Journal of Ethnopharmacology 133 (2011) 687–695 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-B and p38 MAPK signaling and protects mice from lethal endotoxemia Jin-Young Kim a , Hyun Jung Kim a , Sung-Moo Kim a , Kyung-Ran Park a , Hyeung-Jin Jang b , Eun Ha Lee c , Sang Hoon Jung c , Kwang Seok Ahn a,∗ a Department of Oriental Pathology, College of Oriental Medicine and Institute of Oriental Medicine, Kyung Hee University, 1 Hoegidong Dongdaemungu, Seoul 130-701, Republic of Korea b Department of Biochemistry, College of Oriental Medicine and Institute of Oriental Medicine, Kyung Hee University, 1 Hoegidong Dongdaemungu, Seoul 130-701, Republic of Korea c Natural Products Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung 210-340, Republic of Korea article info Article history: Received 2 June 2010 Received in revised form 7 October 2010 Accepted 23 October 2010 Available online 30 October 2010 Keywords: Thuja orientalis NF-B MAPKs Nitric oxide Cyclooxygenase-2 Endotoxemia abstract Aim of the study: Thuja orientalis (TO) has been a recognized herbal medicine across Northeast Asian countries for thousands of years and used for the treatment of various inflammatory diseases through as yet undefined mechanisms. In this study, we set out to determine whether the anti-inflammatory effects of this plant are mediated to suppress mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-B) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Materials and methods: RAW 264.7 cells were pretreated with the methylene chloride fraction of TO (MTO) and stimulated with LPS. Nitric oxide (NO) release was determined by the accumulation of nitrite in the culture supernatants and tumor necrosis factor- (TNF-) and IL-6 secretion were determined by immunoenzymatic assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expres- sion were evaluated via RT-PCR and Western blotting. NF-B activation was also evaluated by reporter gene assay and electrophoretic mobility shift assay (EMSA). In addition, the protective effect of MTO was evaluated by use of the LPS-induced endotoxin shock model in mice. Results: We found that MTO significantly suppressed LPS-stimulated NO and IL-6 production without affecting cell viability. MTO inhibited the expression of LPS-induced iNOS and COX-2 protein and their mRNA expression. Also, TNF- and IL-6 secretion were decreased by MTO in both PMA and ionomycin- stimulated splenocytes. As a result, MTO inhibited pro-inflammatory cytokines such as TNF- and IL-6, which is hypothesized as being due to the suppression of LPS-induced p38 MAPK and NF-B activation. Moreover, MTO improved the survival rate during lethal endotoxemia by inhibiting the production of TNF-  in an animal model and our LC–MS analysis showed that a major component of MTO was pinusolide. Conclusions: We demonstrate here the evidence that the methylene chloride fraction of Thuja orientalis (MTO) potentially inhibits the biomarkers related to inflammation in vitro and in vivo, and might be provided as a potential candidate for the treatment of inflammatory diseases. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Thuja orientalis L. (also known as Platycladus orientalis L.) is a large, dense and compact shrub tree belonging to the Cupressaceae family. It has traditionally been used in the treatment of vari- ous inflammatory diseases (Tang et al., 1999). The plant exhibits a broad variety of biological activities: anti-plasmodial (Asili et al., 2004), platelet activating factor (PAF) receptor binding antagonistic (Yang et al., 1995), improvements for impaired memory acqui- ∗ Corresponding author. Tel.: +82 2 961 2316; fax: +82 2 961 2316. E-mail address: ksahn@khu.ac.kr (K.S. Ahn). sition (Nishiyama et al., 1995), hypouricemic effect (Zhu et al., 2004), fungitoxic (Guleria et al., 2008), molluscicidal (Singh and Singh, 2009), and anti-neurotoxic activities (Ju et al., 2010). In addi- tion, Choi et al. showed that 15-methoxypinusolidic acid from the plant exhibits anti-inflammatory activity in microglial cells (Choi et al., 2008) and Morishige et al. reported that juniperonic acid, a polymethylene-interrupted (PMI) fatty acid which occurs in the plant, has an anti-proliferative activity on the bombesin-induced proliferation of Swiss 3T3 cells (Morishige et al., 2008). However, exactly how the plant mediates these anti-inflammatory effects is not yet completely understood. NF-B is the most pro-inflammatory transcription factor, con- sisting of p50, p65, and IB, that resides in the cytoplasm; and its activation is involved in iNOS, COX-2, and TNF- expression 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.10.051