Synthetic human relaxin-3 was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA). Rabbit anti-human CHOP polyclonal antibody as well as rabbit anti-human cleaved caspase 8 and 12 polyclonal antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-human Beclin 1 polyclonal antibody as well as rabbit anti-human cleaved caspase 9 and 3 antibodies were purchased from Cell Signaling Technologies, Inc. (Beverly, MA, USA). Rabbit anti-human microtubule associated protein 1 light chain 3 (LC3) polyclonal antibodies were purchased from Sigma (St. Louis, MO, USA). All chemicals and reagents used in this study were of analytical grade. The human normal liver cell line L02 was purchased from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China).

Human hepatocyte L02 cells were cultured at 37°C in an incubator (5% CO₂, 95% air). RPMI-1640 medium (Hyclone, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (Sijiqing, Inc., Huzhou, China) was used for the cell cultures. A dose-response experiment was initially conducted on the cells, which were treated with 0, 20, 50, 100, 200, 400, 600, 800 and 1,000 μmol/l H₂O₂ (Sigma); 200 μmol/l was selected as the optimal dose for all subsequent experiments. At ~70–80% confluence, cells were divided into the following five groups: Control; H₂O₂, 200 μmol/l H₂O₂; R10, 200 μmol/l H₂O₂ + 10 ng/ml relaxin-3; R50, 200 μmol/l H₂O₂ + 50 ng/ml relaxin-3; and R100, 200 μmol/l H₂O₂ + 100 ng/ml relaxin-3. Following co-incubation for 24 h, cells from all five groups were collected for analysis.

Relative cell viability was determined using an MTT assay (Sigma). Cells were plated in 96-well microtiter plates (Corning, Inc., New York, NY, USA). Following cell treatment, MTT was added to the culture medium to yield a final MTT concentration of 0.5 mg/ml; cells were then incubated for 4 h at 37°C. The dye was dissolved by adding dimethyl sulfoxide at room temperature for 10 min. The preparations were agitated thoroughly with the cells containing formazan crystals using a plate shaker. Absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Inc., Hercules, CA, USA).

Apoptotic cells were characterized due to a distinctive condensed nuclear structure following staining with Hoechst 33258 (Beyotime, Inc., Haimen, China) and visible chromosomal fragmentation. Treated cells were fixed with 4% paraformaldehyde (Westang, Inc., Shanghai, China) for 15 min at room temperature, washed in phosphate-buffered saline (PBS) and then stained with Hoechst dye for 20 min at room temperature in the dark. Following washing with PBS, blue fluorescent cells were examined under a confocal scanning laser microscope (Nikon, Inc., Tokyo, Japan).

Cells were harvested and fixed using 3.0% glutaraldehyde and 1.5% paraldehyde. Cells were then washed in PBS, fixed in osmium tetroxide (Xiya, Inc., Chengdu, China) and then dehydrated in an ethanol series. Subsequently, the samples were embedded in epoxy resin and examined under a transmission electron microscope (Olympus, Inc., Tokyo, Japan).

Following treatment for 24 h, cells were washed with PBS and resuspended in cold lysis buffer containing phenylmethylsulfonyl fluoride. Cell lysates were incubated on ice for 30 min and then centrifuged at 12,000 xg for 15 min at 4°C. The protein content of the supernatant was determined using a bicinchoninic acid-200 protein assay kit (Beyotime, Inc.). Equal amounts of protein (20 μg) from each group were separated using 12% SDS-PAGE (Sanland, Inc., Xiamen, China) and transferred onto polyvinylidene difluoride (PVDF) membranes (Gelman, Inc., Morgan Hill, CA, USA). The membranes were blocked using 5% skimmed milk (Yili, Inc., Neimenggu, China) for 1 h at room temperature with agitation and then incubated with the corresponding primary antibodies overnight at 4°C. All of the following primary antibodies were diluted in Tris-buffered saline/Tween 20 (TBST) solution (anti-β-actin, 1:2,000; anti-CHOP, 1:500; anti-cleaved caspase-12, 1:500; anti-cleaved caspase-8, 1:1,000; anti-cleaved caspase-9, 1:1,000; anti-cleaved caspase-3, 1:1,000; anti-Beclin-1, 1:1,000; and anti-LC3, 1:1,000). Samples were then washed three times (10 min/wash) with TBST and a secondary antibody (1:2,000) conjugated with alkaline phosphatase (ZSGB-BIO, Inc., Beijing, China) was added to the membranes, which were then incubated at room temperature for 1 h with agitation. The membranes were then washed three times (10 min/wash) prior to visualization using Western Blue^® stabilized substrate for alkaline phosphatase (Promega Corp., Madison, WI, USA). In order to quantify the band intensities, the western blot membranes were scanned using ImageJ software (National Institute of Health, Bethesda, MA, USA). The results were normalized based on the respective levels of β-actin.

GraphPad Prism software version 5.0 (La Jolla, CA, USA) was used for data analysis. Each experiment was repeated a minimum of three times and values are expressed as the mean ± standard deviation. The one-way analysis of variance followed by the Newman-Keuls multiple comparison test were used for comparisons among the five groups. P<0.05 was considered to indicate a statistically significant difference between values.

Cell viability assays, Hoechst staining, electron microscopy and western blot analysis were used in order to determine the effect of treatment with graded concentrations of relaxin-3 on H₂O₂-induced hepatocyte apoptosis.

As shown in Fig. 1A, exposure of human hepatocytes to graded concentrations of H₂O₂ (0–1,000 μmol/l) for 24 h remarkably decreased cell viability in a dose-dependent manner. Concentrations of H₂O₂ produced significant results at 200 μmol/l and reached maximum inhibition between 400–600 μmol. Administration of 10 ng/ml relaxin-3 to cells treated with 200 μmol H₂O₂ was found to significantly enhance cell viability; however, the protective effect of relaxin-3 diminished at higher doses (Fig. 1B).

Nuclear condensation, a hallmark characteristic of apoptotic cells, was observed in cells in the H₂O₂ only group following Hoechst staining. By contrast, cells treated with relaxin-3 were round and homogeneous, with a markedly decreased number of apoptotic nuclei, most notably in the R10 group (Fig. 2A). Cell morphological changes were also observed using electron microscopy (Fig. 2B). The later stages of apoptosis are characterized by nuclear pyknosis, mitochondrial and endoplasmic reticulum distension, as well as the formation of apoptotic bodies. These characteristics of later stage apoptosis were observed in cells in the H₂O₂ group; however, no obvious apoptotic changes were detected in the relaxin-3 treated cells. Quantification of the percentage of apoptotic cells counted in each group by Hoechst staining is shown in Fig. 2C. The results demonstrated that H₂O₂ treatment significantly increased the number of apoptotic cells compared with that of the control. By contrast, the R10 relaxin-3 treatment group (10 ng/ml) showed a significantly decreased number of apoptotic cells compared with that of the H₂O₂ group (P<0.05); however, the decrease in the number of apoptotic cells in the R50 and R100 treatment groups were not significant compared with levels in the H₂O₂ group (Fig. 2C).

Intrinsic and extrinsic apoptotic pathways activate the proenzyme form of caspase-3, which is then cleaved through self-proteolysis and other proteases (21). Therefore, endogenous cleaved (activated) caspase-3 is a common indicator of cellular apoptosis. In the present study, western blot analysis was used to determine the expression levels of cleaved caspase-3. The results demonstrated that H₂O₂ treatment significantly increased the expression of cleaved caspase-3 compared to that of the control. By contrast, the relaxin-3 R10 treatment group (10 ng/ml) showed significantly attenuated cleaved caspase-3 protein levels compared to those of the H₂O₂ group (P<0.05); however, the decrease in caspase-3 protein levels in the R50 and R100 treatment groups was not significant compared to levels in the H₂O₂ group (Fig. 2D).

Caspase-8 and -9 are upstream mediators of the extrinsic and intrinsic apoptotic pathways, respectively; cleavage of these caspases results in the activation of downstream executioner caspases, such as caspase-3 (21). Therefore, in the present study, western blot analysis was used to determine the levels of cleaved caspase-8 and -9 using western blot anlaysis and investigate whether these pathways were involved in the anti-apoptotic effect of relaxin-3 (Fig. 3). The results revealed that the protein expression of cleaved caspase-8 and -9 was significantly increased in the H₂O₂ group compared to that of the control group. However, the R10 and R50 relaxin-3 treatment groups demonstrated significantly attenuated cleaved caspase-8 levels compared to those of the H₂O₂ group; in addition, cleaved caspase-9 protein expression was significantly reduced in the R10 group (P<0.05) .

Sustained or severe ERS leads to apoptosis through several pathways, including the CHOP and caspase-12 pathways (22). Therefore, the present study used western blot analysis to detect protein expression levels of CHOP and cleaved caspase-12 in order to directly examine the role of relaxin-3 in ERS-associated apoptosis (Fig. 4). The results revealed elevated levels of CHOP and cleaved caspase-12 in the H₂O₂ group. However, 10 μg/ml relaxin-3 treatment significantly inhibited the H₂O₂-induced overexpression of CHOP and cleaved caspase-12 (P<0.05), while this effect was not observed at higher doses of relaxin-3.

Autophagy is the process by which cells recycle cytoplasm and degrade surplus or dysfunctional organelles (23). It has been reported that autophagy was the cell survival mechanism of primary human hepatocytes during oxidative stress (24). In order to examine the effect of relaxin-3 on autophagy in H₂O₂-induced hepatocyte apoptosis, the present study aimed to determine the expression levels of LC3-II/LC3-I and Beclin-1. The results showed that the expression of these autophagy markers was unchanged following H₂O₂ treatment alone or in combination with relaxin-3 treatment (Fig. 5).