Anti-Inflammatory Activity of Diterpenoids from Celastrus orbiculatus in Lipopolysaccharide-Stimulated RAW264.7 Cells

Celastrus orbiculatus Thunb has been known as an ethnopharmacological medicinal plant for antitumor, anti-inflammatory, and analgesic effects. Although various pharmacological studies of C. orbiculatus extract has been reported, an anti-inflammatory mechanism study of their phytochemical constituents has not been fully elucidated. In this study, compounds 1–17, including undescribed podocarpane-type trinorditerpenoid (3), were purified from C. orbiculatus and their chemical structure were determined by high-resolution electrospray ionization mass (HRESIMS) and nuclear magnetic resonance (NMR) spectroscopic data. To investigate the anti-inflammatory activity of compounds 1–17, nitric oxide (NO) secretion was evaluated in LPS-treated murine macrophages, RAW264.7 cells. Among compounds 1–17, deoxynimbidiol (1) and new trinorditerpenoid (3) showed the most potent inhibitory effects (IC50: 4.9 and 12.6 μM, respectively) on lipopolysaccharide- (LPS-) stimulated NO releases as well as proinflammatory mediators, such as inducible nitric oxide (iNOS), cyclooxygenase- (COX-) 2, interleukin- (IL-) 1β, IL-6, and tumor necrosis factor- (TNF-) α. Its inhibitory activity of proinflammatory mediators is contributed by suppressing the activation of nuclear transcription factor- (NF-) κB and mitogen-activated protein kinase (MAPK) signaling cascades including p65, inhibition of NF-κB (IκB), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. Therefore, these results demonstrated that diterpenoids 1 and 3 obtained from C. orbiculatus may be considered a potential candidate for the treatment of inflammatory diseases.

The major function of the inflammation is to defend the host from infectious pathogens and repair tissue injury through the action of leukocytes including macrophages, neutrophils, and lymphocytes [21,22]. However, immoderate or prolonged inflammation contribute to the development of chronic inflammation diseases such as arthritis, asthma, Crohn's, and inflammatory bowel disease (IBD), resulting in swelling, pain, and eventually damage of tissue or organ dysfunction [23,24]. Macrophage activated by antigen, pathogens, and endogenous inflammatory stimuli is associated with functional and physiological changes in the cells and generates proinflammatory and cytotoxic mediators such as nitric oxide (NO), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), IL-6, reactive oxygen mediators, and hydrolytic enzymes [25,26]. Excessive NO and inflammatory cytokines released from macrophages are implicated in cytotoxicity by initiating both apoptosis and necrosis of normal tissues as well as destruction of tumor cells and exogenous pathogens [27,28]. Thus, blocking these inflammatory mediators is considered to be effective for prevention of inflammation diseases.
Binding of these inflammatory mediators or bacterial lipopolysaccharide (LPS) to specific receptors including Tolllike receptors (TLRs) lead to the inflammatory responses, through the transmembrane signal transduction and intracellular responses such as nuclear transcription factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) [29,30]. The activation of NF-κB is involved in the phosphorylation of IκB, resulting in the release of NF-κB into the nucleus, which functions as a transcription factor for expressing proinflammatory target genes including inducible nitric oxide synthesis (iNOS), cyclooxygenase 2 (COX-2), TNF-α, IL-1β, and IL-6 [31]. Extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38 kinase are generally known as subfamilies of MAPKs, and this phosphorylation involved in NF-κB activation modulates proinflammation mediators, such as iNOS and COX-2 in activated macrophages [23,32,33]. Therefore, the development of natural sources targeting the NF-κB and MAPK cascades can be a potential therapeutic for inflammatory diseases.
In current study, the chemical structures of phytochemical constituents (1-17) isolated from C. orbiculatus were determined by spectroscopic data including NMR and ESI-MS. Among components obtained from C. orbiculatus, compounds 1 and 3, both of which are podocarpane trinorditerpenoids, exhibited most potent inhibitory activity against LPS-treated NO release, and their anti-inflammatory activity was explored through underlying molecular mechanisms including NF-κB and MAPK signaling pathway.  13 C-, and 2D NMR spectroscopic data were measured on a JEOL JNM-ECA600 or JEOL JNM-EX400 instrument (JEOL, Tokyo, Japan) using TMS as a reference. Optical rotation was recorded on a JASCO P-2000 polarimeter (Jasco Co., Tokyo, Japan). UV spectrum was obtained using SpectraMax M 2 e spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). IR data were acquired using a Spectrum Jas.co FT/IR-4600 spectrometers (Jasco Corp., Tokyo, Japan). HRESIMS data were obtained using a Waters SYNAPT G2-Si HDMS spectrometer (Waters, Milford, MA, USA).
2.6. Immunoblot Analysis. The whole cell lysate were extracted using a Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA, USA). Immunoblot analysis was performed using a previously described method [34]. After transfer to nitrocellulose (NC) membrane, the blocking membrane with 5% skimmed milk powder was incubated overnight at 4°C with primary antibody, including anti-phospho-JNK (

2.7.
Real-Time PCR Using TaqMan Probe. Total RNA was extracted from RAW264.7 cells using the TaKaRa MiniBEST Universal RNA Extraction Kit following the manufacturer's instructions (Takara Bio Inc., Japan). The complementary DNA (cDNA) was synthesized from 1 μg of the total RNA using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio Inc. Japan). Quantitative real-time PCR (qPCR) of IL-1bβ (Mm00434228_m1), IL-6 (Mm00446190_m1), and TNF (Mm00443258_m1) was performed with a TaqMan Gene Expression Assay Kit (Thermo Fisher Scientific, San Jose, CA, USA). To normalize the gene expression, an 18S rRNA endogenous control (Applied Biosystems, Foster City, CA, USA) was used. The qPCR was employed to verify the mRNA expression using a StepOnePlus Real-Time PCR System. To quantify mRNA expression, TaqMan mRNA assay was performed according to the manufacturer's protocol (Applied Biosystems). PCR amplification was analyzed using the comparative ΔΔCT method.

Statistical Analysis.
Half-maximal inhibitory concentration (IC 50 ) values expressed as 95% confidence intervals were calculated by nonlinear regression analysis using GraphPad Prism 5 software (GraphPad software, San Diego, CA, USA). Each experiment, including immunoblot and realtime PCR, was performed independently three times, and these data represent the mean ± SEM. The statistical significance of each value was measured by the unpaired Student t-test. * p < 0:05, * * p < 0:01, and * * * p < 0:001 were considered significant.
In maintenance of homeostasis from various organs systems, NO has been recognized as one of the important biological mediator involved in the various pathophysiological and physiological mechanisms, such as neurotransmitters, host defense against pathogenic microorganism, and regulation of immune systems [50]. However, the overproduction of NO in intracellular levels is associated to inflammatory diseases and carcinogenesis, and measurement of NO content has been employed by various literatures on the anti-inflammatory properties of phytochemicals derived from natural products [51]. To investigate whether NO production stimulated by LPS was inhibited by phytochemicals isolated from C. orbiculatus, compounds 1-17 were tested by NO assay in the RAW264.7 cells. As shown in Table 2, 1-4, 11, and 12 showed potent inhibitory activity against LPStreated NO secretion based on 50% inhibitory effect at 50 μM concentration compared to only LPS-treated control group (IC 50 : 4.9-40.0 μM) (Fig. S17), and all isolates did not affect cytotoxicity at IC 50 concentration, respectively (Fig.  S18). Among isolates showing NO inhibitory effect, 1 and 3, which are podocarpane-type trinorditerpenoid class, were selected to evaluate further anti-inflammatory activity at 5 or 10 μM concentrations, respectively, which are approximately IC 50 values without cytotoxicity effect by compounds.
iNOS is a major downstream mediator of inflammation in several cell types including macrophage cells [52]. During the course of an inflammatory response, large amount of NO formed by the action of iNOS surpass the physiological amounts of NO [53], and consequentially, iNOS overproduction reflects the degree of inflammation [54,55]. COX-2 is an inducible enzyme that has a role in the development of epithelial cell dysplasia, carcinoma, wound edge of tissue, and inflammatory diseases such as arthritis, allergic asthma, and atopic dermatitis [56][57][58]. The expression of COX-2 is a key mediator of inflammatory pathway, which is representatively the NF-κB signaling pathway [59,60].
In order to examine the biological evidence of effectively reduced NO production after treatment with 1 and 3, we performed the immunoblot analysis to investigate whether 1 and 3 suppressed the upregulation of iNOS and COX-2 protein expression after LPS-activated inflammation condition. As shown in Figure 3, 1 and 3 dose dependently inhibited iNOS and COX-2 protein expression on LPS-induced inflammation in RAW264.7 cells. In addition, a comparison of nitric oxide production between compound 1, 3, and celastrol was exhibited (Fig. S19).
Each protein expression level was represented as relative ratio values of iNOS/β-actin and COX-2/β-actin (Figures 3(c) O COSY HMBC  Dexamethasone or nonsteroidal anti-inflammatory drugs (NSAIDs) [61] are well known for blocking the MAPKs and NF-κB signaling cascades and results in potent antiinflammatory activity through the reduction of proinflammatory mediators such as iNOS and COX-2. MAPK (JNK, ERK,   Journal of Immunology Research and p38) and NF-κB are crucial intracellular signaling pathways leading to the inflammatory response. These biological response are mediated by their transcription factors, such as activator protein-(AP-) 1, cAMP response element-binding protein (CREB), and NF-κB, which are phosphorylated and activated in the cytoplasmic or nuclear, resulting in an inflammatory action via the expression of target genes including proinflammatory cytokines IL-1β, IL-6, and TNF-α as well as iNOS and COX-2 proteins [62][63][64].
To further investigate anti-inflammatory effects associated with inhibition of NO production, iNOS, and COX-2, major inflammatory signaling cascades, MAPKs (JNK, ERK, and p38), and NF-κB, were evaluated with treatment of 1 or 3 in LPS-induced murine macrophages. As shown in Figures 4(a) The continuous overexpression of proinflammatory cytokines, IL-1β, IL-6, and TNF-α, is characterized as chronic inflammatory pathogenesis, which results in cell and tissue degeneration [63,65], such as rheumatoid arthritis and inflammatory bowel diseases. Thus, following the hypothesis that these proinflammatory cytokines may be inhibited by 1 and 3, we performed real-time PCR experiments to evaluate the inhibitory effect of IL-1β, IL-6, and TNF-α levels. In accordance with our hypothesis, 1 and 3 revealed a reduction in LPS-induced IL-1β, IL-6, and TNF-α gene expression at mRNA transcription levels ( Figure 6). All taken together, these results indicated that the anti-inflammation activity of 1 and 3 was attributed Values are means ± SEM, and an unpaired Student t-test was used for statistical analysis. # p < 0:05, ## p < 0:01, ### p < 0:001, * p < 0:05, * * p < 0:01, and * * * p < 0:001 represented significant differences from the LPS-treated group. 8 Journal of Immunology Research to blockade of the MAPK and NF-κB signaling pathways via the suppression of p-ERK, p-JNK, p-p38, p-IκB, and p-p65 ( Figure 6(d)).