molecules Article Anti-Inflammatory Effects of Aurantio-Obtusin from Seed of Cassia obtusifolia L. through Modulation of the NF-κB Pathway Jingyi Hou 1,2, Yu Gu 1,2, Shuai Zhao 1,2, Mengqi Huo 1,2, Shifeng Wang 1,2, Yanling Zhang 1,2,*, Yanjiang Qiao 1,2,* and Xi Li 1,2 1 Research Center of Traditional Chinese Medicine Information Engineering, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China; [email protected] (J.H); [email protected] (Y.G.); [email protected] (S.Z.); [email protected] (M.H.); [email protected](S.W.); [email protected] (X.L.) 2 Beijing Key Laboratory of Traditional Chinese Medicine Basics and New Drug Research, Research Center of Traditional Chinese Medicine Information Engineering, Beijing University of Chinese Medicine, Beijing 100102, China * Correspondence: [email protected] (Y.Z.); [email protected] (Y.Q.); Tel.: +86-010-8473-8620 (Y.Q.) Received: 5 November 2018; Accepted: 21 November 2018; Published: 27 November 2018 Abstract: Aurantio-obtusin, an anthraquinone compound, isolated from dried seeds of Cassia obtusifolia L. (syn. Senna obtusifolia; Fabaceae) and Cassia tora L. (syn. Senna tora). Although the biological activities of Semen Cassiae have been reported, the anti-inflammatory mechanism of aurantio-obtusin, its main compound, on RAW264.7 cells, remained unknown. We investigated the anti-inflammatory effect of aurantio-obtusin on lipopolysaccharide- (LPS)-induced RAW264.7 cells in vitro and elucidated the possible underlying molecular mechanisms. Nitric oxide production (NO) and prostaglandin E2 (PGE2) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Protein expression levels of cyclooxygenase 2 (COX-2) were monitored by cell-based ELISA. Interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) synthesis were analyzed using ELISA. The mRNA expression of nitric oxide synthase (iNOS), COX-2, and the critical pro-inflammatory cytokines (IL-6 and TNF-α) were detected by quantitative real-time PCR. Aurantio-obtusin significantly decreased the production of NO, PGE2, and inhibited the protein expression of COX-2, TNF-α and IL-6, which were similar to those gene expression of iNOS, COX-2, TNF-α and IL-6 (p < 0.01). Consistent with the pro-inflammatory gene expression, the Aurantio-obtusin efficiently reduced the LPS-induced activation of nuclear factor-κB in RAW264.7 cells. These results suggested that aurantio-obtusin may function as a therapeutic agent and can be considered in the further development of treatments for a variety of inflammatory diseases. Further studies may provide scientific evidence for the use of aurantio-obstusin as a new therapeutic agent for inflammation-related diseases. Keywords: inflammation; NF-κB; Semen Cassiae; anthraquinone 1. Introduction Inflammation is essential in the complex network of immunological and physiological defense processes which occur following noxious stimulation. The stimulations include infection, toxin exposure, tissue injury, or exposure to endotoxins [1–4]. Unwanted and prolonged inflammatory responses are harmful to the body and function as a potential risk and pivotal drivers, in a number of major diseases. Cancer [5], diabetes [6], rheumatoid arthritis [7], chronic inflammatory bowel Molecules 2018, 23, 3093; doi:10.3390/molecules23123093 www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 2 of 15 Molecules 2018, 23, 3093 2 of 15 responses are harmful to the body and function as a potential risk and pivotal drivers, in a number of major diseases. Cancer [5], diabetes [6], rheumatoid arthritis [7], chronic inflammatory bowel diseasedisease [8 ],[8], cardiovascular cardiovascular disease [9], [9], multiple multiple sclerosis sclerosis [10], [ 10and], psoriasis and psoriasis [11] are [11 some] are of somethe major of the majordiseases diseases that may that be may affected be affected by these byresponses. these responses. One of the key One events of the involved key events in the involvedinflammatory in the inflammatoryprocess includes process the includesexpression the of expressioninflammatory of inflammatorycytokines, chemokines, cytokines, the chemokines, signaling pathway, the signaling and pathway,other mediators and other (inducible mediators NOS) (inducible [12]. NOS) [12]. Macrophages,Macrophages, a a well-known well-known inflammatoryinflammatory and and immune immune effector effector cell cell type, type, play playcrucial crucial roles rolesin in thethe immuneimmune response, in in inflammatory inflammatory diseases, diseases, and and in in host host defense defense mechanisms, mechanisms, through through the the productionproduction of of pro-inflammatory pro-inflammatory cytokines cytokines andand inflammatoryinflammatory mediators mediators [13]. [13]. Lipopolysaccharide Lipopolysaccharide (LPS),(LPS), one one of of the the most most powerful powerful activators activators ofof macrophages,macrophages, activate activate these these cells cells via via the the release release of of inflammatoryinflammatory mediators, mediators, cytokinescytokines and and chemotactic chemotactic factors. factors. Toll-like Toll-like receptor receptor 4 (TLR4), 4 (TLR4), one of onethe of theLPS LPS recognition recognition receptor, receptor, which which triggers triggers downstream downstream of nuclear of nuclearfactor kappaB factor (NF-κB) kappaB [14]. (NF- NF-κB,κB) [14 ]. NF-aκ B,five-membered a five-membered collection collection of transcription of transcription factors, factors, is involved is involved in physiological in physiological and and patho- patho- physiologicalphysiological responses responses [15 [15].]. In In addition, addition, thisthis proteinprotein regulates the the expression expression of of genes genes and and cytokines cytokines whichwhich play play crucial crucial roles roles in in apotheosis, apotheosis, inflammation,inflammation, proliferation, proliferation, and and calcium calcium homeostasis homeostasis [16–18]. [16–18 ]. NF-NF-κBκB p50/p65, p50/p65, two two subunits subunits ofof NF-NF-κBκB[ [19],19], once encountered encountered inflammatory inflammatory stimuli stimuli such such as LPS, as LPS, whichwhich increase increase the the nuclear nuclear p65 p65 protein, protein, decreasesdecreases cytosolic I-κB I-κB protein, protein, promotes promotes the the expression expression of of its downstream pro-inflammatory mediators and cytokines (i.e., inducible NOS, cyclooxygenase-2 its downstream pro-inflammatory mediators and cytokines (i.e., inducible NOS, cyclooxygenase-2 (COX-2), moocyte chemoattractant protein 1(MCP-1), IL-6, IL-1β, tumor necrosis factor—α (TNF-α) (COX-2), moocyte chemoattractant protein 1(MCP-1), IL-6, IL-1β, tumor necrosis factor—α (TNF-α) and others [19,20]), and eventually induces an inflammatory response [21]. Therefore, the NF-κB and others [19,20]), and eventually induces an inflammatory response [21]. Therefore, the NF-κB pathway is emerging as potential therapeutic targets for the remedy the inflammatory diseases [22]. pathway is emerging as potential therapeutic targets for the remedy the inflammatory diseases [22]. OverOver the the past past century, century, natural natural products, products, especiallyespecially anthraquinone compounds, compounds, have have become become valuablevaluable products products for achievingfor achieving chemical chemical diversity diversity in the in moleculesthe molecules used used for inflammation for inflammationrelief relief [23,24 ]. 1,3,7-Trihydroxy-2,8-dimethoxy-6-methyl-9,10-anthracenedione[23,24]. 1,3,7-Trihydroxy-2,8-dimethoxy-6-methyl-9,10-anthracenedione (aurantio-obtusin, (aurantio-obtusin, Figure Figure1), 1), is a reportedis a reported anthraquinone anthraquinone derivative, derivative, and and is theis the major major bioactive bioactive compoundcompound obtained obtained from from the the dried dried seedsseeds of Cassiaof Cassia obtusifolia obtusifoliaL. L (syn.. (syn.Senna Senna obtusifolia obtusifolia; Fabaceae); Fabaceae) and andCassia Cassia tora toraL. L. (syn. (syn. SennaSenna toratora)[) [25–25–27] 27] as well as a phytochemical marker of quality control in the Chinese Pharmacopeia (Version 2015) as well as a phytochemical marker of quality control in the Chinese Pharmacopeia (Version 2015) [28]. [28]. FigureFigure 1. 1.The The chemicalchemical structure of of aurantio-obtusin. aurantio-obtusin. AccumulatedAccumulated evidences evidences havehave shownshown thatthat aurantio-obstusin exhibits exhibits a anumber number of ofbiological biological propertiesproperties including including anti-oxidative, anti-oxidative, anti-hypertension, anti-hypertension, anti-mutagenic, anti-mutagenic, anti-genotoxic, anti-genotoxic, anti-allergic anti-allergic and neuroprotectiveand neuroprotective effects effects [3,29– 32[3,29–32].]. Semen Semen Cassiae Cassiaeis widely is widely used used in Chinese in Chinese folk folk medicine medicine and and has has been demonstratedbeen demonstrated to exhibit to exhibit significant significant anti-inflammatory anti-inflammatory effects effects [33,34 [33,34].]. Whether Whether aurantio-obtusin aurantio-obtusin plays a pivotalplays a role pivotal in the role anti-inflammatory in the anti-inflammatory effects exhibited effects exhibited by Semen by Cassiae Semen, Cassiae remains, remains unclear. unclear. Likewise, theLikewise, underlying the intracellularunderlying intracellular mechanisms mechanisms whereby aurantio-obtusin whereby aurantio-obtusin exhibit its anti-inflammatoryexhibit