HSC-T6 cells were purchased from the Chinese Academy of Sciences (Beijing, China), and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gemini, Australia). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. IL-22 was purchased from R&D Systems (Minneapolis, MN, USA) and TGF-β1 from Cusabio Biotech Co., Ltd. (Wuhan, China).

The effects of IL-22 on HSC-T6 cell viability were evaluated with the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) cell proliferation assay. Cells were seeded in 96-well plates at a density of 1×10⁴ cells/well. After overnight growth, the cells were incubated with various concentrations of IL-22 (0–1,000 pg/ml) (R&D Systems). After cell treatment for 24 h, 10 µl CCK-8 solution was added into each well. Absorbance was measured at 450 nm with background at 655 nm, using a micro plate reader (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were repeated three times.

Apoptosis was assessed, after 24 h of treatment with IL-22, with the APC Annexin V Apoptosis Detection kit and PE Active Caspase-3 Apoptosis kit (both from BD Pharmingen, San Diego, CA, USA) according to the manufacturers' instructions. Briefly, HSC-T6 cells were treated with IL-22, followed by staining in fluorescence activated cell sorter (FACS) buffer (PBS, 2% bovine serum albumin, 0.1% sodium azide) for 10 min at 4°C. Finally, cells were washed and assessed on BD FACSCalibur Flow Cytometer (BD Pharmingen). Data were analyzed with the FlowJo software (Tree Star, Ashland, OR, USA).

RNA was extracted from cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed with a cDNA Reverse Transcription kit (Takara Bio, Inc., Otsu, Japan). RT-qPCR was performed with a SYBR-Green Master Mix kit (Takara Bio, Inc.). Relative mRNA levels were normalized to GAPDH mRNA expression, and calculated by the 2^−∆∆Cq method. The primer sequences are summarized in Table I.

Samples were homogenized in lysis buffer containing Tris-HCl, NP-40, NaCl, ethylene diamine-tetra acetic acid, NaN₃, phenylmethylsulfonyl fluoride, aprotinin, and leupeptin (pH 7.5), and centrifuged at 16,000 rpm at 4°C for 10 min. Protein concentration was determined by BCA (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of total protein (30 µg) were separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. Antibodies against SMA were obtained from Abcam (Cambridge, MA, USA). Anti-GAPDH was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-Notch3 was manufactured by Abcam (Cambridge, UK). Then membranes were incubated with various primary antibodies overnight at 4°C, followed by incubation with secondary antibodies and detection by enhanced chemiluminescence (ECL) (Odyssey; LI-COR Biosciences, Lincoln, NE, USA).

Data are mean ± standard deviation (SD) from three independent experiments. Statistical analysis was performed by unpaired Student's t-test, with two tailed P<0.05 considered statistically significant. Multiple groups were assessed by one-way analysis of variance and Dunnett's post hoc test. The SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

We first assessed the anti-proliferative effects of IL-22 on hepatic cells. Increasing concentrations of IL-22 were respectively applied to HSC-T6 cells, and cell proliferation was determined by CCK-8 assay. Cell viability was significantly decreased at high IL-22 amounts (Fig. 1A). However, IL-22 had no effect on apoptosis inHSC-T6 cells. As shown in Fig. 1B and C, there was no significant difference between IL-22 treatment and control groups. Taken together, these results indicated that IL-22 may exert anti-proliferative effects, independent of apoptosis.

Increased expression levels of α-SMA and collagen are commonly recognized as biomarkers of HSC activation (3). Next, we assessed the expression levels of α-SMA and multiple hepatocyte-associated proinflammatory cytokines to verify the inhibitory effects of IL-22 on HSC-T6 proliferation. Interestingly, application of IL-22 inhibited α-SMA expression in a dose-dependent manner (Fig. 2A). Furthermore, we assessed whether common pro-inflammatory cascades were inhibited by IL-22. TGF-β and tumor necrosis factor-α (TNF-α) are the most important mediators of inflammatory responses in HSCs, while ICAM-1 is an essential cell adhesion molecule (8,9). As shown in Fig. 2B-E, the mRNA levels of TGF-β, TNF-α, and ICAM-1 were simultaneously decreased by IL-22 treatment in HSC-T6 cells, suggesting that these effectors are downstream of IL-22. Collectively, HSC inactivation by IL-22 was confirmed by the downregulation of α-SMA and related-inflammatory cytokines.

We further explored the possible downstream effectors upon TGF-β stimulation. Several studies previously described a functional interaction between the TGF-β superfamily of proteins and Notch signaling in multiple biological processes (15,16). The results demonstrated that Notch3 levels were remarkably increased in HSC-T6 cells after TGF-β1 treatment, in a dose-dependent manner (Fig. 3A), in line with densitometry analysis (Fig. 3B). Furthermore, not only was the expression of Notch3 receptor increased by TGF-β1, but Notch pathway activation was confirmed, as indicated by substantial upregulation of the downstream effectors Hes family basic helix-loop-helix transcription factor-1 (Hes-1), Hes-5 and Hey-1 (Fig. 3C-E). Taken together, these results indicated that TGF-β is responsible for the activation of Notch signaling in HSC-T6 cells.

As TGF-β1-induced Notch3 upregulation, we wondered if such regulation can be altered by IL-22. Intriguingly, Notch3 expression was reduced after incubation with IL-22 (Fig. 4A and B). Meanwhile, the effect of IL-22 on the TGF-β1-induced Notch3 increase was evaluated. The results showed that IL-22 decreased Notch3 expression in a dose dependent way in HSC-T6 cells (Fig. 4A and B), suggesting Notch3 to be a downstream signal transducer of IL-22-related TGF-β1 inhibition. These results further confirmed that both Notch3 expression and pathway activation were impaired by IL-22. Compared with the TGF-β1 group, combined treatment with IL-22 significantly decreased Hes-1, Hes-5 and Hey-1 mRNA levels (Fig. 4C-E). Taken together, these findings suggested that IL-22 plays a role in regulation of the Notch signaling pathway.