Oxysterols from a Marine Sponge Inflatella sp. and Their Action in 6-Hydroxydopamine-Induced Cell Model of Parkinson’s Disease

Four new oxysterols 1–4 along with previously known oxygenated sterols 5–14 were isolated from the sponge Inflatella sp., collected from the Sea of Okhotsk. Structures of 1–4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as by comparison of the corresponding experimental data with those reported in literature. The influence of compounds 1–14 on the viability of neuronal Neuro2a cells treated by 6-hydroxydopamine and reactive oxygen species (ROS) formation in these cells was investigated.


Introduction
Oxysterols, formed either enzymatically or by auto-oxidation, have a second oxygen function in addition to that of C-3 when compared with natural sterols.These compounds are of particular interest due to important biological functions of some of them and various bioactivities, including effects on lipid metabolism, platelet aggregation, apoptosis, different receptors and proteins [1].Generally, oxysterols attract a great attention by the contribution to many physiological processes, including those connected with pathophysiology of the neurodegenerative diseases [2].As a key trend, the roles of two brain sterols, 24S-hydroxycholesterol and 27-hydroxycholesterol, have been studied for years and discussed in multiple reports [3][4][5].Among others, 24S-hydroxycholesterol has been reported to be markedly reduced in the circulation of patients with Parkinson's disease (PD) [4].The treatment with 27-hydroxycholesterol or a combination of 24S-hydroxycholesterol and 27-hydroxycholesterol reduced the levels of noradrenaline, whereas the treatment with 24S-hydroxycholesterol alone had no effect.With these results, the authors demonstrated that oxysterols could regulate the proteins involved in the development of PD [5].
Recent studies highlighted the point that even well-known oxygenated sterols might be a critical source of new medicine leads.Thus, hecogenin and cholest-4-en-3-one showed significant inhibitory activity (EC 50 values of 116.3 and 390.6 µM, respectively) against the human β-site amyloid cleaving enzyme (BACE1), which has been considered as an effective drug target for treatment of Alzheimer's disease [8].
The search for new oxysterols in marine organisms and the studies on their action in model cell systems could be also considered as an approach to the creation of new pharmaceutical leads against neurodegenerative diseases.As a part of our chemical investigations of steroidal compounds from marine sponges [9][10][11], we have studied oxysterols of a cold-water marine sponge Inflatella sp.The sponges belonging to the genus Inflatella are insufficiently studied for their secondary metabolites, though some bioactivities of their extracts were noted [12].The ethanol extract of the studied Inflatella sp.sample was preliminary analyzed by thin-layer chromatography during the screening of sponge samples from PIBOC collection.The results revealed that in contrast with extracts of other sponges, it contained the oxygenated steroids visually detected on chromatograms as a number of bright colored spots (Figure S1).Herein we describe the isolation and structural elucidation of fourteen individual oxysterols of Inflatella sp., including four new compounds as well as the results of their biotesting using a cell model of PD.Part of this work has been presented at the 3rd International Symposium on Life Sciences, Vladivostok, Russia, September 2018.
Encouraged with the short literature review presented above, that shows different attractive bioactivities of oxysterols, we have made an attempt to evaluate the action of the isolated compounds 1-14 on viability of Neuro2a cells and reactive oxygen species (ROS) formation in these cells.In fact, neuroblastoma cells treated by 6-hydroxydopamine (6-OHDA) are used as a cell model of PD [31].
Compounds 4, 5, 9 and 12 did not show any notable effects on Neuro2a cell viability.Compounds 1, 2, 7, 8, 13 and 14 demonstrated slight cytotoxic activity at concentration 100 µM and decreased viability of Neuro2a cells on 25%, 17%, 44%, 27%, 38% and 33%, respectively.It is of interest that all of them have the same structural peculiarity, being the 24 (28)-unsaturated derivatives of ergostane series.No compounds decreasing cell viability more than 50% were found.At concentration of 10 µM, the oxysteroids 1-14 were non-toxic against these neuronal cells, and were used in next experiments at the non-toxic concentrations (Figure S25).Moreover, compounds 3, 6, 10 and 11 increased the viability of Neuro 2a cells in comparison with non-treated cells, when MTT cell viability test was used (Figure 2a and Figure S25).Applied MTT assay is one of the most widely exploited approaches in research for measuring cell proliferation, viability and drug cytotoxicity.In living cells, the water-soluble yellow dye MTT is reduced to a dark purple (blue-magenta) colored formazan precipitate, which can be analyzed colorimetrically after dissolving in an organic solvent.It was shown, that the MTT reduction site is not only mitochondria.Non-mitochondrial, cytosolic and microsomal MTT reduction makes the major contribution to an overall reduction.Changes in the activity of dozens of the mitochondrial and non-mitochondrial oxidoreductases, cellular metabolic and energy perturbations, and oxidative stress may significantly impact the MTT assay read out [32].
To study action of the tested compounds on cells in details, we additionally used fluorescein diacetate (FDA) assay based on nonspecific esterase activity measuring and thus examined the influence of compounds 3, 6, 10 and 11 on proliferation or/and viability of Neuro 2a cells [33].Compounds 3, 6, 10 and 11 did not increase the fluorescence intensity in FDA assay in comparison with control and therefore did not influence significantly on nonspecific esterase activity in Neuro2a cells (Figure 2b).Hence, we could conclude that the observed increasing of MTT reduction was not caused by the influence of tested compounds on cell proliferation.
In fact, the overestimation in MTT assay of the compounds 3, 6, 10 and 11 could be caused by alternative metabolic processes.For example, the overestimation reported for rottlerin was explained by dissipation of the inner mitochondrial membrane potential, acceleration of electron transfer and increasing of dehydrogenases activity, oxygen consumption and NADH oxidation [33].On the other hand the polyphenolic antioxidant resveratrol exhibited increasing of MTT-reducing activity without a corresponding increasing of living cells number [34].The ability of resveratrol to down-regulate NADPH-oxidase leading to decreased ROS production and thereby provide a protective effect in cardiovascular and neurodegenerative diseases is also well known [35].
As the above reviewed reports described the influence of compounds on intracellular ROS formation, we investigated the effect of compounds 1-14 on ROS formation in Neuro 2a cells by short-time 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCF-DA) test.Oxysterols 1-4, 8, 10 and 13 at concentrations of 1 or/and 10 µM slightly decreased the ROS level in Neuro2a cells by 12-16%, while the increasing in ROS formation was not detected for all the studied compounds (Figure S26).
PD is the one of the most common age-related motoric neurodegenerative disease, regardless of countries and regions [36].Pathogenesis of PD includes neuronal death as a result of oxidative stress involved intracellular level of ROS increasing.In this reason, compounds exhibited ROS-scavenger activities could be interesting as neuroprotective agents.All isolated compounds 1-14 were studied in 6-OHDA-induced Neuro2a cell model of PD (Figure S26).Only compounds 3, 4 and 11 affected on viability of 6-OHDA-treated cells (Figure 3a) and ROS formation in these cells (Figure 3b).

Animal Material
The samples of an Inflatella sp.sponge were collected by dredging near Kashevarov Bank, the Sea of Okhotsk (55 • 30 N; 146 • 05 E, Russia) at a depth of 214-197 m and were freeze dried after collection.The species was identified by Dr. Krasokhin V. B. from G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS.A voucher specimen (PIBOC O07-33 (11)) is deposited at the G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS (Vladivostok, Russia).

Extraction and Isolation
The sponge material (dry weight 216 g) was chopped into pieces and extracted with EtOH at room temperature.The ethanol soluble materials (21.0 g) were obtained after concentration of the extract was dissolved in distilled water (300 mL) and partitioned in turn with CHCl 3 (200 mL × 3).Evaporation of the chloroform extracts gave dark green gum (12.7 g) that was further separated on a silica gel column (7 cm × 10 cm) using the mixtures of CHCl 3 /EtOH as stepwise eluent systems to yield the fractions A-F (Figure S1).

Cell Line and Culture Condition
The neuroblastoma cell line Neuro2a was purchased from ATCC.Cells were cultured according to the manufacturer instructions in DMEM medium containing 10% fetal bovine serum (Biolot, Russia) and 1% penicillin/streptomycin (Invitrogen).Cells were incubated at 37 • C in a humidified atmosphere containing 5% (v/v) CO 2 .

Nonspecific Esterase Activity Assay
Cell suspension (1 × 10 3 cells/well) was incubated with different concentrations of the studied compounds during 24 h.A stock solution of the fluorescein diacetate (FDA) (Sigma-Aldrich, USA) in DMSO (1 mg/mL) was prepared.After incubation of the cells with compounds, FDA solution (50 µg/mL) was added to each well and the plate was incubated at 37 • C for 15 min.Cells were washed with phosphate buffer saline and fluorescence was measured with a Fluoroskan Ascent plate reader (ThermoLabsystems, Finland) at λ ex = 485 nm and λ em = 518 nm.Cell viability was expressed as the percent of control.

6-OHDA-Induced In Vitro Model of Parkinson's Disease
The neuroprotective activity of the studied compounds in 6-hydroxydopamine-induced cell model of Parkinson's disease was investigated as described previously [37].Neuroblastoma Neuro2a line cells (1 × 10 3 cells/well) were treated with compounds at concentrations of 1 and 10 µM during 1 h, after that 6-OHDA (Sigma-Aldrich, USA) at concentration of 50 µM was added in each well and neuroblastoma cells were cultivated during 24 h.After that, viability of cells was measured by MTT assay.The results were presented as percent of control data.

Reactive Oxygen Species (ROS) Level Analysis
Cell suspensions (1 × 10 3 cells/well) were incubated during 1 h with 1 and 10 µM solutions of the tested compounds.Non-treated and treated with 6-OHDA at concentration of 50 µM (Sigma-Aldrich, USA) cells were used as negative and positive controls, respectively.The portion (20 µL) of 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCF-DA) stock solution (Molecular Probes, Eugene, OR, USA) with concentration of 100 mM was added in each well and the microplate was incubated for an additional 10 min at 37 • C. The intensity of dichlorofluorescin fluorescence was measured at λ ex = 485 nm, and λ em = 518 nm with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany).The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany).In other experiments, cells were incubated with compounds during 1 h.Then, 6-OHDA (50 µM) was added in each well for 30 min and ROS levels were measured.All obtained results were presented as percent of negative control data.

Conclusions
A sponge Inflatella sp.contains a variety of oxysterols differing from each other in positions of additional hydroxy or oxo groups of their tetracyclic core and in structures of side chains.Fourteen oxidized sterols, including four previously unknown compounds, were isolated.Structures of new oxysterols have been established.Previously known compounds were structurally identified by comparison of their NMR and MS spectra with those reported in literature.All of the obtained compounds were studied in the 6-hydroxydopamine-induced cell model of Parkinson's disease.At least, one new oxysterol, (22E)-24-nor-cholesta-5,22-diene-3β,7β-diol (4), showed a substantial activity in this test and might be used for the further studies as a drug candidate.Supplementary Materials: 1 H and 13 C spectra of new compounds 1-4 are available online at http://www.mdpi.com/1660-3397/16/11/458/s1, Figure S1: Experimental Section, Figure S2: HRESI MS Spectra (Positive Ion Mode) of compounds 1-4 in CDCl 3 , Figure S3: 1 H NMR (500.13MHz) spectrum of 1 in CDCl 3 , Figure S4: 13  Funding: This work was supported by the Grant No. 17-14-01065 from the RSF (Russian Science Foundation).

Figure 2 .
Figure 2. Compounds 3, 6, 10 and 11: (a) Caused a statistically significant overestimation of MTT reduction in MTT cell viability assay; (b) did not statistically significant affect activity of nonspecific esterase in fluorescein diacetate cell viability test.* Statistically significant differences (p ≤ 0.05) between results for control cells and cells incubated with these compounds.

Figure 3 .
Figure 3. Influence of compounds 3, 4 and 11 in 6-OHDA-treated Neuro2a cells: (a) on cell viability; (b) on ROS formation.* Statistically significant differences (p ≤ 0.05) between results for 6-OHDA-treated cells and cells incubated with compounds.As a result, compound 3 increased the viability of 6-OHDA-treated cells by 18% (at the dose of 10 µM) and 22% (1 µM), while compounds 4 and 11 increased cell viability by 28% (10 µM) and 18% (10 µM), correspondingly.All these compounds decreased ROS formation in 6-OHDA-treated cells to normal value in similar manner.Thus, compound 4 exhibits the essential neuroprotective activity in 6-OHDA-induced model of Parkinson's disease, probably due to ROS scavenging effect.Oxysterols 3, 6, 10 and 11 may positively influence on metabolic processes in the Neuro2a cells because they show the overestimation of survival in MTT assay.