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In the canonical activation pathway (Figure 2A), excitatory signaling can be mediated through Toll-like receptors (TLRs), Interleukin-1 receptor (IL-1R), tumor necrosis factor receptor (TNFR) and antigen receptors. Typical stimulating signaling molecules are tumor necrosis factor α (TNFα), lipopolysaccharides (LPS), which are bacterial cell wall components, and interleukin-1 β (IL-1β) [18, 33]. Stimulation through these receptors leads to activation of the IκB kinase (IKK) complex, which in turn phosphorylates IκBα primarily by IKK2.




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Crosstalk of the canonical NF-κB pathway with other signaling processes. (A) Many different kinases can phosphorylate and activate the IKKα and IKKβ subunits of the IKK complex or can enhance NF-κB transcriptional activity. Important examples are glycogen synthase kinase 3β (GSK3β), Protein Kinase B (PKB or Akt), Protein Kinase R (PKR), Protein Kinase C (PKC), Mitogen-Activated Type 3-Protein Kinase 7 (MAP3K7 or TAK1), p38 MAP Kinases or c-Jun N-terminal kinases (JNKs). (B) Various transcription factors such as p53, Ets Related Gene (ERG) or Signal Transducer and Activator of Transcription 3 (STAT3) can influence the transcriptional activity of NF-κB or directly activate transcription of NF-κB target genes. (C) microRNAs (miRNAs) can be target genes of the NF-κB signaling pathways or can affect the expression of NF-κB family members or effector molecules of the NF-κB activation pathway. (D) Prominent target genes of the NF-κB signaling pathway include anti-apoptotic genes as the Baculoviral IAP repeat-containing proteins (BIRCs or cIAPs) and the B-cell lymphoma 2 gene (Bcl-2), cytokines such as Interleukin-1 (IL-1), IL-6, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), adhesion factors including the Vascular Cell Adhesion Molecule 1 (VCAM-1) and the Intercellular Cell Adhesion Molecule 1 (ICAM-1). (E) Another layer of complexity of NF-κB signaling are positive and negative feedback mechanism. Examples for positive feedback molecules are the X-linked inhibitor of apoptosis protein (XIAP) as well as TNFα or IL-1. Important negative feedback circuits are generated by the NF-κB target genes IκBα, Cylindromatosis (CYLD) or A20.


Inflammation plays a crucial role in the pathogenesis of many diseases such as arthritis and atherosclerosis. In the present study, we evaluated anti-inflammatory activity of sterol-rich fraction prepared from Spirogyra sp., a freshwater green alga, in an effort to find bioactive extracts derived from natural sources.


The sterol content of ethanol extract of Spirogyra sp. (SPE) was enriched by fractionation with hexane (SPEH), resulting 6.7 times higher than SPE. Using this fraction, the in vitro and in vivo anti-inflammatory activities were evaluated in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells and zebrafish.


These results demonstrate that SPEH possesses strong in vitro and in vivo anti-inflammatory activities and has the potential to be used as healthcare or pharmaceutical material for the treatment of inflammatory diseases.


Inflammation is a highly regulated biological process that enables the immune system to efficiently eliminate stimuli and injuries (Masresha et al. 2012). Inflammation can be classified into two subtypes, chronic and acute inflammation, depending on the difference in response time and procedure. Acute inflammation is an initial response of bodies against harmful stimuli. During acute inflammatory response, the plasma and leukocytes are moving from the blood into the injured tissues. There is a shift in the type of mononuclear phagocytic cells in the inflammatory tissues during the course of inflammatory response, from pro-inflammatory to anti-inflammatory or wound healing type.


The inflammatory response is associated with the release of the inflammatory mediators, including nitric oxide (NO), prostaglandins, histamine, and bradykinin from various immune cells such as mononuclear phagocytes and mast cells (González Mosquera et al. 2011; Wijesinghe et al. 2014a). Inflammation is crucial in many diseases such as arthritis, atherosclerosis, cancer, and some deadly diseases (Lee and Weinblatt 2001; Firestein 2006; Klegeris et al. 2007; Wen et al. 2016; Wijesinghe et al. 2013; Wijesinghe et al. 2014b). Therefore, there is increasing attention on finding safe and effective anti-inflammatory agents and elucidating their anti-inflammatory mechanisms.


Algae are considered as a potential bio-resource for the development of functional food, cosmeceutical, and pharmaceutical because they are rich in various bioactive compounds, such as proteins, polysaccharides, phenolic compounds, and sterols (Kim et al. 2016; Sanjeewa et al. 2016; Fernando et al. 2017b). These compounds possess broad-spectrum bioactivities including anti-bacterial, anti-inflammatory, antioxidant, and anti-cancer activities (Jung et al. 2008; Heo et al. 2010; Lee et al. 2012; Lee et al. 2013; Oh et al. 2016; Fernando et al. 2017a).


Spirogyra sp. is freshwater green alga. It has been used as a bio-sorbent to remove heavy metal ions from wastewater (Khalaf 2008). Recently, the pharmacological activities of Spirogyra sp, such as antioxidant, ultraviolet (UV)-protective, and anti-hypertension activities, have been reported (Kang et al. 2015; Lee et al. 2016; Wang et al. 2017). However, the anti-inflammatory activities of Spirogyra sp. have not been evaluated so far. Therefore, in the present study, ethanol extract of Spirogyra sp. (SPE) and the fractions from SPE were prepared, and their anti-inflammatory activities were evaluated in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. The effect of sterol-enriched fraction (SPEH) from SPE on the production of pro-inflammatory mediators was investigated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. In addition, the in vivo anti-inflammatory effect of SPEH was evaluated using a zebrafish model.


At 3 dpf, zebrafish were dyed with DCFH2-DA (20 μg/mL), acridine orange (7 μg/mL), and DAF-FM-DA solutions (5 μM) for the detection of ROS generation, cell death, and NO production, respectively. The anesthetized zebrafish were photographed under the microscope equipped with Cool SNAP-Procolor digital camera (Olympus, Japan). The fluorescence intensity of individual zebrafish was quantified using an Image J program.


Algae-derived compounds possess various health benefits (Mayer and Hamann 2003; Athukorala and Jeon 2005; Heo et al. 2005; Kotake-Nara et al. 2005). The sterols including cholesterol, β-sitosterol, campesterol, and polyhydroxylated sterols isolated from algae were shown to strong bioactivities, especially anticancer and anti-inflammatory activities (Kazłowska et al. 2013; Elbagory et al. 2015). The freshwater green alga, Spirogyra sp. contains various bioactive compounds. In our previous studies, we have reported antioxidant, UVB photoprotective, and anti-hypertension activities of phenolic compounds isolated from Spirogyra sp. (Kang et al. 2015; Wang et al. 2017). However, the bioactivities of sterols from Spirogyra sp. have not been evaluated yet. In the present study, we have prepared a sterol-enriched fraction (SPEH) from Spirogyra sp. and evaluated its in vitro and in vivo anti-inflammatory activities.


As shown in Fig. 1, the sterol contents of SPE and its fractions were ranged from 0.50% to 9.08%. This result indicates that the sterol content in hexane fraction (SPEH) was enriched with 6.7 times than SPE. Many reports support sterols isolated from algae possess strong anti-inflammation activity (Ku et al. 2013; Sanjeewa et al. 2016; Suh et al. 2018). Our previous study investigated anti-cancer and anti-inflammatory activities of the sterol-rich fraction of Nannochloropsis oculata. The results indicated that the hexane fraction of methanol extract of N. oculata is rich in sterol content and possesses strong anti-inflammatory activity. In this regard, anti-inflammatory activity can be expected from SPEH prepared in this study, in which the content of sterol was 9.08%.


Sample preparation and anti-inflammatory activity screening. NO generation (b) and cytotoxicity (c) of SPE and its fractions on LPS-stimulated RAW 264.7 cells. NO production was measured by Griess assay and cell viability was measured by MTT assay. The data were expressed as the mean SE (n = 3). *p p p


In order to evaluate the anti-inflammatory activity of SPE and its fractions, their inhibitory effects on NO production were measured. As shown in Fig. 1, all samples significantly and dose-dependently decreased NO production (Fig. 1b). However, SPE and SPEE showed remarkable cytotoxicity in RAW264.7 cells. SPEH and SPEC that contain relative higher sterol contents showed stronger NO inhibitory effect in LPS-induced RAW264.7 cells than other samples. Furthermore, SPEH significantly improved the viability of LPS-induced RAW264.7 cells at high concentration. The viability of the cells treated with 100 μg/mL of SPEH was 99.52%, which is close to the cells not treated with LPS (100%) (Fig. 1c). These results indicate that SPEH showed the most potent anti-inflammatory activity among 5 samples. Thus, SPEH was selected as the target sample for the further study.


iNOS and COX-2 are two inducible enzymes, which synthesize two key inflammatory mediators, NO and PGE2, respectively (Kim et al. 2005; Wijesinghe et al. 2014a). The levels of iNOS and COX-2 are up-regulated in inflammatory response (Wijesinghe et al. 2014a; Wijesinghe et al. 2014b; Xiong et al. 2014). Therefore, we investigated the effect of SPEH on the protein expression levels of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. As shown in Fig. 3, LPS significantly stimulated iNOS and COX-2 expressions, but SPEH remarkably and dose-dependently downregulated the expression level of iNOS and COX-2. These results indicated that SPEH possesses strong in vitro anti-inflammatory activity. 041b061a72


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