The LX2 human HSC cell line originated from the American Type Culture Collection (Manassas, VA, USA) was purchased from Shanghai Fuxiang Biotechnology Co., Ltd. (Shanghai, China). It was cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM-h) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, USA) and maintained in a 5% CO₂ atmosphere at 37°C. OSU-03012 was purchased from Selleck Chemicals (Houston, TX, USA) and was dissolved in dimethyl sulfoxide (DMSO; Sangon Biotech, Co., Ltd., Shanghai, China) at a concentration of 50 μM as stock solution for further use. The rabbit anti-human antibodies against P15INK4B (#4822; polyclonal), p16INK4A (#4824; polyclonal), p21Cip1 (#9932; monoclonal), P27Kip1 (#9932; monoclonal), AKT (#9272; polyclonal), p-AKT (activated AKT; #9275; polyclonal) and β-actin (#4967; polyclonal) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; 1:1,000 dilution) and diluted with Primary Antibody Dilution Buffer (Beyotime Institute of Biotechnology).

The cell counting kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology, Shanghai, China) was used to evaluate cell proliferation. LX2 cells were seeded in 96-well plates at a density of 2×10³ cells/well in DMEM-h supplemented with 10% FBS. Following a resting period of 24 h, cells were washed with phosphate-buffered saline (PBS; Medicago AB, Uppsala, Sweden) and exposed to either DMSO alone or a series of dilutions of OSU-03012 in DMSO (1, 5 or 15 μM). Subsequent to incubation for another 24, 48 or 72 h, the medium was replaced with fresh DMEM-h. A total of 10 μl CCK-8 was added to each well and the cells were incubated in the presence of CCK-8 for 3 h. The absorbencies were determined with a DTX 800 Multimode Detector (Beckman Coulter, Brea, CA, USA) at a wavelength of 450 nm. The absorbance values were normalized by subtracting blank values obtained from untreated cells.

Following a resting period of 0, 24, 48 or 72 h subsequent to the addition of 1 μM OSU-03012, the total RNA in the LX2 cells was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. The RNA concentrations were assessed by spectrophotometry at a wavelength of 260 nm using a Nano-100 spectrophotometer (Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, China). RNA was reverse-transcribed using the PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) and qPCR was performed using the 7500 Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). The PCR primer sequences used were as follows: Type I collagen, F 5′-TGA CGA GAC CAA GAA CTG-3′ and R 5′-CCA TCC AAA CCA CTG AAA-3′; α-smooth muscle actin (α-SMA), F 5′-TTC GTT ACT ACT GCT GAG CGT GAG A-3′ and R 5′-AAA GAT GGC TGG AAG AGG GTC-3′; internal reference gene GAPDH, F 5′-ACC ACA GTC CAT GCC ATC AC-3′ and R 5′-TCC ACC ACC CTG TTG CTG T-3′. The PCR cycling conditions were as follows: 95°C for 30 sec, 40 cycles of 95°C for 5 sec and 60°C for 34 sec.

Approximately 5×10⁵ LX2 cells were seeded onto 10-cm dishes and incubated at 37°C overnight. The cells were treated with either DMSO alone or 1 μM OSU-03012 in DMSO for 48 or 72 h, and then suspended by treatment with trypsin 0.25%-EDTA (Biosera, Boussens, France) and fixed with 70% ice-cold ethanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) overnight at 4°C. Subsequent to washing the cells with PBS and resuspending in the binding buffer (Beyotime Institute of Biotechnology) containing Annexin V-fluorescein isothiocyanate and propidium iodide (PI) according to the manufacturer’s instructions, apoptosis was assessed by flow cytometry using a FACScan cytometer (Beckman, Pasadena, CA, USA).

SA-β-Gal staining was performed using the Senescence-Associated β-Galactosidase Staining kit (Beyotime Institute of Biotechnology). Approximately 3×10⁵ LX2 cells were seeded onto 6-cm dishes and incubated at 37°C overnight. The cells were treated with 1 μM OSU-03012 in DMSO or DMSO alone for 48 h or 72 h. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 15 min at room temperature. Next, the cells were incubated overnight at 37°C in darkness with the working solution containing 0.05 mg/ml X-gal (Beyotime Institute of Biotechnology) and viewed under an optical microscope (TE2000-S; Nikon Corp., Tokyo, Japan).

Approximately 5×10⁵ LX2 cells were seeded onto 10-cm dishes and incubated at 37°C overnight. The cells were treated with 1 μM OSU-03012 in DMSO or DMSO alone for 48 h or 72 h and then suspended by treatment with trypsin and fixed with 70% ice-cold ethanol overnight at 4°C. The cells were then stained with PI in the presence of RNase A (Beyotime Institute of Biotechnology). The DNA content was analyzed by flow cytometry (FACScan cytometer; Beckman) and data were analyzed using Modfit LT software version 3.2 (Verity Software House, Inc., Topsham, ME, USA).

Following the above treatments, the LX2 cells were lysed in protein extraction buffer (Beyotime Institute of Biotechnology) followed by incubation at 95°C for 5 min. Samples were separated using an SDS-PAGE system (MINI-P TET SYS/PPAC BASIC; Bio-Rad Laboratories, Inc., Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories), blocked with 5% nonfat milk in Tris-buffered saline/Tween-20 (Shanghai BioScience Co., Ltd., Shanghai, China) for 1 h and probed with the antibodies at 4°C overnight. The membranes were then incubated with corresponding horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit secondary antibody (#HA1001-100; 1:2000; Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China) for 1 h at room temperature. The immunoblots were visualized using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA).

All data were collected from a minimum of three independent replicates. Analysis was performed using SPSS software, version 11.0 (SPSS, Inc., Chicago, IL, USA). Comparisons between groups were performed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

To examine whether OSU-03012 has an inhibitory effect on LX2 cell proliferation, LX2 cells were treated with a range of doses (0–15 μM) of OSU-03012 and assessed for viability using the CCK-8 assay. As demonstrated in Fig. 1A, the growth of cells was suppressed by OSU-03012 in a dose-dependent manner, indicating that OSU-03012 may inhibit LX2 cell proliferation.

Activated HSCs express type I collagen and α-SMA, which primarily contribute to liver fibrogenesis. To investigate the effect of OSU-03012 on the activation of HSCs, the mRNA levels of the hepatic profibrotic factors type I collagen and α-SMA were analyzed using RT-qPCR. Following 48 or 72 h of exposure, a reduction in the mRNA levels of type I collagen and α-SMA was observed in OSU-03012 cells, as presented in Fig. 1B. These results demonstrate the inhibitory effect of OSU-03012 on the activation of HSCs, indicating its potential function against hepatic fibrosis.

It was reported that the inhibitory effect of OSU-03012 on cell proliferation was mediated via the induction of cell apoptosis in certain cancer cells (6–11). To verify this hypothesis, the ability of OSU-03012 to induce apoptosis in LX2 cells was investigated by flow cytometric analysis. As demonstrated in Fig. 2, apoptosis was not induced in LX2 cells treated with OSU-03012, compared with those treated with DMSO. These results indicated that OSU-03012 did not inhibit the growth of LX2 cells via the induction of apoptosis.

Cell senescence involves normal cells losing the ability to proliferate, thus it was hypothesized that the inhibition of LX2 cells by OSU-03012 is mediated by senescence. LX2 cells were exposed to OSU-03012 and assessed for senescence using the SA-β-Gal assay 48 h later. The cells treated with OSU-03012 displayed a large increase in SA-β-Gal activity (P<0.02; Fig. 3) compared with the control (DMSO). Senescence-associated morphological alterations were observed in the OSU-03012-treated group, with the cells frequently becoming flat with pyknosis of the nucleus. These results demonstrate that OSU-03012 may induce senescence in LX2 cells.

Cell senescence is a stable form of cell cycle arrest in mitotic cells, usually at the G₁ phase. However, certain cells become senescent at the G₂ or S phase (3). Thus, the influence of OSU-03012 on the cell cycle of LX2 cells was investigated. The cells treated with OSU-03012 for 48 or 72 h were stained with PI and then subjected to flow cytometry. As demonstrated in Fig. 4, LX2 cells treated with OSU-03012 exhibited a significant ~20× increase in G₁-arrested cells compared with those treated with DMSO (P<0.05). These results suggest that OSU-03012 may induce G₁ phase arrest in HSCs.

There are several pathways known to be associated with cell senescence. P53 promotes senescence by inhibiting cyclin-dependent kinases 2 and 4 by transactivating p21 or p16 (INK4a) (3,12). In addition, p15 and p27 have been identified to be associated with cell senescence (13,14). Therefore, to elucidate the mechanism of OSU-03012 in the induction of senescence in LX2 cells, total proteins were extracted from cells treated with 1 μM OSU-03012 or DMSO, for 48 or 72 h and analyzed by western blot analysis with the antibodies against p15, p16, p21 and p27. As demonstrated in Fig. 5, at 48 and 72 h, OSU-03012 significantly increased the protein levels of p16, p21 and p27, compared with DMSO-treated cells (P<0.01). No significant difference was observed in the levels of p15 between OSU-03012- and DMSO-treated cells at 48 or 72 h.

Furthermore, the PDK1/AKT signaling pathway, an important senescence regulator, has been suggested to be involved in the inhibition of proliferation in certain cells by OSU-03012 (15,16), but its role in LX2 cells remains unclear. To investigate whether OSU-03012 acts via the inhibition of the PDK1/AKT signaling pathway in LX2 cells, the levels of AKT and p-AKT were also measured in the western blot analysis. As demonstrated in Fig. 5, the level of p-AKT was reduced following treatment with OSU-03012 (P<0.01). These results suggest that OSU-03012 induced senescence of LX2 cells via p16, p21, p27 and the PDK1/AKT signaling pathway.