3β-Hydroxy-Δ5-steroidal congeners from a column fraction of Dendronephthya puetteri attenuate LPS-induced inflammatory responses in RAW 264.7 macrophages and zebrafish embryo model

Bioactive compounds from marine organisms and their action mechanisms have provided new insights into medicinal and natural product research. Here, we report the identification of 3β-hydroxy-Δ5-steroidal congeners from a purified column fraction (DPCMH24) of the soft coral Dendronephthya puetteri harvested from Jeju, South Korea. DPCMH24 exerted strong anti-inflammatory effects through a dose-dependent decrease in the levels of nitric oxide (NO) in LPS-induced RAW 264.7 macrophages (IC50 value = 6.54 ± 0.38 μg mL−1). Further, DPCMH24 attenuated the levels of PGE2 and the pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6. The above effects were mediated via the inhibition of nuclear factor κB activation and mitogen-activated protein kinase pathways. In vivo evaluation indicated that DPCMH24 reduced NO, iNOS, COX-2, ROS production and cell death in LPS-induced zebrafish embryos, confirming its anti-inflammatory potential. The constituent compounds were identified by GC-MS/MS analysis. These findings suggest that the steroidal congeners from D. puetteri may offer ample therapeutic potential against LPS-induced inflammation.


Introduction
Natural products from marine invertebrates have emerged as a focus for investigation owing to their broad spectrum of bioactive functionalities and unusual structural properties. So corals are marine invertebrates thriving in nutrient-rich waters. They are renowned for their constituent terpenoids with notable bioactivity and structural diversity. Many of the steroidal derivatives found in so corals have received special attention for their antiviral, antimicrobial, cytotoxic, and anti-inammatory properties. These steroidal derivatives can be found as 1-or 4-en-3-ones, 1,4-dien-3-ones, 9,10-or 9,11secosteroids, polyhydroxysteroids, steroid peroxides, spiro acetals, hemiacetals, steroidal glycosides, and those with side chains, which oen contain the cyclopropane functional group. 1 So corals belonging to the genus Dendronephthya are among the most abundant and widespread species. Some of the Dendronephthya species are widely exploited for their bioactive natural products. Sarma et al. provided a comprehensive review on triterpenoids in so corals. 2 Although previous studies have reported the anti-inammatory potential of terpenoids from several Dendronephthya species, the present study is the rst to report on the natural products found in Dendronephthya puetteri.
Chronic inammation is a detrimental condition that leads to the pathogenesis of diseases such as cancer, inammatory arthritis, multiple sclerosis, coronary artery diseases, atherosclerosis, obesity, dermatitis, interstitial cystitis, migraines, insulin resistance, and irritable bowel syndrome. [3][4][5][6] Hence, investigation of the anti-inammatory properties of natural products has become a cornerstone of modern biomedical research. Steroidal derivatives, especially glucocorticosteroids, are well known for their potent anti-inammatory properties; they have been found to inhibit a wide range of pro-inammatory cytokines, including tumor necrosis factor (TNF)-a, interleukins (IL)-1, 3, 4, 5, 6, and 8, and granulocyte macrophage colony-stimulating factor (GM-CSF). 7 In addition, most of the steroidal derivatives can inhibit inammatory mediators, such as prostaglandins, leukotrienes, and plateletactivating factor (PAF), which contribute to the observed anti-inammatory activity. 8 The present study is a continuation of our previous screening studies in which we analyzed the anti-inammatory potential of ten different so coral sample crude ethanolic extracts. 9 Current disclosure report the identication of natural products found in D. puetteri which could contribute for the mediation of the anti-inammatory effects, through the use of LPS-induced RAW 264.7 murine macrophages and in vivo zebrash embryo model.

Sample collection and extraction
D. puetteri so coral samples were collected from the Jeju island coast at a depth of 12-15 m during August 2016. The sample was identied by the Jeju Biodiversity Research Institute, and a voucher specimen, JSC16003 was deposited in the laboratory of marine bioresource technology, Jeju National University. The samples were rinsed with tap water to remove attached debris and ground to a powder aer lyophilization. 1.2 kg of the powder was extracted three times using a solvent system of 1 : 1 chloroform and methanol. The solvent was evaporated to dryness by a rotary evaporator obtaining the crude extract (DPCM).

Characterization of compounds by GC-MS/MS analysis
The selected DPCMH24 fraction was analyzed by a Shimadzu GCMS-TQ8040 system (Japan). Capillary column; Rtx-5MS fused-silica, column length 30.0 m, internal diameter 0.25 mm. Sample injection; split mode (ratio 2) injector port temperature 280 C. GC oven program; hold at 260.0 C for 3 minutes, increase to 320.0 C at 6.0 C min À1 , increasing to 330.0 at 5.0 C min À1 and hold for 5 minutes. Carrier gas; helium at a constant ow rate (0.73 mL min À1 ). 10 Cell-culture RAW 264.7 mouse macrophages were cultured and maintained in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin mixture at 37 C in a humidied atmosphere with 5% CO 2 . The cells were subcultured periodically, and the cells at exponential growth were seeded for the experiments. The samples for the cell culture experiments were prepared by rst dissolving the samples in dimethyl sulfoxide (DMSO). Necessary dilutions were carried out using phosphate-buffered saline. The nal DMSO concentration in the culture medium aer treatment were less than 0.0025%. 11 Evaluating the suppressive effects of samples on NO production, and viability of RAW 264.7 macrophages RAW 264.7 macrophages were seeded (1 Â 10 5 cells per mL concentration) and incubated for 24 h in 24 well plates. Different sample concentrations were treated to the wells and aer 1 h, the cells were stimulated by adding LPS (1 mg mL À1 ). Following a 24 h incubation period, a portion of the culture media from each well was withdrawn to a 96 well plate and mixed with a similar volume of Griess regent. NO levels were evaluated by measuring the absorbance at 540 nm. Simultaneously MTT assay was carried out to determine the cell viability. 11 Evaluating the levels of PGE 2 and pro-inammatory cytokines Culture media collected from the above-mentioned sample treated, LPS-stimulated wells were used for the determination of PGE 2 and pro-inammatory cytokines including TNF-a, IL-1b, and IL-6. The experiments were carried out using ELISA kits following the manufacturer instructions.

Western blot analysis
Levels of iNOS, COX-2, NFkB p65, NFkB p50, p44/42 MAPK (Erk1/2), Phospho-p44/42 MAPK (Erk1/2), p38 MAPK, and Phospho-p38 MAPK were determined by Western blot analysis. The cells were seeded in 6 well culture plates (2 Â 10 5 cells per mL) and aer 24 h, treated with different concentrations of the samples. Following 1 h the cells were stimulated with LPS. The cells were harvested at two different intervals to evaluate the different levels of protein expressions. For the analysis of upstream signaling proteins (related with NFkB and MAPK proteins), the cells were harvested 15 min aer the LPS stimulation whereas the levels of iNOS and COX-2 were evaluated 24 h aer the LPSstimulation. Harvested cells were washed with PBS and lysed using a NE-PER® Nuclear and Cytoplasmic extraction kit (Thermo Scientic, Rockford, USA). The protein levels in each cytoplasmic and nuclear extracts were determined using the BCA protein assay kit, and the standardized proteins (50 mg) were loaded on 10% SDS-polyacrylamide gels. The proteins were resolved by gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk for 2 h and incubated with primary (8 h at 4 C under gentle shaking) and secondary antibodies (2 h at room temperature). Aer washing away the excess antibodies using TBST, the bands were visualized using enhanced chemiluminescence (ECL) reagents (Amersham, Arlington Heights, IL, USA). 11

In vivo zebrash embryo experiments
Aer acclimating the adult zebrash to laboratory condition for two weeks, the embryos were obtained by stimulating the fertilization with the onset of light in the morning. Healthy embryos (15 per group) were transferred to separate wells in 12 well culture plates and mounted in 1.0 mL of embryo media. At 8 h post fertilization, different sample concentrations were treated into the wells. Following 1 h, LPS (10 mg mL À1 ) was treated to the wells. The embryonic media was replaced aer 24 h of post-fertilization. At the 3rd day of post fertilization (3dpf), the hatched larvae (three from each sample treatment group) were transfered into 24 well plates, stained with specic uorescence dyes which includes DCFDA, DAF-FM DA, and acridine orange to respectively identify the ROS production, NO production, and cell viability. The uorescence images of the larvae were obtained using Lionheart™ FX Automated Microscope system BioTek Instruments, Inc. (Winooski, Vermont, USA) with the 469/525 (GFP) lter in place. The intensity of green color was quan-tied using the ImageJ soware and compared with the mean values of respective controls. The levels of iNOS and COX-2 proteins in the embryos were determined by Western blot analysis. The proteins were obtained by homogenizing the embryos under frozen conditions. 11 The zebrash experiments were performed in compliance with the guidelines "Ethical principles and guidelines for the care and use of animals" with the approval of Animal Care and Use Committee of Jeju National University of Republic of Korea.

Statistical analysis
Data values are expressed as the mean AE standard deviation of at least three independent determinations. Statistical analysis for the comparison of results was carryout using one way ANOVA by Duncan's multiple range test using the SPSS statistics V20 soware. P-values < 0.05 "*" and P < 0.001 "**" was considered as signicant.

Results
Process of bioassay-guided fractionation, purication, and suppressive effect of DPCMH24 on NO production in LPSinduced RAW 264.7 macrophages A graphical summary of the purication process is provided in Fig. 1(A). The chloroform-methanol (1 : 1) solvent system preferably provides a high extraction yield for the terpenoids. 12 Each step of the purication process was performed aer the evaluation of the reduction in NO levels in LPS-stimulated RAW macrophages. Among the solvent/solvent fractionations, the hexane fraction (DPCMH), which was more active, was selected for further purication instead of the chloroform (DPCMC), ethyl acetate (DPCME), and water (DPCMW) fractions. Silica open column purication of DPCMH produced ve column fractions, with prominent anti-inammatory activity and the best cell viability in the second fraction (DPCMH2). DPCMH2 was obtained through elution of the column with a solvent system containing hexane and ethyl acetate, 4 : 1. Further purication of DPCMH2 obtained ve column fractions, with prominent anti-inammatory activity in the fourth column fraction (DPCMH24). Thin-layer chromatography (TLC) analysis revealed that the column fractions were adequately separated. The presence of dark/maroon color spots on TLC, especially in DPCMH24 and DPCMH25 with 10% H 2 SO 4 in ethanol staining, and their absence under UV light conrmed the presence of sterols. Since reaction with sulfuric acid is highly unspecic, these observations can only suggest presence of sterols. The content of sterols in each of the second column fractions (DPCMH21, DPCMH22, DPCMH23, DPCMH24, and DPCMH25) was 9.60 AE 3.66, 40.79 AE 4.2, 62.07 AE 2.2, 96.49 AE 0.5, and 74.77 AE 1.5% based on the Liebermann-Burchard colorimetric assay, which conrmed the presence of sterols in DPCMH24.
The fractions DPCMH23, DPCMH24, and DPCMH25 reduced NO production in LPS-stimulated RAW macrophages in a dose-dependent manner ( Fig. 1(B)). Among the fractions, the most prominent effects were seen in DPCMH24, which had an IC 50 value of 6.54 AE 0.38 mg mL À1 . A minor decrease in RAW cell viability was observed with an increase in sample concentrations ( Fig. 1(C)). The viability of RAW cells at the IC 50 value was >85%. As shown in ESI materials (Sheet 2 †) the well-known steroidal anti-inammatory agent dexamethasone indicated an IC 50 value of 3.39 AE 0.02 mg mL À1 for the inhibition of NO production in LPS-stimulated RAW macrophages under similar experimental conditions which was approximately twice stronger than DPCMH24.
Compounds characterized by DPCMH24 indicated the presence of 3b-hydroxy-D5-steroidal congeners The GC chromatograms and the identied compounds are presented in Fig. 2. The corresponding mass spectra are provided in the ESI Materials. † DPCMH24 was composed of a mixture of six main 3b-hydroxy-D5-steroidal congeners, DPCMH24 reduced the production levels of PGE 2 and pro-inammatory cytokines in LPS-induced RAW 264.7 macrophages through the inhibition of the NF-kB and MAPK signaling pathways Inammatory responses are mediated via a complex system of signaling pathways. iNOS, COX-2, PGE 2 are some of the key inammatory mediators used for the evaluation of anti-inammatory potential. As shown in Fig. 3(A), treatment with DPCMH24 resulted in a dose-dependent reduction in the levels of iNOS and COX-2 in LPS-induced RAW macrophages. In addition, DPCMH24 dose-dependently reduced the levels of PGE 2 and the levels of pro-inammatory cytokines, including TNF-a, IL-1b, and IL-6 ( Fig. 3(B)-(E)).
Mitogen-activated protein kinases (MAPKs) and nuclear factor kB (NF-kB) proteins are vital upstream signaling molecules that control the production of inammatory mediators. Their activation reaches a maximum within 30 min of the LPS-stimulation and decreases thereaer. As shown in Fig. 3(F), the cytosolic levels of NF-kB p50 and p65 in the LPS-treated groups were lower than those in the control group. However, reciprocal events were observed in the nucleus for the levels of NF-kB p50, and p65, where treatment with DPCMH24 dose-dependently reduced their nuclear translocation (Fig. 3(G)). Simultaneous evaluation of IkB levels could have been crucial for justifying/ validating the increase in the level of cytoplasmic p50 and p65 with increasing DPCMH24 concentration. The levels of Erk1/2, p38, and JNK were constant in the cytosol of RAW cells, regardless of LPS stimulation or sample treatment (Fig. 3(H)). However, the proportion of phosphorylated Erk1/2, p38, and JNK increased by LPS-stimulation and decreased with the increase in sample concentrations.

DPCMH24 reduced inammatory response, in LPS-induced in vivo zebrash embryo model
Many researchers have focused on the use of zebrash animal model to mimic a wide variety of human diseases. It is a rapid and sensitive method to screen the anti-inammatory effects of drug candidates. 13 In the present study, different uorescence probe dyes were used to detect NO, ROS, and cell death in the embryos. LPS stimulation caused a marked increase in NO and ROS levels, and cell death in the zebrash embryos ( Fig. 4(A)); however, treatment with DPCMH24 dose-dependently reduced the uorescence intensity in LPS-induced zebrash embryos. As shown in Fig. 4(B), density of the Western blotting bands for iNOS and COX-2 increased in LPS-stimulated zebrash embryos. Treatment with DPCMH24 dose-dependently decreased the iNOS and COX-2 levels in LPS-induced zebrash embryos. Hence, the observed reduction in NO levels in LPSinduced zebrash embryos could be attributable to the reduction in iNOS levels.

Discussion
Inammation is an essential biological process regulated by complex molecular mechanisms involving inammatory and anti-inammatory cytokines, prostaglandins, leukotrienes, growth factors, and nitric oxide (NO). Macrophages are key elements for the regulation of the immune responses triggered by foreign invaders or deregulated biological processes. Activated macrophages cause the production of pro-inammatory cytokines, such as TNF-a, IL-1b, IL-6, and inammatory mediators, including prostaglandins (PGE 2 ) and NO. Deregulated production of these inammatory mediators causes pathogenesis of inammatory diseases such as rheumatoid arthritis, autoimmune diseases, periodontitis, hearing loss, and bacterial sepsis. 14 Compounds with ability to reduce the levels of these compounds would therefore be promising candidates for the development of anti-inammatory drugs. As shown in Fig. 1(B), DPCMH24 treatment caused a noticeable reduction in NO production in LPS-stimulated RAW macrophages, with an IC 50 value of 6.54 AE 0.38 mg mL À1 . DPCMH24 was found to contain a mixture of six 3b-hydroxy-D5-steroidal congeners, namely cholesta-5,22-dien-3-ol, cholest-5-en-3-ol, ergosta-5,22-dien-3ol, 22,23-methylenecholestene-3-ol, stigmast-5-en-3-ol, and cholesta-5,24-dien-3-ol. In published literature, the reported IC 50 values of the steroidal and non-steroidal anti-inammatory drugs dexamethasone and indomethacin are approximately 3.92 mg mL À1 and 3.58 mg mL À1 , respectively. 15 Hence, the ability of DPCMH24 to reduce NO production in LPS-stimulated RAW macrophages is promising. LPS treatment reduced the viability of RAW cells to approximately 70%, whereas DPCMH24 treatment exerted cytoprotective effects against LPS-induced inammatory responses and restored cell viability. However, as the concentration of DPCMH24 was increased, minor reductions in cell viability were observed. An extensive review by Voultsiadou showed that marine invertebrates, including anthozoans, have been used since the ancient Greek and early Byzantium periods for their therapeutic effects. So corals are well known for their bioactive sesqui-, tri-, and diterpene metabolites. More than 200 types of steroid metabolites have been identied from so corals, with a characteristic cholestane structure containing a 3b-hydroxy-D5-(or D0-) moiety with a side chain of 8-10 carbons. 16 Carlson et al. reported that marine invertebrates contain a wide variety of sterols with unusual structures, some of which are the subject of ongoing investigations. 17 Byju et al. identied six sterols from the so coral Subergorgia reticulata with potential antiproliferative effects, and four compounds (cholesta-5,22-diene-3ol, ergosta-5-22-dien-3ol, cholesterol, and b-sitosterol) were similar to those reported in the present study. 18 Fernando et al. revealed a sterol-rich fraction from Dendronephthya gigantea with anti-inammatory activity (IC 50 ¼ 4.33 AE 0.50 mg mL À1 ) against NO production in LPS-induced RAW macrophages. 19 The compounds cholesta-5,22-dien-3-ol, cholest-5-en-3-ol, ergosta-5,22-dien-3-ol, and stigmasta-5,24(28)-dien-3-ol were found, similar to those in the present study. The synergistic Quantification of green color fluorescence intensities and the densitometric analysis of Western blotting were computed using ImageJ software. The data are expressed as the means AE SE obtained from three replicates in two independent experiments. *P < 0.05 and **P < 0.001 were considered significant compared to the positive control (LPS only). effect of these steroidal metabolites might be responsible for the observed anti-inammatory potential.
Moreover, DPCMH24 dose-dependently inhibited the iNOS, COX-2, and PGE 2 inammatory mediators, as well as pro-inammatory cytokines including TNF-a, IL-1b, and IL-6, which demonstrated a broad range of activity. The observed reduction in NO production could be attributable to the reduction in iNOS levels; moreover, the reduction in COX-2 levels was related to the reduction in PGE 2 . Hence, a detailed investigation was conducted to explore the effect of DPCMH24 on the regulation of upstream signaling molecules that control inammatory responses.
LPS stimulation causes the activation of the intracellular signaling pathways of MAPKs and NF-kB transcription factors. MAPKs, activated by extracellular stimuli (mitogens), mediate a variety of cellular activities related to cell proliferation, survival, differentiation, and apoptosis; MAPKs include extracellular signal-regulated kinase (Erk), Jun N-terminal kinase (JNK), and p38, and play an essential role in the progression of inammatory and immune responses. The activation of MAPKs, in conjunction with the activation of NF-kB and interferon regulatory factors, induce the expression of several genes involved in the regulation of the inammatory responses. 20 The maximal activation (phosphorylation) of Erk1/2, p38, and JNK was attained 30 min aer LPS stimulation and decreased thereaer. However, as a precautionary measure, we harvested the cells 20 min aer LPS stimulation. The ndings of the present study indicated that LPS treatment increased the phosphorylation of MAPKs, including Erk1/2, p38, and JNK, whereas DPCMH24 treatment suppressed the LPS-induced phosphorylation in a dose-dependent manner. There was no change in the levels of non-phosphorylated MAPKs in RAW cells treated with LPS or LPS and DPCMH24.
The NF-kB family of proteins (p50, p52, p65, c-Rel, and RelB) comprises a set of signaling molecules that are involved in the maintenance of the release of pro-inammatory cytokines and inammatory mediators. The phosphorylation of IkB-a proceeds via the activation of MAPKs, NF-kB-inducing kinase (NIK), and IkB kinase (IKK). 21 NF-kB transcription factors, which reside in the cytosol, are composed of dimeric proteins that belong to the Rel homology family (e.g., p50 and p65) and remain bound to the IkB (inhibitor), preventing its nuclear translocation. 22 Upon activation, phosphorylation of IkB causes the breakdown of the IkB-p50 and p65 complex, which translocates the Rel proteins to the nucleus. Rel proteins bind to kB-binding sites in the target genes to induce the transcription of inammatory mediators (iNOS, COX-2, and PGE 2 ) and pro-inammatory cytokines (TNF-a, IL-1b, and IL-6). 23 Many anti-inammatory drugs can suppress NF-kB activation and consequently inhibit the production of pro-inammatory cytokines. 24 In the present study, we observed a decrease in the cytosolic NF-kB p50 and p65 proteins in LPStreated cells compared to the control. This could be attributed to the phosphorylation of IkB-p50 and p65 complex and translocation into the nucleus. The levels of cytosolic p50 and p65 increased with an increase in the sample concentrations. These observations were in agreement with the results observed for the p50 and p65 levels in the nucleus. The highest levels of p50 and p65 were seen in the LPS-treated group, whereas they were almost absent in the control group. Moreover, reductions in p50 and p65 were observed as the concentrations of DPCMH24 increased. Collectively, these observations suggested that the steroidal congeners in DPCMH24 inhibited the phosphorylation of IkB-p50 and p65 complexes in a concentration-dependent manner.
The LPS-induced in vivo zebrash embryo model is a rapid and sensitive animal model for the investigation of anti-inammatory properties of drug candidates. The welldeveloped innate and acquired immune systems in zebrash, with similarity to mammalian systems, provide an excellent environment to mimic the inammatory conditions associated with humans. The increased uorescence intensity of NO and ROS, and cell death in LPS-stimulated zebrash embryos decreased as the concentrations of DPCMH24 was increased, which suggested that the steroidal congeners in DPCMH24 could act as anti-inammatory agents. Further, they indicated protective effects against LPS-induced toxicity. Western blotting analysis ( Fig. 4(B)) indicated that the iNOS and COX-2 levels were increased in zebrash embryos aer LPS treatment, while DPCMH24 concentrations dose-dependently reduced their levels, supporting its potential anti-inammatory effects.
In conclusion, the therapeutic potential of the mixture of six 3b-hydroxy-D5-steroidal congeners against LPS-induced inammation offer advantages for their potential applications in the cosmeceutical and pharmaceutical industries. Further studies are required to elucidate the bioactivity of the individual components of DPCMH24 or their synergistic effects.

Conflicts of interest
There are no conicts of interest to declare.