Effect of curcumin on lung epithelial injury and ferroptosis induced by cigarette smoke

Reagents and antibodies

CUR (C1386), ferrostatin-1 (Fer-1, SML0583), and deferoxamine (DFO, D9533) were purchased from Sigma-Aldrich. Cell counting kit-8 (CCK-8) assay kit (C6005) was purchased from US Everbright Inc. The detection kits for lactate dehydrogenase (LDH, A020-2), ROS (E004-1), malondialdehyde (MDA, A003-1), and tissue iron (A039-2) were purchased from Nanjing Jiancheng Bioengineering Institute. GSH assay kit (S0053) was purchased from Beyotime Biotechnology. Intracellular iron colorimetric assay kit (E1042) was purchased from Applygen Technologies Inc. Calcein-AM/PI double stain kit (40747ES76) was purchased from YEASEN Biotechnology. The human IL-6 enzyme linked immunosorbent assay (ELISA) kit (EK0410), rat IL-6 ELISA kit (EK0412), human TNF-α ELISA kit (EK0525), and rat TNF-α ELISA kit (EK0526) were purchased from BOSTER Biotechnology. The glutathione peroxidase 4 (GPX4) (ab125066) antibody was purchased from Abcam. The antibodies solute carrier family 7 member (SLC7A11, A2413), FTH1 (A1144), TFR1 (A5865), and β-actin (AC004) were purchased from ABclonal.

Preparation of cigarette smoke extract

CSE was extracted from Daqianmen cigarettes (tar: 10 mg, nicotine: 1 mg, carbon monoxide: 12 mg; Tianjin, China) as previously reported with minor modifications. ³ In brief, five cigarettes were burned in turn, and the CS was sucked into the syringe and bubbled into 10 mL of sterile phosphate buffered saline (PBS) in bubble absorption bottle. The CSE solution was filtered (0.22 μm; Millipore, USA) to remove the bacteria and insoluble particles. CSE preparations were designated as a 100% CSE solution.

Cell culture

Human bronchial epithelial cell line BEAS-2B was kindly provided by the Laboratory of Lung Development and Diseases at Nankai University (Tianjin, China). The BEAS-2B cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were incubated under humidified conditions with 5% CO₂ at 37°C. BEAS-2B cells were treated with 5 μM Fer-1 or 80 μM DFO 1 h before 2.5% CSE treatment for 24 h.

Cell death assay

In accordance with the manufacturer’s protocol, the LDH assay kit, and CCK-8 assay kit were used to determine cytotoxicity and cell viability, respectively, to analyze cell death. The BEAS-2B cells were seeded in a 96-well microplate with 5000 cells/well. After 24 h, the cells were incubated in non-FBS DMEM containing CSE (0, 1, 2.5, and 5%) for 24 h. Cell supernatant was collected for the LDH assay. Then, cells were washed with non-FBS DMEM twice, new non-FBS DMEM at 100 μl/well and CCK solution at 10 μl/well were added and incubated for an additional 2 h. The absorbance at 450 nm was detected by a microplate reader (BioTek, Vermont, USA). The calculation methods are as following: cytotoxicity (%) = (A_treatment − A_control)/(A_max − A_control) × 100, cell viability (%) = (A_treatment − A_blank)/(A_control − A_blank) × 100.

Animal models

Sprague-Dawley rats (male, seven weeks old, and 150 ± 20 g) were provided by SiPeiFu Biotechnology Company (Beijing, China). They were divided randomly into four groups of five as follows: normal control (NC), CS, CUR, and CS + CUR group. The rats were housed in a pathogen-free room with a light-dark cycle of 10:14 h, a temperature of 26 ∼ 28°C, and a relative humidity of 40% ∼ 60%. Except during the CS exposure period, rats are free to eat and drink water. After the rats were raised for a week, those in the CS and CS + CUR groups were passively exposed to CS by using a homemade smoke exposure device as described previously. ¹² The rats were exposed to 15 cigarettes for 30 min each time, twice a day (at 9 a.m. and 9 p.m), and 6 days a week for 16 weeks. And the volume fraction of CS in the smoke exposure device was about 15% (V/V). The rats in the NC and CUR groups are exposed to normal air. The rats in the CUR and CS + CUR groups were intragastrically administered with CUR (100 mg/kg/day), while those in the NC and CS groups were given sodium carboxymethyl cellulose at 8 a.m. every day for 16 weeks. All experimental procedures were performed in accordance with the guidelines issued by the Guide for the Care and Use of Laboratory Animals of Tianjin Medical University.

Histologic analysis

The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL/100g) and sacrificed by cardiac puncture and bloodletting. BALF was collected by repeatedly lavaging the lungs with PBS through a tracheostomy cannula. The right lung was resected and frozen with liquid nitrogen for western blot and other experiments, and the left lung was filled with 10% neutral formalin, resected, fixed, paraffin-embedded, and cut into 4 μm sections. Then, the dewaxed sections were stained by hematoxylin and eosin to evaluate lung epithelial injury.

Levels of inflammatory cytokines

The concentrations of IL-6 and TNF-α in supernatants of BEAS-2B cells and BALF of rats were measured using the corresponding ELISA kit in accordance with the manufacturers’ protocol.

Western blot analysis

Total proteins were isolated from BEAS-2B cells and lung tissue of rats by using RIPA buffer and the concentration was measured using a BCA protein kit. The same amount of denatured protein (cells: 30 μg; lung tissues: 50 μg) of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They were transferred onto polyvinylidene fluoride membranes, and the membranes were incubated with primary antibodies at 4°C overnight. Then, the membrane was incubated with goat anti-rabbit/mouse IgG and HRP-linked antibodies as a secondary antibody for 2 h at room temperature. The protein bands were detected by electrochemical luminescence, and the band density was analyzed using the Tanon GIS digital image analysis system (Tanon, Shanghai, China). The relative protein levels were normalized to β-actin.

Determination of reactive oxygen species generation

Intracellular ROS levels were detected with peroxide-sensitive fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA) in accordance with the manufacturer’s instructions. In brief, DCFH-DA was diluted to 10 μM in serum-free medium. The BEAS-2B cells were incubated with diluted DCFH-DA at 37°C for 1 h. Fluorescence intensity was measured via flow cytometry in accordance with the FITC fluorescence detection conditions.

Malondialdehyde, glutathione, and iron assay

BEAS-2B cells were washed with PBS, harvested, and then centrifuged to collect cell precipitates. The lung tissue was frozen in liquid nitrogen and then ground into powder. According to the manufacturer’s instructions, the contents of MDA, GSH, and iron in BEAS-2B cells and lung tissues of rats were measured with corresponding kit.

Statistical analysis

Data were shown as mean ± standard deviation. One-way ANOVA was used for comparison among multiple groups, and LSD was used to compare the differences between two groups. p < 0.05 was considered statistically different. Statistical analysis was performed using GraphPad Prism 7.0 software.

Cigarette smoke extract induces ferroptotic cell death in BEAS-2B cells

BEAS-2B cells were exposed to different concentrations of CSE to verify the effect of CSE on bronchial epithelial injury, inflammation, and ferroptosis in COPD. The results showed that CSE obviously triggered cell death (Figures 1(a) and (b)), increased cytotoxicity (Figure 1(c)), and reduced cell viability in a dose-dependent manner (Figure 1(d)). Meanwhile, CSE treatment could increase the level of inflammatory cytokines, such as IL-6 (Figure 1(e)) and TNF-α (Figure 1(f)), in a dose-dependent manner. Then, the characteristic changes in ferroptosis, including lipid peroxidation, GSH depletion, and iron overload, were detected. The results indicated that with the increase in CS concentration, the contents of MDA (Figure 1(g)) and iron (Figure 1(i)) in the BEAS-2B cells increased, while the content of GSH (Figure 1(h)) decreased. Antioxidant stress-related proteins (SLC7A11 and GPX4) and iron metabolism-related proteins (FTH1 and TFR1) are closely associated with ferroptosis. Western blot results indicated that the protein levels of SLC7A11, GPX4, and FTH1 were down-regulated and that of TFR1 was up-regulated by CSE (Figures 1(j) and (k)). These data strongly revealed that CSE could cause cell damage and death, increase the secretion of inflammatory cytokines, and promote ferroptosis in BEAS-2B cells.OPEN IN VIEWER

Ferroptosis is involved in cigarette smoke extract-caused cell damage and inflammation in vitro

As expected, the up-regulated levels of intracellular ROS (Figures 2(a) and (b)), MDA (Figure 2(c)), and iron (Figure 2(e)) caused by CSE extensively decreased, and the down-regulated level of GSH (Figure 2(d)) increased after the pretreatment of Fer-1 and DFO. Besides, the protein levels of SLC7A11, GPX4, and FTH1 were significantly up-regulated, and TFR1 was down-regulated in the CSE+Fer-1/DFO group compared with that in the CSE group (Figures 2(f) and (g)). Interestingly, Fer-1 and DFO rescued CSE-caused cell damage and death (Figures 2(h) to (k)), and alleviated the increase in cellular inflammatory cytokines induced by CSE (Figures 2(l) and (m)) in BEAS-2B cells, indicating that Fer-1 and DFO could partially protect epithelial cells from lung injury by suppressing ferroptosis.OPEN IN VIEWER

Curcumin inhibits cell damage, inflammation, and ferroptosis-related changes in vitro

BEAS-2B cells were exposed to 2.5% CSE and different concentrations of CUR (5/10/20 μm) to clarify the potential effect of CUR on COPD. The CSE + CUR group had less cell death (Figures 3(a) and (b)), lower cytotoxicity (Figure 3(c)), and higher cell viability (Figure 3(d)) than the CSE group. Moreover, CUR treatment could decrease the levels of IL-6 (Figure 3(e)) and TNF-α (Figure 3(f)). A notable detail that CUR also inhibited characteristic changes in ferroptosis. The levels of MDA (Figure 3(g)), iron (Figure 3(i)), and ROS (Figures 3(j) and (k)) were lower, and the levels of GSH (Figure 3(h)) were higher in BEAS-2B cells treated with CSE and CUR than in those treated with CSE. Furthermore, western blot results displayed that the protein levels of SLC7A11, GPX4, and FTH1 in the CSE + CUR group were up-regulated, and TFR1 was down-regulated compared with those in the CSE group (Figures 3(l) and (m)). These results suggested that CUR could weaken CSE-induced ferroptosis, cellular inflammation, and cell damage in BEAS-2B cells.OPEN IN VIEWER

Cigarette smoke-induced inflammation and ferroptosis is attenuated by curcumin in vivo

Data showed that the body weight of rats in the NC group was higher than that in the other three groups, and the body weight in the CS group was lower than that in the other three groups (Figure 4(a)). In addition, compared with the NC group, the alveolar structure was disordered, the alveolar septum was ruptured, and the infiltration of inflammatory cell around the bronchi was increased in the CS group. However, inflammatory cell infiltration was significantly mitigated in the CS + CUR group (Figure 4(b)). CUR also rescued the increased levels of IL-6 (Figure 4(c)) and TNF-α (Figure 4(d)) in the BALF of rats caused by CS.OPEN IN VIEWER

The levels of MDA (Figure 4(e)) and iron (Figure 4(f)) were higher in the lung tissue of the CS group than those in the NC group. Meanwhile, the protein expression levels of SLC7A11, GPX4, and FTH1 decreased, and the level of TFR1 increased in the CS group (Figures 4(g) and (h)). However, after CUR treatment, the ferroptosis-related changes induced by CS were reversed. Collectively, these findings suggested that CUR could inhibit the inflammatory injury of lung, accompanied by the inhibition of ferroptosis.