β-hydroxysteroid dehydrogenase 1; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor.

11β-HSD1 inhibition decreases liver Hyp content and enzyme activity

Subsequently, whether the Hyp content and 11β-HSD1 activity in the liver tissues of fibrotic rats were altered in response to fibrosis induction was investigated. Hyp content and 11β-HSD1 enzyme activity were significantly increased in the M group compared with the N group (P<0.01; Fig. 2D and E). By contrast, Hyp content and 11β-HSD1 enzyme activity were significantly decreased in the M+I group compared with the M group (P<0.01).

HSC activation decreases following 11β-HSD1 inhibition

Concomitant with serum markers of liver fibrosis, the levels of α-SMA, 11β-HSD1 and CTGF gradually increased in the M group as hepatic fibrogenesis progressed compared with the N group (P<0.01; Fig. 2F-I). By contrast, protein expression levels of α-SMA, 11β-HSD1 and CTGF in the liver tissues of the M+I group were significantly decreased compared with the M group (P<0.01), indicating that the 11β-HSD1 inhibitor prevented HSC activation.

Alteration of 11β-HSD1 during primary HSC activation

To determine whether 11β-HSD1 expression could be altered during HSC activation, primary HSCs were freshly prepared and treated with TGF-β1 in vitro. As shown in Fig. 3, the 11β-HSD1 and α-SMA expression levels were higher after the TGF-β1-mediated activation of primary HSCs compared with the controls.

Figure 3. Upregulation of 11β-HSD1 during primary HSC activation. Primary HSCs were prepared and treated with or without TGF-β1. Immunocytochemical staining of α-SMA and 11β-HSD1 protein expression was performed in HSCs with or without activation. Magnification, ×400. 11β-HSD1, 11β-hydroxysteroid dehydrogenase 1; HSCs, hepatic stellate cells; TGF-β1, transforming growth factor β 1; α-SMA, α-smooth muscle actin.

Silencing of the 11β-HSD1 gene in HSCs via siRNA transfection

As shown in Fig. 4A, the protein expression levels of 11β-HSD1 in LX2-HSCs transfected with siRNA-11β-HSD1 were significantly lower compared with the NC group (P<0.01). Furthermore, siRNA-mediated knockdown of 11β-HSD1 resulted in an 85% decreased in 11β-HSD1 expression levels. No significant difference in the expression levels of 11β-HSD1 protein were observed between the NC and the N groups.

Figure 4. Effects of 11β-HSD1 knockdown and overexpression on LX2-HSC proliferation. (A) 11β-HSD1 knockdown using siRNA-11β-HSD1. (B) 11β-HSD1 overexpression using pSuper-11β-HSD1. ##P<0.01 vs. N. (C) Effects of 11β-HSD1 knockdown and overexpression on LX2-HSC proliferation under normal culture conditions. (D) Effects of 11β-HSD1 knockdown and overexpression on LX2-HSC proliferation following TGF-β1 induction. #P<0.05 and ##P<0.01 vs. N. 11β-HSD1, 11β-hydroxysteroid dehydrogenase 1; HSCs, hepatic stellate cells; siRNA, small interfering RNA; N, normal control group; NC, non-specific control; Vec, group in which LX2-HSCs were transfected with the pSuper-11β-HSD1-vector plasmid; OD, optical density; TGF-β1, transforming growth factor-β1.

Overexpression of 11β-HSD1 in HSCs following transfection of the pSuper-11β-HSD1 expression vector

Eukaryotic expression of pSuper-11β-HSD1 plasmids was identified and the sequencing results demonstrated that the recombinant plasmid had no deletions, mutations or other alterations (data not shown). As shown in Fig. 4B, 11β-HSD1 protein expression in the pSuper group was significantly higher (~x3.4) compared with the Vec group (P<0.01). No significant difference in 11β-HSD1 protein expression was observed between the Vec and N groups.

Effect of alterations in 11β-HSD1 expression on HSC proliferation

HSCs with 3 different expression levels of 11β-HSD1 were challenged with TGF-β1 (5 ng/ml). Proliferation was measured at different time points (12, 24, 48, 72 and 96 h) using the CCK-8 assay.

As observed in Fig. 4C, under normal culture conditions, LX2-HSC proliferation was higher in the pSuper group compared with the N group (P<0.01), whereas LX2-HSC proliferation was decreased in the siRNA group compared with the N group (12 h, P<0.05; 24, 48, 72 and 96 h, P<0.01). However, no significant differences were observed between the NC and Vec groups. Following TGF-β1 induction, LX2-HSC proliferation was enhanced compared with untreated LX2-HSCs. Cell proliferation was significantly increased in the pSuper group compared with the N group (P<0.01). Although LX2-HSC proliferation in the siRNA group was higher following TGF-β1 treatment compared with untreated cells, the effect of TGF-β1 on the LX2-HSC proliferation was significantly reduced compared with the N group (P<0.01). No significant differences were observed between the NC and Vec groups.

Effect of TGF-β1 induction on cell cycle distribution in LX2-HSCs

HSC cycle distribution was detected via flow cytometry (Fig. 5). In the siRNA group (11β-HSD1 knockdown group), the cells displayed increased G0/G1 phase arrest compared with the NC group. No significant differences were observed in the cell cycle distribution between the NC and N groups. In the pSuper group (11β-HSD1 overexpression group), a decreased number of cells were in the G0/G1 phase and an increased number of cells were in the S and G2/M phases compared with the N group. However, no significant differences were observed in cell cycle distribution between the Vec and N groups.

Figure 5. Effect of siRNA-11β-HSD1 or pSuper-11β-HSD1 on LX2-HSC cycle. Cell cycle distribution of unstimulated LX2-HSCs was (A) determined by flow cytometry and (B) quantified. Cell cycle distribution of TGF-β1-stimulated LX2-HSCs was (C) determined by flow cytometry and (D) quantified. *P<0.05, **P<0.01 vs. N. siRNA, small interfering RNA; 11β-HSD1, 11β-hydroxysteroid dehydrogenase 1; HSCs, hepatic stellate cells; TGF-β1, transforming growth factor β1; NC, non-specific control; N, normal control group; Vec, group in which LX2-HSCs were transfected with the pSuper-11β-HSD1-vector plasmid.

Influence of TGF-β1 on the expression of α-SMA, 11β-HSD1 and CTGF in vitro

TGF-β1 treatment significantly increased the protein expression levels of α-SMA, 11β-HSD1 and CTGF in LX2-HSCs cells compared with untreated cells in all experimental groups (Fig. 6A-H). The pSuper group (11β-HSD1 overexpression group) displayed altered the protein expression levels of α-SMA, 11β-HSD1 and CTGF (Fig. 6E-H) to a greater extent compared with the siRNA group (11β-HSD1 knockdown group; Fig. 6A-D), whereas no statistical differences were observed between the NC and Vec groups (Fig. 6A-H).

Figure 6. Effects of siRNA-11β-HSD1 or pSuper-11β-HSD1 on protein levels of α-SMA, 11β-HSD1 and CTGF, enzyme activity of 11β-HSD1 and collagen secretion in LX2-HSCs. Protein expression levels were (A) determined by western blotting and semi-quantified for (B) α-SMA, (C) 11β-HSD1 (D) CTGF following siRNA transfection. Protein expression levels were (E) determined by western blotting and semi-quantified for (F) α-SMA, (G) 11β-HSD1 and (H) CTGF following pSuper transfection. Effects of siRNA-11β-HSD1 or pSuper-11β-HSD1on (I) 11β-HSD1enzyme activity and (J) collagen secretion in LX2-HSCs. ★P<0.05, ★★P<0.01 and ★★★P<0.001; ✩P<0.05 and ✩✩P<0.01; ▼P<0.05 and ▼▼P<0.01; ##P<0.01. siRNA; small interfering RNA; 11β-HSD1, 11β-hydroxysteroid dehydrogenase 1; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; HSCs, hepatic stellate cells; N, normal control group; NC, non-specific control; Vec, group in which LX2-HSCs were transfected with the pSuper-11β-HSD1-vector plasmid; TGF-β1, transforming growth factor β1.

Effect of TGF-β1 on collagen release and 11β-HSD1 enzyme activity in HSCs in culture

As shown in Fig. 6I and J, LX2-HSCs in the pSuper group displayed increased collagen secretion, which was associated with enhanced 11β-HSD1 enzyme activity, compared with the control group. By contrast, LX2-HSC collagen secretion was significantly reduced in the siRNA group, which was accompanied by a decrease in 11β-HSD1 enzyme activity compared with the control group. No significant differences were observed between the NC and Vec groups. Following TGF-β1 induction, collagen secretion and 11β-HSD1 enzyme activity was increased in all experimental groups. Significant increases in these parameters were observed in the pSuper group.

Discussion

Consistent with previous studies (27,32,33), the present study indicated that there was an increase in collagen fibers in the liver, which was associated with significantly elevated levels of Hyp, and serum Col IV, HA and PIIINP in an experimental rat model of hepatic fibrosis compared with control rats.

11β-HSD1 is a key enzyme that catalyzes GC synthesis and promotes the conversion of GCs to active forms (34). It is a bidirectional catalytic enzyme with redox capacity, which catalyzes the conversion between the biologically inactive forms of cortisol (cortisone) and the biologically active form (hydrocortisone) (35). 11β-HSD1 is distributed in the liver, fat and kidney, as well as other organs, and serves various physiological roles, such as the intracellular metabolism of GCs and oxysterol metabolism, while its aberrant expression is associated with insulin resistance, visceral obesity and hypotension (36). In the present study, a hepatic fibrosis rat model was established by intraperitoneal injection of porcine serum. Alterations in 11β-HSD1 expression and biochemical parameters associated with the development of hepatic fibrosis were systematically evaluated. The results demonstrated that, in addition to the progression of liver fibrosis, 11β-HSD1 expression levels increased in the liver, which may be attributed to enhanced 11β-HSD1 activity and increased levels of markers of liver fibrosis, including Col IV, HA, PIIINP, α-SMA and CTGF. The use of BVT.233, an inhibitor of 11β-HSD1, markedly reduced levels of the biochemical markers of liver fibrosis and the severity of hepatic fibrosis. Subsequently, the results suggested that 11β-HSD1 overexpression promoted cell cycle progression, enhanced LX2-HSC proliferation, increased α-SMA, CTGF and collagen secretion, and induced TGF-β1 in LX2-HSCs. By contrast, 11β-HSD1 knockdown displayed the opposite effects, including diminishing TGF-β1-mediated stimulation of LX2-HSCs. The results suggested that 11β-HSD1 may participate in the development and progression of hepatic fibrosis by activating HSCs and regulating the expression of profibrotic markers.

Several studies on hepatic 11β-HSD1 have generated conflicting results. For example, Konopelska et al (37) reported that there was no association between hepatic 11β-HSD1 expression and the pathology of fatty liver or non-alcoholic steatohepatitis (NASH) in humans, whereas Ahmed et al (38) suggested that in the early stages of nonalcoholic fatty liver disease (NAFLD), with steatosis alone, hepatic 11β-HSD1 activity decreases with the progression to NASH, which is associated with increased 11β-HSD1 levels. Notably, a more recent study conducted by Zou et al (39) demonstrated that specific deletion or inhibition of 11β-HSD1 enhances the activation of HSCs in a murine model of liver fibrosis induced by carbon tetrachloride (CCl4), which contradicts the results of the present study that used a rat model of liver fibrosis induced by persistent immune injury. In the face of this disagreement, independent experiments were carefully performed, and the findings were confirmed in the present study. There is a possibility that the measured outcomes varied due to the differences in animal models of liver fibrosis induced by chronic and acute liver injury. Chemical liver injury induced by CCl4 is the usual fibrosis model of acute liver injury; however, in the present study, porcine serum was used to induce liver fibrosis. Chronic viral hepatitis, especially chronic hepatitis B, is the major cause of liver fibrosis in China, and liver fibrosis caused by viral hepatitis is a process of chronic immune injury rather than a simple, acute chemical injury (40). Therefore, heterogeneous serum samples were selected to induce hepatic fibrosis in rats in the present study. Notably, Chen et al (41) reported that gossypol, an inhibitor of 11β-HSD1, ameliorates liver fibrosis in a rat model of type 2 diabetes-related liver fibrosis, which was consistent with the observations of the present study, suggesting the antifibrotic effects of the 11β-HSD1 specific siRNA and inhibitor in rats. In addition, another study demonstrated that gossypol had an antifibrotic effect on lung fibrosis (42). Chen et al (41) demonstrated that the number of activated HSCs is significantly reduced following treatment with the 11β-HSD1 inhibitor. Indeed, the present study demonstrated that a decrease in 11β-HSD1 expression significantly inhibited LX2 cell proliferation, with cell cycle arrested in the G0/G1 phase. In support of the role of 11β-HSD1 in cell cycle, 11β-HSD1 overexpression significantly promoted LX2 cell proliferation with increased numbers of cells in the S and G2/M phases. Given the key events in liver fibrosis, including HSC activation and proliferation, the present study hypothesized that there may be a mechanism that partially explains why the inhibition of 11β-HSD1 led to an antifibrotic effect. The results of the present study and the aforementioned studies have provided scientific evidence that inhibiting 11β-HSD1 holds therapeutic potential for chronic inflammatory liver fibrosis.

As 11β-HSD1 blocks hydroxycortisone, the active form that can activate HSCs, it is worth determining whether the hydroxycortisone could rescue 11β-HSD1 inhibition. Collectively, the study of 11β-HSD1 in liver fibrosis requires further investigation to improve the understanding of the role of 11β-HSD1 and its underlying mechanisms in liver fibrosis.

In conclusion, the results suggested that upregulation of 11β-HSD1 in the liver may be associated with the progression and development of liver fibrogenesis, and its mechanism may be related to HSC activation and proliferation, as well as increased CTGF and α-SMA production.

Acknowledgements

Not applicable.

Funding

The present study was financially supported by the National Natural Science Foundation of China (grant no. 81771827).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

WX and ZGL designed the study. WX, MHL, PFR, HYZ, JG and YQP performed the experiments. WX collected the data. WX, MHL, HYG and ZGL analyzed the data. WX, PFR, HYZ, JG and YQP interpreted the data. WX and ZGL drafted the manuscript, which was revised for content by MHL and HYG. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Institutional Animal Care and Use Committee [Xiang license no. SCXK (Xiang) 2011-0003]. All procedures involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References