Fifteen male Sprague-Dawley rats (12–16-week-old) were provided by the Shanghai Experimental Animal Center (Shanghai, China). The rats were housed at 22±2°C with 55±5% humidity and a 12 h alternating light/dark cycle. Rats had access to water and food ad libitum. The present study was approved by the Animal Care Committee of Fujian Medical University (Fuzhou, China) and experiments were performed in accordance with the Guidelines on Animal Experiments in Fujian Medical University (Fuzhou, China).

Rat HSC isolation was performed as previously described (11). Rat livers were perfused in situ and HSCs were isolated by density gradient centrifugation. Rat livers were perfused via the portal vein with D-Hanks buffer (PYG-0079, Boster Biological Technology, Wuhan, China) and subsequently with 0.075% type I collagenase for 10 min (Worthington Biochemical Corporation, Lakewood, NJ, USA). Livers were further digested by shaking for 20 min at 200 rpm and 37°C. Cell suspension was filtered to remove undigested debris and Hanks buffer was added to make up the solution to 50 ml. This was centrifuged at 50 × g for 5 min at room temperature to remove residual hepatocytes. Supernatant was subsequently transferred to a clean 50 ml tube and centrifuged at 500 × g for 5 min at 4°C. Cell pellets were resuspended in 12% Histodenz (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and centrifuged by density gradient for HSC isolation. The newly isolated HSCs were seeded in 6-well plates (2×10⁶ cells/well) and cultivated in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 20% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) at 37°C in an environment containing 5% CO₂ for 48 h. Following seeding, HSCs were cultured in DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences) and 1% penicillin and 1% streptomycin.

An equal volume (0.1 ml) of DMEM-suspended HSCs were mixed with 0.4% Trypan blue solution. An aliquot of the mixture was analyzed in a Neubauer chamber following 5 min at room temperature and viable and non-viable cells within four big squares in the four corners were counted under a phase contrast microscopy (magnification, ×100) The cell viability (%) was calculated as follows: Total viable cells (unstained)/total cells (stained and unstained) ×100%.

Primary HSCs were seeded in 6-well plates (2×10⁵ cells/well) and cultured in DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences). Vitamin A lipid droplet autofluorescence in primary rat HSCs was detected at a wavelength of 328 nm using a fluorescence microscope (magnification, ×200; Nikon Corporation, Tokyo, Japan).

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed with ice cold PBS and stained with 0.3% oil red O solution for 10 min at the room temperature. Stained slides were washed and counterstained with hematoxylin Staining Solution (Beyotime Institute of Biotechnology, shanghai, China) for 30 sec at room temperature and washed again in PBS. Positive cells were observed from ten random fields under a phase contrast microscope (magnification, ×200).

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed twice with ice cold PBS, and then permeabilized with 0.3% Triton X-100 for 15 min. Endogenous peroxidase was removed with 30 ml/l H₂O₂. They were incubated with mouse anti-rat α-smooth muscle actin (α-SMA, 1:400, cat. no. BM0002; Boster Biological Technology) and rabbit anti-rat desmin (1:200, cat. no. BM0036; Boster Biological Technology) primary antibodies in a humidified chamber overnight at 4°C. Cells were treated with instant Polink-2 plus^® Polymer HRP Detection System (cat. no. PV-9001/PV-9002; ZSGB-BIO; OriGene Technologies, Inc.) and incubated in a buffer solution containing 3,3-diaminobenzidine tetrahydrochloride (DAB) to produce a brown reaction product. Finally, cells were counterstained with hematoxylin Staining Solution (C0107; Beyotime Institute of Biotechnology) dehydrated, plated on coverslips and visualized under a phase contrast microscopy (magnification, ×200). Counting the total 200 cells on the cover glass, the purity of hepatic stellate cells (%) = Desmin positive cells/total (positive and negative) ×100%. the degree of HSC activation (%) = a-SMA positive cells/total (positive and negative) ×100%.

Primary rat HSCs were seeded in 6-well plates (2×10⁵ cells/well) and cultured in DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences). When cells reached 60–70% confluence, the culture medium was replaced with serum-free DMEM for 24 h and subsequently divided into the control and IL-10 treatment groups. Control group HSCs were cultured with DMEM supplemented with 10% FBS, whereas cells in the IL-10 treatment group were cultured with increasing concentrations of IL-10 (10, 20 or 40 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) for a further 24 h at 37°C.

The Senescence β-Galactosidase Staining kit (C0602; Beyotime Institute of Biotechnology) was used to detect the senescence-associated β-galactosidase (SA-β-Gal) activity of activated HSCs. Briefly, HSCs treated with or without IL-10 were fixed with the staining fixative for 15 min at room temperature, washed with PBS and stained overnight with X-Gal solution at 37°C. Cells were washed twice with PBS and incubated with 4′,6-diamidino-2-phenlindole dihydrochloride (0.5 µg/ml; Roche Diagnostics, Indianapolis, IN, USA) for 10 min. SA-β-Gal-positive cells were detected in at least three fields with a phase contrast microscope (magnification, ×200) using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

The Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to examine cell viability. Primary rat HSCs were seeded in 96-well plates (3×10³ cells/well) in DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences) for 24 h and subsequently cultured without serum for 16 h prior to culture with either DMEM supplemented with 10% FBS alone or with increasing concentrations of IL-10 for 24 h. The medium was replaced with fresh DMEM and 10 µl CCK-8 was added to each well for 2 h. The absorbency was determined with a microplate reader (Epoch^™; BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm. The absorbance values were normalized by subtracting the blank values obtained from untreated cells.

The FITC Annexin V Apoptosis Detection kit (cat. no. 556547; BD Biosciences, San Jose, CA, USA) was used to analyze the apoptosis of activated primary rat HSCs. Activated primary rat HSCs following treatment with or without IL-10 were washed twice with cold PBS and suspended in 1X Binding Buffer at a concentration of 1×10⁶ cells/ml. Subsequently, 100 µl of the solution (1×10⁵ cells) were added to 5 ml culture and add 5 µl of FITC Annexin V and 5 µl PI for 15 min at room temperature in the dark, 400 µl of 1X Binding Buffer were added to each tube and analyzed using a FACScan cytometer (BD Accuri C6; BD Biosciences).

Primary rat HSCs were treated with or without IL-10 for 24 h. Cell culture supernatant was collected and assayed for IL-6 (cat. no. R6000B), IL-8 (cat. no. D8000C) and tumour necrosis factor (TNF)-α (cat. no. RTA00) expression levels using the respective ELISA kits, according to the manufacturer's protocol (R&B Systems, Minneapolis, MN, USA).

Total RNA was isolated from HSCs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse transcribed to cDNA using a Prime Script Reverse Transcription System (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. cDNA samples were amplified using the SYBR-Green master mix kit (Takara Biotechnology Co., Ltd.). The primer sequences used were as follows: p53 forward, 5′-CTCCTCTCCCCAGCAAAAG-3′ and reverse, 5′-CCTGCTGTCTCCTGACTCCT-3′; p21 forward, 5′-TGTGGTAGTTGGAGCTGGTG-3′ and reverse, 5′-TGACCTGCTGTGTCGAGAAT-3′; and β-actin forward, 5′-GGCATCCTGACCCTGAAGTA-3′ and reverse, 5′-AGGCATACAGGGACAACACA-3′. The PCR cycling conditions were as follows: 95°C for 2 min and 45 cycles of 95°C for 15 sec, 63°C for 15 sec, 72°C for 20 sec. All procedures were performed in triplicate. Alterations in mRNA expression were calculated relative to the expression control β-actin using the 2^−ΔΔCq method (12).

HSCs were washed twice with PBS and lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and blocked in a phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology) and PhosSTOP (Roche Diagnostics) cocktail on ice. The supernatant was obtained by centrifugation at 12,000 × g for 15 min at 4°C. Protein concentration was determined by the enhanced BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (40 µg) of protein were separated on a 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Following blocking with 5% skim milk (cat. no. 1172GR500; BioFroxx GmbH, Einhausen, Germany; www.neofroxx.com/en/home/) in Tris-buffered saline/Tween-20 for 1 h at room temperature, the membranes were incubated with the primary antibodies (p53 (cat. no. 2524, 1:1,000; CST Biological Reagents, Co., Ltd., Shanghai, China), GAPDH (cat. no. 2118, 1:1,000; CST Biological Reagents, Co., Ltd.), and p21 (cat. no. ab109199, 1:1,000; Abcam, Cambridge, UK) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated homologous secondary antibody (cat. no. ZB-2301/ZB2305, 1:3,000; ZSGB-BIO; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. The signals were visualized using an enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The band density was determined by densitometry, and quantified using ChemiDoc^™ Touch Imageing System with Image Lab^™ Touch Software 5.2 version. (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data are expressed as the mean ± standard error. Statistical analysis was performed with one-way analysis of variance followed by Tukey's Multiple Comparison Test, using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To examine the effects of IL-10 on primary rat HSC senescence, isolation and culture of the HSCs was conducted. The collagenase perfusion and density-gradient centrifugation method (13) was used to isolate a high-purity and high-yield of quiescent rat HSCs. These were visible as a thin white layer in the interface between the Histodenz solution and the overlay with DMEM following isolation from density-gradient centrifugation (Fig. 1A). A total of 2–5×10⁷ cells/rat were harvested. Cell viability as detected by Trypan blue staining was >95%. Phase contrast microscopy revealed that the morphology of plated HSCs was round and bright (Fig. 1B).

A typical feature of HSCs is the presence of cytoplasmic, triacylglycerol rich droplets (14). In the present study, rat HSCs emitted a striking, rapidly fading blue-green autofluorescence when excited by ultraviolet light at 328 nm (Fig. 1C). Quiescent HSCs may be identified with the Oil Red O stain. The retinoid droplets of HSCs cultured for 72 h were observed to be bright red under the light microscope (Fig. 1D). HSCs were identified using desmin, a reliable HSC marker (15) and α-SMA, an activated HSC marker. HSCs were cultured for 7 days and immunocytochemical analysis demonstrated that desmin (Fig. 1E) and α-SMA (Fig. 1F) were expressed in the HSC cytoplasm. The purity of the isolated HSCs was 93% and the degree of HSC activation was 96%.

The most widely used assay for cell senescence is the histochemical detection of β-galactosidase activity at pH 6.0, which is known as SA-β-Gal (4). In the present study, the effect of IL-10 on the senescence of primary rat HSCs was examined by SA-β-Gal staining. Cells were incubated in serum-free medium for 24 h, followed by incubation with different concentrations of IL-10 for a further 24 h prior to staining. The senescent primary rat HSCs cytoplasm was stained blue (Fig. 2A). The number of SA-β-Gal positive cells was significantly increased in HSCs treated in all IL-10 groups compared with the cells in the control group (P<0.01). The number of SA-β-Gal positive cells peaked in the 20 ng/ml IL-10 treatment group and subsequently decreased (Fig. 2B).

To investigate if IL-10 promoted primary rat HSCs senescence by decreasing viability or promoting apoptosis, flow cytometry analysis and a CCK-8 assay were performed, respectively. The apoptotic rate of primary rat HSCs increased significantly in the IL-10 treatment groups compared with the control group (P<0.001; Fig. 3A and B) and HSC viability decreased significantly in the IL-10 treatment groups compared with the control group (P<0.01; Fig. 3C).

p53 and p21 have a key role in cellular senescence, responding to a range of cellular damage signals (4). To examine the role of p53 and p21 in IL-10-induced senescence, p53 and p21 expression was detected in the absence or presence of IL-10 by RT-qPCR and western blot analysis. The expression of p53 mRNA was significantly increased in the IL-10 treatment groups compared with the control group (P<0.05; Fig. 4A), particularly in the 10 ng/ml group (P<0.001). p21 mRNA expression was also significantly increased in all IL-10 treatment groups (P<0.001; Fig. 4B). Western blot analysis demonstrated that that p53 and p21 protein expression was increased in the IL-10 treatment groups (Fig. 4C). Relative quantification of protein expression revealed that p53 expression increased in a dose-dependent manner (Fig. 4D). Relative expression of p21 was highest in the 10 and 20 ng/ml IL-10 treatment groups (Fig. 4E).

Senescent cells secrete a number of inflammatory cytokines and chemokines that reinforce and propagate senescence in an autocrine and paracrine manner (4). The present study detected the expression of IL-6, IL-8 and TNF-α in HSCs treated with increasing concentrations of IL-10 for 24 h. Compared with the control, IL-6 expression was markedly inhibited by IL-10 in a dose-dependent manner (P<0.001; Fig. 5A). IL-8 levels were also significantly decreased in the 20 and 40 ng/ml IL-10 groups (P<0.001; Fig. 5B). Conversely, TNF-α levels significantly increased in IL-10 treatment groups, particularly at 40 ng/ml (P<0.001; Fig. 5C).