Establishment of a new animal model of azithromycin-induced liver injury and study the molecular pathological change during the process

Abstract

The purpose of the present study is to establish a new animal model of azithromycin (AZ)-induced liver injury and study the molecular pathological change during the process. First, mice were respectively injected intraperitoneally with AZ of different high doses. Our results showed that 800 mg/kg AZ injection significantly induced liver injury in the mice, which reflected an ideal process of liver injury and repair. In this study, we analyzed the molecular pathological changes during the process by hematoxylin and eosin staining, immunohistochemistry, Western blot, and quantitative real-time reverse transcription polymerase chain reaction in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg injection. Our results showed that the expression of heat shock protein 70, proliferating cell nuclear antigen, vascular endothelial growth factor, caspase 3, and cytochrome P450 2E1 were significantly differently expressed during liver injury induced by 800 mg/kg AZ in mice. Our results will be conducive for further study of the pathogenesis and prevention of drug-induced liver injury.

Keywords

Azithromycin drug-induced liver injury HSP70 PCNA VEGF caspase 3

Introduction

Drug-induced hepatotoxicity is a common cause of liver injury and accounts for approximately half of the cases of acute liver failure while mimicking all forms of acute and chronic liver diseases. Many prescription drugs currently in use have side effects. This contributes to hepatotoxicity, which is becoming a serious health challenge since the liver is the major site of drug metabolism.¹ Animal models of drug-induced liver injury have been limited, which is not conducive for studying the pathogenesis and prevention of drug-induced liver injury.

Azithromycin (AZ) is one of the new generation macrolides with numerous advantages, such as a high oral bioviability, a rapid cell absorption and wide distribution in the organism, and administration only once a day.² AZ is used for the treatment of multiple bacterial infections, most often those causing middle ear and upper respiratory tract infections, bronchitis, and community-acquired pneumonia.³ But some reports have shown that high-dose AZ treatment or the normal use of azithromycin in some situations could induce liver injury.^3

–8 But the concrete molecular process of AZ-induced liver injury has not been studied.

In order to establish a new animal model of drug-induced liver injury, mice were given intraperitoneal (i.p.) injection of AZ with different concentrations and recover at room temperature for different times. After this, we determined the optimal concentration of AZ which induced liver injury in mice. In addition, the molecular pathological change was studied during the process in the liver of mice induced by AZ of optimal concentration, which will be helpful to understand the pathogenesis of AZ-induced liver injury at the molecular level.

Materials and methods

Chemicals

AZ was purchased from GuangZhou Bai Yun Shan Pharmaceutical General Factory (Guangzhou, China). All other reagents and solvents used in this study were purchased commercially and were of analytical pure grade.

Animals

Male BALB/c mice (approximately aged 6–8 weeks and weighing 22 ± 2 g) were purchased from the experimental animals center of Henan province and maintained in an air-conditioned animal room at 25°C with free access to water and food under 12-h light/12-h dark cycles for the experiments. All animals were allowed to adapt to the environment for 1 week before the experiment and fed on laboratory chow. All protocols conformed to the guidelines from the National Animal Care and Use Committee of China. All animals received care in compliance with the Principles of Laboratory Animal Care.

Experimental design

Mice were divided into five groups, with 30 mice in each group. The mice in each group respectively received a single i.p.injection of PBS, 400 mg/kg AZ, 600 mg/kg AZ, 800 mg/kg AZ, and 1000 mg/kg AZ. AZ was dissolved in PBS at a concentration of 10 mg/ml. The mice of each group were killed at 0, 12, 24, 48, and 72 h by carbon dioxide asphyxiation after the AZ injection. At each time point, six mice were killed. After killing, blood and liver samples were collected for further analysis. The experiment was repeated three times. In addition, the same five groups received a single i.p. injection of PBS, 400 mg/kg AZ, 600 mg/kg AZ, 800 mg/kg AZ, and 1000 mg/kg AZ, respectively, and the mortality was counted for each group.

Serum AST and ALT levels detection

Blood was drawn through orbital vein of mice at 0, 12, 24, 48, and 72 h in the mice of all the five groups after AZ injection. Each time point contained six mice. The coagulated blood is left to clot at room temperature for approximately 15–30 min. After complete clotting, it is rimmed using an applicator stick and then centrifuged for approximately 5–10 min at 2500 r/min. Then the supernatant fluid is separated. Serum aspartate transaminase (AST) and alanine transaminase (ALT) activities were determined using a commercial assay kit (Nanjing Jiancheng Biological Technology, Inc., China) at 0, 6, 24, 42, and 72 h in the mice of five groups after AZ injection. Enzyme activities were expressed as international units per liter.

Histopathological examination

Liver specimens were obtained from the mice at 0, 12, 24, 48, and 72 h after AZ injection in the mice of each group. Samples of liver were fixed in 10% formaldehyde for 24 h and then dehydrated and embedded in paraffin. Six micrometer thick sections were cut from each paraffin-embedded tissue and stained with hematoxylin–eosin (HE). To evaluate the degree of necrosis after acute liver injury, we referred to an injury grading score (grades 0 to 4) based on severity of necrotic lesions in the liver parenchyma.⁹ The grades were as follows: grade 0 = normal histology; grade 1 = presence of degenerated hepatocytes with only rare foci of necrosis; grade 2 = mild centrilobular necrosis around the central vein, occupying only a part of Rappaport’s zone 3; grade 3 = established necrosis limited to zone 3; and grade 4 = extensive, confluent centrilobular necrosis involving Rappaport’s zone 3 and 2.

Immunohistochemistry of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg injection

Our results showed that 800 mg/kg AZ injection significantly induced liver injury in the mice and the process reflected the ideal process of liver injury and repair. So, 800 mg/kg AZ proved to be a suitable dose to establish an animal model of drug-induced liver injury. Then, the molecular pathological change during the process induced by 800 mg/kg AZ in the mice was analyzed. Six micrometer thick sections were cut from each paraffin-embedded tissue as prepared above. The sections of liver from the mice in different groups were immunostained with a monoclonal antibody to mouse heat shock protein 70 (HSP70), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), caspase 3, and cytochrome P450 2E1 (CYP2E1; dilution 1:300; Santa Cruz Biotechnology, Dallas, Texas, USA) as described by Xu et al.¹⁰ The signal was detected using the Polink-2 plus polymer horseradish peroxidase (HRP) detection system (Zhongshan, Beijing, China) using 3,3′-diaminobenzidine (DAB; Sigma, St Louis, Missouri, USA). A negative control was carried out on each slide by omitting the primary antibody. Sections were examined microscopically for specific staining, and photographs were taken using a digital image capture system (Olympus, Tokyo, Japan).

Apoptosis analysis by TUNEL

Apoptosis in the hepatocytes were further analyzed at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ injection in the mice using a commercial kit (GENMED, Shanghai, China) based on the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatase-digoxigenin nick end labeling (TUNEL) of apoptotic cells. Sections were examined microscopically for specific staining, and photographs were taken using a digital image capture system (Olympus).

Western blot of HSP70, PCNA, VEGF, caspase 3, and CYP2E1

Protein samples of 70 μg from the liver of mice at different time points after 800 mg/kg AZ injection were adjusted to the composition of the electrophoresis sample buffer (50 mM Tris, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 0.1% bromphenol blue) and boiled for 5 min prior to analysis. SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gels) in 1 mm slab gel was performed as described by Li et al.¹¹ The proteins were transferred from the gel to the nitrocellulose membranes. Then the membrane was probed with a monoclonal antibody to mouse HSP70, PCNA, VEGF, caspase 3, and CYP2E1 (Santa Cruz), respectively. The signal was detected by HRP detection system using DAB. Protein bands were quantified with Gel Pro Analyzer software 4.0 (Media Cybernetics Inc., Bethesda, Maryland, USA), and the intensities of the bands were normalized against β-actin. Each experiment was repeated three times.

Quantitative real-time RT-PCR for mRNA expression detection of HSP70, PCNA, VEGF, caspase 3, and CYP2E1

The specific primers for these genes were designed using Primer3¹² and are listed in Table 1. Samples of RNA extracted from individual mice were used in the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. Briefly, complementary DNA (cDNA) was synthesized from 1 µg of RNA in the presence of ribonuclease inhibitor (BBI, Toronto, Canada), deoxynucleotide triphosphates (Sangon, Shanghai, China), Oligo(dT)18 primers, and RevertAid™ M-Mulv reverse transcriptase (Fermentas, Hanover, Maryland, USA) in a total of 25 µl reaction mix. Real-time RT-PCR was conducted using Applied Biosystems PRISM 7900HT system (Applied Biosystems, Foster City, California, USA) and SYBR Green I chemistry (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The final concentration of each primer was 200 nM. The signal of the target gene was normalized to β-actin and compared with the control by a 2^−ΔΔC_T method.¹³ A plot of the log cDNA dilution versus ΔC_T was made to calculate the amplification efficiencies of the target and reference.¹³

Table 1.

Oligonucleotide primers used in quantitative real-time RT-PCR.

Gene name	GenBank accession No.	Forward primer	Reverse primer
Actb	NM_007393	5′-CTGTCCCTGTATGCCTCTG	5′-ATGTCACGCACGATTTCC-3′
HSP70	NM_031165	5′-AAATCATCAGCTGGCTGGATAAGAA	5′-CATGCCACCTGCACTCTGGTA-3′
PCNA	AK034723	5′-CTGAAGAAGGTGCTGGAGGCT	5′-TTTGGACATGCTGGTGAGGTT-3′
VEGF	NM_001025250.3	5′-GGCAGACTATTCAGCGGACTC	5′-CTCAAACCGTTGGCACGA-3′
Casp3	NM_009810	5′-CTGCCGGAGTCTGACTGGAA	5′-ATCAGTCCCACTGTCTGTCTCAATG-3′
CYP2E1	NM_021282	5′-CCACCCTCCTCCTCGTAT-3′	5′-CTTGACAGCCTTGTAGCC-3′

Actb: β-actin; HSP70: heat shock protein 70; AZ: azithromycin; VEGF: vascular endothelial growth factor; PCNA: proliferating cell nuclear antigen; Casp3: caspase 3; CYP2E1: cytochrome P450, family 2, subfamily e, polypeptide 1.

Statistical analysis

All data were presented as the mean ± standard deviation. Statistical comparisons were made using one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. All statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, Illinois, USA).

Results

Alterations of serum AST and ALT levels and survival rate in the mice

Figure 1 shows that 400 mg/kg AZ and 600 mg/kg AZ injection did not significantly induce the increase of serum AST and ALT levels from 0 h to 72 h after AZ injection in the mice compared with those that received PBS injection (p > 0.05). Serum AST and ALT levels were significantly upregulated from 0 h to 24 h (p < 0.05 or p < 0.01), then gradually declined, and finally reached near the basal value at 72 h after 800 mg/kg AZ injection in mice compared with those that received PBS injection. The dosage of 1000 mg/kg AZ injection always remarkably induced the increase of serum AST and ALT levels and reached the peak levels at 72 h after AZ injection in mice compared with those that received PBS injection (p < 0.05 or p < 0.01). The serum AST and ALT levels in 1000 mg/kg AZ-treated mice were significantly higher than those in 800 mg/kg AZ-treated mice at 24, 48, and 72 h after AZ injection in the mice (p < 0.05 or p < 0.01). The survival rate of 800 mg/kg AZ-treated mice (90%) and 1000 mg/kg AZ-treated mice (70%) were significantly lower than those in PBS (100%), 400 mg/kg AZ (100%), and 600 mg/kg AZ (100%)-treated mice (p < 0.05 or p < 0.01).

Figure 1.

Serum AST (a) and ALT (b) levels in the mice at 0, 12, 24, 48, and 72 h after PBS or azithromycin treatment. PBS, 400 mg/kg AZ, 600 mg/kg AZ, 800 mg/kg AZ, 1000 mg/kg AZ: The mice in each group respectively received a single i.p. injection of PBS, 400 mg/kg, 600 mg/kg, 800 mg/kg, or 1000 mg/kg AZ. **p < 0.01, *p < 0.05: significant difference in each AZ treatment group as compared to PBS treatment group; #p < 0.05, ##p < 0.01: significant difference in 1000 mg/kg AZ-treated group as compared to 800 mg/kg AZ-treated group. Each experiment was repeated three times. AZ: azithromycin; AST: aspartate transaminase; ALT: alanine transaminase.

Histology change in the liver of mice after AZ injection

Table 2 and Figure 2 show that 400 mg/kg AZ and 600 mg/kg AZ injection did not significantly induce liver injury (p > 0.05), while 800 mg/kg AZ and 1000 mg/kg AZ injection remarkably induced liver injury (p < 0.01) compared with those received PBS injection at 24 h after AZ injection in mice. The liver injury was very severe in 1000 mg/kg AZ-treated mice than 800 mg/kg AZ-treated mice (p < 0.05) at 24 h after AZ injection. After 24 h, liver injury was ameliorated, and liver began to repair until 72 h in 800 mg/kg AZ-treated mice (Table 2). But the liver injury was still aggravated from 0 h to 72 h in 1000 mg/kg AZ-treated mice (Table 2).

Figure 2.

Histologic examination of liver injury in mice at 24 h after PBS or AZ administration by hematoxylin–eosin staining. (a, b, c, d, and e) Represent the degree of liver injury in mice with PBS, 400 mg/kg AZ, 600 mg/kg AZ, 800 mg/kg AZ, and 1000 mg/kg AZ injection, respectively and (f) necrotic areas. Representative findings from at least 10 mm² tissue sections were counted for each mouse. **p < 0.01: significant difference in each AZ treatment group as compared to PBS treatment group; #p < 0.05, ## p < 0.01: significant difference in 1000 mg/kg AZ group as compared to other AZ-treated groups; Δp < 0.05: significant difference in 800 mg/kg AZ-treated group as compared to 400 mg/kg AZ and 600 mg/kg AZ-treated groups. Each experiment was repeated three times (scale bar: 50 µm). AZ: azithromycin.

Table 2.

Liver injury degree.^a

Group of mice	12 h	24 h	48 h	72 h
PBS	0	0	0	0
400 mg/kg AZ	0	0	0	0
600 mg/kg AZ	0–1	0–1	0–1	0
800 mg/kg AZ	1–2^b	2–3^c,d	1^b,e	0–1 ^e
1000 mg/kg AZ	2 ^b	3 ^c	3–4 ^c	4 ^c

AZ: azithromycin; i.p.: intraperitoneal.

^aTime (hours) after AZ i.p. injection in the mice. Injury degree according to severity of necrosis in the liver parenchyma: grade 0 = normal histology; grade 1 = presence of degenerated hepatocytes with only rare foci of necrosis; grade 2 = mild centrilobular necrosis around the central vein, occupying only a part of Rappaport’s zone 3; grade 3 = established necrosis limited to zone 3; grade 4 = extensive, confluent centrilobular necrosis involving Rappaport’s zone 3 and 2. PBS, 400 mg/kg AZ, 600 mg/kg AZ, 800 mg/kg AZ, 1000 mg/kg AZ: The mice in each group respectively received a single i.p. injection of PBS, 400 mg/kg, 600 mg/kg, 800 mg/kg, or 1000 mg/kg AZ and recovered at room temperature for 0, 12, 24, 48, and 72 h. Each experiment was repeated three times.

^bp < 0.05: significant difference in each AZ treatment group as compared to PBS treatment group.

^cp < 0.01: significant difference in each AZ treatment group as compared to PBS treatment group.

^dp < 0.05: significant difference in 1000 mg/kg AZ-treated group as compared to the 800 mg/kg AZ-treated group.

^ep < 0.01: significant difference in 1000 mg/kg AZ-treated group as compared to the 800 mg/kg AZ-treated group.

Immunohistochemistry of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ injection

Figures 3, 5, 6, and 7 show that HSP70, VEGF, caspase 3, and CYP2E1 were mostly expressed in the cytoplasm of hepatocytes of mice. Figure 4 shows that PCNA was mainly expressed in the nucleus of hepatocytes. The expression of HSP70, caspase 3, and CYP2E1 were significantly upregulated from 0 h to 24 h and got to the peak level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. After that, the expression of HSP70, caspase 3, and CYP2E1 were gradually downregulated and reached near the normal level at 72 h compared with those at 0 h (Figures 3, 6, and 7). On the contrary, the expression of PCNA and VEGF were first decreased from 0 h to 24 h and got to the lowest level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. Then, the expression of PCNA and VEGF were gradually increased and reached near the normal level at 72 h (Figures 4 and 5).

Figure 3.

Photomicrographs of immunohistochemical staining of HSP70 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. (a, b, c, d, and e) Represent the immunohistochemical staining in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. The arrowheads indicate the HSP70-positive cells in the liver of mice. (f) Numbers of HSP70-positive cells in the liver of mice at different time points, and tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA). **p < 0.01, *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h; #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. Each experiment was repeated three times (scale bar: 50 µm). HSP70: heat shock protein 70; AZ: azithromycin.

Figure 4.

Photomicrographs of immunohistochemical staining of PCNA in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. (a, b, c, d, and e) Represent the immunohistochemical staining in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment, respectively. The arrowheads indicate the PCNA-positive cells in the liver of mice; (f) numbers of PCNA-positive cells in the liver of mice at different time points, and tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA). **p < 0.01 or *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h. #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. Each experiment was repeated three times (scale bar: 50 µm). PCNA: proliferating cell nuclear antigen; AZ: azithromycin.

Figure 5.

Photomicrographs of immunohistochemical staining of VEGF in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. (a, b, c, d, and e) Represent the immunohistochemical staining in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment, respectively. The arrowheads indicate the VEGF-positive cells in the liver of mice. (f): Numbers of VEGF-positive cells in the liver of mice at different time points, and tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA). **p < 0.01, *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h; #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &&p < 0.01, &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. Each experiment was repeated three times (scale bar: 50 µm). VEGF: vascular endothelial growth factor; AZ: azithromycin.

Figure 6.

Photomicrographs of immunohistochemical staining of caspase 3 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. (a, b, c, d, and e) Represent the immunohistochemical staining in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment, respectively. The arrowheads indicate the caspase 3-positive cells in the liver of mice. (f): Numbers of caspase 3-positive cells in the liver of mice at different time points, and tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA). **p < 0.01, *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h; #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. Each experiment was repeated three times (scale bar: 50 µm). AZ: azithromycin.

Figure 7.

Photomicrographs of immunohistochemical staining of CYP2E1 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. (a, b, c, d, and e) Represent the immunohistochemical staining in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment, respectively. The arrowheads indicate the CYP2E1-positive cells in the liver of mice. (f): Numbers of CYP2E1-positive cells in the liver of mice at different time points, and tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA). **p < 0.01, *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h; #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. Each experiment was repeated three times (scale bar: 50 µm). CYP2E1: cytochrome P450 2E1; AZ: azithromycin.

Hepatocyte apoptosis analysis during liver injury after 800 mg/kg AZ injection

Our results showed that hepatocytes apoptosis was significantly increased from 0 h to 24 h and got to the peak level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. Then, hepatocytes apoptosis was gradually decreased and reached near the normal level at 72 h compared with those at 0 h (Figure 8).

Figure 8.

Photomicrographs of results of TUNEL assay of liver sections prepared from the mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. (a, b, c, d, and e) Represent the TUNEL assay in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment, respectively. The arrowheads indicate the TUNEL-positive cells in the liver of mice. (f) Percentages of TUNEL-positive cells among total hepatocytes at least 12 mm² tissue sections were counted for each mouse by ImagePro Plus 6.0 software (Media Cybernetics, Bethesda, Maryland, USA). **p < 0.01, *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h; #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &&p < 0.01 &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. Each experiment was repeated three times (scale bar: 50 µm). TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatase-digoxigenin nick end labeling; AZ: azithromycin.

Analysis of the expression of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 by Western blot

The results of Western blot show that the expression of HSP70, caspase 3, and CYP2E1 were significantly increased from 0 h to 24 h and got to the peak level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. After that, the expression of HSP70, caspase 3, and CYP2E1 were gradually decreased and reached near the normal level at 72 h compared with those at 0 h (Figure 9(a), (b), (e), and (f)). At the same time, the expression of PCNA and VEGF were first decreased from 0 h to 24 h and got to the lowest level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. Then, the expression of PCNA and VEGF were gradually increased and reached near the normal level at 72 h (Figure 9(a), (c), and (d)).

Figure 9.

The expression of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. The expression of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 were detected by Western blot (a). The protein bands were quantified for HSP70 (b), PCNA (c), VEGF (d), caspase 3 (e), and CYP2E1 (f) with the Gel-Pro Analyzer 4.0 software (Media Cybernetics Inc.), and the intensities of the bands were normalized against β-actin. AU represents arbitrary unit. Each experiment was repeated three times. All data were presented as the mean ± standard deviation. **p < 0.01, *p < 0.05: significant difference at 12, 24, 48, and 72 h as compared to 0 h; #p < 0.05, ##p < 0.01: significant difference at 24 h as compared to 12, 48, and 72 h; &&p < 0.01 &p < 0.05: significant difference at 12 h as compared to 48 and 72 h; Δp < 0.05: significant difference at 48 h as compared to 72 h. HSP70: heat shock protein 70; AZ: azithromycin; VEGF: vascular endothelial growth factor; PCNA: proliferating cell nuclear antigen; CYP2E1: cytochrome P450 2E1.

Analysis of the mRNA expression of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 in the liver of mice by qRT-PCR

Figure 10 shows that 800 mg/kg AZ injection remarkably induced the messenger RNA (mRNA) expression of HSP70, caspase 3, and CYP2E1 (p < 0.05 or p < 0.01) but inhibited the mRNA expression of PCNA and VEGF from 0 h to 24 h (p < 0.05 or p < 0.01) compared with those at 0 h. With the restoration after liver injury, the mRNA expression of HSP70, caspase 3, and CYP2E1 were decreased (p < 0.05 or p < 0.01) and the mRNA expression of PCNA and VEGF were increased (p < 0.05 or p < 0.01) and reached near the normal level compared with those at 0 h.

Figure 10.

Fold changes in mRNA expression of HSP70, PCNA, VEGF, caspase 3, and CYP2E1 in the liver of mice at 0, 12, 24, 48, and 72 h after 800 mg/kg AZ treatment. The expression of HSP70 (a), PCNA (b), VEGF (c), caspase 3 (d), and CYP2E1 (e) were detected by quantitative real-time RT-PCR and normalized against β-actin. Fold changes in mRNA expression was relative to that in 0 h. Each experiment was repeated three times. All data were presented as the mean ± standard deviation. mRNA: messenger RNA; HSP70: heat shock protein 70; AZ: azithromycin; VEGF: vascular endothelial growth factor; PCNA: proliferating cell nuclear antigen; CYP2E1: cytochrome P450 2E1; RT-PCR: reverse transcription polymerase chain reaction.

Discussion

In this study, we first establish a new animal model of drug-induced liver injury induced by azithromycin and study the molecular pathological change during the process.

Some reports have shown that high-dose AZ treatment or the normal use of azithromycin in some situations could induce liver injury.^3

–8 But the concrete molecular process of AZ-induced liver injury has not been studied. Our results showed that 800 mg/kg and 1000 mg/kg AZ injection significantly induced the increase of serum AST and ALT levels (p < 0.05 or p < 0.01; Figure 1) and liver injury (Table 2 and Figure 2; p < 0.05 or p < 0.01) compared with those that received PBS injection. But the process of 800 mg/kg AZ-induced liver injury reflected the ideal process of liver injury and repair than that induced by1000 mg/kg AZ. So we determined that an animal model of 800 mg/kg AZ-induced liver injury is an ideal animal model of drug-induced liver injury.

Our results showed that the expression of HSP70 were significantly upregulated from 0 h to 24 h and got to the peak level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. After that, the expression of HSP70 were gradually downregulated and reached near the normal level at 72 h compared with those at 0 h (Figures 3, 9(a) and (b), and 10(a)). It has been suggested that the primary function of HSPs is to serve as molecular chaperones in which they recognize and bind nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding, or as chaperones that facilitate protein folding directly.^14,15 Molecular chaperones are necessary not only for the folding of newly synthesized proteins in the cells but also for the protection of proteins during exposure to stressful situations such as heat shock, which causes proteins folded previously to unfold.^16,17 It has also been speculated that HSPs reduce the oxidative injury that is caused by oxygen free radicals and occurs during the early phase of reperfusion.^18,19 So we think that HSP70 may play important roles in protection of proteins during exposure to stressful situations of AZ-induced liver injury in mice. With the liver repair after injury, the expression of HSP70 was gradually decreased and reached near the normal level in mice (Figures 3, 9(a) and (b), and Figure 10(a)).

The expression of PCNA was first decreased from 0 h to 24 h and got to the lowest level at 24 in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h. Then, the expression of PCNA was gradually increased and reached near the normal level at 72 h (Figures 4, 9(a) and (c), and Figure 10(b)). PCNA is a subunit of the mammalian DNA polymerase delta and is synthesized primarily during the S phase of the cell cycle.²⁰ PCNA is a relay or anchoring molecule that functions as a molecular integrator for proteins involved in the control of the cell cycle, DNA replication, DNA repair, and cell death.^21,22 PCNA has been shown to be a good marker to distinguish proliferating cells.^10,23 Our results indicated that the inhibition of hepatocytes proliferation were increased with the liver injury aggravated from 0 h to 24 h after 800 mg/kg AZ injection. After that, the proliferation of hepatocytes were significantly increased with the liver repair after injury.

Our results also showed that the expression change of VEGF was similar to that of PCNA during liver injury induced by 800 mg/kg AZ injection (Figures 5, 9(a) and (d), and 10(c)). The formation of new blood vessels is critical for normal development and tissue repair as well as for pathological events such as retinal neovascularization, rheumatoid arthritis, and tumor growth.^24

–29 During physiological angiogenesis, VEGF expression in response to local hypoxia drives the development of new blood vessels.^30
–32 In addition, angiogenesis is known to play a critical role in pathological settings like chronic inflammatory and tumor growth.^33
–35 VEGF is considered to be the central angiogenic factor during chronic liver injury. Our results indicated that 800 mg/kg AZ injection first inhibited angiogenesis in the liver of mice from 0 h to 24 h. Then, angiogenesis was promoted with the liver repair after injury.

Our results showed that hepatocytes apoptosis and the expression of caspase 3 were significantly increased from 0 h to 24 h and got to the peak level at 24 h in the liver of mice after 800 mg/kg AZ injection (p < 0.05 or p < 0.01) compared with those at 0 h, then gradually decreased, and reached near the normal level at 72 h compared with those at 0 h (Figures 6, 8, 9(a) and (e), and Figure 10(d)). Caspase 3 is a member of the interleukin 1β-converting enzyme or cell death effector 3 family, which is involved in the induction of apoptosis and has been considered to be correlated with apoptosis because of the most downstream enzyme in their apoptosis-inducing pathway.^36,37 These results indicate that hepatocytes apoptosis aggravated liver injury in the mice from 0 h to 24 h after 800 mg/kg AZ injection. Then, liver began to repair with hepatocytes apoptosis reducing.

The expression of CYP2E1 was also increased from 0 h to 24 h, followed by a decrease during liver injury induced by 800 mg/kg AZ in mice (Figure 7, 9(a) and (f), and 10(e)).

CYP2E1, a microsomal enzyme involved in xenobiotic metabolism and generation of oxidative stress, has been implicated in promoting liver injury.³⁸ Wong et al.³⁹ has demonstrated that CYP2E1 plays a major role in carbon tetrachloride toxicity based on a previous study with Cyp2e1-null mice. Dey and Cederbaum⁴⁰ reported that induction of CYP2E1 promoted liver injury in obese mice. Dey and Kumar³⁸ think that changes in regulation of CYP2E1 under hyperglycemic conditions are tightly linked with increased oxidative stress and injury in liver. CYP2E1 may be an important marker for liver injury in many conditions.⁴¹ Our results illustrated that CYP2E1 may play important roles in promoting liver injury induced by AZ in the mice. The significant increasing of CYP2E1 expression may be an important marker in AZ-induced liver injury.

In summary, we establish a new animal model of drug-induced liver injury induced by 800 mg/kg AZ in mice. HSP70, PCNA, VEGF, caspase 3, and CYP2E1 were significantly differently expressed during liver injury induce by 800 mg/kg AZ in mice, which may play important roles in AZ-induced liver injury. Our results will be conducive for further study of the pathogenesis and prevention of drug-induced liver injury.

Footnotes

Acknowledgments

The authors thank all the members in the laboratory when this work was carried out. The experiments comply with the current laws of China.

Declaration of Conflicing Interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (#U1204802) to SQL, Program for Science & Technology Innovation Talents in Universities of Henan Province (#13HASTIT025) to SQL, foundation for the Henan Province’s Key Project to Tackle Key Problems of Science and Technology (#122102310030).

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