The present study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Tianjin Medical University (Tianjin, China). The protocol was approved by the Animal Care Committee of Tianjin Medical University (Permit no. 2010-0002). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering of animals.

TRIzol reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The TIANScript RT kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). SYBR Green polymerase chain reaction (PCR) core reagents were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). RPMI 1640 medium was purchased from Gibco; Thermo Fisher Scientific, Inc. Fetal calf serum was purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA). Dexamethasone (Dex) was purchased from Sigma-Aldrich; Merck Millipore (Billerica, MA, USA). RU486 (mifepristone), a specific antagonist of glucocorticoid receptor, was purchased from Sigma-Aldrich; Merck Millipore.

Male Wistar rats weighing 180±20 g and aged 6 weeks were purchased from the Model Animal Center of the Radiological Medicine Research Institute, Chinese Academy of Medical Science (Beijing, China). The rats were housed in standard laboratory cages (n=5) at 22°C with a 12 h light/dark cycle and free access to food and water. A total of 30 rats were divided into two groups (15 per group), comprising a cigarette smoking-exposed group and an unexposed control group. As described previously (24), the smoking group received whole-body exposure to the smoke of five unfiltered cigarettes (Daqianmen™; tar ≤15 mg, nicotine ≤1.1 mg and CO ≤13 mg) for 30 min, twice daily (prior to 9:00 a.m and after 5:00 p.m), for 14 weeks inside a 0.6 m³ custom plexiglass chamber (constructed in-house). The protocol for the control group was identical, but without smoke exposure.

LO2 cells (a gift from Chenghu Liu lab, NanKai University, Tianjin, China) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO₂. In the experiments, 10 nM Dex and 10 µM RU486 were added into the culture medium when the cells were 80% confluence.

At the end of smoking exposure period, the rats were anesthetized with sodium pentobarbital and sacrificed by cervical dislocation. The abdominal cavity was opened and the liver tissues were excised, rinsed in ice-cold PBS (pH 7.4), and then either stored at −80°C for the analysis of gene expression or fixed in 10% neutral-buffered formalin for the analysis of histology.

Following fixation in 10% neutral-buffered formalin, the liver tissues were embedded in paraffin and 5 µm thick sections were cut. The sections were then stained with HE solution (Solarbio Science & Technology Co., Ltd., Beijing, China) and images of the staining were captured under an Olympus IX71 microscope with a DP80 camera (Olympus Corporation, Tokyo, Japan).

RNA was extracted from either the liver tissues or the LO2 cells using TRIzol reagent. The RNA (3 µg) was then reverse transcribed using oligo (dT) primers for 1 h at 50°C using the TIANScript RT kit (Tiangen Biotech, Co., Ltd.) according to the manufacturer's protocol. The qPCR analysis was performed using the SYBR Green method. Specific gene primers were designed using Primer-Quest SM software (http://www.idtdna.com/Scitools/Applications/PrimerQuest; Integrated DNA Technologies, Inc., Coralville, IA, USA), and then commercially produced (BGI Tech, Shenzhen, China; listed in Tables I and II). The DNA amplification reactions were performed on a Light Cycler 96 real-time PCR system (Roche Diagnostics, Indianapolis, IN, USA) under the following reaction conditions: An initial heating cycle of 95°C for 2 min; and 40 cycles alternating between denaturation at 95°C for 25 sec, primer annealing at 60°C for 25 sec and extension at 72°C for 20 sec. Melting curves were used to clarify the identity of amplicons, and the housekeeping gene, GADPH, served as an internal control. The relative mRNA expression levels of targeted genes were calculated using the comparative Cq (quantification cycle) method normalized to GAPDH mRNA in the same sample. Briefly, specific ΔCq was calculated as follows: ΔCq = (CqGAPDH) - (Cqtarget); and relative expression was defined as: 2^−ΔCq (24).

The data were analyzed using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). Data from three or more independent experiments were collected and analyzed as the mean ± standard error of the mean. The significance of the results was assessed using a paired t-test between two groups. P<0.05 was considered to indicate a statistically significant difference.

Evidence has been accumulating, which indicates that the progression of liver disease is associated with cigarette smoking (25). To assess the inflammatory status in the liver upon smoking exposure, the present study used a smoking-exposed rat model. Examination of liver tissues by HE staining showed no significant structural alterations or inflammatory infiltrates in the rats exposed to smoke for 14 weeks (smoke group), compared with the control group (Fig. 1A and B). However, the mRNA expression of IL1β was higher in the livers from the smoke group, compared with those from the control group (Fig. 1C). Unlike IL1β, the mRNA expression levels of IL6 and TNFα remained unchanged following smoke exposure (Fig. 1D and E).

Inflammation is shown to involve cross-talk with autophagy in the liver (23). To evaluate the effects of smoking on autophagic activity in the liver, RT-qPCR analysis was performed. The results revealed that the expression of mammalian target of rapamycin (mTOR), an inhibitor of autophagy, was decreased in the smoke group, compared with the control group, although this was not significant (data not shown). However, the expression of AMP-activated protein kinase (Ampk), a negative regulator of the mTOR pathway, was elevated when the rats were exposed to smoke (Fig. 2A). In addition, the expression of Wip1, a positive regulator of the mTOR pathway, was decreased in the smoke group, compared with the control group (Fig. 2B). These findings indicated that autophagy in the liver may be induced by smoking. To confirm this, the present study examined the effects of smoking on the expression of autophagy-associated genes in the liver tissues. The expression levels of Atg5 and Atg12 were comparable between the smoke group and control group (Fig. 2C and D). However, the expression levels of Ulk1 and annexin A3 (Anx3), a homolog of human microtubule associated protein 1 light chain 3 (LC3) were significantly higher in the smoke group, compared with those in the control group (Fig. 2E and F). Taken together, these data suggested that autophagy was upregulated in the liver upon smoking exposure.

Cyp enzymes possess the capacity to catalyze the oxidative biotransformation of the majority of drugs. To examine whether smoking-induced inflammation alters hepatic function by regulating the expression of Cyp, the present study measured the expression of Cyp genes in the liver. A significant reduction in the mRNA expression level of Cyp1A2 was observed in the smoke group, compared with the control group (Fig. 3A). For the Cyp2 family, the expression of Cyp2D4, but not Cyp2C9 or Cyp2C19, was significantly reduced following smoke exposure (Fig. 3B-D). As for the Cyp3 family, the expression of Cyp3A2 was decreased in the smoke group (Fig. 3E). In concordance with previous reports showing a reduction in the expression of Cyp during inflammation (26,27), these data suggested that smoking-induced inflammation altered the catalyzing capacity of the liver.

It is known that the gene expression of Cyp is regulated by nuclear receptors, including PXR and CAR (28–30). The present study investigated effects of smoking exposure on the expression of these nuclear receptors in the liver. It was found that the hepatic mRNA expression of CAR was comparable between the control and smoke groups (Fig. 4A). However, the expression levels of PXR and GR were significantly reduced in the smoke group, compared with the control group (Fig. 4B and C). These results suggested that the reduction in the hepatic expression of Cyp in the smoking-exposed rats may have been attributed to the reduced synthesis of nuclear receptors, including GR, in the liver.

The present study used the LO2 human hepatocyte cell line to evaluate the role of GR in regulating the expression of CYP. The LO2 cells were treated with Dex alone or with Dex and the GR inhibitor, RU486 for 2 days. LO2 cells are sensitive to Dex. Compared with the control cells (Fig. 5A), treatment with Dex alone resulted in morphological alterations, for example the LO2 cells became longer and more stretched (Fig. 5B). These morphological changes were absent when the LO2 cells were treated with Dex and RU486 (Fig. 5C). Dex exhibited differential effects on the expression of CYP by LO2 cells. Treatment of the LO2 cells with Dex alone led to an increase in the expression of CYP3A4, however, this increase was reduced when the LO2 cells were incubated with Dex and RU486 (Fig. 5D). Similarly, Dex treatment resulted in upregulation of thee expression of CYP2C19. The expression of CYP2C19 was decreased, although not significantly, in cells treated with Dex and RU486, compared with the Dex group (Fig. 5E). By contrast, treatment with Dex alone resulted in a significant reduction in the expression of CYP2C9 in LO2 cells (Fig. 5F). The expression of CYP2C9 in the Dex and RU486 group returned to a level, which was comparable with that of the control cells. However, Dex had no effect on the expression of CYP2D6 (Fig. 5G). Similar to CYP3A4, LO2 cells with Dex alone led to an increase in the expression of CYP1A2, however, this increase was reduced when the LO2 cells were incubated with Dex and RU486 (Fig. 5H). Collectively, these data suggested that GR exhibited a differential role in regulating hepatic CYP expression.

To evaluate effects of GR on autophagic activity in human heptocytes, the present study measured autophagic genes in LO2 cells in response to Dex. No significant change in the RNA expression levels of mTOR, ATG5, ATG12, LC3 or ULK1 were found in the Dex-treated cells, compared with the control cells or the Dex and RU486-treated cells (Fig. 6A-E). Among the genes selected in the present study, only the expression of Beclin 1 was reduced in the Dex-treated cells, however, the addition of RU486 did not recover the expression level (Fig. 6F). Taken together, these data suggested that GR had minimal effect on the regulation of autophagy in LO2 cells.