Aging-associated oxidative stress inhibits liver progenitor cell activation in mice

Liver progenitor cell activation decreases with age

To determine the effects of aging on LPC activation and liver regeneration, we fed mice with modified CDE diet to induce liver damage [21], and examined activation and proliferation of LPCs in young (Y) and old (O) mice. Mice fed with normal diet were used as control. Following CDE diet, young mice were resistant to CDE diet and survived for the 3-week period of the study, consistent with previous report [21]. In contrast, old mice exhibited a high sensitivity to CDE diet, with approximately 80% of them died (Fig. 1A). These CDE diet-fed O-mice (O-CDE mice) showed a dramatic increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels at day 21 compared with CDE diet-fed Y-mice (Y-CDE mice) (Fig. 1B). Histology showed that ductular reactions (formation of ductular structures) were induced in Y-CDE mice as reported, whereas not so obvious in O-CDE mice (Fig. 1C). These results suggest that O-mice have impaired capacity in liver regeneration in response to CDE diet.

Figure 1. Liver injury in old mice with CDE diet. Y/O mice were fed with normal/CDE diet for 3 weeks. (A) Cumulative survival rates of Y/O mice with CDE diet were analyzed (n = 5 mice per group). (B-C) Serum and liver tissues from Y/O mice fed with normal/CDE diet were sampled on day 21. (B) Serum levels of AST and ALT were measured. Results are mean ± SEM from three independent experiments (n = 6 mice per group). (C) Liver tissues were sectioned for histological examination. Scale bar = 100 μm. Representative images from one experiment out of three are shown. *P < 0.05.

To test this possibility, we first measured mRNA expressions of LPC markers (including EpCAM, CD133 and AFP) in livers of normal/CDE diet-fed Y/O mice. As compared to normal mice, expressions of EpCAM, CD133 and AFP were increased in livers of Y-CDE mice (Fig. 2A). Of note, O-CDE liver had marked lower levels of EpCAM, CD133 and AFP than Y-CDE liver (Fig. 2A). Furthermore, FCM analysis demonstrated that percentages of EpCAM⁺CD45^- LPC cells in NPCs were lower in O-CDE mice than those in Y-CDE mice (Fig. 2B).

Figure 2. LPC activation and liver regeneration are impaired in old mice. Y/O mice were fed with normal/CDE diet for 3 weeks. At day 21, liver tissues from Y/O mice were collected and analyzed. (A) mRNA levels of EpCAM, CD133 and AFP in livers were measured by Q-PCR. Results are mean ± SEM from three independent experiments (n > 6 mice per group). (B) EpCAM⁺CD45^- cells in NPCs from Y/O mice with normal/CDE diet were analyzed by FCM. Percentage of EpCAM⁺CD45^- cells was quantified. Results are mean ± SEM from three independent experiments (n > 3 mice per group). (C) EpCAM(red)/Ki67(green)/DAPI(blue) staining of liver tissues. a-d, scale bar = 100 μm; e-h, scale bar = 20 μm. Numbers of EpCAM⁺ and EpCAM⁺Ki67⁺ cells were quantified. Results are mean ± SEM from three independent experiments (n > 3 mice per group). (D) mRNA level of cyclin E1 in EpCAM⁺CD45^- cells from Y/O mice with normal/CDE diet was measured by Q-PCR. Results are mean ± SEM from three independent experiments (n > 6 mice per group). *P < 0.05, **P < 0.01.

IF staining demonstrated that after CDE diet feeding, O-CDE mice had significantly lower numbers of EpCAM⁺ and Ki67⁺EpCAM⁺ LPCs compared with Y-CDE mice, suggesting decreased level of LPC proliferation in O-CDE mice (Fig. 2C). Next, we sorted EpCAM⁺CD45^- LPCs to test the expressions of cyclin A2/B1/D1/E1. As expected, expression of cyclin E1 was increased in LPCs from Y-CDE mice compared with that of Y-normal mice, indicating stronger LPC proliferation in Y-CDE mice. In contrast, O-CDE mice did not show elevated expression of cyclin E1, which was consistent with lower levels of Ki67-positive cells in the O-CDE mice (Fig. 2D). Taken together, these results indicate that LPC activation and proliferation decrease with age.

LPCs retain functional capacity during aging

To dissect if the decrease of LPC activation and proliferation in old mice is cell-intrinsic or extrinsic, we next determined the functional capacity of freshly isolated EpCAM⁺CD45^- LPCs in Y/O-CDE mice. We plated LPCs isolated from Y/O mice in equal numbers to assess their clonogenic capacity. Primary LPCs from Y-CDE mice produced significantly more colonies than O-CDE mice. Furthermore, the colony size of LPCs from Y-CDE was larger than that of O-CDE (Fig. 3A).

Figure 3. Comparison between LPCs isolated from Y/O mice. (A) Clonogenic colony-forming assay of freshly isolated EpCAM⁺CD45^- cells from Y/O mice with normal/CDE diet. Pictured are wells of each condition. Scale bar = 500 μm. Colony number was quantified. Results are mean ± SEM from three independent experiments. (B) Morphology of cultured LPC lines from Y/O-CDE mice. Scale bar = 500 μm. (C) LPC lines from Y/O-CDE mice were analyzed for the indicated markers by FCM. Fluorescence intensities of markers were analyzed. MFI indicates mean fluorescence intensity. (D) Proliferation of LPC lines in vitro culture were analyzed by CCK-8 assay. Results are mean ± SEM from three independent experiments. (E) Clonogenic colony-forming assay of LPC lines from Y/O-CDE mice. Pictured are wells of each condition. Colony number was quantified. Results are mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01.

To further assess the proliferative capabilities of LPCs, we established LPC lines from Y/O-CDE mice. In contrast to freshly isolated LPCs, LPC lines showed similar morphology, regardless of their origins, young or old mice (Fig. 3B). In addition, both LPC lines displayed similar phenotypes, as EpCAM⁺CD44⁺CD49f⁺CD45^- (Fig. 3C). Furthermore, both Y/O-LPC lines exhibited comparable rate of proliferation (Fig. 3D) and clonogenic capacity (Fig. 3E). Altogether, these results demonstrate that although primary LPCs freshly isolated from old mice have reduced capacity to undergo proliferation in vivo, LPC cell lines derived from old mice, which had been passaged for 5 to 15 passages, re-acquired the potent clonogenic and proliferative capacity, indicating the notion that the reduced activation and proliferation capacity might result from alteration of stem cell niche in old mice, although we could not definitively rule out the possibility of an intrinsic defect.

Increased infiltration of neutrophils in livers of old mice

Microenvironments have been shown to play essential roles in regulating an array of biological events. Since previous studies have demonstrated that immune cells and cytokines, such as T cells, macrophages, and proinflammatory cytokines (TNF-α and LT-β) play important roles in regulating the activation of LPCs in murine CDE model [30], we then determined the expression of TNF-α and LT-β in livers from O-CDE mice using real-time PCR. Of note, we did observe a slight increase for expression of TNF-α and LT-β in CDE mice compared to control normal mice, but no significant difference between Y-CDE and O-CDE mice (Fig. 4A).

Figure 4. Increased neutrophil infiltration in livers of O-CDE mice. (A) mRNA levels of cytokines in livers were measured by Q-PCR. Results are mean ± SEM from three independent experiments (n > 4 mice per group). (B) Quantification of liver-infiltrating leukocytes from Y/O mice with normal/CDE diet by FCM: neutrophils (CD11b⁺Gr-1^high), macrophages (CD11b⁺Gr-1^low), CD4⁺ T cells (CD3⁺CD4⁺) and CD8⁺ T cells (CD3⁺CD8⁺). Results are mean ± SEM from three independent experiments (n > 4 mice per group). (C) mRNA level of MPO in livers was measured by Q-PCR. Results are mean ± SEM from three independent experiments (n > 4 mice per group). (D) MPO(green)/DAPI(blue) staining of liver tissues from Y/O mice with normal/CDE diet. a-d, scale bar = 100 μm; e, scale bar = 20 μm. *P < 0.05, **P < 0.01.

Then we focused on the presence of immune cells in liver, and examined the cells by FCM analysis. Our data showed no significant differences in numbers of macrophages (Ly6G^lowCD11b⁺) or T cells (CD4⁺/CD8⁺) between Y-CDE and O-CDE mice. Strikingly, neutrophils (Ly6G^highCD11b⁺) were greatly increased in livers of O-CDE mice compared with those of Y-CDE mice (Fig. 4B). Consistently, Q-PCR and IF staining showed that expression of myeloperoxidase (MPO), a peroxidase enzyme most abundantly expressed in neutrophil granulocytes, was significantly increased in livers from O-CDE mice compared with that of Y-CDE mice (Fig.4 C,D). Together, these results suggest that neutrophils were increased in livers of O-CDE mice.

Neutrophils mediate the impairment of LPC proliferation in O-CDE mice

To assess the possible impact of infiltrating neutrophils in regulating LPC response in old mice, we administered anti-Ly6G antibody, which could deplete neutrophils [31], to O-CDE mice by i.p. injection (Fig. 5A). We found that depletion of neutrophils improved the survival of O-CDE mice considerably (Fig. 5B). Further, depletion of neutrophils restored the LPC response in O-CDE mice as evidenced by the increased percentages of EpCAM⁺CD45^- cells in FCM analysis (Fig. 5C). Thus, increased infiltration of neutrophils to the liver of old mice is critical for reducing the capacity of LPCs to proliferate in response to CDE diet.

Figure 5. Neutrophils are required for inhibiting LPC proliferation in old mice. Old mice with CDE diet were i.p. injected with 500 μg of 2A3 (n/a) or 1A8 (anti-mLy6G) mAb every other day to deplete neutrophils. (A) Representative FACS scatterplots of liver-infiltrating neutrophils (CD11b⁺Gr-1^high) in liver tissues from O-CDE mice treated with 2A3 or 1A8 mAb. Percentages of liver-infiltrating neutrophils were quantified. Results are mean ± SEM from three independent experiments (n = 3 mice per group). (B) Cumulative survival rates of mice were analyzed. n > 8 mice per group. (C) Representative FACS scatterplots and summarized percentages of EpCAM⁺CD45^- cells in liver tissues from O-CDE mice treated with 2A3 or 1A8 mAb. Results are mean ± SEM from three independent experiments (n > 5 mice per group). *P < 0.05, **P < 0.01.

Hepatic stellate cells from old mice produce high levels of chemokine CXCL7 to recruit neutrophils

As expressions of chemokines CXCL1 [32] and CXCL7 [33] were reported to be associated with migration of neutrophils in mice, so we tested the expression of CXCL1/CXCL7 in livers in Y/O mice with normal/CDE diet. Q-PCR results revealed that mRNA levels of CXCL1/CXCL7 were upregulated in livers of O-CDE mice compared with those of Y-CDE (Fig. 6A), especially CXCL7, suggesting CXCL7 might be a crucial factor inducing neutrophil infiltration. To investigate from which type of cells was CXCL7 derived, we isolated hepatic stellate cells and leukocytes from liver, the cell populations in liver reported to express chemokines [34,35]. Gene expression analysis demonstrated that both hepatic stellate cells and leukocytes expressed CXCL7 (Fig. 6B). However, expression of CXCL7 in hepatic stellate cells from O-CDE mice was higher than that in Y-CDE mice, suggesting hepatic stellate cell-derived CXCL7 was responsible for neutrophil infiltration in O-CDE mice. Q-PCR analysis also confirmed this (Fig. 6C).

Figure 6. Hepatic stellate cell-derived CXCL7 induced neutrophil infiltration into livers of O-CDE mice. (A) mRNA levels of CXCL1/CXCL7 in livers from Y/O mice with normal/CDE diet were measured by Q-PCR. Results are mean ± SEM from three independent experiments (n = 3 mice per group). (B) PCR analysis of CXCL7 expression in hepatic stellate cells and leukocytes isolated from livers of Y/O-CDE mice. (C) mRNA level of CXCL7 in hepatic stellate cells and leukocytes isolated from Y/O-CDE mice with CDE diet were measured by Q-PCR. Results are mean ± SEM from three independent experiments (n = 3 mice per group). (D) Levels of CXCR2 expression on neutrophils derived from livers were analyzed by FCM. Fluorescence intensity of CXCR2 was analyzed. Results are mean ± SEM from three independent experiments (n > 3 mice per group). (E) Mobility of neutrophils in response to conditioned medium from Y/O-CDE hepatic stellate cells was analyzed in the absence or presence of SB225002 (100 nM). Numbers of migrated neutrophils were determined. Results are mean ± SEM from three independent experiments (n = 3 per group). *P < 0.05, **P < 0.01. (F) Quantification of liver-infiltrating neutrophils (CD11b⁺Gr-1^high) from Y/O mice with normal/CDE diet treated with SB225002 (2 mg/kg) or control by FCM. Results are mean ± SEM from three independent experiments (n > 4 mice per group).

As CXCR2 is the receptor for CXCL7 and are expressed by neutrophils (Fig. 6D), we next investigated whether hepatic stellate cell-derived CXCL7 recruited neutrophils through CXCR2. We isolated and cultured hepatic stellate cells from Y/O-CDE mice, and conditioned media were collected. In vitro chemotaxis assay was conducted to assess the neutrophil migration induced with media from hepatic stellate cells, and data demonstrated that the medium from O-CDE hepatic stellate cells possessed stronger chemotaxis ability on neutrophils. By contrast, neutrophils pretreated with CXCR2 antagonist SB225002 was deprived of migration capability (Fig. 6E). In addition, SB225002 treatment in vivo significantly decreased the number of neutrophils (Ly6G^highCD11b⁺) in the livers of O-CDE mice (Fig. 6F). These data combined show that hepatic stellate cells are responsible for recruitment of neutrophils by producing CXCL7, working through CXCR2 on neutrophils.

ROS derived from infiltrating neutrophils cause DNA damage to LPCs

Studies on primary biliary cirrhosis have already demonstrated that infiltrating MPO⁺ or CD68⁺ inflammatory cells, mainly neutrophils or macrophages, participated in bile duct damage through nitric oxide and ROS [36]. We reasoned that ROS production might contribute to changes of LPC niches in livers of O-CDE mice. To test it, first we measured the oxidative stress in liver samples from normal/CDE diet-fed Y/O mice using MDA, a marker of oxidative stress, as a readout, and we found that MDA level in O-CDE mice was significantly higher than that in Y-CDE mice (Fig. 7A), indicating higher level of ROS in O-CDE mice.

Figure 7. Oxidative stress in livers of O-CDE mice. (A) Hepatic lipid peroxidation was measured by MDA assay. Results are expressed as nmol MDA/mg liver protein. Results are mean ± SEM from three independent experiments (n > 6 mice per group). (B) Level of ROS as assessed by APF fluorescence in leukocytes isolated from Y/O-CDE mice. Results are mean ± SEM from three independent experiments (n = 4 mice per group). *P < 0.05, **P < 0.01.

It has been proposed that free radicals promote aging [16], and studies have shown that ROS limited the lifespan of hematopoietic stem cells, leading to the exhaustion of the stem cell population [37]. We then tested levels of ROS within liver leukocytes isolated from CDE diet-fed Y/O mice. As expected, levels of ROS in neutrophils from O-CDE mice were higher than those in Y-CDE (Fig. 7B). ROS promotes DNA damage, and the resulted cumulative oxidative DNA damage is considered the key factor in aging and senescence [38]. To understand the mechanism of aging by ROS, we studied the DNA damage in LPCs using the marker, phosphorylation of histone variant H2A.X on serine 139 (γ-H2A.X). As revealed by IF staining, few γ-H2A.X⁺EpCAM⁺ LPCs with DNA double-strand breaks were observed in Y-CDE mice. However, a large percentage of γ-H2A.X⁺EpCAM⁺ LPCs were present in O-CDE mice, suggesting DNA double strand damage in O-CDE LPCs (Fig. 8A).

Figure 8. Neutrophils inhibit LPC response through oxidative stress and induce DNA damage during aging. (A) EpCAM(red)/γ-H2A.X(green)/DAPI(blue) staining of liver tissues from Y/O mice with normal/CDE diet. Numbers of EpCAM⁺γ-H2A.X⁺ cells were quantified. Results are mean ± SEM from three independent experiments (n > 3 mice per group). (B) Old mice with CDE diet were subjected to NAC drinking (1 g/L). Cumulative survival rates of mice were analyzed. n > 5 mice per group. (C) Representative FACS scatterplots and summarized percentages of EpCAM⁺CD45^- cells in liver tissues from O-CDE mice treated with NAC drinking. Results are mean ± SEM from three independent experiments (n > 3 mice per group). (D) EpCAM(red)/γ-H2A.X(green)/DAPI(blue) staining of liver tissues from O-CDE mice treated with NAC drinking. Numbers of EpCAM⁺γ-H2A.X⁺ cells were quantified. Results are mean ± SEM from three independent experiments (n > 6 mice per group). (E) Representative FACS scatterplots of liver-infiltrating neutrophils (CD11b⁺Gr-1^high) in liver tissues from O-CDE mice treated with NAC drinking. (F) Hepatic lipid peroxidation was measured by MDA assay. Results are mean ± SEM from three independent experiments (n > 6 mice per group). *P < 0.05, **P < 0.01.

To further verify the ROS in inducing DNA damage in LPCs from old CDE mice, we administrated antioxidant NAC to the mice, and uncovered the treatment using NAC improved the survival of O-CDE mice (Fig. 8B). Meanwhile, percentages of EpCAM⁺CD45^- LPCs were increased in O-CDE mice (Fig. 8C), whereas numbers of γ-H2A.X⁺EpCAM⁺ cells were decreased, as determined by IF staining (Fig. 8D). NAC treatment had no effect on neutrophil infiltration in liver (Fig. 8E). In addition, MDA levels in O-CDE mice were also downregulated after NAC treatment (Fig. 8F). Taken together, we conclude that neutrophils dampen LPC activation through producing excessive ROS in old mice.

Mice

Six- to eight-week-old C57BL/6 mice were obtained from the Shanghai Laboratory Animal Center at the Chinese Academy of Sciences, and maintained under specific pathogen-free conditions at the Chinese Academy of Sciences for up to 2 years. Mice used at 2 and 24 months of age were considered as young or old (Y/O) mice accordingly [7]. All animal experiments were performed in accordance with the guidelines from the Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The protocol used was reviewed and approved by the Animal Experiment Administration Committee of the Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All efforts were made to minimize the suffering of mice during experiments.

LPC isolation and culture

Y/O mice were fed with a choline-deficient, ethionine-supplemented (CDE) diet (Trophic Diet, Nantong, Jiangsu, China) to induce LPC activation [21]. In some groups, mice were treated with drinking water containing N-Acetyl Cysteine (NAC, 1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). In some groups, mice were i.p. injected with Ly6G neutralizing Ab (1A8)/control Ab (2A3) (Bio-X-Cell, West Lebanon, NH, USA). In some groups, mice were i.p. injected with 2 mg/kg of CXCR2 antagonist SB225002/control once a day (Sigma-Aldrich). After 3 weeks on the CDE diet, mice were anaesthetized by i.p. injection of pentobarbital sodium. Liver nonparenchymal cells (NPCs) and LPCs were then harvested by a modification of a multi-step digestion protocol as previously described [22–24]. NPCs were separated from the hepatocytes by several episodes of low-speed centrifugation (40 × g for 3 min) and were then collected by centrifugation (1500 rpm for 5 min). LPCs were enriched by centrifugation of NPCs through a discontinuous gradient of 20% and 50% PercollTM (Amersham Biosciences, Pittsburgh, PA, USA) in PBS at 1400 × g for 20 min.

Enriched LPCs were then incubated for 30 min on ice with APC-conjugated anti-EpCAM and FITC-conjugated anti-CD45 (eBioscience, San Diego, CA, USA). Then, EpCAM+CD45- cells were isolated by fluorescence-activated cell sorting (FACS) and plated in type I collagen-coated dishes (BD Biosciences, San Jose, CA, USA). The standard culture medium for LPCs is DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovin serum, insulin (1 μg/mL; Sigma-Aldrich), dexamethasone (1 × 10^-7 mol/L; Sigma-Aldrich), penicillin/streptomycin (1% vol/vol; Invitrogen, Carlsbad, CA, USA), hepatocyte growth factor (HGF, 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), epidermal growth factor (EGF, 20 ng/mL; PeproTech), fibroblast growth factor (FGF, 20 ng/mL; PeproTech) and Insulin-Transferrin-Selenium-Ethanolamine (ITS-X, 1 ×; Invitrogen).

Histology and Immunofluorescence

Liver samples for histology analysis were fixed in 4% paraformaldehyde and paraffin-embedded. Deparaffinized sections (5-10 μm) were stained with hematoxylin and eosin (H&E; Beyotime, Nantong, Jiangsu, China) as previously described [25]. Immunofluorescence staining of liver sections was also performed as previously described [25]. Confocal images were acquired with LSM 510 microscope (Zeiss, Oberkochen, Germany).

Flow cytometry (FCM)

For the determination of LPC percentages within liver NPCs, NPCs were incubated for 30 min on ice with the following antibodies: APC-conjugated anti-EpCAM and FITC-conjugated anti-CD45 (eBioscience). For the determination of the subtypes of liver leukocytes, liver leukocytes were incubated for 30 min on ice with the following antibodies: APC/FITC-conjugated anti-Ly6G, PE-conjugated anti-CD11b and FITC/APC-conjugated anti-CD45; FITC/PE-conjugated anti-CD8, PE-conjugated anti-CD4 and APC-conjugated anti-CD45 (eBioscience, San Diego, CA, USA). For the characterization of LPCs cultured in vitro, LPCs were trypsinized and incubated for 30 min on ice with the following antibodies: APC-conjugated anti-EpCAM, PE-conjugated anti-CD44, APC-conjugated anti-CD49f or FITC-conjugated anti-CD45 (eBioscience). For the detection of ROS, liver leukocytes were incubated with 2-[6-(4’-amino)phenoxy-3H-xanthen-3-on9-yl]benzoic acid (APF; Invitrogen, Carlsbad, CA, USA) fluorescence as reported before [26]. FCM measurements were performed with Aria II (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

Quantitative real-time PCR (Q-PCR)

Total RNA was extracted from livers at indicated times and was subsequently reverse-transcribed using PrimeScript RT Master Mix (Takara Bio Inc., Otsu, Shiga, Japan). Quantitative real-time polymerase chain reaction was performed using SYBR Green PCR mix (Roche, Basel, Switzerland) on an ABI Prism 7900HT (Applied Biosystems, Carlsbad, CA, US) as previously reported [27]. β-actin was used as an internal control to normalize for difference in the amount of total RNA in each sample. Relative expression of genes was calculated and expressed as 2^-ΔΔCT. Primers are listed in Table 1.

In vitro colony assay

For analysis of primary EpCAM⁺CD45^- LPCs sorted from NPCs, cells were inoculated at a low density (200 cells per cm2) into 6-well plates coated with type I collagen and cultured in the standard culture medium supplemented with Y27632 (20 μmol/L; Sigma-Aldrich, St. Louis, MO, USA) [24]. After 10-13 days of culture, colonies were stained with crystal violet (Beyotime) and assessed. For cultured LPC lines, cells were inoculated at a low density of 300 cells per well of 6-well plates and subjected to different treatment. After 7 days of culture, colonies were also assessed by crystal violet staining.

Cell proliferation assays

For proliferation assessed by CCK-8 assay, LPC lines from Y/O mice were cultured in type I collagen-coated 96-well plates at an initial density of 5000 cells per well, in 100 μl of standard culture medium. After 24, 48, 72 and 96 hours, the proliferation of LPCs was determined by using a CCK-8 kit (Dojindo Laboratories, Mashikimachi, kamimashiki gun Kumamoto, Japan).

For proliferation assessed by BrdU staining, LPCs were subjected to different treatment, and BrdU (40 μg/ml) was added 2 hours before the end of the coculture. Immunofluorescent staining of incorporated BrdU was performed according to the BrdU Flow Kit instruction manual (BD Biosciences). Percentages of BrdU⁺ cells were analyzed by FCM.

Hepatic stellate cell isolation and culture

Hepatic stellate cells from Y/O mice with CDE diet were isolated according to the published protocol [28]. Isolated hepatic stellate cells were plated at a density of 2 × 104 cells/cm². After 48 hours of culture, conditioned medium of hepatic stellate cells were collected for subsequent experiments. And hepatic stellate cells were collected for Q-PCR analysis.

Neutrophil isolation and chemotaxis assay

Neutrophil were isolated from bone marrow and were incubated with cytochalasin-B (5 μg/ml, 30 min; Sigma-Aldrich), then N-formylmethionyl-leucyl-phenylalanine (fMLP; 10 μM, 1 hour; Sigma-Aldrich) [29]. For chemotaxis assay, freshly isolated neutrophils (1.5 × 10⁶) were placed in the upper chamber of the Transwell inserts (3 μm pore size; Corning Costar, Corning, NY, USA). Inserts were placed in wells containing medium alone (control) or Y/O-hepatic stellate cell-conditioned medium with or without CXCR2 antagonist SB225002 (100 nM). After 120 min, inserts were removed, and migrated neutrophils were collected and counted.

Western blot

Western blot was performed as previously described [25]. Primary antibody against γ-H2A.X and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Malondialdehyde (MDA) assay

A whole liver homogenate was prepared from frozen liver pieces to measure hepatic lipid peroxidation, using the Lipid Peroxidation (MDA) Assay Kit in accordance with the manufacturer’s protocol (Sigma-Aldrich).

Statistical analysis

Differences were evaluated using Statistical Package for Social Science software (version 16.0, SPSS Inc., Chicago, IL, USA). Mantel-Cox test was performed to estimate cumulative survival rates of mice. Differences between 2 groups were compared using unpaired Student t test or Mann-Whitney U test. Multiple treatment groups were compared within individual experiments by ANOVA. Values of p less than 0.05 were considered significant. All data were presented as mean ± SEM.