All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Wuhan University (Wuhan, China) and adhered to the ethical guidelines of the International Association for the Study of Pain (20). A total of 20 male Sprague-Dawley rats, aged 6–8 weeks old and weighing 180–200 g, were purchased from the Center for Animal Experiment, Wuhan University (Wuhan, China) and maintained in specific pathogen-free conditions. The animals were housed at a constant temperature (20–24°C) and humidity (45–50%) under a 12-h light/dark cycle, and provided with food and water ad libitum. Rats were randomly assigned to two groups: The bile duct ligation (BDL) group and the control group (10 rats/group). Biliary hepatic cirrhosis was induced by ligation of the common bile duct. Briefly, rats were anesthetized with 10% (w/v) chloral hydrate (3.5 ml/kg; Jiangsu Lianshui Pharmaceutical Co., Ltd., Lianshui, China) through intraperitoneal injection. In the BDL group, a 1.5 cm midline incision was made and the common bile duct was located and double ligated with 3.0 mm silk ligatures. Sham-operated animals in the control group received a midline incision and manipulation of the common bile duct, without ligation. All rats in each group survived the procedure. At 8 weeks following surgery, the animals were anesthetized and sacrificed by carbon dioxide euthanasia. At the time of death, the BDL was confirmed to be intact with proximal dilatation of the common bile duct. Portions of the right and left liver lobes and the portal vein were fixed in 10% buffered formalin for 24 h at room temperature and embedded in paraffin for histological examination.

Formalin-fixed, paraffin-embedded portions of the liver lobes and portal vein were cut into 3 mm sections and stained with hematoxylin and eosin (H&E) as previously described (21). Briefly, sections were deparaffinized, hydrated, stained in alum hematoxylin, differentiated with acid alcohol, washed with tap water, stained with eosin and dehydrated. Sections were then observed under a light microscope (BX51; Olympus Corporation, Tokyo, Japan), by a pathologist who was blinded to the groups. Ten high-power fields were randomly collected. The thickness of portal vein wall was quantified using Image J software version 1.45 (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry was performed on sections of formalin-fixed, paraffin-embedded liver to detect expression of TMEM16A, extracellular signal-regulated kinase 1 and 2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2). Briefly, the 3-µm-thick sections were prepared as described above and incubated with 3% hydrogen peroxide for 10 min at room temperature to block the endogenous peroxidase activity. The sections were then boiled in sodium citrate buffer (pH 6.0) to retrieve antigen. The sections were incubated with goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 15 min at room temperature. TMEM16A polyclonal antibody (cat. no. sc-69343; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:500 dilution), ERK1/2 monoclonal antibody (cat. no. 4695; 1:400 dilution) and p-ERK1/2 monoclonal antibody (cat. no. 4370; 1:400 dilution) (both, Cell Signaling Technology, Inc., Danvers, MA, USA) were added and incubated overnight at 4°C. The following day, the sections were rinsed in PBS three times and incubated with horseradish-peroxidase conjugated secondary antibody (cat. no. HAF008; 1:1,000 dilution; R&D Systems, Inc., Minneapolis, MN, USA) for 15 min at room temperature. A 3,3′-diaminobenzidine color developing substrate was added and the sections were examined microscopically for color development for 5–10 min, mounted and visualized under a light microscope (BX51; Olympus Corporation).

Primary single PVSMCs were isolated as previously described (22,23). Briefly, the portal vein was dissected under sterile conditions. The connective tissues surrounding the outer membrane and endothelial cells of tunica intima were carefully stripped off under a dissecting microscope. The smooth muscle layer of the tunica media was separated from the tunica adventitia with a blunt dissection technique and then cut into small fragments (1–2 mm³), which were placed in 25 cm³ culture plates (~20/plate). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1X strength Antibiotic/Antimycotic (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was carefully added to the culture plates so as not to disturb adhered explants. Culture plates were placed in a 37°C incubator (5% CO₂). Cells started growing from explants within 1 week and became confluent in 4 weeks. Cells were fixed for 10 min in glacial acetate at room temperature and immersed in 0.3% H₂O₂ for 30 min to quench endogenous peroxidase activity at room temperature. They were then incubated with primary antibodies directed against smooth muscle α-actin (cat. no. SAB2500963; 1:1,000 dilution; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 1 h and secondary antibodies conjugated to FITC (cat. no. SAB3700002; 1:1,000 dilution; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Morphometry was performed from three random fields of each slide using the Image Pro Plus software version 6.0 (Media Cybernetics Inc., Rockville, MD, USA). An Olympus-BX53 microscope (Olympus Corporation, Tokyo, Japan) was used at a magnification of ×200 to observe the slides and calculate the number of smooth muscle α-actin positive PVSMCs.

Recombinant pEGFP-TMEM16A and pEGFP-N1 GV358 lentivirus plasmids were packaged using Lenti-Easy Packaging mix (all Shanghai GeneChem Co., Ltd, Shanghai, China) and the virus titer was determined using methods of fluorescence enumeration, as previously described (24). Briefly, PVSMCs were plated on a 6-well plate with DMEM supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO₂ at a density of 1–1.5×10⁵/ml. After 24 h, pEGFP-TMEM16A or pEGFP-N1 plasmids were transfected into the cells with Lipofectamine^™ 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in OPTI-MEM I reduced serum medium (Gibco; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After 6 h the cells were rinsed with PBS and switched to 5% fetal bovine serum-containing DMEM. The expression of green fluorescence protein was observed under an inverted fluorescent microscope (Olympus-IX71; Olympus Corporation) 3 days later and the transfection efficiency was calculated. The average rate of transfection=positive cells number/total number of cells ×100% (one visual field of the microscope) (25).

Stock solutions (5 mM) of the TMEM16A inhibitor, T16Ainh-A01 (cat. no. SML0493; Sigma-Aldrich; Merck KGaA) were made in dimethyl sulfoxide (DMSO; cat. no. D-2650-5; Sigma-Aldrich) and stored at −20°C. The compound was freshly diluted on the day of the experiment to a final concentration of 10 µM. DMSO was used at a final concentration of 1:500 for the vehicle control. Cells were treated with 10 µM T16Ainh-A01 at 37°C in 5% CO₂ for 6 h.

Following treatment with plasmid transfection or the TMEM16A inhibitor, T16Ainh-A01 the cells were detached from the 6-well plates with 0.2% trypsin, harvested, washed twice with PBS and centrifuged twice at 300 × g for 5 min at 4°C. Then, the supernatant was discarded and the pellet was resuspended in 400 µl Annexin V binding buffer (Beyotime Institute of Biotechnology, Haimen, China) at 20°C for ≥12 h. Cells were subsequently treated in PBS with RNase A for 30 min at room temperature and stained with propidium iodide. Flow cytometric analysis was performed using EPICS XL-MCL^™ software (Beckman Coulter, Inc., Brea, CA, USA) and a FACScan Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to determine the DNA contents. A concentration of 1–5×10⁶/ml cells were analyzed for each sample, and the experiment was repeated ≥3 times. The S-phase cell ratio and proliferation index were calculated at the same time based on the following equation: S-phase cell ratio=S/(G₀/G₁+S+G₂/M); proliferation index=(S+G₂/M)/(G₀/G₁ +S+G₂/M) (26).

Western blotting analysis was performed as previously described (27). In brief, Portal vein samples were lysed in RIPA lysis buffer (50 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 10 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l EDTA, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate) for 20–30 min on ice. Protein concentrations were determined using the Lowry protein assay. Samples were then boiled in loading buffer and separated by 10% SDS-PAGE, 20 µg of protein was loaded per lane. After electrophoresis, protein was transferred onto a nitrocellulose membrane, which was incubated with blocking solution [10% non-fat dry milk in TBS containing 0.05% Tween-20 (TBST)] for 2 h. Membranes were immunoblotted with primary antibodies, including TMEM16A (1:1,000 dilution), ERK 1/2, p-ERK 1/2 (both 1:2,000 dilution) and β-actin (cat. no, ab8229; 1:10,000 dilution; Abcam, Cambridge, UK) overnight at 4°C. After washing with TBST, the membranes were incubated with anti-rabbit immunoglobulin conjugated to horseradish peroxidase secondary antibody (cat. no. sc-362261; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The membranes were developed using an enhanced chemiluminescence western blotting kit (EMD Millipore, Billerica, MA, USA), then exposed to X-ray film. The bands of interest were quantified by Image Pro Plus version 6.0 analysis software.

Data are presented as the mean ± standard deviation. SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used for data analysis. Statistical analysis was performed using Student's t-test for the comparison of two groups, or one-way analysis of variance followed by a Dunnett's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

First, it was verified that biliary hepatic cirrhosis and portal vein remodeling rats by BDL had been established successfully (Fig. 1A). H&E staining of liver pathological sections indicated that lymphocyte infiltration and pseudolobuli formation were obvious in the liver of the BDL group. Normal structures were observed in the sham-operated group. The portal vein remodeling in PHT was characterized by thickening of the vein. The thickness of portal vein was significantly increased in the BDL group compared with the sham-operated group (P<0.05; Fig. 1A).

Immunohistochemistry results indicated that the TMEM16A protein expression level was decreased in BDL rats compared with the control (Fig. 1B). Western blot analysis indicated that the protein expression level of TMEM16A was significantly decreased in BDL rats (P<0.05; Fig. 2). p-ERK1/2 expression was indicated to be increased in BDL rats in the immunohistochemistry results (Fig. 1B) and significantly increased in the results of western blot analysis (P<0.05; Fig. 2). These results suggested that TMEM16A may have a negative association with PVSMC proliferation and portal vein remodeling.

PVSMCs were successfully isolated and cultured. Then, pEGFP-TMEM16A and pEGFP-N1 plasmids were used to transfect PVSMCs (Fig. 3A and B). The expression of TMEM16A was significantly upregulated in the pEGFP-TMEM16A transfection plasmids group compared with the control and pEGFP-N1 plasmids groups (Fig. 3C and D).

The expression of TMEM16A was upregulated by pEGFP-TMEM16A transfection plasmids as described above and CaCCs were inhibited by T16Ainh-A01 (10 µM) as described previously (28). Compared with the negative control pEGFP-N1 plasmid group, the S rate (34.44±3.72 vs. 15.56±2.36; P<0.05) and proliferation index (45.06±0.09 vs. 20.40±0.02; P<0.001) of pEGFP-TMEM16A plasmid-transfected cells were significantly increased (Fig. 4). The S rate (4.51±0.45 vs. 16.72±0.21) and proliferation index (8.59±0.02 vs. 22.8±0.01) of T16Ainh-A01 treated cells were significantly reduced compared with the control group (P<0.01; Fig. 4). It was identified that overexpression of TMEM16A facilitated PVSMC proliferation, while inhibition of TMEM16A reduced PVSMC proliferation. TMEM16A contributed to PVSMC proliferation in vitro, but in vivo, it may be a negative regulator of cell proliferation. The contradictory results in vivo and in vitro require further research.