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In vitro and in vivo anti-inflammatory activities of a sterol-enriched fraction from freshwater green alga, Spirogyra sp.

In vitro and in vivo anti-inflammatory activities of a sterol-enriched fraction from freshwater... Background: 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. Methods: 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. Results: SPEH effectively and dose-dependently decreased the production of nitric oxide (NO) and prostaglandin E (PGE ). SPEH suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β through downregulating nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells without cytotoxicity. The in vivo test results indicated that SPEH significantly and dose-dependently reduced reactive oxygen species (ROS) generation, cell death, and NO production in LPS-stimulated zebrafish. Conclusions: 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. Keywords: Spirogyra sp., Sterol-enriched fraction, Anti-inflammatory activity, RAW 264.7 cells, Zebrafish Background inflammatory tissues during the course of inflammatory Inflammation is a highly regulated biological process response, from pro-inflammatory to anti-inflammatory that enables the immune system to efficiently eliminate or wound healing type. stimuli and injuries (Masresha et al. 2012). Inflammation The inflammatory response is associated with the re- can be classified into two subtypes, chronic and acute in- lease of the inflammatory mediators, including nitric flammation, depending on the difference in response oxide (NO), prostaglandins, histamine, and bradykinin time and procedure. Acute inflammation is an initial re- from various immune cells such as mononuclear phago- sponse of bodies against harmful stimuli. During acute cytes and mast cells (González Mosquera et al. 2011; inflammatory response, the plasma and leukocytes are Wijesinghe et al. 2014a). Inflammation is crucial in many moving from the blood into the injured tissues. There is diseases such as arthritis, atherosclerosis, cancer, and a shift in the type of mononuclear phagocytic cells in the some deadly diseases (Lee and Weinblatt 2001; Firestein 2006; Klegeris et al. 2007; Wen et al. 2016; Wijesinghe * Correspondence: jikim@pknu.ac.kr et al. 2013; Wijesinghe et al. 2014b). Therefore, there is Department of Food Science and Nutrition, Pukyong National University, increasing attention on finding safe and effective anti- Busan 48513, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 2 of 9 inflammatory agents and elucidating their anti- in the present study, ethanol extract of Spirogyra sp. (SPE) inflammatory mechanisms. and the fractions from SPE were prepared, and their anti- Algae are considered as a potential bio-resource for inflammatory activities were evaluated in lipopolysaccha- the development of functional food, cosmeceutical, and rides (LPS)-stimulated RAW264.7 cells. The effect of pharmaceutical because they are rich in various bioactive sterol-enriched fraction (SPEH) from SPE on the produc- compounds, such as proteins, polysaccharides, phenolic tion of pro-inflammatory mediators was investigated by compounds, and sterols (Kim et al. 2016; Sanjeewa et al. enzyme-linked immunosorbent assay (ELISA) and western 2016; Fernando et al. 2017b). These compounds possess blot analysis. In addition, the in vivo anti-inflammatory ef- broad-spectrum bioactivities including anti-bacterial, fect of SPEH was evaluated using a zebrafish model. anti-inflammatory, antioxidant, and anti-cancer activities (Jung et al. 2008; Heo et al. 2010; Lee et al. 2012; Lee Methods et al. 2013; Oh et al. 2016; Fernando et al. 2017a). Chemicals and regents Spirogyra sp. is freshwater green alga. It has been used LPS, acridine orange, 2′,7′-dichlorofluorescin diacetate as a bio-sorbent to remove heavy metal ions from waste- (DCFH -DA), dimethyl sulfoxide (DMSO), and water (Khalaf 2008). Recently, the pharmacological activ- diaminofluorescein-FM diacetate (DAF-FM DA) were ities of Spirogyra sp, such as antioxidant, ultraviolet purchased from Sigma-Aldrich (St. Louis, MO, USA). (UV)-protective, and anti-hypertension activities, have Fetal bovine serum (FBS), penicillin–streptomycin (P/S), been reported (Kang et al. 2015; Lee et al. 2016; Wang and Dulbecco’s modified Eagle’s medium (DMEM) were et al. 2017). However, the anti-inflammatory activities of purchased from Gibco/BRL (Grand Island, NY, USA). Spirogyra sp. have not been evaluated so far. Therefore, The ELISA kits for measuring prostaglandin E (PGE ), 2 2 Fig. 1 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 < 0.05, p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 3 of 9 interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), were obtained. Samples were concentrated and stored at and IL-1β levels were purchased from R&D Systems Inc. – 20 °C until used. (Minneapolis, MN, USA). Antibodies against β-actin, cyclooxygenase-2 (COX-2), and inducible nitric oxide Sterol content analysis synthase (iNOS) were purchased from Santa Cruz Bio- The sterol contents of samples were measured by Lie- technology (Santa Cruz, CA, USA). Secondary anti- bermann Burchard method described by Xiong et al. rabbit and anti-mouse IgG antibodies were purchased (2007). In brief, the samples (2 mg) were dissolved in from Cell Signaling Technology (Beverly, MA, USA). glacial acetic acid (1 mL) and mixed with prepared Lie- Other chemicals and regents used in the present study bermann Burchard regent. The mixture was reacted in were analytical grade. the dark at the room temperature for 10 min. After reac- tion, the absorbance of the mixture was recorded at 625 nm using a micro-plate reader (BioTek, Synergy, HT, Extraction and fractionation USA). Cholesterol was used as standard sterol com- Sampling, processing, extraction, and fractionation were pound to prepare the standard curve. performed following the protocols described in the pre- vious study (Wang et al. 2017). In brief, lyophilized Cell culture Spirogyra sp. powder was extracted by 70% ethanol; the RAW 264.7 cells (ATCC® TIB-71™) were cultured in 70% ethanol extract of Spirogyra sp. (SPE) was fraction- DMEM containing FBS (10%) and P/S (1%) under a hu- ated by hexane, chloroform, and ethyl acetate, respect- midified atmosphere containing 5% CO at 37 °C. RAW ively. Then, the hexane (SPEH), chloroform (SPEC), 264.7 cells were seeded as a density of 1 × 10 cells/mL ethyl acetate (SPEE), and water (SPEW) fractions of SPE for experiments. Fig. 2 Inhibitory effect of SPEH on PGE (a), TNF-α (b), IL-1β (c), and IL-6 (d) production in LPS-induced RAW 264.7 cells. The PGE and pro- 2 2 inflammatory cytokines levels were determined by ELISA kits according to the manufacturer’s instructions. The data were expressed as the mean * ** ## ±SE(n = 3). p < 0.05, p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 4 of 9 Determination of NO, pro-inflammatory cytokines, PGE with the primary antibodies at 4 °C. After 6 h incuba- production, and cell viability tion, the membranes were incubated with HRP- RAW 264.7 cells were seeded and incubated for 24 h. conjugated secondary antibody and visualized using ECL Cells were treated with different concentrations of sam- reagent (Amersham, Arlington Heights, IL, USA). ples and incubated for 1 h. After incubation, LPS (1 μg/ mL) was introduced to cells. The culture medium was Measurement of survival rate and heart-beating rate on collected and used for measuring NO, PGE , TNF-α, IL- LPS-stimulated zebrafish 6, and IL-1β production levels. The NO production Approximately 7–9 h post-fertilization (hpf), zebrafish levels of LPS-treated RAW264.7 cells were measured embryos (15 embryos/group) were treated with different using Griess reagent. The PGE , TNF-α, IL-6, and IL-1β concentrations of SPEH for 1 h. Then, LPS (10 μg/mL) production levels were evaluated using the commercial was added into the embryo medium. The survival rate ELISA kits following the manufacturer’s instructions. was measured at 3 days post-fertilization (dpf) by count- The cell viability was measured by MTT assay based on ing live embryos, and heart-beating rate was measured the method described by Wang et al. (2018). following the protocol described by Sanjeewa et al. (2018). Western blot analysis The LPS-stimulated RAW 264.7 cells were harvested Measurement of reactive oxygens species (ROS) and lysed. The lysates contain 50 μg of protein were sub- generation, cell death, and NO production on LPS- jected to electrophoresis on SDS–polyacrylamide gels stimulated zebrafish (12%). Then, the proteins were transferred onto nitrocel- At 3 dpf, zebrafish were dyed with DCFH -DA (20 μg/ lulose membranes, and the membranes were incubated mL), acridine orange (7 μg/mL), and DAF-FM-DA Fig. 3 Inhibitory effects of SPEH on iNOS and COX-2 expression in LPS-induced RAW 264.7 cells. a iNOS and COX-2 expression in LPS-induced RAW 264.7 cells. b The relative amount of iNOS and COX-2. Relative amounts of iNOS and COX-2 expressions were compared to β-actin. The data ** ## were expressed as the mean ± SE (n = 3). p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 5 of 9 solutions (5 μM) for the detection of ROS generation, sterols including cholesterol, β-sitosterol, campesterol, cell death, and NO production, respectively. The anes- and polyhydroxylated sterols isolated from algae were thetized zebrafish were photographed under the micro- shown to strong bioactivities, especially anticancer and scope equipped with Cool SNAP-Procolor digital camera anti-inflammatory activities (Kazłowska et al. 2013; Elba- (Olympus, Japan). The fluorescence intensity of individ- gory et al. 2015). The freshwater green alga, Spirogyra ual zebrafish was quantified using an Image J program. sp. contains various bioactive compounds. In our previ- ous studies, we have reported antioxidant, UVB photo- Statistical analysis protective, and anti-hypertension activities of phenolic The data were expressed as the mean ± standard error compounds isolated from Spirogyra sp. (Kang et al. (SE). One-way ANOVA test (using SPSS 11.5 statistical 2015; Wang et al. 2017). However, the bioactivities of software) was used to compare the mean values of each sterols from Spirogyra sp. have not been evaluated yet. treatment. The significant differences were established In the present study, we have prepared a sterol-enriched as *p < 0.05, **p < 0.01 as compared to LPS only-treated fraction (SPEH) from Spirogyra sp. and evaluated its ## group and p < 0.01 as compared to control group. in vitro and in vivo anti-inflammatory activities. As shown in Fig. 1, the sterol contents of SPE and its Results and discussion fractions were ranged from 0.50% to 9.08%. This result Sterol content of SPE and its fractions indicates that the sterol content in hexane fraction Algae-derived compounds possess various health bene- (SPEH) was enriched with 6.7 times than SPE. Many re- fits (Mayer and Hamann 2003; Athukorala and Jeon ports support sterols isolated from algae possess strong 2005; Heo et al. 2005; Kotake-Nara et al. 2005). The anti-inflammation activity (Ku et al. 2013; Sanjeewa Fig. 4 SPEH improved the survival rate and reduced heart-beating rate in LPS-stimulated zebrafish. a The survival rate. b the heart-beating rate. * ** ## The data were expressed as the mean ± SE (n = 3). p < 0.05, and p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group. Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 6 of 9 et al. 2016; Suh et al. 2018). Our previous study investi- treated with 100 μg/mL of SPEH was 99.52%, which is gated anti-cancer and anti-inflammatory activities of the close to the cells not treated with LPS (100%) (Fig. 1c). sterol-rich fraction of Nannochloropsis oculata. The re- These results indicate that SPEH showed the most po- sults indicated that the hexane fraction of methanol ex- tent anti-inflammatory activity among 5 samples. Thus, tract of N. oculata is rich in sterol content and possesses SPEH was selected as the target sample for the further strong anti-inflammatory activity. In this regard, anti- study. inflammatory activity can be expected from SPEH pre- We next examined the inhibitory effects of SPEH on pared in this study, in which the content of sterol was the production of PGE and pro-inflammatory cytokines 9.08%. in LPS-stimulated RAW264.7 cells. As Fig. 2a shows, LPS induced PGE level in RAW264.7 cells to 319.85% In vitro anti-inflammatory effect of SPEH compared to the control group (100%). However, the In order to evaluate the anti-inflammatory activity of PGE levels of LPS-treated cells were decreased to SPE and its fractions, their inhibitory effects on NO pro- 307.83%, 301.38%, and 278.90% by 25, 50, and 100 μg/ duction were measured. As shown in Fig. 1, all samples mL of SPEH treatment, respectively. In addition, SPEH significantly and dose-dependently decreased NO pro- inhibited 17.70%, 33.01%, and 65.76% of TNF-α (Fig. duction (Fig. 1b). However, SPE and SPEE showed re- 2b), 113.31%, 173.91%, and 179.67% of IL-1β (Fig. 2c), markable cytotoxicity in RAW264.7 cells. SPEH and 50.233%, 91.22%, and 252.14% of IL-6 expressions (Fig. SPEC that contain relative higher sterol contents showed 2d) at the concentration of 25, 50, and 100 μg/mL, stronger NO inhibitory effect in LPS-induced RAW264.7 respectively. cells than other samples. Furthermore, SPEH signifi- iNOS and COX-2 are two inducible enzymes, which cantly improved the viability of LPS-induced RAW264.7 synthesize two key inflammatory mediators, NO and cells at high concentration. The viability of the cells PGE , respectively (Kim et al. 2005; Wijesinghe et al. Fig. 5 The protective effect of SPEH against LPS-induced ROS generation in zebrafish. a Zebrafish under fluorescence microscope. b The levels of ** ROS. ROS levels were measured using Image J software. The data were expressed as the mean ± SE (n = 3). p < 0.01 as compared to LPS only- ## treated group and p<0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 7 of 9 2014a). The levels of iNOS and COX-2 are up-regulated shown in Fig. 4a, while the survival rate of zebrafish was in inflammatory response (Wijesinghe et al. 2014a; Wije- significantly reduced by LPS, it was remarkably and singhe et al. 2014b; Xiong et al. 2014). Therefore, we in- dose-dependently increased by SPEH-treatment. In vestigated the effect of SPEH on the protein expression addition, the heart-beating rate of LPS-treated zebrafish levels of iNOS and COX-2 in LPS-stimulated RAW was significantly increased (112.93%) compared to non- 264.7 cells. As shown in Fig. 3, LPS significantly stimu- treated zebrafish (100%) (Fig. 4b). However, it was re- lated iNOS and COX-2 expressions, but SPEH remark- stored to the normal level in the zebrafish treated with ably and dose-dependently downregulated the 100 μg/mL of SPEH (101.65%) (Fig. 4b). The ROS gener- expression level of iNOS and COX-2. These results indi- ation and cell death levels of SPEH-treated zebrafish cated that SPEH possesses strong in vitro anti- were significantly and dose-dependently reduced inflammatory activity. compared to the zebrafish treated with LPS only (Figs. 5 and 6). Furthermore, SPEH pretreatment reduced 26.04, In vivo anti-inflammatory effect of SPEH 39.08, and 50.88% of NO production in LPS-stimulated In vivo evaluation for anti-inflammation activity of SPEH zebrafish pretreated with 25, 50, and 100 μg/mL, re- was adopted in zebrafish due to its advantages such as spectively (Fig. 7). These results indicated that SPEH similarity to mammals, short life span, and the ability of possesses strong in vivo anti-inflammatory activity. the female fish to produce a large number of eggs (Kishi et al. 2003; Ko et al. 2011). In the present study, we in- Conclusion vestigated the survival rate, heart-beating rate, ROS gen- In the present study, we prepared a sterol-enriched frac- eration, cell death, and NO generation in zebrafish. As tion from Spirogyra sp. (SPEH) and evaluated its in vitro Fig. 6 The protective effect of SPEH against LPS-induced cell death in zebrafish. a Zebrafish under fluorescence microscope. b The levels of cell ** death. Cell death levels were measured using Image J software. The data were expressed as the mean ± SE (n = 3). p < 0.01 as compared to ## LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 8 of 9 Fig. 7 The protective effect of SPEH against LPS-induced NO production in zebrafish. a Zebrafish under fluorescence microscope. b The levels of ** NO production. NO production levels were measured using ImageJ software. The data were expressed as the mean ± SE (n = 3). p < 0.01 as ## compared to LPS only-treated group and p < 0.01 as compared to control group and in vivo anti-inflammatory activities in LPS- Consent for publication Not applicable stimulated RAW 264.7 cells and zebrafish. The results indicated that SPEH possesses strong anti-inflammatory Competing interests activity in both in vitro and in vivo models and has the The authors declare that they have no competing interests. potential to be developed as a healthcare product or a Author details drug to treat inflammatory diseases. 1 Department of Marine Life Sciences, Jeju National University, Jeju, Self-Governing Province 63243, Republic of Korea. 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In vitro and in vivo anti-inflammatory activities of a sterol-enriched fraction from freshwater green alga, Spirogyra sp.

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Abstract

Background: 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. Methods: 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. Results: SPEH effectively and dose-dependently decreased the production of nitric oxide (NO) and prostaglandin E (PGE ). SPEH suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β through downregulating nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells without cytotoxicity. The in vivo test results indicated that SPEH significantly and dose-dependently reduced reactive oxygen species (ROS) generation, cell death, and NO production in LPS-stimulated zebrafish. Conclusions: 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. Keywords: Spirogyra sp., Sterol-enriched fraction, Anti-inflammatory activity, RAW 264.7 cells, Zebrafish Background inflammatory tissues during the course of inflammatory Inflammation is a highly regulated biological process response, from pro-inflammatory to anti-inflammatory that enables the immune system to efficiently eliminate or wound healing type. stimuli and injuries (Masresha et al. 2012). Inflammation The inflammatory response is associated with the re- can be classified into two subtypes, chronic and acute in- lease of the inflammatory mediators, including nitric flammation, depending on the difference in response oxide (NO), prostaglandins, histamine, and bradykinin time and procedure. Acute inflammation is an initial re- from various immune cells such as mononuclear phago- sponse of bodies against harmful stimuli. During acute cytes and mast cells (González Mosquera et al. 2011; inflammatory response, the plasma and leukocytes are Wijesinghe et al. 2014a). Inflammation is crucial in many moving from the blood into the injured tissues. There is diseases such as arthritis, atherosclerosis, cancer, and a shift in the type of mononuclear phagocytic cells in the some deadly diseases (Lee and Weinblatt 2001; Firestein 2006; Klegeris et al. 2007; Wen et al. 2016; Wijesinghe * Correspondence: jikim@pknu.ac.kr et al. 2013; Wijesinghe et al. 2014b). Therefore, there is Department of Food Science and Nutrition, Pukyong National University, increasing attention on finding safe and effective anti- Busan 48513, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 2 of 9 inflammatory agents and elucidating their anti- in the present study, ethanol extract of Spirogyra sp. (SPE) inflammatory mechanisms. and the fractions from SPE were prepared, and their anti- Algae are considered as a potential bio-resource for inflammatory activities were evaluated in lipopolysaccha- the development of functional food, cosmeceutical, and rides (LPS)-stimulated RAW264.7 cells. The effect of pharmaceutical because they are rich in various bioactive sterol-enriched fraction (SPEH) from SPE on the produc- compounds, such as proteins, polysaccharides, phenolic tion of pro-inflammatory mediators was investigated by compounds, and sterols (Kim et al. 2016; Sanjeewa et al. enzyme-linked immunosorbent assay (ELISA) and western 2016; Fernando et al. 2017b). These compounds possess blot analysis. In addition, the in vivo anti-inflammatory ef- broad-spectrum bioactivities including anti-bacterial, fect of SPEH was evaluated using a zebrafish model. anti-inflammatory, antioxidant, and anti-cancer activities (Jung et al. 2008; Heo et al. 2010; Lee et al. 2012; Lee Methods et al. 2013; Oh et al. 2016; Fernando et al. 2017a). Chemicals and regents Spirogyra sp. is freshwater green alga. It has been used LPS, acridine orange, 2′,7′-dichlorofluorescin diacetate as a bio-sorbent to remove heavy metal ions from waste- (DCFH -DA), dimethyl sulfoxide (DMSO), and water (Khalaf 2008). Recently, the pharmacological activ- diaminofluorescein-FM diacetate (DAF-FM DA) were ities of Spirogyra sp, such as antioxidant, ultraviolet purchased from Sigma-Aldrich (St. Louis, MO, USA). (UV)-protective, and anti-hypertension activities, have Fetal bovine serum (FBS), penicillin–streptomycin (P/S), been reported (Kang et al. 2015; Lee et al. 2016; Wang and Dulbecco’s modified Eagle’s medium (DMEM) were et al. 2017). However, the anti-inflammatory activities of purchased from Gibco/BRL (Grand Island, NY, USA). Spirogyra sp. have not been evaluated so far. Therefore, The ELISA kits for measuring prostaglandin E (PGE ), 2 2 Fig. 1 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 < 0.05, p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 3 of 9 interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), were obtained. Samples were concentrated and stored at and IL-1β levels were purchased from R&D Systems Inc. – 20 °C until used. (Minneapolis, MN, USA). Antibodies against β-actin, cyclooxygenase-2 (COX-2), and inducible nitric oxide Sterol content analysis synthase (iNOS) were purchased from Santa Cruz Bio- The sterol contents of samples were measured by Lie- technology (Santa Cruz, CA, USA). Secondary anti- bermann Burchard method described by Xiong et al. rabbit and anti-mouse IgG antibodies were purchased (2007). In brief, the samples (2 mg) were dissolved in from Cell Signaling Technology (Beverly, MA, USA). glacial acetic acid (1 mL) and mixed with prepared Lie- Other chemicals and regents used in the present study bermann Burchard regent. The mixture was reacted in were analytical grade. the dark at the room temperature for 10 min. After reac- tion, the absorbance of the mixture was recorded at 625 nm using a micro-plate reader (BioTek, Synergy, HT, Extraction and fractionation USA). Cholesterol was used as standard sterol com- Sampling, processing, extraction, and fractionation were pound to prepare the standard curve. performed following the protocols described in the pre- vious study (Wang et al. 2017). In brief, lyophilized Cell culture Spirogyra sp. powder was extracted by 70% ethanol; the RAW 264.7 cells (ATCC® TIB-71™) were cultured in 70% ethanol extract of Spirogyra sp. (SPE) was fraction- DMEM containing FBS (10%) and P/S (1%) under a hu- ated by hexane, chloroform, and ethyl acetate, respect- midified atmosphere containing 5% CO at 37 °C. RAW ively. Then, the hexane (SPEH), chloroform (SPEC), 264.7 cells were seeded as a density of 1 × 10 cells/mL ethyl acetate (SPEE), and water (SPEW) fractions of SPE for experiments. Fig. 2 Inhibitory effect of SPEH on PGE (a), TNF-α (b), IL-1β (c), and IL-6 (d) production in LPS-induced RAW 264.7 cells. The PGE and pro- 2 2 inflammatory cytokines levels were determined by ELISA kits according to the manufacturer’s instructions. The data were expressed as the mean * ** ## ±SE(n = 3). p < 0.05, p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 4 of 9 Determination of NO, pro-inflammatory cytokines, PGE with the primary antibodies at 4 °C. After 6 h incuba- production, and cell viability tion, the membranes were incubated with HRP- RAW 264.7 cells were seeded and incubated for 24 h. conjugated secondary antibody and visualized using ECL Cells were treated with different concentrations of sam- reagent (Amersham, Arlington Heights, IL, USA). ples and incubated for 1 h. After incubation, LPS (1 μg/ mL) was introduced to cells. The culture medium was Measurement of survival rate and heart-beating rate on collected and used for measuring NO, PGE , TNF-α, IL- LPS-stimulated zebrafish 6, and IL-1β production levels. The NO production Approximately 7–9 h post-fertilization (hpf), zebrafish levels of LPS-treated RAW264.7 cells were measured embryos (15 embryos/group) were treated with different using Griess reagent. The PGE , TNF-α, IL-6, and IL-1β concentrations of SPEH for 1 h. Then, LPS (10 μg/mL) production levels were evaluated using the commercial was added into the embryo medium. The survival rate ELISA kits following the manufacturer’s instructions. was measured at 3 days post-fertilization (dpf) by count- The cell viability was measured by MTT assay based on ing live embryos, and heart-beating rate was measured the method described by Wang et al. (2018). following the protocol described by Sanjeewa et al. (2018). Western blot analysis The LPS-stimulated RAW 264.7 cells were harvested Measurement of reactive oxygens species (ROS) and lysed. The lysates contain 50 μg of protein were sub- generation, cell death, and NO production on LPS- jected to electrophoresis on SDS–polyacrylamide gels stimulated zebrafish (12%). Then, the proteins were transferred onto nitrocel- At 3 dpf, zebrafish were dyed with DCFH -DA (20 μg/ lulose membranes, and the membranes were incubated mL), acridine orange (7 μg/mL), and DAF-FM-DA Fig. 3 Inhibitory effects of SPEH on iNOS and COX-2 expression in LPS-induced RAW 264.7 cells. a iNOS and COX-2 expression in LPS-induced RAW 264.7 cells. b The relative amount of iNOS and COX-2. Relative amounts of iNOS and COX-2 expressions were compared to β-actin. The data ** ## were expressed as the mean ± SE (n = 3). p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 5 of 9 solutions (5 μM) for the detection of ROS generation, sterols including cholesterol, β-sitosterol, campesterol, cell death, and NO production, respectively. The anes- and polyhydroxylated sterols isolated from algae were thetized zebrafish were photographed under the micro- shown to strong bioactivities, especially anticancer and scope equipped with Cool SNAP-Procolor digital camera anti-inflammatory activities (Kazłowska et al. 2013; Elba- (Olympus, Japan). The fluorescence intensity of individ- gory et al. 2015). The freshwater green alga, Spirogyra ual zebrafish was quantified using an Image J program. sp. contains various bioactive compounds. In our previ- ous studies, we have reported antioxidant, UVB photo- Statistical analysis protective, and anti-hypertension activities of phenolic The data were expressed as the mean ± standard error compounds isolated from Spirogyra sp. (Kang et al. (SE). One-way ANOVA test (using SPSS 11.5 statistical 2015; Wang et al. 2017). However, the bioactivities of software) was used to compare the mean values of each sterols from Spirogyra sp. have not been evaluated yet. treatment. The significant differences were established In the present study, we have prepared a sterol-enriched as *p < 0.05, **p < 0.01 as compared to LPS only-treated fraction (SPEH) from Spirogyra sp. and evaluated its ## group and p < 0.01 as compared to control group. in vitro and in vivo anti-inflammatory activities. As shown in Fig. 1, the sterol contents of SPE and its Results and discussion fractions were ranged from 0.50% to 9.08%. This result Sterol content of SPE and its fractions indicates that the sterol content in hexane fraction Algae-derived compounds possess various health bene- (SPEH) was enriched with 6.7 times than SPE. Many re- fits (Mayer and Hamann 2003; Athukorala and Jeon ports support sterols isolated from algae possess strong 2005; Heo et al. 2005; Kotake-Nara et al. 2005). The anti-inflammation activity (Ku et al. 2013; Sanjeewa Fig. 4 SPEH improved the survival rate and reduced heart-beating rate in LPS-stimulated zebrafish. a The survival rate. b the heart-beating rate. * ** ## The data were expressed as the mean ± SE (n = 3). p < 0.05, and p < 0.01 as compared to LPS only-treated group and p < 0.01 as compared to control group. Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 6 of 9 et al. 2016; Suh et al. 2018). Our previous study investi- treated with 100 μg/mL of SPEH was 99.52%, which is gated anti-cancer and anti-inflammatory activities of the close to the cells not treated with LPS (100%) (Fig. 1c). sterol-rich fraction of Nannochloropsis oculata. The re- These results indicate that SPEH showed the most po- sults indicated that the hexane fraction of methanol ex- tent anti-inflammatory activity among 5 samples. Thus, tract of N. oculata is rich in sterol content and possesses SPEH was selected as the target sample for the further strong anti-inflammatory activity. In this regard, anti- study. inflammatory activity can be expected from SPEH pre- We next examined the inhibitory effects of SPEH on pared in this study, in which the content of sterol was the production of PGE and pro-inflammatory cytokines 9.08%. in LPS-stimulated RAW264.7 cells. As Fig. 2a shows, LPS induced PGE level in RAW264.7 cells to 319.85% In vitro anti-inflammatory effect of SPEH compared to the control group (100%). However, the In order to evaluate the anti-inflammatory activity of PGE levels of LPS-treated cells were decreased to SPE and its fractions, their inhibitory effects on NO pro- 307.83%, 301.38%, and 278.90% by 25, 50, and 100 μg/ duction were measured. As shown in Fig. 1, all samples mL of SPEH treatment, respectively. In addition, SPEH significantly and dose-dependently decreased NO pro- inhibited 17.70%, 33.01%, and 65.76% of TNF-α (Fig. duction (Fig. 1b). However, SPE and SPEE showed re- 2b), 113.31%, 173.91%, and 179.67% of IL-1β (Fig. 2c), markable cytotoxicity in RAW264.7 cells. SPEH and 50.233%, 91.22%, and 252.14% of IL-6 expressions (Fig. SPEC that contain relative higher sterol contents showed 2d) at the concentration of 25, 50, and 100 μg/mL, stronger NO inhibitory effect in LPS-induced RAW264.7 respectively. cells than other samples. Furthermore, SPEH signifi- iNOS and COX-2 are two inducible enzymes, which cantly improved the viability of LPS-induced RAW264.7 synthesize two key inflammatory mediators, NO and cells at high concentration. The viability of the cells PGE , respectively (Kim et al. 2005; Wijesinghe et al. Fig. 5 The protective effect of SPEH against LPS-induced ROS generation in zebrafish. a Zebrafish under fluorescence microscope. b The levels of ** ROS. ROS levels were measured using Image J software. The data were expressed as the mean ± SE (n = 3). p < 0.01 as compared to LPS only- ## treated group and p<0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 7 of 9 2014a). The levels of iNOS and COX-2 are up-regulated shown in Fig. 4a, while the survival rate of zebrafish was in inflammatory response (Wijesinghe et al. 2014a; Wije- significantly reduced by LPS, it was remarkably and singhe et al. 2014b; Xiong et al. 2014). Therefore, we in- dose-dependently increased by SPEH-treatment. In vestigated the effect of SPEH on the protein expression addition, the heart-beating rate of LPS-treated zebrafish levels of iNOS and COX-2 in LPS-stimulated RAW was significantly increased (112.93%) compared to non- 264.7 cells. As shown in Fig. 3, LPS significantly stimu- treated zebrafish (100%) (Fig. 4b). However, it was re- lated iNOS and COX-2 expressions, but SPEH remark- stored to the normal level in the zebrafish treated with ably and dose-dependently downregulated the 100 μg/mL of SPEH (101.65%) (Fig. 4b). The ROS gener- expression level of iNOS and COX-2. These results indi- ation and cell death levels of SPEH-treated zebrafish cated that SPEH possesses strong in vitro anti- were significantly and dose-dependently reduced inflammatory activity. compared to the zebrafish treated with LPS only (Figs. 5 and 6). Furthermore, SPEH pretreatment reduced 26.04, In vivo anti-inflammatory effect of SPEH 39.08, and 50.88% of NO production in LPS-stimulated In vivo evaluation for anti-inflammation activity of SPEH zebrafish pretreated with 25, 50, and 100 μg/mL, re- was adopted in zebrafish due to its advantages such as spectively (Fig. 7). These results indicated that SPEH similarity to mammals, short life span, and the ability of possesses strong in vivo anti-inflammatory activity. the female fish to produce a large number of eggs (Kishi et al. 2003; Ko et al. 2011). In the present study, we in- Conclusion vestigated the survival rate, heart-beating rate, ROS gen- In the present study, we prepared a sterol-enriched frac- eration, cell death, and NO generation in zebrafish. As tion from Spirogyra sp. (SPEH) and evaluated its in vitro Fig. 6 The protective effect of SPEH against LPS-induced cell death in zebrafish. a Zebrafish under fluorescence microscope. b The levels of cell ** death. Cell death levels were measured using Image J software. The data were expressed as the mean ± SE (n = 3). p < 0.01 as compared to ## LPS only-treated group and p < 0.01 as compared to control group Wang et al. Fisheries and Aquatic Sciences (2020) 23:27 Page 8 of 9 Fig. 7 The protective effect of SPEH against LPS-induced NO production in zebrafish. a Zebrafish under fluorescence microscope. b The levels of ** NO production. NO production levels were measured using ImageJ software. The data were expressed as the mean ± SE (n = 3). p < 0.01 as ## compared to LPS only-treated group and p < 0.01 as compared to control group and in vivo anti-inflammatory activities in LPS- Consent for publication Not applicable stimulated RAW 264.7 cells and zebrafish. The results indicated that SPEH possesses strong anti-inflammatory Competing interests activity in both in vitro and in vivo models and has the The authors declare that they have no competing interests. potential to be developed as a healthcare product or a Author details drug to treat inflammatory diseases. 1 Department of Marine Life Sciences, Jeju National University, Jeju, Self-Governing Province 63243, Republic of Korea. 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