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Pyroptosis, a type of programmed cell death mediated by caspases-1 or −11, may play an important role in airway epithelial injury and airway remodeling, thereby promoting the occurrence of asthma and chronic obstructive pulmonary disease (COPD). Studies have suggested that hydrogen sulfide (H2S) plays a protective role against COPD by inhibiting the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The present study established a rat model of cigarette smoke (CS)-induced COPD to observe the effects of H2S on cell pyroptosis. A 16HBE cell model was also used to further examine the effects of H2S on the Toll-like receptor 4 (TLR4)/NF-κB signaling pathway is affected by, and to determine the underlying mechanisms. The results revealed that cell pyroptosis was significantly promoted in the model of CS-induced COPD. The cellular experiments also revealed that CS induced the pyroptosis of the cells in a NLRP3/gasdermin D (GSDMD)-dependent manner. In addition, H2S significantly attenuated the effects of CS extract (CSE) on pyroptosis, cell viability and the expression levels of pyroptosis-related proteins, indicating that H2S inhibited pyroptosis by decreasing NLRP3 expression and promoting GSDMD activation. It was also identified that CSE activated TLR4 protein in 16HBE cells, while this was inhibited by H2S. Furthermore, TLR4 and NF-κB overexpression significantly abolished the effects of H2S on cell pyroptosis. On the whole, the findings of the present study demonstrate the role of pyroptosis in the development of COPD and provide an experimental basis for the use of H2S and drugs targeting the TLR4/NF-κB pathway to exert protective effects against COPD.
Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disorder; it is a common respiratory illness and the third highest cause of mortality in China (
Cell pyroptosis is a type of programmed cell death mediated by Caspase-11 or −1. Under physiological conditions, pyroptosis is crucial for organ development (
Hydrogen sulfide (H2S) is a toxic gas with an odor of rotten egg (
A total of 48 male Sprague-Dawley rats, weighing 250–270 g (9–10 weeks old), were purchased from the Hebei Chest Hospital Animal Center. The animal procedures were approved by the Animal Ethics Committee of the Hebei Chest Hospital Animal Center and complied with the Guide of the Care and Use of Laboratory Animals published by the National Institutes of Health (
The rats were randomly assigned into different groups (n=12 per group) as follows: The H2S, CS + H2S, CS and control groups. The rat model of COPD was established according to the study by Ke
During H2S inhalation, the rats were placed in a plastic 20-liter container and permitted to inhale air combined with H2S for 8 h every day for 7 days following the establishment of the model of CS-induced COPD. H2S flowed through a flowmeter and regulator, and blended into the air. The flow of air, including H2S, was regulated to maintain the H2S concentration at 40 ppm. Non-COPD model rats also breathed 40 ppm H2S for 8 h every day for 7 days. H2S was purchased from the Shijiazhuang Zhongyuan Specialty Gas Co., Ltd. The concentration of H2S was measured using a H2S concentration monitor (HG-BX-H2S, Haigu Co.;
The rats were anesthetized with pancuronium bromide [0.6 mg/kg, intraperitoneal (i.p.)] and sodium pentobarbital (100 mg/kg, i.p.) following the inhalation of H2S. Cervical dislocation was used as the method of sacrifice and a thoracotomy was performed to expose the lungs. The lungs were perfused via the right ventricle with 30 ml ice-cold sterile phosphate-buffered saline (PBS). The left lung lobes were extracted and preserved at 25°C with 4% formalin for 48 h. The lung was then sliced into 4-µm-thick sections for further histological analysis.
For hematoxylin and eosin (H&E) staining, the protocol used was as previously described (
Paraffin-embedded sections, as well as block fabrication, were conducted in the same manner as described above for H&E staining (
First, RIPA buffer (Sigma-Aldrich, Merck KGaA) was used to extract protein from the cultured cells or rat tissues. The protein lysate was then separated using 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a difluoride polyvinylidene membrane. The amount and quality of the protein was examined using BCA assay (Beyotime Institute of Biotechnology) in a Synergy H1 microplate reader (BioTek Instruments, Inc.). Equal amounts of protein per lane (mass of 20 µl) were then loaded onto a 12% gel from a TGX Stain-Free FastCast Acrylamide kit (Bio-Rad Laboratories, Inc.) and finally transferred onto a 0.45 µm PVDF membrane (GE Healthcare, Inc.). The membrane was blocked with 5% non-fat milk-PBS for 1 h at room temperature and was then incubated overnight at 4°C with the following antibodies: NLRP3 (dilution, 1:1,000; cat. no. sc-134306; Santa Cruz Biotechnology, Inc.), cleaved Caspase-1 (dilution, 1:1,500; cat. no. YC0002; ImmunoWay Biotechnology Company), Caspase-1 (dilution, 1:800; cat. no. PA5-99477; Invitrogen; Thermo Fisher Scientific, Inc.), cleaved IL-1β (dilution, 1:1,000; cat. no. 83186; Cell Signaling Technology, Inc.), IL-1β (dilution, 1:1,000; cat. no. P420B; Thermo Fisher Scientific, Inc.), cleaved GSDMD (dilution, 1:1,000; cat. no. ab215203; Abcam, Inc.), pro-GSDMD (dilution, 1:1,000; cat. no. sc-81868; Santa Cruz Biotechnology, Inc.), TLR4 (dilution, 1:1,000; cat. no. sc-293072; Santa Cruz Biotechnology, Inc.), inhibitor κB-α (IκBα, dilution, 1:1,000; cat. no. sc-1643; Santa Cruz Biotechnology, Inc.), phosphorylated (p)-IκBα (dilution, 1:1,000; cat. no. sc-8404; Santa Cruz Biotechnology, Inc.), NF-κB p-p65 (dilution, 1:1,000; cat. no. sc-166748; Santa Cruz Biotechnology, Inc.), NF-κB p50 (dilution, 1:1,000; cat. no. sc-8414; Santa Cruz Biotechnology, Inc.), GAPDH (dilution, 1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), Histone H3 (dilution, 1:1,000; cat. no. ab1791; Abcam, Inc.) and NF-κB p65 (dilution, 1:1,000; cat. no. sc-8008; Santa Cruz Biotechnology, Inc.). The membranes were washed and incubated with with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (dilution, 1:10,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) or anti-mouse secondary antibody (dilution, 1:10,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Finally, the membranes were washed and antibodies were identified using a SuperSignal West Pico Chemilluminescent Substrate (Thermo Fisher Scientific, Inc.). The band intensity was quantified using ImageJ software (version 1.6.0; National Institutes of Health).
Immortalized 16HBE cells were acquired from Procell Life Science & Technology Co., Ltd. A total of 10 generations of 16HBE cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Biological Industries) in room air with 5% CO2 at 37°C. The preparation of CSE was performed as previously described (
The 16HBE cells were plated at a density of 2×103 cells/well in a 96-well culture plate and treated with 0.5–10% CSE for 24 h. A WST-1 cell proliferation assay kit (Beyotime Institute of Biotechnology) was used to evaluate 16HBE cell viability. To fully dissolve the WST-1 powder and form its solutions, 5 ml electronic coupling substance was added. Subsequently, 100 µl solution containing 2,000 cells was added to each well. Subsequently, 10 µl WST-1 solution was added to each well followed by incubation for 2 h at room temperature. Finally, a microplate reader (BioTek Instruments, Inc.) was used to measure the optical density at a wavelength of 450 nm.
The LDH Assay kit (Beyotime Institute of Biotechnology) was used to detect the rate of pyroptosis. Briefly, the culture supernatant of 16HBE cells (120 µl/well) was collected and incubated for 30 min at 25°C with 60 µl LDH reaction buffer in the dark. The absorbance was measured using a microplate reader (BioTek Instruments, Inc.) at 490 nm.
Similar to the study of Ding
Briefly, the 16HBE cells were cultured into 6-well plates until the cells were ~50% confluent. The cells were then transfected with the 10 µl GV492-TLR4/NC-EGFP, 10 µl GV492-NF-κB/NC-EGFP or 10 µl packing vector (MOI: 50–80). The medium was changed 6 h later, and the cells continued to be cultured for 72 h. The lentivirus-treated 16HBE cells were then treated with 5% CSE and NaHS for 24 h. The transfection efficiency was measured using reverse transcription-quantitative PCR (RT-qPCR).
The total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Reverse transcription was then performed using the One-Step SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd.). The reaction was performed using the ABI PRISM 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 42°C for 5 min, 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec, 55°C for 30 sec and 72°C for 30 sec. A total of three independent experiments were conducted each time. The data were then analyzed by comparing the 2−ΔΔCq value (
Similar to the study of Zhang
All data were evaluated using SPSS 17.0. Software (SPSS, Inc.). One-way ANOVA with the Bonferroni post hoc test was performed to examine the quantitative data. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
The lung histopathological changes as detected using H&E staining are presented in
First, the 16HBE cells were exposed to various concentrations of CSE (0.5–10% of the CSE) for 24 h, and pyroptosis and cell viability were then measured using LDH and WST-1 assay, respectively. The LDH activity was enhanced by CSE in a concentration-dependent manner (
The 16HBE cells were then exposed to various concentrations of CSE (0.5–10%) for 24 h to validate the effects on pyroptosis. The expression of NLRP3, cleaved Caspase-1, cleaved IL-1β, Caspase-1, pro-IL-1β, cleaved GSDMD and pro-GSDMD in the cells was then detected using western blot analysis. Representative results are shown in
To examine the function of NLRP3 in 16HBE cells undergoing CSE-induced pyroptosis, the cells were exposed to 5% CSE and treated with the NLRP3 inhibitor, CRID3, for 24 h, and pyroptosis and cell viability, as well as the expression levels of pyroptosis-related proteins, were then examined. As shown in
To examine the effects of H2S on the CSE-induced pyroptosis of 16HBE cells, the cells were exposed to 5% CSE and treated with NaHS (an H2S donor) for 24 h. The LDH activity, cell viability and the expression levels of pyroptosis-related proteins were then measured. As shown in
To examine the role of the TLR4/NF-κB pathway in the effects of H2S on the CSE-induced pyroptosis of 16HBE cells, the cells were exposed to 5% CSE and NaHS (H2S donor) for 24 h, and the expression levels of IκBα, p-IκBα, NF-κB p50, NF-κB p65 and NF-κB p-p65 were measured (
To examine the role of the TLR4/NF-κB pathway in the effects of NaHS on CSE-induced pyroptosis, the 16HBE cells were exposed to 5% CSE, and treated with NaHS and lentivirus overexpressing TLR4 or NF-κB. The transfection efficiency was determined using RT-qPCR (
Pyroptosis is a type of Caspase-1-dependent programmed cell death and is accompanied by an inflammatory reaction (
GSDMD is the executor of cell pyroptosis. To demonstrate that GSDMD was cleaved by Caspase-11 at the preserved residual of d276, Kayagaki
NLRP3, as an important inflammatory body, can be activated via two mechansims. On the one hand, it can stimulate TLR4 and NF-κB and can modulate the expression levels of adhesion and inflammatory factors, thus producing pro-IL-18 and pro-IL-1β (
Several studies have reported that H2S plays a protective role against cell injury by deterring NLRP3 inflammasome activation. In human acute monocytic leukemia cells, H2S has been shown to inhibit NLRP3 protein expression, as well as the LPS-induced Caspase-1 expression (
The contribution of the TLR4/NF-κB signaling pathway to the inflammatory response and other pathological changes has been demonstrated (
NF-κB is an ubiquitous transcription factor mediating the cytoplasmic-nuclear signaling pathway. NF-κB exists in the form of a dimer and its role in the development of different inflammation-related illnesses has been proven, such as via its involvement in cell apoptosis and proliferation (
In the present study, to examine the function of the NF-κB pathway, the cells were exposed to 5% CSE and treated with NaHS, and the expression levels of IκBα, p-IκBα, NF-κB p50, NF-κB p65 and NF-κB p-p65 were measured in the 16HBE cells. The ratios of NF-κB p-p65/p65 and p-IκBα/IκBα, as well as the expression levels of NF-κB p50 in 16HBE cells were increased by CSE, which these were all partly reversed by NaHS. These findings suggested that CSE induced the activation of the TLR4/NF-κB pathway in 16HBE cells, while this was inhibited by H2S. A recent study reported that NF-κB was the prominent GSDMD transcription factor (
In conclusion, the present study demonstrated that H2S alleviated lung injury and pyroptosis in a model of CS-induced COPD by inhibiting the activation of the TLR4/NF-κB signaling pathway. These findings suggest the importance of pyroptosis in the development of COPD and provide an experimental framework in which H2S and drugs targeting the TLR4/NF-κB pathway may be utilized for protection against COPD.
Not applicable.
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
CY and YL contributed to the conception of the study. LW and JM performed all of the experiments. LW and JM contributed significantly to the analysis of the results of the experiments and to manuscript preparation. CW performed the data analyses and wrote the manuscript. YW helped perform the analysis of the proofs manuscript and provided constructive discussions. LW, CW and JM confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The animal procedures were approved by the Animal Ethics Committee of the Hebei Chest Hospital Animal Center and complied with the Guide of the Care and Use of Laboratory Animals published by NIH (NIH Pub. no. 85-23, revised 1996).
Not applicable.
The authors declare that they have no competing interests.
Effect of H2S inhalation on histopathological appearance of the lung tissue of rats and the expression of pyroptosis-related proteins. (A) H&E staining results. (B) GSDMD-N expression was detected in the lung using immunohistochemistry. (C and D) Representative protein bands of NLRP3, cleaved Caspase-1, Caspase-1, cleaved pro-IL-1β, IL-1β, cleaved GSDMD and pro-GSDMD expression. (E-H) Quantitative results of western blot analysis. Values are presented as the mean ± SD and are representative of three independent experiments. *P<0.05 compared with the control group; @P<0.05 compared with the CS group. H&E, hematoxylin and eosin; GSDMD, gasdermin D; NLRP3, NLR family pyrin domain containing 3; CS, cigarette smoke; H2S, hydrogen sulfide.
Effects of CSE on the pyroptosis and proliferation of 16HBE cells. The 16HBE cells were exposed to 0.5–10% CSE for 24 h. (A) Pyroptosis was measured using LDH release assay. (B) Cell viability was measured using WST-1 assay. Subsequently, 5% CSE was selected to challenge the 16HBE cells, and the GSDMD inhibitor, NSA, was added to the culture medium. (C) Pyroptosis and (D) cell viability were measured using LDH and WST-1 assay, respectively. Values are presented as the mean ± SD and are representative of three independent experiments. *P<0.05 compared with the control group; @P<0.05 compared with the CSE group. CSE, cigarette smoke extract; GSDMD, gasdermin D; NSA, necrosulfonamide; LDH, lactate dehydrogenase.
Effects of CSE on the expression of pyroptosis-related proteins in 16HBE cells. (A and B) Representative protein bands. (C-F) Results of quantitative analysis. Data are presented as the mean ± SD and are representative of three independent experiments. *P<0.05 compared with the control group. CSE, cigarette smoke extract; GSDMD, gasdermin D; NLRP3, NLR family pyrin domain containing 3.
The NLRP3 inhibitor, CRID3, reverses the effects of CSE on the pyroptosis of 16HBE cells. To examine the role of NLRP3 in the CSE-induced pyroptosis of 16HBE cells, the cells were exposed to 5% CSE and treated with the NLRP3 inhibitor, CRID3, for 24 h. (A and B) LDH activity and cell viability, (C-H) as well as the expression levels of pyroptosis-related proteins were examined. Data are presented as the mean ± SD and are representative of three independent experiments. *P<0.05 compared with the control group; @P<0.05 compared with the CSE group. NLRP3, NLR family pyrin domain containing 3; GSDMD, gasdermin D; CRID3, cytokine release inhibitory drug 3; CSE, cigarette smoke extract; LDH, lactate dehydrogenase.
NaHS reverses the CSE-induced pyroptosis of 16HBE cells. To examine the effect of H2S on the CSE-induced pyroptosis of 16HBE cells, the cells were exposed to 5% CSE and treated with NaHS (an H2S donor) for 24 h. (A) LDH activity, (B) cell viability and (C-H) the expression levels of pyroptosis-related proteins were then measured. Data are presented as the mean ± SD and are representative of three independent experiments. *P<0.05 compared with the control group; @P<0.05 compared with the CSE group. CSE, cigarette smoke extract; H2S, hydrogen sulfide; LDH, lactate dehydrogenase; GSDMD, gasdermin D; NaHS, sodium hydrosulfide.
NaHS reduces CSE-induced TLR4/NF-κB pathway activation in 16HBE cells. (A-D) To analyze the role of the TLR4/NF-κB pathway in the effects of H2S on the CSE-induced pyroptosis of 16HBE cells, the cells we exposed to 5% CSE and treated with NaHS (H2S donor) for 24 h, and the expression levels of IκBα, p-IκBα, NF-κB p50, NF-κB p65 and NF-κB p-p65 were then measured in 16HBE cells. Data are presented as the mean ± SD. *P<0.05 compared with the control group; @P<0.05 compared with the CSE group. CSE, cigarette smoke extract; TLR4, Toll-like receptor 4; NaHS, sodium hydrosulfide.
Overexpression of TLR4/NF-κB abolishes the suppressive effects of NaHS on the CSE-induced pyroptosis of 16HBE cells. (A-G) 16HBE cells were exposed to 5% CSE, and treated with NaHS and lentivirus overexpressing TLR4 or NF-κB. (A) LDH activity, (B) cell viability and (E-G) the expression levels of pyroptosis-related proteins were then measured. (C and D) The transfection efficiency was measured using RT-qPCR. (H-N) 16HBE cells were exposed to 5% CSE, and then treated with NaHS and transfected with lentivirus to knockdown TLR4 or NF-κB. (H) LDH activity, (I) cell viability and (L-N) the expression levels of pyroptosis-related proteins were then measured. (J and K) The transfection efficiency was measured using RT-qPCR. Data are presented as the mean ± SD and are representative of three independent experiments. #P<0.05 compared with the 5% CSE + NaHS + Lenti-NC group; *P<0.05 compared with the control + Lenti-NC group; @P<0.05 compared with the 5% CSE + Lenti-NC group; &P<0.05 between lenti-NC and transfection groups. TLR4, Toll-like receptor 4; CSE, cigarette smoke extract; LDH, lactate dehydrogenase; GSDMD, gasdermin D; NaHS, sodium hydrosulfide; RT-qPCR, reverse transcription-quantitative PCR.