Abstract

Cigarette smoke (CS) is the major cause of chronic obstructive pulmonary disease (COPD). CS heightens inflammation, oxidative stress and apoptosis. Ergosterol is the main bioactive ingredient in Cordyceps sinensis (C. sinensis), a traditional medicinal herb for various diseases. The objective of this work was to investigate the effects of ergosterol on anti-inflammatory and antioxidative stress as well as anti-apoptosis in a cigarette smoke extract (CSE)-induced COPD model both in vitro and in vivo. Our results demonstrate that CSE induced inflammatory and oxidative stress and apoptosis with the involvement of the Bcl-2 family proteins via the nuclear factor kappa B (NF-κB)/p65 pathway in both 16HBE cells and Balb/c mice. CSE induced epithelial cell death and increased the expression of nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), malondialdehyde (MAD) and the apoptosis-related proteins cleaved caspase 3/7/9 and cleaved-poly-(ADP)-ribose polymerase (PARP) both in vitro and in vivo, whereas decreased the levels of superoxide dismutase (SOD) and catalase (CAT). Treatment of 16HBE cells and Balb/c mice with ergosterol inhibited CSE-induced inflammatory and oxidative stress and apoptosis by inhibiting the activation of NF-κB/p65. Ergosterol suppressed apoptosis by inhibiting the expression of the apoptosis-related proteins both in vitro and in vivo. Moreover, the usage of QNZ (an inhibitor of NF-κB) also partly demonstrated that NF-κB/p65 pathway was involved in the ergosterol protective progress. These results show that ergosterol suppressed COPD inflammatory and oxidative stress and apoptosis through the NF-κB/p65 pathway, suggesting that ergosterol may be partially responsible for the therapeutic effects of cultured C. sinensis on COPD patients.

Introduction

Chronic obstructive pulmonary disease (COPD) is an incurable but preventable respiratory disease [1]. Currently, COPD is the fourth leading cause of deaths worldwide, and it is expected to become the third leading cause of deaths worldwide by 2020 [2,3]. It is a chronic and progressive inflammatory pulmonary disease characterized by chronic bronchitis and emphysema. Cigarette smoke (CS) is identified as the main causative agent in inducing chronic lung inflammatory, protease/anti-protease imbalance and oxidative stress involving nuclear factor kappa B (NF-κB) activation [4,5]. Oxidative stress is generated as a consequence of the intra/extracellular metabolism of toxins or oxidants [6]. The reactive species generated by oxidative stress will activate resident cells in the lung, such as alveolar macrophage and epithelial cells, which further generate chemotactic molecules that recruit the additional inflammatory cells (neutrophils, monocytes and lymphocytes) into the lungs to release inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interlukin-1β (IL-1β) [1,7]. It has been reported that, in smokers or patients with COPD, the oxidative stress increases the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and decreases airway levels of glutathione, superoxide dismutase (SOD) and catalase (CAT) [8,9].

Dysregulated cell death processes are possibly involved in CS-induced COPD [10,11]. The increased numbers of apoptotic alveolar, bronchiola, and endothelia cells in lung tissues from patients with COPD have been observed [12]. Studies have shown that lung epithelial cells undergo apoptotic cell death upon CS exposure, and subsequently induced innate immune response [6]. The increase of hydrogen peroxide (H2O2) induced by tobacco smoke also leads to caspase-3-dependent apoptosis in airway epithelial cells [13]. Ting Yuan et al. [12] demonstrated that endoplasmic reticulum (ER) stress plays an important role in cigarette smoke extract (CSE)-induced HBE cell apoptosis via a PERK-elF2α pathway.

NF-κB is a family of transcription factors that play important roles in regulating cell differentiation, proliferation, immune response and apoptosis [14]. It protects cells against inducers of apoptosis, possibly through the activation of the anti-apoptotic genes [14]. Numerous studies show that CSE-induced lung inflammatory response and injury are linked to the activation of NF-κB/p65 [1,15,16]. Roscioli et al. [17] demonstrated that wildfire smoke extract (WFSE) and CSE induced airway epithelial cell apoptosis via activating NF-κB/p65 and poly-(ADP)-ribose polymerase (PARP), and down-regulating Bcl-2 activity. Therefore, drugs that can regulate NF-κB/p65 activation have potential applications in the treatment of COPD.

Studies suggest that ergosterol and its peroxidation products may contribute to potential health benefits and significant pharmacological activities, such as anti-tumor, anti-inflammation, antioxidation and immunomodulatory activity [18–20]. However, no available study has evaluated the anti-apoptotic effects of ergosterol treatment on CSE-induced COPD in the airway epithelial cells and mouse model. Recently, a number of studies on the active ingredients and underlying mechanism for the COPD therapy have been initiated in our laboratory. The objective of this work was to explore the anti-inflammatory, antioxidative stress and anti-apoptotic effects as well as the mechanisms of ergosterol from Cordyceps sinensis on 16HBE cells and COPD mice stimulated by CSE.

Materials and methods

Regents

Ergosterol (Purity of 99%) was purchased from Aladdin Regents CO., Ltd. (Shanghai, China). RPMI-1640 and fetal bovine serum (FBS) were purchased from Biological Industries (Beit Haemek Ltd., Israel). Penicillin and streptomycin were purchased from Solarbio Biotechnology (Beijing, China). Nitric oxide (NO), CAT, MDA and SOD commercial assay kits were purchased from Nanjing Jiancheng Biology Engineering Institute (Nanjing, Jiangsu, China). TNF-α, IL-6 Enzyme-linked Immunosorbent Assay Kits were purchased from Shanghai MultiSciences (Lianke) Biotech Co., Ltd. (Shanghai, China). QNZ (EVP4593, an inhibitor of NF-κB) was purchased from MedChemExpress (MCE, China). Anti-inducible nitric oxide synthase (iNOS), NF-κB/p65, IκB and cyclooxygenase-2 (COX-2) antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). Anti-Bcl-2, Bax, caspase 3, cleaved-caspase 3, caspase 7, cleaved-caspase 7, caspase 9, cleaved-caspase 9 and cleaved-PARP antibodies were provided by Abcam (Cambridge, U.K.). Anti-GADPH antibodies were provided by Proteintech Biotechnology (Rocky Hill, CT, U.S.A.). Horseradish peroxidase (HRP)-conjugated antibodies were bought from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, U.S.A.). NF-κB/p65 Transcription Factor Assay Kit was got from Cayman chemical company (Cayman, Ann Arbor, U.S.A.). Other reagents used in the experiment were all of analytical grade.

Preparation of aqueous CSE

CSE was prepared as previously reported [1]. Briefly, three cigarettes (Taishan brand, Jinan, China) were burned and the smoke was collected by a vessel containing the phosphate-buffered saline (PBS, 10 ml) using a vacuum pump. This 100% CSE was adjusted to pH 7.4 and was sterile filtered through a 0.22-µm filter. CSE was freshly prepared for each experiment and diluted with the culture medium containing 10% FBS immediately before use although the filtered CSE could be used within 24 h. The nicotine content in the range of 36-39 μg/ml in the CSE was determined by high performance liquid chromatography and used as a quality control.

Cells culture

The 16HBE cell line was a generous gift from the Department of Pulmonary Disease, Qilu Hospital, Shandong University (Shandong, China). 16HBE cells were cultured in the RPMI-1640 medium supplemented with 100 U/ml of penicillin G, 100 μg/ml of streptomycin, and 10% (v/v) inactivated FBS. 16HBE cells were grown at 37°C in a humidified 5% CO2 incubator.

Determination of NO, IL-6 and TNF-α secretion in 16HBE cells

The cells were pre-incubated in 12-well plates for 24 h at 37°C in a humidified incubator with 5% CO2. After cells were cultured with or without 5% CSE in the absence or presence of ergosterol (5, 10 and 20 μM) for 24 h, levels of NO, IL-6 and TNF-α in the culture supernatants were then measured by a commercial assay kit or ELISA kit according to the manufacturer’s instructions.

Measurement of CAT, MDA and SOD production in 16HBE cells

The cells were treated with ergosterol after 1 h treatment with 5% CSE. After 24 h incubation, culture supernatant was collected and centrifuged for 10 min at 2500 rpm. The CAT, MDA and SOD activities were measured in the supernatant using an assay kit based on the specified manufacturer’s instructions.

Measurement of ROS generation

The generation of ROS was measured using 2′,7′-Dichlorodi-hydrofluorescein diacetate (DCFH-DA, Sigma–Aldrich St. Louis, MO, U.S.A.) as described previously [21]. Briefly, cells were treated with or without ergosterol for 24 h after 1 h of 5% CSE treatment. Afterward, the cells were harvested, washed and suspended in the serum-free medium containing 10 µM of DCFH-DA at 37°C for 20 min in the dark. Finally, cells were washed and resuspended in the PBS and transferred to flow acquisition tubes and quantified using Beckman Coulter FC500 (Beckman Coulter Commercial Enterprise, U.S.A.).

Cell apoptosis assay

The 16HBE cells were incubated with the different concentrations of ergosterol for 24 h after 1 h of 5% CSE treatment. For DAPI staining via microscopy, the 16HBE cells were fixed with cold methanol/acetone (1:1) for 5 min, and then incubated with the DAPI solution for 10 min. The apoptotic morphology of the cells was observed using Olympus fluorescence microscope. For quantification, the 16HBE cells were washed with the cold PBS buffer and harvested. Apoptotic cell death was identified by double staining with PI and Annexin V-FITC solutions following the protocol of the manufacturer (Invitrogen, Carlsbad, U.S.A.). The cell apoptosis rate was measured immediately by flow cytometry (Beckman FC 500, Brea, CA, U.S.A.).

Animal experiment

Male SPF Balb/c mice weighing 20 g were purchased from Jinan Pengyue Experimental Animal Breeding Co. LTD (Certificate No. SCXK 20140007, Shandong, China). Mice were housed in a temperature-controlled room (25 ± 2°C) with the relative humidity of 40–70%, and had free access to the standard commercial diet and water. All experimental procedures involving animals were performed in accordance with the institutional guidelines of the Animal Care and Use Committee of Shandong University (No. 2016020, Jinan, China) and conducted in the Center for Pharmaceutical Research and Drug Delivery System of Shandong University. After 1-week acclimatization to the laboratory conditions, the mice were randomly divided into 5 groups (n=7) as follows: control group (C), model group (M), positive control group (P, budesonide, 2 mg/kg), ergosterol (E-L, 20 mg/kg) and ergosterol (E-H, 40 mg/kg). Each mouse in the model group was intraperitoneally injected with the 100% CSE (0.3 ml) on days 1, 8 and 15. Mice in the positive control and ergosterol groups were intraperitoneally injected with the 100% CSE (0.3 ml) on days 1, 8 and 15 along with the daily oral gavage of budesonide or ergosterol, respectively. Budesonide and ergosterol were freshly prepared by suspending in the PBS containing 20% hydroxypropyl-β-cyclodextrin (w/v). Mice in the control group were treated with PBS containing 20% hydroxypropyl-β-cyclodextrin. All mice were fed under the same rearing condition for 21 days. On the 21st day after the experiment, all mice were killed.

Bronchoalveolar lavage fluid collection

The lungs were lavaged via a cannula inserted into the trachea and then instilled with 0.8 ml aliquots of saline. All aliquots were collected and centrifuged at 2000 rpm for 10 min at 4°C. The supernatants were obtained and stored at −80°C for the further analysis of MDA, SOD and CAT using commercial kits.

The cells were resuspended in the PBS solution (100 µl) and counted using a hemocytometer. The cell differential was determined from an aliquot of the cell suspension by centrifugation on a slide and Wright-Giemsa stain (Solarbio Biotechnology, Beijing, China). Differential cell counts were calculated based on the morphological criteria.

Hematoxylin and Eosin staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling assay for lung tissues

The lung tissues were fixed with the 4% formaldehyde phosphate buffer overnight and then dehydrated and paraffin embedded and sliced into 4-µm section and stained with Hematoxylin and Eosin (H&E). The slides were observed under a morphometric microscope (Nikon, Japan) at 100 and 400 magnification to evaluate the morphological changes in the lungs. The level of lung alveolarization was evaluated by mean linear intercept (MLI). Six non-overlapping areas were examined randomly for per lung. The formula to calculate was as following: MLI = total length of a line drawn across the lung section/total number of the encountered alveolar septa [22]. The percentage of destroyed alveoli and lung parenchymal destruction was evaluated by the destructive index (DI) according to previous studies [23]. The DI was quantified by dividing the number of destroyed alveoli by the total number of counted alveoli.

The cell death in lung was evaluated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay with an apoptosis detection kit (Roche, California, U.S.A.) [24]. In brief, the lung sections were deparaffinated and permeabilized, then incubated with a TUNEL reaction mixture for 60 min and Converter-POD solution for 30 min at 37°C in dark. Then, the sections were visualized by exposing to diaminobenzidine (DAB) substrate and counterstained with Hematoxylin.

Finally, the slides were observed under a morphometric microscope (Nikon, Japan) at 200 magnification to evaluate the apoptosis in the lungs.

Western blot analysis

The 16HBE cells and lung tissues were homogenized in the RIPA lysis buffer, and then total proteins were extracted and determined by a BCA kit (Beyotime Institute of Biotechnology, Beijing, China). Equal quantities of proteins were separated on 8–12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, MA, U.S.A.). Membranes were incubated with primary antibodies overnight at 4°C, followed by the incubation with HRP-conjugated anti-rabbit or anti-mouse antibodies for 1 h at room temperature. The signals were detected by enhanced chemiluminescence detection reagents. The relative optical densities of the bands were quantified using AlphaView SA software. All Western blot analysis was carried out at least three times.

NF-κB/p65 activity assay

The activity of the nuclear NF-κB/p65 both in 16HBE cells and lung tissues were evaluated by the NF-κB/p65 transcription factor assay kit. First, the nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) based on the specified manufacturer’s instructions. Then, the NF-κB/p65 activity was analyzed according to the specified manufacturer’s instructions.

Immunohistochemistry for Bax, cleaved-caspase 3 and NF-κB/p65

Bax, cleaved-caspase 3 and NF-κB/p65 in the lung tissues of mice were determined by immunohistochemistry. In brief, the lung tissue slice embedded with paraffin was deparaffinized and rehydrated. The antigen was retrieved using sodium citrate with heat-induced retrieval. After blocking with goat serum, Bax, cleaved-caspase 3 and NF-κB/p65 antibodies were applied overnight at 4°C. Then HRP-conjugated anti-rabbit antibody was applied, and Bax, cleaved-caspase 3 and NF-κB/p65 were finally visualized using a DAB detection kit. Images of Bax, cleaved-caspase 3 and NF-κB/p65 were obtained and photographed using a microscope (Olympus Corporation, Tokyo, Japan).

Statistical analysis

Data are presented as mean ± SEM. The statistical significance of the difference was determined by one-way ANOVA followed by the Tukey’s post-hoc multiple comparison test using Prism version 5.0 (GraphPad Software, Inc.). Values of P<0.05 were considered statistically significant.

Results

Anti-inflammatory effect of ergosterol in CSE-induced 16HBE cells and mice

To evaluate the anti-inflammatory activity of ergosterol, the levels of NO, TNF-α and IL-6 in the CSE-induced 16HBE cells were determined (Figure 1A–C). Results show that the incubation of the 5% CSE had significantly increased the levels of NO, TNF-α and IL-6 as opposed to the control group in the CSE-treated 16HBE cells. Compared with the CSE-treated group, ergosterol significantly reversed these changes.

Effects of ergosterol on the inflammatory markers in CSE-induced 16HBE cells and mice

Figure 1
Effects of ergosterol on the inflammatory markers in CSE-induced 16HBE cells and mice

Ergosterol decreased the levels of NO (A), TNF-α (B), and IL-6 (C) in vitro. (D) The inflammatory cells in BALF in mice (200 magnification). Scale bar, 50 μm. (E) Quantification histograms of the inflammatory cells in BALF. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. ** P<0.01, *** P<0.001 the CSE or model group versus the control group; # P<0.05, ## P<0.01, ### P<0.001, the ergosterol or budesonide treatment groups versus the CSE or model group. All the experiments in vitro were repeated three times.

Figure 1
Effects of ergosterol on the inflammatory markers in CSE-induced 16HBE cells and mice

Ergosterol decreased the levels of NO (A), TNF-α (B), and IL-6 (C) in vitro. (D) The inflammatory cells in BALF in mice (200 magnification). Scale bar, 50 μm. (E) Quantification histograms of the inflammatory cells in BALF. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. ** P<0.01, *** P<0.001 the CSE or model group versus the control group; # P<0.05, ## P<0.01, ### P<0.001, the ergosterol or budesonide treatment groups versus the CSE or model group. All the experiments in vitro were repeated three times.

Furthermore, the numbers of inflammatory cells in the bronchoalveolar lavage fluid (BALF) were measured by Wright-Giemsa staining. As presented in Figure 1D,E, the CSE challenge resulted in an increased number of the total cells and neutrophil counts in the BALF compared with the control group. In contrast, ergosterol led to a significant reduction in the counts of inflammatory cells compared with the model group.

Effect of ergosterol on the redox imbalance both in vitro and in vivo

The levels of MDA, SOD and CAT in the CSE-induced 16HBE cells and mice were determined in order to evaluate the antioxidative activity of ergosterol (Figure 2A–F). Results show that the CSE treatment had significantly increased the level of MDA, while decreased the levels of SOD and CAT as opposed to the control group both in the CSE-treated 16HBE cells and mice (model group). However, ergosterol significantly reversed these changes comparing with the CSE-treated group.

Antioxidant effect of ergosterol in the CSE-induced cell and mouse models

Figure 2
Antioxidant effect of ergosterol in the CSE-induced cell and mouse models

(A) MDA, (B) SOD, (C) CAT in the CSE-induced 16HBE cells; and (D) MDA, (E) SOD, (F) CAT in BALF in CSE-induced mice; (G) ROS measured by flow cytometry, and (H) corresponding quantification histograms in the CSE-induced 16HBE cells. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. ** P<0.01, *** P<0.001, the CSE or model group versus the control group; # P<0.05, ## P<0.01, ## P<0.001, the ergosterol or budesonide treatment groups versus the CSE or model group. All the experiments in vitro were repeated three times.

Figure 2
Antioxidant effect of ergosterol in the CSE-induced cell and mouse models

(A) MDA, (B) SOD, (C) CAT in the CSE-induced 16HBE cells; and (D) MDA, (E) SOD, (F) CAT in BALF in CSE-induced mice; (G) ROS measured by flow cytometry, and (H) corresponding quantification histograms in the CSE-induced 16HBE cells. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. ** P<0.01, *** P<0.001, the CSE or model group versus the control group; # P<0.05, ## P<0.01, ## P<0.001, the ergosterol or budesonide treatment groups versus the CSE or model group. All the experiments in vitro were repeated three times.

Moreover, the ROS production in the CSE-induced 16HBE cells was measured by flow cytometry. As shown in Figure 2G,H, results show that the CSE challenge markedly elevated the generation of ROS in 16HBE cells, whereas the ergosterol treatment clearly reversed this change.

Ergosterol inhibited CSE-induced cell apoptosis both in vitro and in vivo

The cell apoptosis ratio in the CSE-induced 16HBE cells was measured by flow cytometry. As shown in Figure 3A,B, cellular apoptosis markedly increased in the CSE-induced 16HBE cells compared with the control group. However, the ergosterol treatment significantly reversed this change.

Effects of ergosterol on apoptosis both in vitro and in vivo

Figure 3
Effects of ergosterol on apoptosis both in vitro and in vivo

(A) Ergosterol defense CSE-induced cell apoptosis measured by flow cytometry, and (B) is its corresponding quantification histograms. (C) Ergosterol reduced the number of apoptotic bodies in CSE-induced 16HBE cells detected by optical microscope. (D) The cell apoptosis in the lung of CSE-induced mice was assessed by TUNEL and examined under a fluorescence microscope (200 magnification). Scale bar, 50 μm. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. *** P<0.001, the CSE or model group versus the control group; ## P<0.01, ### P<0.001, the ergosterol or budesonide treatment groups versus the CSE or model group.

Figure 3
Effects of ergosterol on apoptosis both in vitro and in vivo

(A) Ergosterol defense CSE-induced cell apoptosis measured by flow cytometry, and (B) is its corresponding quantification histograms. (C) Ergosterol reduced the number of apoptotic bodies in CSE-induced 16HBE cells detected by optical microscope. (D) The cell apoptosis in the lung of CSE-induced mice was assessed by TUNEL and examined under a fluorescence microscope (200 magnification). Scale bar, 50 μm. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. *** P<0.001, the CSE or model group versus the control group; ## P<0.01, ### P<0.001, the ergosterol or budesonide treatment groups versus the CSE or model group.

Besides, the effect of ergosterol on morphological changes of the CSE-induced 16HBE cells was determined by a fluorescence microscope (Figure 3C). Results show that the fragmented nuclei and apoptotic bodies (shown by the red arrow) appeared in the 5% CSE-induced 16HBE cells. However, when the cells were treated with ergosterol, fewer apoptotic bodies were observed.

Furthermore, the TUNEL results show a significant increase of apoptotic cells in the CSE-treated group, whereas the number of apoptotic cells markedly reduced in the ergosterol and budesonide treatment group (Figure 3D). Interestingly, from TUNEL results, the high-dose ergosterol group showed a better anti-apoptosis effect than the budesonide group in mouse lungs.

Pathological change of lung tissues in CSE-induced mice

The morphometric assay was performed to evaluate the effect of ergosterol on the CSE-induced lung damage in mice. As shown in Figure 4, the lung parenchyma of the model group showed increased inflammatory cell infiltration, thickened small airways and alveolar space collapse as opposed to the control group. Compared with the model group, these histopathological changes were notably alleviated in the ergosterol-treated groups.

Effects of ergosterol on lung histological changes in the CSE-induced mice

Figure 4
Effects of ergosterol on lung histological changes in the CSE-induced mice

(A) The lung histological changes of the CSE-induced mice were assessed by HE (hematoxylin and eosin) staining and examined under a fluorescence microscope (200 and 400 magnification). Scale bar, 50 μm. Morphometric chages of MLI (B) and DI (C) were measured after the ergosterol treatment. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. * P<0.05, the model group versus the control group; # P<0.05, the ergosterol treatment groups versus the model group.

Figure 4
Effects of ergosterol on lung histological changes in the CSE-induced mice

(A) The lung histological changes of the CSE-induced mice were assessed by HE (hematoxylin and eosin) staining and examined under a fluorescence microscope (200 and 400 magnification). Scale bar, 50 μm. Morphometric chages of MLI (B) and DI (C) were measured after the ergosterol treatment. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. * P<0.05, the model group versus the control group; # P<0.05, the ergosterol treatment groups versus the model group.

Effect of ergosterol on NF-κB/p65, IκB, iNOS and COX-2

Oxidative stress (e.g. ROS) activates NF-κB/p65-mediated transcription of pro-inflammatory mediators through activation of its activating inhibitory κB kinase (IKK) in COPD [1,25]. iNOS and COX-2 were also increased both in human bronchial epithelial cells and CS-exposed mice [26,27]. Therefore, NF-κB/p65, IκB, iNOS and COX-2 were analyzed using Western blot in order to evaluate the mechanistic pathway. As expected, NF-κB/p65, iNOS and COX-2 both in vitro (Figure 5A,B) and in vivo (Figure 5C,D) was obviously elevated in the CSE groups, while IκB was reduced compared with the control groups. On the contrary, the ergosterol treatment reduced the levels of NF-κB/p65, iNOS and COX-2, and increased that of IκB compared with the CSE only groups. Moreover, the ergosterol treatment decreased the NF-κB/p65 activity both in vitro (Figure 5E) and in vivo (Figure 5F). In addition, we performed the immunohistochemistry analysis to evaluate the effect of ergosterol on the protein expression of NF-κB/p65, results of which (Figure 7A) are in consistent with the above data.

Effects of ergosterol on NF-κB, IκB, iNOS and COX-2 proteins induced by CSE both in vitro and in vivo

Figure 5
Effects of ergosterol on NF-κB, IκB, iNOS and COX-2 proteins induced by CSE both in vitro and in vivo

Ergosterol suppressed the expression of NF-κB, iNOS and COX-2 in CSE-induced 16HBE cells (A) and mice (C), whereas increased that of IκB. (B) and (D) are the corresponding quantification histograms of (A) and (C), respectively. The NF-κB/p65 activity in 16HBE cells (E) and mouse lungs (F). Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. * P<0.05, ** P<0.01, *** P<0.001, the CSE or model group versus the control group; # P<0.05, ## P<0.01, the treatment group versus the CSE or model group. The values shown represent means ± SEM (n=3).

Figure 5
Effects of ergosterol on NF-κB, IκB, iNOS and COX-2 proteins induced by CSE both in vitro and in vivo

Ergosterol suppressed the expression of NF-κB, iNOS and COX-2 in CSE-induced 16HBE cells (A) and mice (C), whereas increased that of IκB. (B) and (D) are the corresponding quantification histograms of (A) and (C), respectively. The NF-κB/p65 activity in 16HBE cells (E) and mouse lungs (F). Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. * P<0.05, ** P<0.01, *** P<0.001, the CSE or model group versus the control group; # P<0.05, ## P<0.01, the treatment group versus the CSE or model group. The values shown represent means ± SEM (n=3).

Effect of ergosterol on the apoptosis-associated proteins

It is reported that ergosterol attenuates LPS-induced myocardial injury by regulating the apoptosis associated proteins (Bcl-2, Bax, cleaved-caspase 3, cleaved-caspase 9 and cleaved-PARP) in rats [28]. So, we further measured the apoptosis associated-proteins both in CSE-induced human bronchial epithelial cells (16HBE) (Figure 6A,B) and COPD mice (Figure 6C,D) using Western blot to evaluate the effect of ergosterol. Results show that the CSE treatment markedly increased the levels of Bax, cleaved-caspase 3, cleaved-caspase 7, cleaved-caspase 9 and cleaved-PARP, while decreased Bcl-2 activities compared with the control group. In contrast, the administration of ergosterol reversed the effects of CSE on the apoptosis-related proteins. Interestingly, from the Western blot results, the high-dose ergosterol group showed a better inhibition effect than that of the budesonide group on the CSE-induced apoptosis in mouse lungs. Furthermore, we performed the immunohistochemistry analysis to evaluate the effect of ergosterol on the Bax and cleaved-caspase 3, results of which (Figure 7A) are consistent with the above data.

Effects of ergosterol on apoptosis-associated proteins induced by CSE both in vitro and in vivo

Figure 6
Effects of ergosterol on apoptosis-associated proteins induced by CSE both in vitro and in vivo

Ergosterol suppressed the expression of pro-apoptotic proteins, including Bax, cleaved-caspase 3/7/9 and cleaved PARP in CSE-induced 16HBE cells (A) and mice (C), whereas increased that of Bcl-2. (B) and (D) are the corresponding quantification histograms of (A) and (C), respectively. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. * P<0.05, ** P<0.01, *** P<0.001, the CSE or model group versus the control group; # P<0.05, ## P<0.01, the treatment group versus the CSE or model group. The values shown represent means ± SEM (n=3).

Figure 6
Effects of ergosterol on apoptosis-associated proteins induced by CSE both in vitro and in vivo

Ergosterol suppressed the expression of pro-apoptotic proteins, including Bax, cleaved-caspase 3/7/9 and cleaved PARP in CSE-induced 16HBE cells (A) and mice (C), whereas increased that of Bcl-2. (B) and (D) are the corresponding quantification histograms of (A) and (C), respectively. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after the CSE challenge; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. * P<0.05, ** P<0.01, *** P<0.001, the CSE or model group versus the control group; # P<0.05, ## P<0.01, the treatment group versus the CSE or model group. The values shown represent means ± SEM (n=3).

Effects of ergosterol on apoptosis-associated proteins in the CSE-induced mice and 16HBE cells

Figure 7
Effects of ergosterol on apoptosis-associated proteins in the CSE-induced mice and 16HBE cells

(A) The apoptosis-associated proteins (Bax, cleaved-caspase3 and NF-κB/p65) in the lung of CSE-induced mice were assessed by immunohistochemistry assay and examined under a fluorescence microscope (400 magnification). Scale bar, 50 μm. (B) Western blot analyses and (C) related histograms showed that the inhibition of NF-κB/p65 enhanced the protective effect of ergosterol in CSE-induced 16HBE cells. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. ** P<0.01, the CSE or model group versus the control group; # P<0.05, ## P<0.01, the treatment group versus the CSE or model group. The values shown represent means ± SEM (n=3).

Figure 7
Effects of ergosterol on apoptosis-associated proteins in the CSE-induced mice and 16HBE cells

(A) The apoptosis-associated proteins (Bax, cleaved-caspase3 and NF-κB/p65) in the lung of CSE-induced mice were assessed by immunohistochemistry assay and examined under a fluorescence microscope (400 magnification). Scale bar, 50 μm. (B) Western blot analyses and (C) related histograms showed that the inhibition of NF-κB/p65 enhanced the protective effect of ergosterol in CSE-induced 16HBE cells. Mouse groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; 20 and 40: ergosterol dosed at 20 and 40 mg/kg, respectively. ** P<0.01, the CSE or model group versus the control group; # P<0.05, ## P<0.01, the treatment group versus the CSE or model group. The values shown represent means ± SEM (n=3).

Effect of ergosterol on apoptosis-related protein expression were regulated by NF-κB/p65 Pathway

The 16HBE cells were pretreated with 5% CSE for 1 h and subsequently treated with a specific NF-κB/p65 antagonist (QNZ, 10 nM), and/or ergosterol (20 µM) for 24 h. The Western blot results (as shown in Figure 7B,C) show that the ergosterol or QNZ treatment markedly down-regulated the elevation of Bax, cleaved-caspase 3, cleaved-caspase 7 and cleaved-PARP in the CSE-induced 16HBE cells. Furthermore, the ergosterol only group showed no obvious effect on the expression of these proteins. Taken together, our findings indicated that the NF-κB/p65 signaling pathway might be involved in the ergosterol protection effects on the CSE-induced cell apoptosis.

Discussion

Our results demonstrate that ergosterol significantly protected mice against the CSE-induced COPD by exerting antioxidative and anti-inflammatory effects as well as inhibiting cell apoptosis. In addition, the ergosterol effects on the CSE-induced COPD model were enhanced by the NF-κB/p65 inhibitor, suggesting that the ergosterol protective effects might be related to NF-κB/p65 signaling pathway.

COPD is characterized by persistent respiratory symptoms and airflow limitation, which is caused by the combination of small airway diseases (e.g. obstructive bronchiolitis) and parenchymal destruction (emphysema), as well as the apoptosis of the human bronchial/alveolar epithelial cells induced by risk factors [24,29,30]. Cigarette smoking (CS) is a prevalent risk factor of COPD [1,23]. CS exposure and intraperitoneal injection of CSE can induce emphysema and the effectiveness of both model establishing methods are similar while the latter is 3–6 times shorter in terms of challenging time [31], thus the CSE-induced 16HBE cells and COPD mice model were used in the present study. Our results show that ergosterol had a beneficial effect on the emphysema mice by airway remodeling and attenuating cell apoptosis. Our TUNEL results reveal that more apoptosis cells in the CSE-induced mice were observed than that in control group, whereas ergosterol partly reversed it. Moreover, the similar results were observed in the CSE-induced 16HBE cells using flow cytometry and DAPI staining.

All patients with COPD have the characteristics of elevated oxidative stress and inflammation [32]. Several previous studies have demonstrated that increased oxidative stress in smokers contributes to lung damage as shown in oxidative markers such as ROS, MDA and SOD [24,32–34]. In line with the previous studies, the levels of ROS and MDA were markedly increased in the BALF of the COPD mice, as well as in the CSE-induced 16HBE cells compared with the control group in this work. Besides, the levels of SOD and CAT were decreased in the CSE-induced group as opposed to the control group both in vivo and in vitro. Interestingly, the ergosterol treatment reversed these changes. Moreover, oxidative stress leads to inflammation and the related inflammatory mediators are elevated in the COPD lung tissues [34]. It has reported that the CS-exposure increases the levels of pro-inflammatory cytokines including TNF-α and IL-6 in sera and lungs in mice, while the ergosterol treatment effectively reversed them via JAK3/STAT3/ NF-κB pathways [34]. In the present study, we further demonstrated that the administration of ergosterol alleviated the increased level of NO and inflammatory factors (TNF-α and IL-6) in the CSE-induced 16HBE cells. Besides, ergosterol reduced the airway inflammation by reducing the influx of total inflammatory cells including macrophages and neutrophils in the airways of the CSE-treated mice. Those results suggest that ergosterol showed protective effects both in vitro and in vivo.

An increasing number of studies indicated that pulmonary epithelial cells and vascular endothelial cells apoptosis occurs in the lungs of COPD patients, resulting in airspace enlargement and alveolar wall damage [4,32]. Xia et al. [32] demonstrated that Bax and cleaved-caspase 3 were up-regulated in the lungs of the rats exposed to CS, while Bcl-2 is down-regulated. Moreover, Xu et al. [28] demonstrated that ergosterol can inhibit apoptosis in the LPS-induced rats, by decreasing the expression of Bax, cleaved-caspase 3, cleaved-caspase 9 and cleaved-PARP via a Nrf2 pathway. In a similar fashion, we demonstrated that ergosterol attenuated the elevated Bax/Bcl-2 ratio and pro-apoptosis proteins, including cleaved-caspase 3/7/9 and cleaved-PARP, both in the CSE-induced 16HBE cells and mice.

Previous studies demonstrated that smoke exposure can activate NF-κB, which is consequently translocated into nucleus to activate its downstream genes underlying innate immunity, neutrophilic inflammation and apoptosis [7,35,36]. NF-κB plays a great role in the protection of cells against inducers of apoptosis through the inhibition of apoptosis-associated genes [37]. In this study, we demonstrated that the combination treatment of ergosterol and NF-κB inhibitor QNZ strongly attenuated the elevated pro-apoptosis proteins, including Bax, cleaved-caspase 3/7 and cleaved-PARP in the CSE-induced 16HBE cells. This result also suggests that the NF-κB pathway might be involved in the anti-apoptosis effect of ergosterol.

In conclusion, we evaluated the therapeutic effects of ergosterol on the CSE-induced COPD models. Ergosterol decreased the CSE-induced elevation of inflammatory cytokines and oxidative stress. In addition, ergosterol decreased the apoptosis-associated proteins both in the CSE-induced 16HBE cells and mice. The mechanistic study results show that ergosterol exerted protection effects might be associated with the partial suppression of NF-κB/p65 activation, the proposed scheme of which is shown in Figure 8. Therefore, our study gave a relatively complete evidence that ergosterol had the potential in treating COPD.

Proposed therapeutic mechanism of ergosterol for COPD

Figure 8
Proposed therapeutic mechanism of ergosterol for COPD

Exposure of CSE increased ROS in 16HBE cells and in lung of mice. Then, an imbalance of redox status, including the destruction of the scavenger system (e.g., SOD, CAT and MDA), and a boosted inflammatory response occurred. Besides, it also led to the activation of apoptosis-associated proteins (e.g., Bax, cleaved-caspase 3/7/9, cleaved PARP), finally resulting in cell death in the COPD model. The ergosterol treatment reversed these changes, and NF-κB/p65 pathway might be involved in the protective progress.

Figure 8
Proposed therapeutic mechanism of ergosterol for COPD

Exposure of CSE increased ROS in 16HBE cells and in lung of mice. Then, an imbalance of redox status, including the destruction of the scavenger system (e.g., SOD, CAT and MDA), and a boosted inflammatory response occurred. Besides, it also led to the activation of apoptosis-associated proteins (e.g., Bax, cleaved-caspase 3/7/9, cleaved PARP), finally resulting in cell death in the COPD model. The ergosterol treatment reversed these changes, and NF-κB/p65 pathway might be involved in the protective progress.

Clinical perspectives

  • Numerous studies have shown that C. sinensis have a therapeutic effect on patients with COPD [38,39]. Ergosterol is the main bioactive ingredient in C. sinensis, and studies suggest that ergosterol may contribute to potential health benefits and significant pharmacological properties, such as anti-tumor, anti-inflammation, antioxidation and immunomodulatory activities. Thereby, the objective of this work was to investigate the effects of ergosterol on anti-inflammation and antioxidative stress as well as anti-apoptosis in the CSE-induced COPD models both in vitro and in vivo.

  • In brief, our results demonstrate that ergosterol alleviated the CSE-induced inflammation, oxidation and apoptosis via the NF-κB/p65 pathway in both 16HBE cells and Balb/c mice.

  • These results suggest that ergosterol might be partly responsible for the therapeutic effects of cultured C. sinensis on COPD patients. The present study might provide a theoretical basis of the ergosterol-containing remedies for COPD treatment and future drug development.

Funding

This work was supported by the National Major Science and Technology Project–Prevention and Treatment of AIDS, Viral Hepatitis and Other Major Infectious Diseases [grant number 2013ZX10005004]; the Major Project of Science and Technology of Shandong Province [grant numbers 2015ZDJS04001, 2018CXGC1411]; the Science and Technology Enterprise Technology Innovation Fund of Jiangsu Province [grant number BC2014172]; the Small and Medium Enterprise Technology Innovation Project of Lianyungang City [grant number CK1333].

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

X.S., S.-Y.L. and Z.-X.Z. conceived the study design. X.S. and X.-L.F. designed the experiments. X.S., X.-L.F. and D.-D.Z. acquired, analyzed and interpreted the data. X.S. drafted the manuscript. A.L. and C.-Y.L. reviewed the manuscript and provided comments.

Abbreviations

     
  • CAT

    catalase

  •  
  • COPD

    chronic obstructive pulmonary disease

  •  
  • COX-2

    cyclooxygenase-2

  •  
  • CS

    cigarette smoke

  •  
  • CSE

    CS extract

  •  
  • DAB

    diaminobenzidine

  •  
  • DAPI

    4′,6-diamidino-2-phenylindole

  •  
  • DCFH-DA

    dichlorodi-hydrofluorescein diacetate

  •  
  • DI

    destructive index

  •  
  • FBS

    fetal bovine serum

  •  
  • HE

    hematoxylin and eosin

  •  
  • HRP

    horseradish peroxidase

  •  
  • IL-6

    interleukin-6

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • MDA

    malondialdehyde

  •  
  • MLI

    mean linear intercept

  •  
  • PARP

    poly-(ADP)-ribose polymerase

  •  
  • PBS

    phosphate-buffered saline

  •  
  • PERK

    phosphorylated extracellular regulated kinase

  •  
  • QNZ

    N4-[2-(4-phenoxyphenyl]-4,6-quinazolinediamine

  •  
  • ROS

    reactive oxygen species

  •  
  • SOD

    superoxide dismutase

  •  
  • SPF

    Specific pathogen free

  •  
  • TNF-α

    tumor necrosis factor α

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling

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