Inhibition Effect of Eucommia ulmoides Leaf Extract on Interleukin 8 Production by A549 Cells

Abstract

Asthma is characterized by chronic inflammation of the airway mucosa. As Eucommia ulmoides Oliv. leaf extract (ELE) has been known to have anti-inflammatory properties, herein, we investigated the effect of ELE on interleukin (IL-) 8 production in A549 cells, a human airway epithelial cell line. The addition of ELE 1 h before tumor necrosis factor-alpha (TNFα) stimulation inhibited IL-8 production by A549 cells in a concentration-dependent manner. The addition of geniposidic acid, the main component of ELE, also inhibited IL-8 production. To further investigate the mechanism by which ELE inhibits IL-8 production, the effect of ELE or geniposidic acid on TNFα-stimulated p38 phosphorylation was examined by Western blotting. After 30 min of TNFα stimulation, p38 phosphorylation was inhibited by the addition of ELE or geniposidic acid, suggesting that ELE inhibited IL-8 production in TNFα-stimulated A549 cells by suppressing one of the signal transducers of p38 phosphorylation. These results indicate that ELE can be used as an effective measure against asthma, particularly neutrophilic asthma.

INTRODUCTION

Asthma is caused by chronic inflammation of the airway mucosa, with symptoms such as convulsive coughing, spitting, and stifling by slight irritation. The causes of inflammation involve allergens such as ticks, house dust, pet dandruff, and mold. In addition to allergens, exhaust gases in the atmosphere, overwork, and stress cause inflammation. Asthma can be classified as eosinophilic asthma and non-eosinophilic asthma (mainly neutrophilic asthma).¹⁾ In eosinophilic asthma, eosinophils, mast cells, and B cells activated by interleukin (IL-) 4, 5, 9, or 13 produced by T helper type 2 (Th2) cells play an important role. In addition, innate lymphoid cells (ILC2) produce IL-5 by releasing thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 secreted from the airway epithelium. Thereafter, more eosinophils are recruited and activated, and the allergic inflammation is exacerbated.^2,3) In neutrophilic asthma, neutrophil production is increased by the infiltration of IL-8 secreted from the airway epithelium, causing severe airway inflammation. IL-17 produced by Th17 cells and ILC3 is also involved in the infiltration of neutrophils.^3,4)

IL-8 is a CXC chemokine known as a neutrophil chemotactic factor. In neutrophilic asthma, sputum IL-8, neutrophil esterase concentration, and mRNA levels of IL-8 and IL-1β are elevated compared to that in non-neutrophilic asthma.⁵⁾ Furthermore, in non-eosinophilic asthma, high levels of IL-8 are observed from blood neutrophils.⁶⁾ Stimulants such as viruses, tobacco, and pollutants induce chemotactic factors including IL-8 and accumulate neutrophils in the respiratory tract. In the asthma model, IL-17A and IL-17F produced from activated Th17 cells induce the production of neutrophil chemotactic factors such as CXCL1 and IL-8 from the airway epithelium.⁷⁾ Significant increases in Th17-related cytokines and chemokines such as CXCL1 and IL-8 are observed in the sputum of severe asthma patients with neutrophilia compared to that in other patients with severe inflammatory asthma without neutrophilia.⁸⁾

Previous reports have shown that Eucommia ulmoides Oliv. has various antioxidant, antihypertensive, and antihyperglycemic activities.^9–11) The hot water extract of E. ulmoides Oliv. leaves (ELE) contains various substances such as iridoids, phenol compounds, flavonoids, and terpenoids.¹²⁾ Geniposidic acid present in ELE has been known to relieve hypertension and hyperlipidemia¹⁰⁾ and suppress the production of inflammatory cytokines and infiltration of neutrophils in the colitis model.¹³⁾ Furthermore, administration of ELE decreased tumor necrosis factor-α (TNFα) levels in the plasma of rats on a high-fat diet.¹⁴⁾ Thus, it is conceivable that ELE has anti-inflammatory activity. Therefore, this study aimed to investigate the effect of ELE on asthma, especially neutrophilic asthma, by monitoring IL-8 production from cells of the human airway epithelial cell line, A549.

MATERIALS AND METHODS

Reagents

ELE (Lot No. 180724M; geniposidic acid content: 5.898%) was provided by Kobayashi Pharmaceutical Company (Osaka, Japan). Two tons of Eucommia leaves (Sichuan, China) were boiled in 10 t of water at 90 °C for 1 h. The extract was filtered and concentrated and then powdered by vacuum-drying (yield: 18%). Geniposidic acid was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, U.S.A.). Asperuloside was purchased from ChemFaces (Wuhan, Hubei, China). Recombinant human TNF-α was purchased from Peprotech (Rocky Hill, NJ, U.S.A.). Anti-phospho-Syk, anti-Syk, anti-phospho-p38, anti-p38, anti-phospho-c-Jun N-terminal kinase (JNK), anti-JNK, horseradish peroxidase (HRP) conjugated anti-β-Actin, and HRP conjugated anti-rabbit immunoglobulin G (IgG) were purchased from Cell Signaling Technologies (Danvers, MA, U.S.A.).

Cells

The human airway epithelial cell line A549 was purchased from the Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan). A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal bovine serum, 20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C.

Induction of IL-8 Production

A549 cells (1 × 10⁵ cells/mL) were dispensed into 96-well microplates at 200 µL/well and cultured overnight at 37 °C. The next day, the supernatant from each well was discarded; the cells were washed and 100 µL/well of DMEM/Nutrient Mixture F-12 Ham medium supplemented with 0.1% bovine serum albumin (BSA) (0.1% BSA medium) was added to each well. Then, 50 µL/well of ELE, geniposidic acid, or asperuloside diluted to each concentration in 0.1% BSA medium was added to each well and incubated for 1 h at 37 °C. After incubation, 50 µL/well of TNF-α (200 ng/mL) diluted in 0.1% BSA medium was added to each well to stimulate the cells. The culture supernatant on the third day of culture was collected and used for IL-8 measurement.

Enzyme-Linked Immunosorbent Assay (ELISA)

IL-8 levels in cell culture supernatants were assayed using Human IL-8/CXCL8 DuoSet ELISA (R&D Systems, Minneapolis, MN, U.S.A.).

Western Blot Analysis

Cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing both protease and phosphatase inhibitors (Roche, Indianapolis, IN, U.S.A.). Protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). Equal amounts of protein were loaded onto a 12% sodium dodecyl-sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked using 5% skim milk for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Then, the membranes were incubated with HRP conjugated secondary antibodies for 1 h at room temperature and detected by enhanced chemiluminescence reagents using Amersham Imager 680 blot and gel imager (GE Healthcare, Buckinghamshire, U.K.).

Statistical Analysis

Results are expressed as mean ± standard deviation (S.D.). The statistical significance of the difference between groups was determined by one-way ANOVA followed by Tukey’s test. A p-value of less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

We examined the anti-inflammatory mechanisms of ELE using the human airway epithelial cell line, A549. IL-8 levels markedly increased in the supernatants of A549 cells on day 3 after TNFα stimulation (Fig. 1). A549 cells were treated with ELE 1 h before TNFα stimulation. ELE significantly inhibited IL-8 production from TNFα-stimulated cells in a dose-dependent manner.

Fig. 1. Effect of ELE on IL-8 Production by TNFα Stimulated A549 Cells

A549 cells were stimulated with TNFα (50 ng/mL) 1 h after the addition of each concentration of ELE. IL-8 production in the culture supernatant was measured by ELISA on the third day after stimulation. Results are shown as mean ± S.D. (n = 6). * p < 0.01 vs. the 0 mg/mL of ELE treated group (using Tukey’s test). ND: not detected.

Treatment with 10  and 100 µM geniposidic acid significantly inhibited IL-8 production from TNFα-stimulated cells (Fig. 2). Asperuloside is another major component of ELE. We also examined the effect of asperuloside on IL-8 production from TNFα-stimulated cells. Asperuloside had no inhibitory effect on IL-8 production (data not shown).

Fig. 2. Effect of Geniposidic Acid on IL-8 Production by TNFα Stimulated A549 Cells

A549 cells were stimulated with TNFα (50 ng/mL) 1 h after the addition of each concentration of geniposidic acid. IL-8 production in the culture supernatant was measured by ELISA on the third day after stimulation. Results are shown as mean ± S.D. (n = 6). * p < 0.01 vs. the 0 µM of geniposidic acid treated group (using Tukey’s test). ND: not detected.

In this study, we used ELE containing 5898 mg/100 g of geniposidic acid. Therefore, 1 mg/mL of ELE contained approximately 160 µM as geniposidic acid (molecular weight of geniposidic acid is 374.34). According to the present results, IL-8 production was significantly inhibited by 1 mg/mL of ELE and 100 µM of geniposidic acid. Therefore, geniposidic acid was one of the components of ELE, causing an inhibitory effect on IL-8 production.

We proceeded to determine whether ELE suppressed the mitogen-activated protein kinase (MAPK) signaling pathways in TNFα-stimulated A549 cells. ELE and geniposidic acid suppressed phosphorylation of p38 and JNK (Fig. 3). The TNF receptors trimerize when activated by TNFα and bind to the death domain in the cytoplasmic region of the TNF receptors. Aggregation of death domains triggers the assembly of TNF receptor-associated signaling molecules such as TNF receptor-associated death domain (TRADD) and TNF Receptor Associated Factor 2 (TRAF2), which activates the MAPK signaling pathway to produce IL-8.^15–17)

Fig. 3. Effect of ELE or Geniposidic Acid on Phosphorylation of p38 and JNK by TNFα Stimulated A549 Cells

A549 cells were stimulated with TNFα (50 ng/mL) 1 h after the addition of each concentration of ELE or geniposidic acid. The cells were harvested 30 min after TNFα stimulation and Western blotting was performed.

In conclusion, the present results suggest that ELE can be effective against asthma, especially neutrophilic asthma.

Acknowledgments

The authors thank Ms. Haruka Sato and Ms. Kanami Sugiyama for technical assistance with the experiments. This work was supported by the 14th Research Grant from the Japanese Society of Eucommia.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat. Med., 18, 716–725 (2012).
• 2) Chung KF. Targeting the interleukin pathway in the treatment of asthma. Lancet, 386, 1086–1096 (2015).
• 3) Holgate ST. Innate and adaptive immune responses in asthma. Nat. Med., 18, 673–683 (2012).
• 4) Simpson JL, Grissell TV, Douwes J, Scott RJ, Boyle MJ, Gibson PG. Innate immune activation in neutrophilic asthma and bronchiectasis. Thorax, 62, 211–218 (2007).
• 5) Wood LG, Baines KJ, Fu J, Scott HA, Gibson PG. The neutrophilic inflammatory phenotype is associated with systemic inflammation in asthma. Chest, 142, 86–93 (2012).
• 6) Baines KJ, Simpson JL, Bowden NA, Scott RJ, Gibson PG. Differential gene expression and cytokine production from neutrophils in athma phenotypes. Eur. Respir. J., 35, 522–531 (2010).
• 7) Newcomb DC, Peebles RS Jr. Th17-mediated inflammation in asthma. Curr. Opin. Immunol., 25, 755–760 (2013).
• 8) Manni ML, Trudeau JB, Scheller EV, Mandalapu S, Elloso MM, Kolls JK, Wenzel SE, Alcorn JF. The complex relationship between inflammation and lung function in severe asthma. Mucosal Immunol., 7, 1186–1198 (2014).
• 9) Yen GC, Hsieh CL. Reactive oxygen species scavenging activity of du-zhong (Eucommia ulmoides Oliv.) and its active compounds. J. Agric. Food Chem., 48, 3431–3436 (2000).
• 10) Hosoo S, Koyama M, Kato M, Hirata Y, Yamaguchi Y, Yamasaki H, Wada A, Wada K, Nishibe S, Nakamura K. The restrictive effects of Eucommia ulmoides Oliver leaf extract on vascular function in spontaneously hypertensive rats. Molecules, 20, 21971–21981 (2015).
• 11) Lee MK, Kim MJ, Cho SY, Park SA, Park KK, Jung UJ, Park HM, Choi MS. Hypoglycemic effrct of Du-zhong (Eucommia ulmoides Oliv.) leaves in streptozotocin-induced diabetic rats. Diabetes Res. Clin. Pract., 67, 22–28 (2005).
• 12) He X, Wang J, Li M, Hao D, Yang Y, Zhang C, He R, Tao R. Eucommia ulmoides Oliv.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J. Ethnopharmacol., 151, 78–92 (2014).
• 13) Xu B, Li YL, Xu M, Yu CC, Lian MQ, Tang ZY, Li CX, Lin Y. Geniposide ameliorates TNBS-induced experimental colitis in rats via reducing inflammatory cytokine release and restoring impaired intestinal barrier function. Acta Pharmacol. Sin., 38, 688–698 (2017).
• 14) Fujikawa T, Hirata T, Wada A, Kawamura N, Yamaguchi Y, Fujimura K, Ueda Y, Yurugi Y, Soya H, Nishibe S. Chronic administration of Eucommia leaf stimulates metabolic function of rats across several organs. Br. J. Nutr., 104, 1868–1877 (2010).
• 15) Bezzerri V, Borgatti M, Finotti A, Tamanini A, Gambari R, Cabrini G. Mapping the transcriptional machinery of the IL-8 gene in human bronchial epithelial cells. J. Immunol., 187, 6069–6081 (2011).
• 16) Amrani Y, Ammit AJ, Panettieri RA Jr. Tumor necrosis factor receptor (TNFR) 1, but not TNFR2, mediates tumor necrosis factor-α-induced interleukin-6 and RANTES in human airway smooth muscle cells: role of p38 and p42/44 mitogen-activated protein kinases. Mol. Pharmacol., 60, 646–655 (2001).
• 17) Shi JH, Sun SC. Tumor necrosis factor receptor-associated factor regulation of nuclear factor κB and mitogen-activated protein kinase pathways. Front. Immunol., 9, 1849 (2018).