Article pubs.acs.org/JAFC
Protective Effect of Ginkgo biloba leaves extract, EGb761, on Endotoxin-Induced Acute Lung Injury via a JNK- and Akt-Dependent NFκB Pathway Chien-Ying Lee,†,‡,§ Jiann-Jou Yang,∥ Shiuan-Shinn Lee,⊥ Chun-Jung Chen,# Yi-Chun Huang,$ Kuang-Hua Huang,& and Yu-Hsiang Kuan*,†,‡ †
Department of Pharmacology, Chung Shan Medical University, No. 110, Sec. 1, Jianguo North Road, Taichung 40201, Taiwan Department of Pharmacy and §Department of Integrated Chinese and Western Medicine, Chung Shan Medical University Hospital, No. 110, Sec. 1, Jianguo North Road, Taichung 40201, Taiwan ∥ Department of Biomedical Sciences and ⊥School of Public Health, Chung Shan Medical University, No. 110, Sec. 1, Jianguo North Road, Taichung 40201, Taiwan # Department of Education and Research, Taichung Veterans General Hospital, 1650 Taiwan Boulevard Sect. 4, Taichung 40705, Taiwan $ School of Health, National Taichung University of Science and Technology, No. 129 Sec. 3, Sanmin Road, Taichung 404, Taiwan & Department of Health Service Administration, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan ‡
ABSTRACT: Acute lung injury (ALI) is a clinical syndrome mainly caused by Gram-negative bacteria which is still in need of an effective therapeutic medicine. EGb761, an extract of Ginkgo biloba leaves, has several bioeffects including anti-inflammation, cardioprotection, neuroprotection, and free radical scavenging. Preadministration of EGb761 inhibited lipopolysaccharide (LPS)-induced histopathological changes and exchange of arterial blood gas. In addition, LPS-induced expression of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were suppressed by EGb761. The activation of nuclear factor (NF)κB, a transcription factor of proinflammatory mediators, and phosphorylation of IκB, an inhibitor of NFκB, were also reduced by EGb761. Furthermore, we found the inhibitory concentration of EGb761 on phosphorylation of JNK and Akt was less than those of ERK and p38 MAPK. In conclusion, EGb761 is a potential protective agent for ALI, possibly via downregulating the JNK- and Akt-dependent NFκB activation pathway. KEYWORDS: EGb761, LPS, acute lung injury, MAPK, Akt, NFκB
■
INTRODUCTION Acute lung injury (ALI) may be provoked by various pathogenic factors, including severe sepsis, transfusions, pneumonia, gastric aspiration, smoke, and toxic gas inhalation.1 The incidence of ALI in United States, Scandinavia, and Australia is estimated to range from 17.9 to 78.9 cases per 100000 person years, and the mortality reaches about 32−50%.2 Thus, ALI not only is a lifethreatening critical illness but is also accompanied by high healthcare cost. Up to now, the primary treatment for ALI is still stopped at supportive care mainly focused on avoiding complications and treating the underlying diseases that may magnify the severity of ALI, and no effective therapeutic strategy has been established as yet.3 Sepsis is a major risk factor accounting for approximately 23−40% cases of ALI.4 Lipopolysaccharide (LPS) is an endotoxin which acts as a pathological determinant in sepsisrelated ALI.5 Administration of LPS into lungs causes acute inflammatory responses such as hypoxaemia, hypothermia increased pulmonary vascular permeability, pulmonary edema, neutrophil infiltration, hyaline membrane formation, and proinflammatory cytokine and chemokine generation.6 The known molecular mechanism of LPS-triggered ALI has demonstrated the participation of nuclear factor (NF)κB.7−9 Activation © 2014 American Chemical Society
of NFκB is regulated by protein kinase B (PKB/Akt) and mitogen-activated protein kinase (MAPK) pathways which include p38 MAPK, extracellular-signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNK).8−12 The standard extract of Ginkgo biloba leaves code named EGb761 is a trademark composed of 24% ginkgo flavone glycosides (containing quercetin, kaempferol, isorhamnetin, and derivatives), 6% terpenoids (containing ginkgolides and bilobalide), 5−10% organic acids, and other constituents.13 Ginkgo biloba is one of the oldest living tree species on earth and has been the most popular herb in traditional Chinese medicine for thousands of years. In traditional Chinese medicine, the seeds of Ginkgo are frequently used to ameliorate syndromes often accompanying respiratory disease, such as cough and asthma. Recently, several pieces of evidence have proposed that EGb761 possesses many beneficial biological activities and pharmacological effects, such as prevention of vascular occlusion and cochleovestibular disorders, cardioprotective properties, anticerebrovascular Received: Revised: Accepted: Published: 6337
April 23, 2014 June 17, 2014 June 23, 2014 June 23, 2014 dx.doi.org/10.1021/jf501913b | J. Agric. Food Chem. 2014, 62, 6337−6344
Journal of Agricultural and Food Chemistry
Article
Wet to Dry Weight Ratio of Lung Tissues. The lungs were dissected immediately, and the wet weight was recorded. Then the tissues were baked in an incubator at 80 °C for 24 h to obtain the dry weight. The ratio of wet weight to dry weight was calculated to evaluate the extent of lung edema. Measurement of Cytokines and Chemokines. Measurement of cytokines and chemokines, such as tumor necrosis factor (TNF)α, macrophage inflammatory protein (MIP)-2, and interleukin (IL)-6, in BALF was performed with commercially available ELISA assay kits. The quantification of cytokines and chemokines was performed according to the manufacturer’s instructions. Western Blot Analysis of Lung Tissue. As previously described, lungs were harvested, rinsed with saline, and frozen in liquid nitrogen immediately until homogenization.17 Tissue extracts were homogenized in a tissue protein extraction solution (T-PER; Pierce, Rockford, IL, USA) containing 1% proteinase inhibitor cocktail and phosphatase inhibitor cocktail. Protein samples, 100 μg each, were separated by SDSPAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) and 5% nonfat milk (w/v) for 1 h at room temperature. After they were washed with PBST, the membranes were probed with antibodies including iNOS, COX-2, β-actin, phosphory-IκB, and phosphorylated and nonphosphorylated forms of JNK, ERK, p38 MAPK, and Akt. The membranes were washed again with PBST, then a 1/10000 (v/v) dilution of horseradish peroxidase labeled IgG was added at room temperature for 1 h, and the blots were developed using ECL Western blotting reagents. NFκB Activation. The preparation of nuclear extract from lung homogenates and measurement of NFκB activation in nuclear extract were performed with a nuclear extraction kit and NFκB p65 Transcription Factor Kit (both from NEPER Nuclear and Cytoplasmic Extraction Reagents, Thermo Science), respectively, according to the manufacturer’s instructions. Statistical Analysis. Statistical analyses were performed using ANOVA followed by the Bonferroni t test for multigroup comparisons; p < 0.05 was considered significant for all tests. Data are expressed as mean ± standard deviation (SD).
disease, antidementia, anti-inflammation, antitumor, antiaging, and free radical scavenging.13,14 EGb761 has been one of the most commonly used herbal dietary remedies in many countries, including China, the United States, France, and Germany. In rats with osteoarthritis on the knee and LPS-treated chondrocytes, EGb761 was found to reduce the inflammatory effects by production of prostaglandin (PG)E2 and nitric oxide (NO), expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), and phosphorylation of NFκB.15 In murine macrophages, Ginkgo biloba extract reduces LPS-induced production of NO and PGE2 through expression of iNOS and COX-2 via NFκB phosphorylation.16 Previously, we have demonstrated that EGb761 attenuates LPS-induced ALI via inhibition of oxidative stress and the NFκB-dependent matrix metalloproteinase-9 pathway.17 In the present study, we aimed to verify the protective mechanism of EGb761 via MAPK and Akt pathways on LPS-induced ALI in an in vivo animal model.
■
MATERIALS AND METHODS
Materials. EGb761 was obtained from the laboratory of Dr. Willmar Schwabe (Karlsruhe, Germany). Antibodies against p38 MAPK, ERK, JNK, Akt, IκB, phospho-Akt (Ser473), phospho-IκB (Ser32), phosphoERK (Thr212/Tyr204), phospho-JNK (Thr183/Tyr185), COX-2, iNOS, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against phospho-p38 MAPK (Thr180/Tyr182) was purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were obtained from Jackson Immuno Research Laboratories (Baltimore, MD, USA). NFκB p65 Transcription Factor Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from Cayman (Ann Arbor, MI, USA). LPS from Escherichia coli Serotype 0111:B4, dimethyl sulfoxide (DMSO), and other reagents, unless specifically stated elsewhere, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The concluding volume of DMSO in the reaction mixture was
