ABSTRACT
Aster yomena (Kitam.) Honda has been widely used as a traditional herbal medicine for centuries to treat cough, asthma, insect bites, etc. Recent reports indicate that A. yomena possesses a wide spectrum of pharmacological activities; however, few experiments have described its anti-inflammatory properties. The present study examined the anti-inflammatory effects of an ethanol extract of A. yomena leaves (EEAY) on lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. Treatment with EEAY significantly reduced the secretion of pro-inflammatory molecules, such as nitric oxide and interleukin-1ß, in LPS-stimulated RAW 264.7 cells, without incurring any significant cytotoxicity. These protective effects were accompanied by a marked reduction in the expression of regulatory genes at the transcription level. Treatment with EEAY also inhibited the DNA-binding activity of nuclear factor-κB (NF-κB) by suppression of nuclear translocation of NF-κB and by degradation of the inhibitor of NF-κB; these effects were associated with suppression of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. The EEAY treatment also potently suppressed LPS-induced toll like receptor (TLR) 4 expression and attenuated the binding of LPS to the macrophage cell surface. In addition, EEAY treatment markedly inhibited LPS-induced accumulation of intracellular reactive oxygen species in RAW 264.7 macrophages. Therefore, the inhibitory effects of EEAY on LPS-stimulated inflammatory responses in RAW 264.7 macrophages were apparently associated with suppression of the TLR-mediated NF-κB signaling pathway. More work is needed to fully understand the critical role and clinical usefulness of EEAY treatment, but the findings of the present study provide some insights into the potential of EEAY as a therapeutic agent for treatment of inflammatory disorders.
Subject(s)
Asteraceae/chemistry , Ethanol/chemistry , Inflammation/pathology , Macrophages/pathology , Plant Extracts/pharmacology , Animals , Cell Death/drug effects , Cell Survival/drug effects , Enzyme Activation/drug effects , Interleukin-1beta/metabolism , Lipopolysaccharides , MAP Kinase Signaling System/drug effects , Macrophages/drug effects , Mice , NF-kappa B/metabolism , Nitric Oxide/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Plant Leaves/chemistry , Proto-Oncogene Proteins c-akt/metabolism , RAW 264.7 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
The Kalopanax septemlobus leaf (Thunb.) Koidz. has been used as a traditional medicine herb for the treatment of various human diseases for hundreds of years. In this study, we investigated the mechanism underlying the inhibitory effects of an ethanol extract of K. septemlobus leaf (EEKS) on proliferation of HepG2 hepatocellular carcinoma cells. For this study, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DAPI (4,6-diamidino-2-phenylindole) staining, agarose gel electrophoresis, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether HepG2 cell death occurred by apoptosis. Treatment of HepG2 cells with EEKS concentration-dependently reduced cell survival while significantly increasing the ratio of apoptotic cells. EEKS treatment increased the levels of the death receptors (DRs), DR4 and DR5, and activated caspases, as well as promoting proteolytic degradation of poly(ADP-ribose)-polymerase associated with the downregulation of protein expression of members of the inhibitor of apoptosis protein family. Treatment with EEKS also caused truncation of Bid, translocation of pro-apoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. However, treatment of HepG2 cells with a pan-caspase inhibitor reversed EEKS-induced apoptosis and growth suppression, indicating that EEKS appears to induce apoptosis though a caspase-dependent mechanism involving both intrinsic and extrinsic apoptotic pathways. In addition, the phosphorylation level of AMP-activated protein kinase (AMPK) was elevated when cells were exposed to EEKS. A specific inhibitor for AMPK attenuated the EEKS-induced activation of caspases, and consequently prevented the EEKS-induced apoptosis and reduction in cell viability. Overall, our findings suggest that EEKS inhibits the growth of HepG2 cells by inducing AMPK-mediated caspase-dependent apoptosis, suggesting the potential therapeutic application of EEKS in the treatment or prevention of cancers.
