Introduction

Chronic obstructive pulmonary disease (COPD) is featured by progressive airway obstruction and chronic inflammation, thereby resulting into emphysema and chronic bronchiolitis.¹ If no timely treatment is provided, this disease can generate irreversible damage to breathing and even lead to patient’s death.² COPD is estimated to produce over 3 million deaths annually, and projected to result into about 8 million deaths by 2030.^3,4 Inflammation in COPD immensely promotes the occurrence of comorbidities, including lung cancer.⁵ Smoking influences the immune system, and it is one most important inducement in COPD.^6,7 Multiple negative effects of cigarette smoke on airways include immunologic aspects and second-hand exposure.^8,9 Although some treatments were utilized in treating COPD patients,^10,11 these were not very ideal. Therefore, it is indispensable to understand further the pathogenesis of COPD, seek useful drugs, and produce novel therapeutic approaches.

β-elemene, a type of sesquiterpenoid, is extracted from Curcuma wenyujin,¹² and is proved to retard tumor growth. For example, in non-small cell lung cancer, β-elemene evokes the focal adhesion kinase–steroid receptor coactivator (FAK-Src) pathway to restrain cell migration and invasion.¹³ Moreover, in colorectal cancer, β-elemene modulates the reactive oxygen species–adenosine monophosphate-activated protein kinase–mechanistic target of rapamycin (ROS/AMPK/mTOR) pathway to trigger cell apoptosis and autophagy.¹⁴ Besides, β-elemene affects the FAK/claudin-1 pathway in gastric cancer to refrain peritoneal metastasis.¹⁵ Importantly, it was shown that β-elemene affected anti-inflammation and anti-oxidative stress. For instance, β-elemene retards the Janus kinase 2–signal transducer and activator of transcription 3–nuclear factor kappa B (JAK2/STAT3/NF-κB) pathway to mitigate hyperglycemia-evoked cardiac inflammation and remodeling.¹⁶ β-elemene weakens ROS generation and inflammation in atherosclerosis.¹⁷ Furthermore, β-elemene modulates nitric oxide (NO) level and relieves oxidative stress to improve atherosclerosis.¹⁸ However, the regulatory effects of β-elemene in the development of COPD were kept vague.

In this work, it is manifested that β-elemene alleviated cigarette smoke-triggered inflammation, apoptosis, and oxidative stress in human bronchial epithelial cells, and refrained the phosphatidylinositol 3-kinase–protein kinase B–mTOR (PI3K/AKT/mTOR) signaling pathway. This work could offer useful insights in the treatment of COPD.

Materials and Methods

Cell culture and treatment

Human bronchial epithelial cell line (BEAS-2B) was acquired from iCell Bioscience (Shanghai, China). In one incubator (37°C, 5% CO₂), BEAS-2B cells were cultivated with Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Miami, FL, USA), including 10% fetal bovine serum (FBS; Gibco).

β-elemene (5, 10, and 20 μg/mL; Apexbio, Boston, MA, USA) was utilized to treat BEAS-2B cells.

Treatment of cigarette smoke extract (CSE)

The culture medium of BEAS-2B cells was added with CSE to induce COPD cell model. Through one modified syringe-driven apparatus, one cigarette (Marlboro; Longyan Tobacco Industrial Co. Ltd., Fujian, China) was combusted to generate smoke, and smoke was pumped into DMEM (5 mL) to produce 100% CSE solution, which was filtered using aseptic 0.22-μm filter, and diluted into 5% CSE solution for incubating BEAS-2B cells. The cell survival rate of 50% was confirmed to induce COPD cell model successfully.

Cell counting kit-8 (CCK-8) assay

BEAS-2B cells (1000 cells/well) was set in a 96-well plate. Next, CCK-8 solution (10 μL; Dojindo Laboratories, Kumamoto, Japan) was mixed in each well for 2 h. Eventually, the cell survival rate was notarized through spectrophotometer (Thermo Fisher Scientific, MA, USA).

Flow cytometry

The fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, USA) was used. After washing, BEAS-2B cells were resuspended. Staining (5-μL FITC Annexin V and 10-μL propidium iodide) was performed for BEAS-2B cells in the dark. Eventually, cell apoptosis was inspected through flow cytometer (FACSCalibur™; BD Biosciences, San Jose, CA, USA) with the FlowJo software (Tree Star, Inc., Ashland, USA).

Western blot analysis

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was utilized for separating proteins extracted from BEAS-2B cells. Then, proteins were moved to polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China). After sealing, membranes were placed with primary antibodies for 12 h. The primary antibodies were as follows: Bcl-2-associated X (Bax; 1/1000; ab32503), B-cell lymphoma-2 (BCL-2; 1/2000; ab182858), cleaved caspase-3 (1/500; ab32042), phosphorylated phosphatidylinositol 3-kinase (p-PI3K; 1/1000; ab235266), PI3K (1/1000; ab191606), phospho-protein kinase B (p-Akt; 1/1000; ab38449), Akt (1/500; ab8805), phosphorylated (p)-mTOR (1/1000; ab109268), mTOR (1/1000; ab32028), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1/500; ab8245). Next, membranes were placed with secondary antibodies (1/2000; ab7090; Abcam, Shanghai, China) for 2 h. The chemiluminescence detection kit (Thermo Fisher Scientific) was used for examining protein blots.

Detection of malondialdehyde (MDA), superoxide dismutase (SOD), and ROS

MDA (ab118970; Abcam), SOD (ab65354), and ROS (ab186027) commercial kits were used for examination.

Enzyme-linked Immunosorbent Serological Assay (ELISA)

Tumor necrosis factor-α (TNF-α; ab181421; Abcam), interleukin-6 (IL-6; ab178013), and IL-1β (ab214025) commercial ELISA kits were used for measurements.

Statistical analysis

Data were displayed as mean ± standard deviation (SD). The GraphPad Prism Software 9 (GraphPad Software, USA) was used for statistical analysis. Differences in groups were evaluated by using one-way analysis of variance (ANOVA). Data followed the normal distribution; P < 0.05 was deemed as statistically significant.

Results

β-elemene aggrandized cell viability and reduced cell apoptosis in BEAS-2B cells stimulated by cigarette smoke

First, the chemical formula of β-elemene was exhibited, and different concentrations (5, 10, and 20 μg/mL) of β-elemene had no cytotoxicity (Figure 1A). The cell survival rate was lessened after 5% CSE treatment (from 100 to 49; P < 0.001), but this impact was rescued after β-elemene treatment (5, 10, and 20 μg/mL) (from 49 to 71, 82, and 90, respectively; P < 0.001) (Figure 1B). Besides, cell apoptosis increased with 5% CSE induction (from 6.18 to 36.13; P < 0.001), but this change was counterbalanced by different concentrations of β-elemene treatment (5, 10, and 20 μg/mL) (from 36.13 to 25.78, 21.13, and 15.02, respectively; P < 0.01) (Figures 1C–D). Bax and cleaved caspase-3 protein expressions decreased and that of BCL-2 increased after 5% CSE stimulation (P < 0.001), but these effects were reversed after β-elemene treatment (P < 0.05) (Figure 1E). To sum up, β-elemene aggrandized cell viability and reduced cell apoptosis in BEAS-2B cells stimulated by cigarette smoke.

β-elemene alleviates cigarette smoke-evoked oxidative stress

Next, the regulatory impacts of β-elemene on oxidative stress were explored. Levels of MDA and ROS were aggrandized and that of SOD was decreased after 5% CSE induction (P < 0.001); however, these changes were counteracted after β-elemene treatment (P < 0.001) (Figures 2A–C). Taken together, β-elemene alleviates cigarette smoke-evoked oxidative stress.

β-elemene attenuated cigarette smoke-induced inflammation

The influence of β-elemene on inflammation was investigated through ELISA. The levels of TNF-α, IL-6, and IL-1β elated after 5% CSE stimulation (P < 0.001), but these impacts decreased after β-elemene treatment (P < 0.001) (Figures 3A–C). In general, β-elemene attenuated cigarette smoke-induced inflammation.

β-elemene refrained the PI3K/AKT/mTOR pathway

The protein expressions of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/m-TOR elevated after 5% CSE induction (P < 0.001), but these phenomena attenuated after β-elemene treatment (P < 0.001) (Figure 4). Briefly, β-elemene refrained the PI3K/AKT/mTOR pathway.

Discussion

Results of the study demonstrated that β-elemene exaggerated cell viability and reduced cell apoptosis in BEAS-2B cells stimulated by cigarette smoke. β-elemene alleviated cigarette smoke-evoked oxidative stress and attenuated cigarette smoke-induced inflammation. In addition, β-elemene refrained the PI3K/AKT/mTOR pathway.

β-elemene displays its effects of suppressing tumor growth, inflammation, and oxidative stress.^13–19 However, the regulatory effects of β-elemene in COPD development are not reported. COPD is accompanied by airway damage and repair, and apoptosis in airway epithelial cells takes part into these processes to modulate COPD development.²⁰ Similarly, in this study, it was illustrated that β-elemene aggrandized cell viability and reduced cell apoptosis in BEAS-2B cells stimulated by cigarette smoke.

Oxidative stress and inflammation are pivotal processes in the development of COPD.²¹ Many researchers have focused on the modulation of oxidative stress and inflammation in COPD progression. For example, alantolactone restrains inflammation and oxidative stress in COPD.²² In addition, surfactant protein D suppresses lung inflammation in COPD.²³ Astaxanthin mitigates oxidative stress and inflammation triggered by cigarette smoking in COPD.²⁴ Furthermore, the LINC00987/let-7b-5p/SIRT1 axis affects oxidative stress, autophagy, and inflammation evoked by lipopolysaccharides (LPS) in COPD.²⁵ Similar to these findings, this work revealed that oxidative stress was heightened after 5% CSE induction, but this impact was neutralized after β-elemene treatment. Besides, the augmentative inflammation induced by cigarette smoke is attenuated after β-elemene treatment.

The PI3K/AKT/mTOR pathway was verified as a key pathway in progression of COPD. For example, puerarin evokes the PI3K/AKT/mTOR pathway to suppress mitochondrial autophagy and cell apoptosis in CSE-triggered human bronchial epithelial cells.²⁶ Moreover, S-allylmercapto-N-acetylcysteine (ASSNAC) retards the PI3K/Akt/mTOR pathway to improve elastase-stimulated COPD.²⁷ Endothelial alpha-enolase (ENO1) modulates the PI3K/Akt/mTOR pathway to relieve pulmonary hypertension.²⁸ Importantly, it was clarified that β-elemene blocks the PI3K/AKT/mTOR pathway in non-small cell lung cancer and renal-cell carcinoma.^29,30 However, the regulatory function of β-elemene in the regulation of the PI3K/AKT/mTOR pathway in COPD progression remains unclear. Similarly, our data indicated that the triggered PI3K/AKT/mTOR pathway mediated by cigarette smoke was refrained after β-elemene treatment.

Conclusion

The study was the first to manifest that β-elemene alleviates cigarette smoke-evoked inflammation, apoptosis, and oxidative stress in human bronchial epithelial cells, and refrains the PI3K/AKT/mTOR signaling pathway. Our results provide novel clinical perspectives of β-elemene for treating COPD. However, this work also has some limitations, such as the scarcity of animal model, clinical investigations, and exploration of other cellular phenotypes. In the future, a thorough exploration of β-elemene in the progression of COPD progression must be endorsed.