Primary antibodies against phosphorylated (p)-ERK1/2 (cat. no. 4370S; 1:1,000), (p)-p38 (cat. no. 4511S; 1:1,000), (p)-JNK (cat. no. 9251S; 1:1,000), (p)-ERK5 (cat. no. 3371S; 1:500), (p)-c-Jun (cat. no. 9164S; 1:500), (p)-c-Fos (cat. no. 5348S; 1:500), Fos-like 2 activator protein-1 (AP-1) transcription factor subunit (Fra-2) (cat. no. 19967S; 1:1,000), E-cadherin (cat. no. 3195S; 1:1,000), N-cadherin (cat. no. 4061S; 1:500) and vimentin (cat. no. 3932S; 1:1,000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Zona occludens (ZO)-1 (cat. no. sc-8146; 1:500) primary antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The primary antibody for GAPDH (cat. no. 5014; 1:3,000) was from Biogot Technology Co., Ltd. (Nanjing, China). E-cadherin, ZO-1, N-cadherin, vimentin and GAPDH primers for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were synthesized according to published sequences from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Curcumin (purity, >99%) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

A total of 52 male BALB/c mice (4-weeks-old; weight, 18–22 g) were purchased from the Animal Research Center of Nanjing Medical University (Nanjing, China). All mice were allowed one week acclimatize to circumstances prior to experimental exposure. Mice were raised in polypropylene cages at 22±0.5°C and 40–60% relative humidity with a 12-h light/dark cycle, and water and basal diet were provided ad libitum. All mice procedure protocols were approved by the Animal Care and Welfare Committee of Nanjing Medical University.

Mice were randomly divided into tobacco smoke-exposure and control groups (n=10/group). The tobacco smoke-exposure group was treated with tobacco smoke for 6 h daily for 12 weeks. The control group animals were exposed to filtered air. Mice in the tobacco smoke group were exposed to tobacco smoke in a smoking apparatus designed by the authors. A smoke machine was used to combust filterless commercial cigarettes (12 mg tar and 1.1 mg nicotine per cigarette, Hongtashan brand; Hongta Group, Yuxi, China) to generate tobacco smoke. The smoke machine pumped the cigarette smoke regularly from burning filterless cigarettes (5 min/cigarette). The target concentration of smoke was delivered to whole-body exposure chambers in total particulate matter of 85 mg/m³. The components were monitored and characterized as: Carbon monoxide (14.72±2.89 mg/m³) and total particulate matter TPM (0 mg/m³) for the control group; and carbon monoxide (184.07±23.51 mg/m³) and TPM (83.53±5.63 mg/m³) for the tobacco smoke exposure group. Mice were sacrificed at the end of the final tobacco smoke exposure, and the lung tissues were isolated, frozen and stored at −80°C until analysis.

Doses were selected based on those employed by previous studies involving the treatment of mice with curcumin, which were 50 or 100 mg/kg body weight (BW) per day in animal models (30,31). BALB/c mice were randomly assigned into the following four groups (n=8 per group): Filtered air group, in which mice were exposed to filtered air and under a controlled diet; a tobacco smoke-exposed group, in which mice were exposed to tobacco smoke and under a controlled diet; tobacco smoke + curcumin 50 mg group, in which mice were exposed to tobacco smoke and under a controlled diet supplemented with curcumin at a dose of 50 mg/kg BW/day; and tobacco smoke + curcumin 100 mg group, in which mice were exposed to tobacco smoke and under a controlled diet supplemented with curcumin at a dose of 100 mg/kg BW/day. The mice were exposed to filtered air or tobacco smoke at a target concentration of 85 mg/m³ total particulate matter for 6 h daily for 12 weeks. Dietary consumption levels and body weight were measured and recorded every 3 days to determine the administration dosages of curcumin. The diets were prepared weekly for each group. At the end of the exposure, mice were sacrificed and the lung tissues were frozen and stored at-80°C until analysis.

Western blot analyses were performed following standard procedures. Briefly, lung tissues were lysed (lysate buffer, 5 mmol/l EDTA; 50 mmol/l Tris pH 7.5, 1% sodium dodecyl sulfate (SDS), 10 µg/ml aprotinin, 1% sodium deoxycholate, 1% NP-40, 1% Triton-X 100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) and the lysate supernatants were obtained by centrifugation at 12,000 × g for 20 min at 4°C and the pellets were discarded. A BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to measure protein concentrations. Electrophoresis was performed to fractionate 50 µg proteins by 7.5–10% SDS-PAGE. Subsequently, proteins were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (cat. no. 00001-2; 1:5,000) from Biogot Technology Co., Ltd. (Nanjing, China) for 1 h at room temperature. The membranes were developed with SignalFire Elite ECL Reagent (Cell Signaling Technology, Inc.). GAPDH served as the loading control. ImageJ k 1.45 (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometric analysis on protein bands.

Total RNA was extracted from the mouse lung tissues using RNAiso Plus (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. Subsequently, 2 µg purified total RNA was reverse transcribed to cDNA using PrimeScript RT Master mix (Takara Bio, Inc.) according to the manufacturer's protocol. The RT reaction was performed as follows: 37°C 15 min, 85°C 5 sec, and hold at 4°C, using AMV Reverse Transcriptase (Promega Corporation, Madison, WI, USA). qPCR was performed according to the manufacturer's protocol: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 30 sec, 72°C for 30 sec using the SYBR^® Premix Ex Taq^™ II (Takara Bio, Inc.) and an ABI prism 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers (Invitrogen; Thermo Fisher Scientific, Inc.) used were as follows: E-cadherin forward, 5′-TCGACACCCGATTCAAAGTGG−3′ and reverse, 5′-TTCCAGAAACGGAGGCCTGAT−3′; ZO-1 forward, 5′-GCAGCCACAACCAATTCATAG−3′ and reverse, 5′-GCAGACGATGTTCATAGTTTC-3′; vimentin forward, 5′-CCTTGACATTGAGATTGCCA-3′ and reverse, 5′-GTATCAACCAGAGGGAGTGA-3′; N-cadherin forward, 5′-ATCAAGTGCCATTAGCCAAG-3′ and reverse, 5′-CTGAGCAGTGAATGTTGTCA-3′; GAPDH forward, 5′-GCTGCCCAACGCACCGAATA-3′ and reverse, 5′-GAGTCAACGGATTTGGTCGT-3′. The specificity of the PCR products was confirmed using melting curve analysis. Relative gene expression levels, normalized to GAPDH expression, were calculated by a comparative threshold cycle (Cq) method using the formula 2^−(ΔΔCq) (32). Each sample was performed in triplicate.

Statistical differences were analyzed using one-way analysis of variance for comparison of statistical differences among multiple groups and the Fisher's least significant difference test. Unpaired Student's t-test was also used for comparisons between two groups. SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Tobacco smoke is a major risk factor for lung cancer. EMT has important functions in various types of cancer, including lung cancer, and tobacco smoke-induced EMT is implicated in tobacco smoke-associated malignant transformations. To determine whether alterations in EMT were induced by tobacco smoke in an animal model, mice were exposed to tobacco smoke for 12 weeks, and the expression of the epithelial markers E-cadherin and ZO-1, and the mesenchymal markers vimentin and N-cadherin, in lung tissues was examined. Tobacco smoke exposure led to reduced E-cadherin and ZO-1 mRNA levels in mice lungs, and the mRNA expression of vimentin and N-cadherin was increased, compared with the filtered air-treated group, as determined by RT-qPCR (Fig. 1A). Western blot analysis further revealed that tobacco smoke exposure also led to similar effects at the protein level, with downregulated E-cadherin and ZO-1 protein expression levels, and upregulated N-cadherin and vimentin protein expression levels compared with the filtered air-treated group (Fig. 1B and C).

The expression levels of p-ERK1/2, p-JNK, p-p38 and p-ERK5 were also measured to establish whether the observed tobacco smoke-induced pulmonary EMT alterations are associated with changes in MAPK activation. The results demonstrated that tobacco smoke increased the activation of ERK1/2, JNK and p38 pathways, while it suppressed the ERK5 MAPK pathway (Fig. 2A). Additionally, tobacco smoke exposure upregulated the levels of p-c-Fos, p-c-Jun and Fra-2, which are indicators of the AP-1 activation status (Fig. 2B).

To investigate whether curcumin may reverse tobacco smoke-mediated EMT in the mouse lung, mice were treated with curcumin (50 or 100 mg/kg BW) and exposed to tobacco smoke for 12 weeks. Tobacco smoke-induced alterations in the mRNA and protein expression EMT markers were subsequently investigated. As expected, the results demonstrated that curcumin, particularly 100 mg/kg BW curcumin, treatment attenuated the tobacco smoke-induced decreases in E-cadherin and ZO-1 expression, and increases in vimentin and N-cadherin expression. These results indicate that curcumin reversed tobacco smoke-induced alterations in pulmonary EMT in vivo (Fig. 3).

To gain an improved understanding of the influence of curcumin on tobacco smoke-mediated pulmonary activation of the MAPK/AP-1 pathways, the changes in MAPK/AP-1 activation following curcumin treatment were examined. Western blot analysis revealed that curcumin (100 mg/kg BW) suppressed tobacco smoke-induced ERK1/2, p38 and JNK activation, and increased ERK5 activation in the lung tissues of mice exposed to tobacco smoke (Fig. 4A). The results also demonstrated that treatment with 100 mg/kg BW curcumin significantly decreased tobacco smoke-induced AP-1 activation, as indicated by reduced expression of p-c-Fos, p-c-Jun and Fra-2 compared with the tobacco smoke-treated group without curcumin treatment (Fig. 4B).