Modulation of the Soluble Epoxide Hydrolase Enzyme As a Therapeutic Target Against Alzheimer’S Disease

Modulation of the soluble Epoxide Hydrolase enzyme as a therapeutic target against Alzheimer’s Disease

Lidia Almenara Fuentes 53877833B

Master of Biomedicine

Master student Supervisors

Lidia Almenara Dra. Coral Sanfeliu Dr. Rubén Corpas

Aging and Neurodegeneration Research Group Institut d’Investigacions Biomèdiques de Barcelona Academic course: 2017-2018 BARCELONA, July 2018

The master’s thesis of Lidia Almenara Fuentes:

- [email protected] - 626964598

Modulation of the soluble Epoxide Hydrolase enzyme as a therapeutic target against Alzheimer’s Disease

Was performed in:

Institut d’Investigacions Biomèdiques de Barcelona (IIBB) –CSIC

Supervised by:

Dr. Coral Sanfeliu and Dr. Rubén Corpas from IIBB-CSIC and IDIBAPS

Funding resources:

SAF2016-77703-C2-2-R of the Ministerio de Economía y Competitividad, Spain and the European Regional Development Fund (ERDF); AGAUR 2017-SGR-106 and the CERCA Programme of the Generalitat de Catalunya; R. Copas and C. Sanfeliu belong to Group 05 of CIBER Epidemiología y Salud Pública (CIBERESP) of the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain.

ACKNOWLEDGEMENTS

First, I would like to thank Dr. Coral Sanfeliu for giving me the opportunity to work in the Neurodegeneration and Aging laboratory and opened me the door of research into Alzheimer’s disease. I appreciate your advices, your help and all the effort and time you have dedicated me

Also, thanks to Dr. Rubén Corpas for having taught me everything with patience and to guided me throughout the project.

And finally, thanks to Elisa García, Alaó Gatius and Júlia Senserrich for the laughs, the cafes and the great moral support you have given me.

I have learned a lot from all of you, thanks for everything!

III

ABSTRACT

Alzheimer’s Disease (AD) represents a serious problem for the population health due to the increase in the life expectancy. Inflammation in AD is widely present and it is considered as a contributor to the cognitive loss and neurodegeneration. Epoxyeicosatrienoic acids (EETs) have anti-inflammatory properties which disappear when the enzyme soluble Epoxide Hydrolase (sEH) metabolizes them to the corresponding dihydroxyeicosatrienoic acids (DHETs). SEH is emerging as a promising pharmacological target because allows the increase of EETs and keep them active. The aim of the present study was to calculate the half maximal inhibitory concentration (IC50) of a newly synthetized sEH inhibitors (UB21, UB23, UB24, UB28, EPB50 and EV52), study their toxicity and the anti-inflammatory effects. SH-SY5Y cell cultures were used to the IC50 calculation and the toxicity of each sEHi. To study the inflammatory effects, lipopolysaccharide (LPS) stimulated microglial, BV2 cells were used to obtain conditioned media with the proinflammatory mediators. Modulation of the inflammatory pathways iNOS and NFĸB, and the release of proinflammatory cytokines were analyzed by techniques of Western Blot, immunocytochemistry and ELISA. All agents showed a higher inhibitory potency than the reference compound 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), being the IC50 of UB23 and UB28 at nanomolar range. EV52 could not been tested due to their random activity values. All sEHi were not cytotoxic at the anticipated concentrations, except EV52. The preliminary testing of anti-inflammatory effects unveils UB23 as the best candidate.

Keywords: Soluble epoxide hydrolase, inhibitor, epoxyeicosatrienoic acids (EET’s), neuroinflammation

ABBREVIATIONS

AD Alzheimer’s Disease AEPU 1-Adamantanyl-3-{5-[2-(2-ethoxyethoxy) ethoxy] pentyl]} urea APAU 1-(1-acetypiperidin-4-yl)-3-adamantanyl urea APP Amyloid Precursor Protein ARA Arachidonic acid AUDA 12-(3 Adamantan-1-yl-UreidodoDecanoic Acid Aβ Amyloid-β CNS Central Nervous System COX-2 Cycllooxygenase-2 CYP Cytochrome P450 DHET Dihydroxyeicosatrienoic acid DMSO Dimethylsulfoxide EET Epoxyeicosatrienoic acid EpFA Epoxy-fatty acids EPHX2 Epoxide hydrolase 2 FBS Fetal Bovine Serum FDA Fluorescein diacetate IC50 Half maximal inhibitory concentration ICAM-1 Intercellular adhesion molecule 1 IKK IĸB kinase IL-1β Interleukin 1β iNOS Calcium insensitive nitric oxide synthase LOX-5 Lipoxygenase-5 LPS Lipopolysaccharide MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NFĸB Nuclear factor kappa-light-chain-enhancer of activated B cells NO Nitric oxide O/N Overnight

PGE2 Prostaglandin E2 PI Propidium iodide PPAR Peroxisome proliferator activated receptor PVDF PolyVinylidene DiFluoride SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gels Electrophoresis sEH Soluble Epoxide Hydrolase sEHi Soluble epoxide hydrolase inhibitor STAT3 Transducer and activator of transcription 3 t-AUCB Trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid TNF-α Tumor necrosis factor α TPPU 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea VCAM-1 Vascular cell adhesion molecule 1

VII

VIII

INDEX Acknowledgements ...... III Abstract ...... V Abbreviations...... VII 1. Introduction ...... 1 1.1. Alzheimer’s Disease ...... 1 1.1.1. Neuroinflammation in Alzheimer’s Disease ...... 1 1.2. Soluble Epoxide Hydrolase ...... 2 1.2.1. Characteristics ...... 2 1.2.2. Physiological role ...... 2 1.2.3. EET’s functions ...... 3 1.3. The potential of soluble Epoxide Hydrolase inhibitors ...... 4 1.3.1. SEH as a target for inflammatory diseases ...... 4 1.3.2. SEH as a target for Alzheimer’s Disease ...... 5 2. Background and objectives ...... 6 3. Materials and methods ...... 7 3.1. Cell culture ...... 7 3.2. Chemicals ...... 7 3.3. Western Blotting ...... 7

3.4. Soluble Epoxide Hydrolase activity and IC50 calculation ...... 8 3.5. Cell mortality and viability...... 8 3.6. Microglia conditioned medium preparation and treatment ...... 9 3.7. Griess assay ...... 9 3.8. ELISA analyses ...... 9 3.9. Immunofluorescence staining ...... 9 3.10. Co-culture cell viability ...... 9 3.11. Statistical analysis ...... 10 4. Results ...... 10 4.1. Quantification of sEH levels in cell lines ...... 10

4.2. SEH activity and IC50 calculation ...... 10 4.3. SEHi safety in SH-SY5Y cells ...... 11 4.4. Inflammation assays ...... 13 4.4.1. Microglia conditioned medium and SH-SY5Y cell viability ...... 13 4.4.2. Nitric oxide release by microglia cell line ...... 14 4.4.3. Detection of TNF-α release ...... 15 4.4.4. Quantification of iNOS and IL-1β proteins ...... 15

4.4.5. Quantification and immunodetection of NFĸB ...... 16 4.4.6. SEHi protective role in co-cultures ...... 17 5. Discussion ...... 18 6. Conclusions ...... 20 7. Bibliography ...... 21

1. INTRODUCTION

1.1. Alzheimer’s Disease Dementia is an emerging global public health challenge for our generation: over 46.8 million people are affected by this condition worldwide, and that number is expected to double by 2030 and more than triple by 2050. (Prince et al., 2016). Alzheimer’s disease (AD) is the most common form of dementia, accounting for about 60% of cases. AD is a fatal neurodegenerative disorder that is characterized by progressive cognitive and functional impairment and memory loss (Kumar et al., 2015).

Primary brain hallmarks of AD pathology include senile plaques of fibrillary amyloid beta peptides (Aβ), which are extracellular deposits derived from the β-amyloid precursor protein (APP), and neurofibrillary tangles, which are primarily composed of hyperphosphorylated tau (Hardy & Selkoe, 2002). Other hallmarks of AD include the presence of reactive gliosis and neuroinflammation, oxidative damage, metabolism alterations, synaptic loss and neuronal apoptosis. The etiology of the major AD cases is unknown (sporadic AD), but it encompasses many genetic and environmental risk factors that jointly with age-related frailty causes the upregulation of multiple AD pathogenic pathways. Only small percentage of AD cases show early-onset (<5%) and some of them (<1%) inherit a monogenic autosomal dominant mutation causing AD (Alva & Potkin, 2003).

1.1.1. Neuroinflammation in Alzheimer’s Disease For more than a decade, there have been data indicating that the immune system may have a role in AD. However, the importance of inflammation to AD pathogenesis has only very recently been appreciated, and inflammation is now thought to contribute to and exacerbate AD pathology (Krstic & Knuesel, 2013; Sudduth et al., 2013).

The inflammatory reaction observed in AD is driven primarily by central nervous system (CNS)-resident immune cells (microglia, perivascular myeloid cells and other reactive elements such as astrocytes). Microglia are cells derived from primitive myeloid precursors in the yolk sac, and its functions include survey the brain for pathogens and support CNS homeostasis and plasticity (Prinz & Priller, 2014). It has been demonstrated the dual roles of microglia in the pathogenesis of AD. On the one hand, microglia play a beneficial role in generating anti-Aβ antibodies and stimulating the clearance of amyloid plaques. On the other hand, microglia is involved in AD pathogenesis by releasing inflammatory mediators such as inflammatory cytokines, complement components, chemokines, and free radicals that are all known to contribute to β-amyloid production and accumulation (Cai et al., 2014). This reaction is driven when soluble Aβ oligomers and Aβ fibrils bind to various receptors that express microglia, including CD14, CD36, CD47 and toll like receptors (TLRs) (Patel et al., 2005). The interaction makes microglia susceptible to a secondary stimulus and/or promotes their activation, sustaining chronic activation. Finally, is produced a microglia impairment that affects surrounding CNS resident cells, possibly aggravating tau pathology and causing neurodegeneration and neuron loss. If these processes perpetuate over a prolonged period, it

1 forces microglia into a senescent, ‘burn-out’-like (dystrophic) phenotype, which is thought to be irreversible (Heppner et al., 2015).

Activation of the immune system in AD can act perpetuating and accelerating the course of the disease. Although, it is not generally thought to be the trigger of the disease process, it cannot be excluded that immune actions may also have an important role in initiating the disease process.

1.2. Soluble Epoxide Hydrolase 1.2.1. Characteristics Human soluble Epoxide Hydrolase (sEH) is a 62,5 kDa enzyme composed of two domains separated by a short proline-rich linker (Newman et al., 2005). In the intracellular environment, sEH is presented as an antiparallel homodimer. Each monomer has two domains, the N-terminal domain that exhibits a Mg+2 dependent phosphatase activity that hydrolyzes lipid phosphates and the C-terminal domain with an epoxide hydrolase activity that converts epoxides to their corresponding diols. The catalytic mechanism has two steps (Figure 1). First, there is an attack of nucleophilic aspartic acid on the epoxide carbon to give a stable covalent intermediate. Next step consists in a hydrolysis by activated water to give a diol product. The two domains function independently of one another, and inhibition of one activity does not affect the function of the other (Morisseau & Hammock, 2013).

Figure 1. Catalytic mechanism of soluble Epoxide Hydrolase (sEH).

The sEH is widely distributed throughout the body, but it is in high concentration in the liver, kidney, intestine and vasculature in mammals (Morisseau and Hammock, 2008; Enayetallah et al., 2004). In addition, it is implicated in important physiological processes in the brain, lung, and other organs (Morisseau & Hammock, 2013). Certainly, in hindsight, its tissue distribution can be rationalized with a role in the regulation of blood pressure and inflammation, and increasingly with analgesia and neural function. On the subcellular fraction, sHE is localized in the cytosolic or soluble fraction and also can be found in the peroxisomes (Morisseau & Hammock, 2008).

1.2.2. Physiological role One of the principal functions of the sEH is to hydrolyze epoxy-fatty acids (EpFAs) such as epoxyeicosatrienoic acids (EET’s) to their corresponding dihydroxyeicosatrienoic acids (DHETs) (Spector & Norris, 2007). EETs are autocrine and paracrine lipid signalling molecules produced from arachidonic acid (ARA) through the epoxidation of the double bonds by cytochrome P450 enzymes (CYPs) (Konkel & Schunck, 2011). These enzymes are in the endoplasmic reticulum, and they use ARA hydrolyzed from phospholipids when the Ca+2 dependent type IV phospholipase

A2 is activated and translocated from the cytosol to intracellular membranes. The CYP epoxygenases add the oxygen atom across the double bond of ARA producing four EET regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, each with somewhat different properties and

2 functions (Figure 2). Each regioisomer represent two EET isomers, because the epoxide group can attach at each of the double bonds in two different configurations. Although these

Figure 2. Four EET regioisomers formed by CYP epoxygenases that add an oxygen atom across the double bond of ARA (Spector & Norris, 2007).

epoxygenases synthesize all four EETs, most of the enzymes produce mainly 11,12- and 14,15- EET (Fitzpatrick & Soberman, 2001).

EETs main metabolic pathways are three: the incorporation into phospholipids, phospholipase A2 catalyzed hydrolysis from phospholipids and the hydration to form the corresponding diol by sEH. Of the four EETs regioisomers, 14,15-EET possess the highest affinity for sEH, while increasing distance between the epoxide ring and the methyl-terminal carbon reduces the affinity for sEH (Fang et al., 2001).

1.2.3. EET’s functions EETs are implicated in critical biological processes throughout the body in most cells, tissues and organs (Funk, 2001). The initial mechanistic steps that mediate these effects remain uncertain. One possibility is that EETs bind to a membrane receptor linked to an intracellular signal transduction pathway that regulates ion channels or gene expression, producing a change in cell properties and function. The other is an intracellular mechanism in which EETs directly interact inside the cell with an active ion channels, signal transduction components or transcription factors to produce functional responses (Spector & Norris, 2007).

The final actions can be very diverse, EETs produce vasodilatation, angiogenic, fibrinolytic, and Ca+2 -signalling effects, but one of the most relevant is the anti-inflammatory effects (Figure 3). The molecular mechanisms of the EETs regioisomers to reduce the inflammation consist in the inhibition of VCAM-1, E-selectin and ICAM-1 expression in endothelial cells which blocks the adherence and infiltration of activated monocytes. EETs also decrease the tumor necrosis factor alpha (TNFα) from monocytic cells and may also inhibit their adherence (Node et al., 1999). EETs also reduce inflammation by blocking the nuclear translocation of the nuclear factor (NFĸB), by the inhibition of IĸB kinase (IKK). This results in the downregulation of several enzymes including calcium-insensitive nitric oxide synthase (iNOS), lipoxygenase-5 (LOX-5), and cycllooxygenase-2 (COX-2) that are upregulated in inflammation. The downregulation of COX-2 also limits the production of prostaglandin E2 (PGE2) which is a potent inflammatory agent (Schmelzer et al., 2005). Activation of signal transducer and activator of transcription 3 (STAT3) and other nuclear

3 receptors such as peroxisome proliferator activated receptor (PPAR) alpha and gamma are additional anti-inflammatory mechanisms that have been described for EETs in blocking downstream inflammation (Ng et al., 2007).

Figure 3. EET’s block inflammation through several mechanisms (Wagner et al., 2017)

1.3. The potential of soluble Epoxide Hydrolase inhibitors 1.3.1. SEH as a target for inflammatory diseases Many of these potentially beneficial actions of the bioactivity of EETs are attenuated when they are converted to DHETs because they lack the activity of the epoxidized precursors. Also, DHETs are less lipophilic, move rapidly out of the site of action, and are easily conjugated and excreted (Greene et al., 2000)(Morisseau & Hammock, 2013). Targeting the sHE is a novel strategy because its inhibition causes the accumulation and retention for long periods of EETs and helps to attain their biological effects. In addition, is generally assumed that the inhibition of the DHET formation should not impair any vital physiological process (Spector & Norris, 2007).

Because of this, molecule inhibitors of sEH (sEHi) have become as a promising pharmacological target to altering disease pathologies including cardiovascular diseases, inflammation, neurodegenerative disorders and chronic pain among others (Wagner et al., 2017). Several orally bioavailable sEHi have been identified and pharmacologically characterized in vitro, but only a few inhibitors have reached clinical trials (Table 1).

Table 1. Commonly used sEH inhibitors.

Reached the clinical Phase IIa, but now is used as an experimental sEH reference AUDA inhibitor. It has a moderate potency on sEH from a wide variety of species but has a short half-life (Simpkins et al., 2009).

TPPU is a potent sEHi commonly used as an experimental reference because has high activity on the primate sEH and good activity with rodent sEH. It has a high TPPU oral availability and good PK-ADME. The negative point is that has lipophilic and high melting properties so requires a long dissolution time and careful formulation (Inceoglu et al., 2013).

It is in human Phase I and II trials and showed no toxicity at high doses. Also, it APAU has high water solubility and rapid dissolution despite being less active in primate sEH than in rodent sEH (Chen et al., 2012).

It has a good potency on sEH from a variety of species. PK-ADME known in t-AUCB multiple species and half-life shorter than TPPU. Good water solubility off-sets lower potency than TPPU (Sung et al., 2007).

Moderate potency on sEH and short half-life. However, it has a very water AEPU solubility (Sung et al., 2007).

Abbreviations: AUDA, 12-(3 adamantan-1-yl-ureidododecanoic acid; TPPU, 1-Trifluoromethoxyphenyl-3-(1- propionylpiperidin-4-yl) urea; APAU, 1-(1-Acetypiperidin-4-yl)-3-adamantanylurea; t-AUCB, trans-4-[4-(3- Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid; AEPU, 1-Adamantanyl-3-{5-[2-(2-ethoxyethoxy) ethoxy] pentyl]} urea.

1.3.2. SEH as a target for Alzheimer’s Disease Nowadays, the incidence of neurodegenerative diseases and chronic inflammatory conditions are both increasing. Therefore, a new approach to ameliorate neuroinflammation is needed. EETs have the role to regulate inflammatory conditions and will be a potential target beside sEHi to improve their action in brain (Wagner et al., 2017). Studies in neurodegenerative processes are yet scarce, but the whole sum of evidences is very convincing of the neuroprotective potential of sEH inhibition to treat neurodegenerative diseases with an inflammatory component.

There is an emerging a board involvement of EETs signalling in the CNS function and disease, where EETs play additional functions than in the peripheral tissues. They modulate specific processes for neural and glial cells and the cross-talk between the different cell types (Iliff et al., 2010). For instance, 14, 15-EET promoted the production of brain derived neurotrophic factor (BDNF) from astrocytes, exerting neuroprotection during ischemic injury (Guo et al., 2016). SEH immunoreactivity has been shown in neuronal cell bodies and processes, and to a lesser degree in cerebral blood vessels. However, immunoreactivity of sEH was found enhanced in microvessels of affected areas in vascular cognitive impairment suggesting a possible link of vascular dementia with reduced EET signalling (Nelson et al., 2014). Axonal localization of sEH suggested a regulation of 14, 15-EET induced axon growth by this enzyme and therefore its

5 inhibition would promote axonal regeneration. Noteworthy, sEH inhibition induced an enhancement of synaptic plasticity and neurotransmission as measured by long-term potentiation response and glutamate receptor expression in brain slices (Wu et al., 2017). Furthermore, has been shown that β-amyloid plaques inhibited EET generation in rat microsomes form the brain areas more sensitive to AD neurodegeneration whereas EETs supplementation prevented Aβ induced mitochondrial dysfunction in cultures of astrocytes (Sarkar et al., 2014).

All these studies suggest a neuroprotective role of EETs against AD neurodegeneration and warrant the investigation of pharmacological approaches with last generation of specific sEH inhibitors in validated AD experimental models.

2. BACKGROUND AND OBJECTIVES

Currently neurodegenerative diseases represent a serious problem for the population due to the increase in the life expectancy. Inflammation in these diseases is present and it is considered as a contributor to the cognitive loss and AD neurodegeneration. Despite intensive research in multiple models, no clinically effective pharmacological treatments have been found yet for the cure. Therefore, the development of new anti-inflammatory pharmacological strategies could represent an advance in the treatment of AD.

The enzyme sEH is emerging as a promising pharmacological target for neuroprotection. Keeping in mind that endogenous anti-inflammatory EETs are metabolized and consequently inactivated by the sEH, we hypothesize that inhibiting the enzyme will increase EET levels and keep them active. In this way, the beneficial effects of EETs will be maintained leading a reduction of the neuroinflammation. A pilot experiment had confirmed that sEH inhibitor UC1770 has beneficial effects against cognitive impairment in AD mice models (SAMP8). Also, sEHi reduced p-tau in the hippocampus of treated mice. Regarding oxidative stress and inflammation, sEHi reduced COX-2 and TNF-α levels. Considering the positive outcome of the pilot study, a series of new sEHi had been rational designed and synthesized with several drug- like properties: high potency, BB-permeability, non-cytotoxicity, and better solubility and lower melting points than known sEHi.

In that context, the principal objectives of this project are:

1. Confirm the suitability of the neuroblastoma SH-SY5Y for studies of sEH.

2. Determine the half maximal inhibitory concentration (IC50) of the series of newly synthesized sEHi in SH-SY5Y cells and the most suitable one for later use. 3. Test the safety of the analyzed sEHi in SH-SY5Y cells at a range of concentration from

IC50 up to that reaching maximum inhibition. 4. Analyze the anti-inflammatory potency of the analyzed sEHi in an in vitro system composed of microglia-like BV2 and SH-SY5Y cells.

3. MATERIALS AND METHODS

3.1. Cell culture A human neuroblastoma cell line SH-SY5Y (ATCC Number: CRL-2266) was obtained and cultured in a mixture of 50% Minimum Essential Medium (Biowest, Riverside, MO, USA), 50% Ham’s-F12 medium (Biowest), 10% fetal bovine serum (FBS), 1% L-glutamine, 1% MEM non- essential amino acids and 0.1% of gentamicin. In addition, a murine microglia cell line (BV2) was maintained in 90% RPMI medium (Sigma-Aldrich, St Louis, MO, USA) containing 10% FBS and 0.01% of gentamycin. The kidney embryonic cell line, HEK293, was cultured with 90% Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher, Waltham, MA, USA), 10% FBS, 100 units/mL penicillin-streptomycin and blasticidin. The human liver cancer cell line, HepG2, was cultured with 90% RPMI medium (Sigma-Aldrich), 10% FBS, 10mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (Hepes) and 100 units/mL of penicillin-streptomycin. All cells 2 were grown in 75 cm cell culture-treated flasks (37 °C, 5% CO2), and passaged before the confluence.

3.2. Chemicals The sEHi compounds to study were provided to the group by Carlos Galdeano’s group, specialized in clinical chemistry, from the University of Barcelona. A total of six compounds selected from different structure families were used (UB21, UB23, UB24, UB28, EPB50 and EV52). TPPU, that is well stablished as an sEHi (Inceoglu et al., 2013), was used as the control to compare with the rest of them. Every sEHi was dissolved in Dimethylsulfoxide (DMSO) (Life technologies, Carlsbad, CA, USA) and freshly diluted with correspondent medium to the final concentration to perform the assays (0.01% DMSO).

3.3. Western Blotting After 24 hours incubation, cells were lysed using RIPA lysis buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisher) with protease and phosphatase inhibitors. Protein concentrations were determined by the colorimetric Bradford assay, according to manufacturer’s protocol (Bio-Rad, Hercules, CA, USA), and absorbances measured in the Multiskan Spectrum Spectrophotometer (ThermoFisher) at 595 nm. A total of 60 μg protein was fractionated on 10 or 15% Sodium Dodecyl Sulphate-Polyacrylamide Gels Electrophoresis (SDS-PAGE) and transferred to PolyVinylidene DiFluoride membranes (PVDF) (Millipore, Burlington, MA, USA).

Membranes were blocked with 5% non-fat milk in TBS-T buffer (Tris-buffered saline containing 0.1% Tween 20) and then primary antibody incubation overnight (O/N) at 4°C. Western blotting was performed with antibodies mentioned in Table 2. After incubation with corresponding HRP-conjugated IgG secondary antibody (1:2000), protein bands were visualized using enhanced chemiluminescence ECL system (GE Healthcare, UK). The signal was captured and digitalized with VersaDoc Imaging System 500 (Bio-Rad). Image Lab Software (Bio-Rad) was

7 used to quantify the pixel intensities of immunoreactive bands. Protein was normalized relative to actin or β-tubulin.

Table 2. List of primary antibodies used in Western Blot.

Antibody Dilution Reference Host Incubation

EPHX2 1:1000 Abcam Rabbit O/N, 4°C iNOS 1:4000 BD Transduction Laboratories Mouse O/N, 4°C NFĸB 1:1000 Rockland Rabbit O/N, 4°C IL-1β 1:1000 Cell Signalling Mouse O/N, 4°C Actin 1:10000 Sigma Rabbit 1 hour, RT β-Tubulin 1:10000 Sigma Mouse 1 hour, RT Note: O/N, Overnight; RT, Room Temperature.

3.4. Soluble Epoxide Hydrolase activity and IC50 calculation For determining the sEH inhibitory activity of every compound, SH-SY5Y cells were seeded at the concentration of 3 × 105 cells/mL per well on the 96-well plates and cultured for 24 hours. Then the medium of each well was replaced by 100 μL of the corresponding concentration of treatment. Appropriate DMSO control wells were included for all experiments. After 24 hours of incubation the treated cells were processed as is indicated in the Soluble Epoxide Hydrolase Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) and were read in the Gemini XPS Microplate reader (Molecular Devices, San Jose, CA, USA). AUDA was used as a sEHi control as the kit indicates. To normalize the levels of activity the protein levels were digested with NaOH and measured by Bradford assay. Finally, when the levels of sEH activity at different concentrations of the compounds were determined, the IC50 (concentration required to produce 50% inhibition) of every compound was calculated by regression of at least four datum points, with a minimum of two points in the linear region of the curve using GraphPad 7.0 (GraphPad Software, Inc, La Jolla, CA).

3.5. Cell mortality and viability Propidium Iodide (PI) assay to test cytotoxicity and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay to test cell growth and viability, were performed under the effects of the sEHi. First, the cells were seeded in 96-well plate with a concentration of 3·105 cells/mL. After 24 hours, the compounds were incubated for further 24 hours. At termination, 5 µL of PI was added to each well and incubated for one hour. The PI only can cross the cellular membrane of death cells, so the fluorescence measured by Gemini XPS Microplate reader (Molecular devices) gave the relative number of cell death.

After that, 10 µL of the MTT reagent was added to each well and incubated for 2 hours. 100 µL of lysis buffer was also added and incubated at 37°C overnight (O/N). This permit to solubilize the formazan crystals derived from MTT generated by the living cells. This assay gave us a colorimetric reaction assessing cell metabolic activity. Finally, the plates were read in Multiskan Spectrum Spectrophotometer (ThermoFisher) at 570 nm and a reference 630 nm wavelength.

3.6. Microglia conditioned medium preparation and treatment First, BV2 cells, after 24 hours seeded at 3·105 cells/mL, were treated with different concentrations of the sEHi compounds for 1 hour and then were stimulated with lipopolysaccharide (LPS) (E. coli: Serotype O11: B4; Sigma-Aldrich) at 0.1 and 1 µg/mL for 24 hours to produce inflammation reactions. The culture media was collected as a conditioned media that contains the proinflammatory cytokines generated by the microglia-like cells. Cellular debris was eliminated by centrifugation and supernatant was collected and immediately used. For that, SH-SY5Y cells had been seeded in a 96-well plate at density of 3·105 cells/mL 24 hours before. The freshly obtained conditioned media was added to the SH-SY5Y cells, which were incubated for 24 hours, and then the cell mortality and viability were measured by PI and MTT assay.

3.7. Griess assay Supernatants collected from the experiment above were collected and also assayed for oxide - (NO) release. Nitrite (NO2 ) is the terminal product of NO oxidation and can be measured in cell culture media via Griess assay to assess NO release from cells. The colorimetric reaction is achieved by mixing (1:1) the reagent A (1% sulfanilamide, 5% orthophosphoric acid and miliQ water) and the reagent B (0.1% N-( 1-napthyl)ethylenediamine dihydrochoride and miliQ water). Nitrite levels of each condition were determined using Multiskan Spectrum Spectrophotometer (ThermoFisher) at 540 nm.

3.8. ELISA analyses Supernatants collected from the experiment above were also analyzed for the presence of released pro-inflammatory cytokines. The levels of TNF-α in culture media were measured using the Mouse TNF-α ELISA kit (ThermoFisher), according to the manufacturer’s instructions.

3.9. Immunofluorescence staining The BV2 microglia cell line was seeded in a 48 well plate at 3·105 ells/mL and treated with the sEHi in the absence or presence of LPS (0.1 and 1 µg/mL). After 24 hours of incubation cells were fixed with methanol for 10 minutes. Next the blocking with Normal Goat Serum (NGS) at 3%, the primary antibody was added (NFĸB) at 1:1000 dilution, O/N at 4°C. Next step was the incubation with the secondary antibody Alexa Fluor 488 (1:1000). Finally, fluorescent images were captured by an Olympus IX-70 Inverted Fluorescence Microscope at 20x.

3.10. Co-culture cell viability The BV2 microglial cells were co-cultured with SH-SY5Y cell line to study the cytotoxicity of conditioned media from microglia on SH-SY5Y cells. The SH-SY5Y cells were seeded in a 96-well plate at concentration of 3·105 cells/mL 24 hours before the addition of BV2 cells at 3·105

9 cells/mL in each well. After 24 hours, cells where treated with the sEHi compounds and incubated for 24 hours. Next step was the fluorescein diacetate (FDA) (Sigma-Aldrich) assay that was performed in combination with PI to determine viability and mortality cell cultures. The FDA stains viable cells because they take up the non-fluorescent FDA and the intracellular esterases transform it into the green fluorescent metabolite. After 5 minutes incubation, the pictures were taken in an Olympus IX-70 Inverted Fluorescence Microscope at 10x.

3.11. Statistical analysis Results are expressed as the mean ± SEM. Significance of difference between two groups was determined by t-test. For multiple comparisons, one-way or two-way analysis of variance (ANOVA) was used. Turkey’s test was used for pot-hoc comparison between groups. Data were analyzed using GraphPad Prism 7.0 (GraphPad Software, Inc, La Jolla, CA). Differences were considered significant when p<0.05. Experiments were performed in several replicate wells and repeated with cultures of 3-5 different passages, otherwise results were considered preliminary.

4. RESULTS

4.1. Quantification of sEH levels in cell lines It is well established the presence of active sEH is at high levels in kidney and liver cells (Morisseau & Hammock, 2008), and it has been previously tested in vitro in the cell lines HEK295 and HepG2. To know if sEH levels in the cell line to work (SH-SY5Y) was enough, a comparison of the relative levels of enzyme was performed by a Western Blot. As expected, SH-SY5Y cell line has similar protein levels of sEH as HEK295 and slightly lower respect HepG2 cell line (Figure 4). No significative variations were found between the three cell lines.

Figure 4. SH-SY5Y cells has similar protein levels as HEK295 cell line and slightly lower respect to HepG2 cell line. Results of the protein levels quantification of the three cell lines by Western Blot. Statistical analysis was performed with one-way ANOVA. n=3

4.2. SEH activity and IC50 calculation In order to evaluate the sEH inhibition activity of the six candidate compounds, we used the kit Soluble Epoxide Hydrolase Cell-Based Assay Kit (Cayman chemical) that measures the product formation during the reaction. Non-linear regression curves were calculated from a range of concentrations in independent experiments. Results of the inhibitory activity were summarized in Figure 5.

Figure 5. Non-linear regression curves of each sEHi. Simulated (lines) and observed values (points) for sEH % activity after preincubation cells with sEHi compounds are represented. Non-linear regression was performed. n=3, in triplicate wells.

The IC50 of each compound was calculated as a mean from the IC50 of each curve (Table 4). We found that 3 compounds (TPPU, UB21 and UB24) exhibited potent sEH inhibitory activity, with IC50<10 µM. Among them, compound EPB50 exhibited good inhibitory activity at 339nM, while compounds UB23 and UB28 demonstrated excellent sEH inhibition activity, with IC50<

5nM. The compound EV52 exhibited random values, for this reason the IC50 can not be calculated. Values of EV52 are not shown. UB23, UB24 and UB28 showed lower solubility in the culture media and formed microscopically visible crystals into the SH-SY5Y culture at concentration of 100 µM.

Table 4. IC50 values of each sEHi.

TPPU UB21 UB23 UB24 UB28 EPB50

Assay 1 11.000 2.377 0.0059 3.813 0.0049 0.419 Assay 2 9.146 2.613 0.0023 8.931 0.0051 0.156 Assay 3 7.722 1.881 0.0008 2.323 0.0006 0.443 Mean (µM) 9.2893 2.2903 0.0030 5.0223 0.0035 0.3392 SEM 1.6437 0.3736 0.0026 3.4660 0.0026 0.1587

Note: The IC50 values were calculated as a mean ± SEM from the corresponding IC50 curves for each compound. n=3 4.3. SEHi safety in SH-SY5Y cells To determine the compound safety of each sEHi PI and MTT assays were performed. The IP results (Figure 6) showed that EV52 sEHi has clearly toxic properties to SH-SY5Y cells from 30- 50 µM concentrations, even at 100 µM, induced more than 40% of cell death (p<0.001). Additionally, in the Figure 6 (H) we observe that cell death increased proportionally to the EV52 concentration, triton at 0.04% was considered the 100% of cell death. Interestingly, UB24 also showed a significant increasing in cell death at high concentrations (p<0.001) and UB21 at 100 µM showed a significative increase (p<0.005). The rest of the sEHi compounds did not show significative changes.

Figure 6. PI assay at treated SH-SY5Y cells with each sEHi compound. (A-G) Graphic representation of % cell death at different concentrations of sEHi. (H) PI assay images of SH-SY5Y cells treated with different concentrations of EV52 (representative data). Pictures were taken with Olympus IX-70 Inverted Fluorescence Microscope at 10x. Statistical analysis was performed with one-way ANOVA, effect of sEHi concentration ***p<0.0005 and ****p<0.0001. n=5-20 from 3-5 independent experiments.

MTT assay results in Figure 7 show that at high concentrations of EV52 cell survival suffered a significant decrease in cell survival (p<0.001), which correlates with the increase of cell death measured with PI assay. This correlation can also be seen in Figure 7 (G) where the reduction of cell survival at high concentrations of UB24 was significantly reduced (p<0.001). The others sEHi compounds did not produce any significant variation.

Figure 7. MTT assay at treated SH-SY5Y cells with each sEHi compound. Graphic representation of % cell survival at different concentrations of sEHi. Statistical analysis was performed with one-way ANOVA, effect of sEHi concentration *p<0.05, ***p<0.0005 and ****p<0.0001. n=5-20 from 3-5 independent experiments.

4.4. Inflammation assays In order to investigate a possible anti-inflammatory effect of sEHi compounds against the neuroinflammation produced by microglia, different assays were performed with TPPU, as a

control of the group, and UB23 and UB28 selected because of the excellent IC50 and the absence of toxicity. 4.4.1. Microglia conditioned medium and SH-SY5Y cell viability Secreted proinflammatory mediators form activated microglia could induce neurotoxic effects in diverse neurodegenerative disease such as AD. We investigated whether sEHi compounds attenuated activated microglia-induced neurotoxicity. BV2 cells were treated with TPPU (10 and 100 µM) or the selected sEHi for 2 hours. Then, LPS (0.1 and 1 µg/mL) was added and the incubation was maintained for 24 hours in the presence of sEHi compounds or the vehicle (DMSO 0.01%). The culture media of BV2 cells as conditioned media was collected and added to SH-SY5Y cells. PI and MTT assay of SH-SY5Y cells under the effects of the conditioned media were performed. Values were processed, and two-way ANOVA statistical analysis was done to discern effect of factors LPS and sEHi agents. Effects of LPS were marginal indicating a lack of neurotoxicity by the conditioned media, although some statistical effects were detected, always under 10% of cell death. PI assay on cells with TPPU treated BV2 cells conditioned media demonstrated that there is an LPS dose cell death signification (p<0.001) (Figure 8).

Figure 8. PI assay at SH-SY5Y cells with sEHi treated BV2 cells conditioned media. Graphic representation of % cell death at different concentrations of LPS and sEHi. Statistical analysis was performed with two-way ANOVA, effect of LPS dose *p<0.05 and **p<0.01 in TPPU, n=10 from 3 independent experiments; results were preliminary for UB23 and UB28, 4-5 replicate wells in one experiment (no statistics performed).

In Figure 9, MTT assay revealed that viability SH-SY5Y cells under the TPPU treated BV2 conditioned media has an LPS dose effect (p<0.01) and a significant TPPU dose effect (p<0.05). Cells under 0.1 µM UB23 treatment had a significant (p<0.005) increment in cell survival respect to the control (0µg/mL LPS dose) but not significant variations between LPS doses. UB28 showed any relevant variation in LPS or sEHi dose. Conditioned media with LPS in the presence of TPPU, UB23 and UB28 showed no neurotoxic protective effects.

Figure 9. MTT assay at SH-SY5Y cells with sEHi treated BV2 cells conditioned media. Graphic representation of % cell survival at different concentrations of LPS and sEHi. Statistical analysis was performed with two-way ANOVA. n=2-3

4.4.2. Nitric oxide release by microglia cell line Activated microglia play a central role in neuroinflammation by secreting various neurotoxic factors, such as NO. Therefore, we investigate the possibility that the sEHi compounds could reduce NO production in LPS-induced microglial cells with Griess assay. Microglial cells were treated for 2 hours with different concentrations of TPPU (10 and 100 µM), as the reference agent, and UB23 and UB28 (1 and 10 µM) previously to the addition of LPS. Levels of NO were determined in the culture media after 24 hours incubation with LPS (0.1 and 1 µg/mL) in the presence or absence of sEHi compounds. Results showed that, in all conditions, at LPS 0.1 µg/mL concentrations the levels of NO were significantly lower (p<0.001) than at LPS 1 µg/mL showing the progression of the inflammatory response to LPS (Figure 10). It was also observed that TPPU had no effects decreasing NO release at both concentrations, being the tendency to increase. In contrast, UB23 at 1 µM showed a significant decrease in NO levels when cells were stimulated with 1 µg/mL LPS (p<0.01) and the same tendency with the 10 µM dose. Results also showed a significant effect of the UB28 dose at 1 µM (p<0.01).

Figure 10. Nitrite release in treated BV2 cells conditioned media measured by Griess assay. Graphic representation of NaNO2 (µM) at different concentrations of LPS and sEHi. Statistical analysis was performed with two-way ANOVA, effect of LPS dose *p<0.05, **p<0.01, ***p<0.005 and ****p<0.001; effect of sEHi concentration $p<0.05. n=3

4.4.3. Detection of TNF-α release Because TNF-α is a representative proinflammatory factor produced by microglia cells, we measured the levels of TNF-α by using ELISA. The results showed, that with all sEHi, the secretion of TNF-α was significantly increased by stimulation with LPS (0.1 and 1 µg/mL) in comparison with the undetectable release in control media (Figure 11) (p<0.001). However, the cytokine release was not dose dependently inhibited by treatment with sEHi compounds, TPPU (10 and 100 µM) and UB28 (0.1 and 1 µM), showing minor changes. UB23 statistical analysis showed an sEHi dose effect (p<0.01).

Figure 11. TNF-α release levels in treated BV2 cells conditioned media measured by ELISA analyse. Graphic representation of TNF-α levels (pg/mL) at different concentrations of LPS and sEHi. Statistical analysis was performed with two-way ANOVA, effect of LPS dose *p<0.05 and **p<0.01. Preliminary results, n=2

4.4.4. Quantification of iNOS and IL-1β proteins NO production is mediated by iNOS, for this reason, we examined whether sEHi compounds had any effect on expression of iNOS protein. In Figure 12 (A) results showed that the protein expression levels of iNOS dose-dependently increases in the presence of any sEHi compared with that after treatment with LPS alone (p<0.005). TPPU showed a significant increase in relation with the non-treated cells, showing a significant variation of sEHi effect (p<0.005). However, focusing in the 0.1 µg/mL LPS dose, UB3 and UB28 seems to decrease the protein levels respect to the control. Interleukin 1β (IL-1β) is also a well-known proinflammatory cytokine (Horvath et al., 2008). The protein levels of the cleaved mature form were studied preliminary by immunodetermination. As is shown in Figure 12 (B), the expression of IL-1β tends to increase dependently to LPS dose in correlation with the inflammatory response without protective effect by the sEHi agents. The decrease tendency at 0.1 µg/mL LPS dose can also be observed in the three sEHi.

Figure 12. Relative protein levels of treated BV2 cells measured by Western Blot. (A) iNOS relative protein levels vary correlatively with LPS dose and there is a significant TPPU dependent increase. (B) IL-1β relative protein levels show a tendency to increase in correlation with LPS dose in each condition. Statistical analysis for iNOS was performed with two-way ANOVA, significance **p<0.01 and ***p<0.005. Preliminary results, n=2. No statistics were performed for IL-1β, n=1.

4.4.5. Quantification and immunodetection of NFĸB NFĸB signalling pathway is critical for LPS-stimulated microglial activation (Oh et al., 2010). NFĸB complexes in normal cells are retained in the cytoplasm by their inhibitors IƘBs. When an inflammatory stimulus arrives, activation of NFĸB, by the inhibitor degradation, results in the translocation to the nucleus (Perkins, 2007). The protein expression levels were measured by Western Blot (Figure 13). Immunodetection of NFĸB in the whole cells did not allowed to detect changes of relative protein levels after LPS proinflammatory injury, or in the treated BV2 cells with different sEHi.

Figure 13. Relative protein levels of NFĸB in treated BV2 cells measured by Western Blot. The protein levels do not change significantly. Statistical analysis was performed with two-way ANOVA. Preliminary results, n=2

Therefore, the effect of sEHi compounds on the translocation of NFĸB to the nucleus was examined by immunocytochemistry (Figure 14). The sEHi to test were also TPPU (100 µM), UB23 (10 µM) and UB28 (10 µM). Melatonin (500 µM) was added to the assay because of its immunomodulatory properties, which acts on immune system by regulating cytokine production of immunocompetent cells (Esposito & Cuzzocrea, 2010). In Figure 14 is shown that NFĸB translocated to the nucleus under the LPS dose effect in BV2 cells. LPS treated cells generally had the nucleus immunostained indicating the translocation NFĸB, whereas control

treated cells showed only immunostaining in the cytoplasm. That translocation was poorly prevented in BV2 cells treated with the sEHi, but with UB23, some cells with free nucleus are observed. As expected, under the melatonin effects the translocation was not produced even when the dose of LPS was high.

Figure 14. NFĸB staining of treated BV2 cells. First row represents the 0 µg/mL LPS dose, in all conditions NFĸB remains in cytoplasm. Second and third row are the 0.1 and 1 µg/mL LPS dose respectively. Each column represents the treatment: Control, TPPU (100 µM), UB23 (10 µM), UB28 (10 µM) and melatonin (500 µM). NFĸB migrates to the nucleus except with melatonin treatment. Pictures were taken with the Olympus IX-70 Inverted Fluorescence Microscope at 20x.

4.4.6. SEHi protective role in co-cultures Co-cultures were performed to study the inflammatory microenvironment of BV2 cells on SH-SY5Y cells. Because of cell mortality and viability wanted to be studied microscopically, a double staining assay with PI and FDA was performed. In Figure 15 is shown that LPS has a clear mortality dose effect on cells. Mortality under the UB23 effects seems to be slightly decreased, although more studies would be needed to confirm these preliminary results.

Figure 15. FDA/PI staining at co-cultures of SH-SY5Y and BV2 cells. FDA satins alive cells with green fluorescence and PI stains damaged cells with red fluorescence. First row represents the 0 µg/mL LPS dose. Second and third row are the 0.1 and 1 µg/mL LPS dose respectively. Each column represents the treatment: Control, TPPU (100 µM), UB23 (10 µM) and UB28 (10 µM). Pictures were taken with the Olympus IX-70 Inverted Fluorescence Microscope at 10x.

5. DISCUSSION

Over the past three decades, there have been no effective new treatments for neurodegenerative diseases, notably AD. This lack of progress is most likely to be due to the lack of understanding of the basic mechanism that underpins the disease. One possibility may be that neuroinflammation play a critical role in the AD development. Nowadays, there is an increasing interest in the development of sEH inhibitors, which block the degradation of EET’s and lead their accumulation. In this way, the beneficial effects of EETs will be maintained leading a reduction of the neuroinflammation

The aim of the present study was to examine the sEH inhibitory effect of the newly synthetized compounds (UB21, UB23, UB24, UB28, EPB50 and EV52) in comparison with the well-known sEHi, TPPU (Inceoglu et al., 2013). With the purpose to test the sEHi in an appropriate model, we compared the sEH protein levels of the neuroblastoma SH-SY5Y cell line with HepG2 and HEK295, both cell lines with high levels of active sEH (Morisseau & Hammock, 2008). Results showed that SH-SY5Y cells had enough sEH protein levels and the enzyme was responsive to the inhibitory agents. Therefore, the human neuroblastoma SH-SY5Y is a good neuronal model to perform sEH inhibitory experiments.

Analysis of the sEH inhibitory activity allowed to calculate the half maximal inhibitory concentration, IC50, of each sEHi. TPPU’s IC50 in this study was 9.3±1.6 µM. In previous studies

TPPU reached, directly with the enzyme, IC50 concentrations of 3.7±1.0 nM (Hwang et al., 2013). This variation may be due to parameters as substrate concentration, target accessibility, cell permeability, duration of incubation and type of cells used. Respect to the other compounds is remarkable the EPB50 good inhibitory activity (0.33±0.16 µM) and the excellent UB23 and UB28

IC50 (3±2.6 nM and 3.5±2.6 nM respectively). EV52 IC50 could not be calculated because values of activity were random, may be due to toxicity or interference with the measure wavelength. Research also has shown that all sEHi, except UB24 and EV52, can be considered safe because do not produce cell mortality and do not affect to the cell viability in SH-SY5Y cell cultures, including cell growth and metabolic activity. However, UB23, UB24 and UB28 showed crystal formations in the media of SH-SY5Y cultures at 100 µM suggesting poor solubility at high concentrations. Despite this, UB23 and UB28 were selected as the most suitable sEHi to study their anti-inflammatory properties because the concentration at which crystals were formed was much higher than the IC50 concentration. TPPU continued as the control group although the

IC50 was the highest because of their confirmed sEH inhibitory activity. Despite of the good

EPB50 IC50 and the no toxicity effects on the SH-SY5Y cells, this sEHi was not included in the anti- inflammatory assays because arrived late to the laboratory.

Accumulating evidences suggest that neuroinflammation is commonly involved in neurodegenerative diseases as AD, and this is mediated mainly by microglia cells. Microglia cells are specialized macrophage-like immune cells in the brain that play a critical role in host defence and neuronal repair for maintenance of CNS homeostasis (Prinz & Priller, 2014). However, persistent microglia activation leads to undesirable damage in the CNS through the production and accumulation of proinflammatory mediators (Heppner et al., 2015). Therefore, development of specific inhibitors of microglial hyperactivation may serve as a therapeutic agent for the AD progression.

This study also investigated the possibility that TPPU, UB23 and UB28 reduce the inflammatory response of LPS activated microglia. The used BV2 cell line is derived from murine

18 primary microglial cultures transformed with retrovirus. This immortalized cells exhibit the morphological, phenotypical, and functional properties of activated microglial cells (Blasi et al., 1990). The purpose of the conditioned BV2 media experiments was to detect a reduction of the proinflammatory mediators where cells were treated with the selected sEHi. TPPU concentration dose was 100 µM because of the high IC50 value. The dose used with UB23 and

UB24 was 10 µM, higher than the IC50 because BV2 cells are murine and there is a possibility that

IC50 between species changes. To detect if these proinflammatory mediators affected to the SH- SY5Y cell viability, the conditioned media was added to these cultured cells. It is reported the release of neurotoxic factors in LPS stimulated BV2 cells that affect to the SH-SY5Y cell viability (Seo et al., 2017). Unexpectedly, our results did not show any variation in cell death dependently of LPS. Possibly indicating that BV2 were not releasing enough concentration of NO and cytokines in our culture conditions.

Our efforts then focused on study directly the proinflammatory mediators in the microglial cells. Exposure to LPS has been believed to stimulate intracellular signalling pathways mediated by NFĸB through activation of toll-like receptor-4 (TLR4) and induce iNOS expression causing NO production (Pålsson-McDermott & O’Neill, 2004). In addition, specifically BV2 cells are reported to increase the NO levels by the administration of LPS (Seo et al., 2017). In our results, LPS dose effect correlated with the amount of NO released. Interestingly, UB23 and UB28 seems that they reduced the NO released indicating a possible anti-inflammatory property. To confirm this protective mechanism, we measured iNOS protein levels. At low LPS doses, a decrease in iNOS relative protein levels was observed with UB23 and UB28 treated BV2 cells regarding control. The decrease of NO release and iNOS protein levels of the BV2 cells may indicate an anti- inflammatory response conducted by UB23 and UB24.

Regarding to the TNF-α release, anti-inflammatory effects of sEH inhibitors are associated with the decrease in the levels of TNF-α (Zhang et al., 2012). In addition, it has previously seen that 1 µg/mL LPS stimulated BV2 cells have high TNF-α levels (Horvath et al., 2008). In ours study has been seen that at 0.1 µg/mL LPS dose, TNF-α release levels are similar to the 1 µg/mL dose. These results can indicate that TNF- α release starts at very low LPS concentrations and reaches the maximum release. The preliminary IL-1β protein quantification of the cleaved mature form showed an increase of its expression levels similar to iNOS in BV2 treated with LPS. This results agree with other studies done with BV2 cells, where there is an IL-1β protein increase with LPS stimulation (Horvath et al., 2008).

NFĸB signalling pathway is critical for LPS-stimulated microglial activation (Oh et al., 2010). Our results showed no differential changes in NFĸB protein quantification due to any of the treatments, but in the immunofluorescence assay the translocation to the nucleus can be observed (Perkins, 2007). Our preliminary results can indicate that UB23 and UB28 has slight effect against the LPS induced inflammatory response in the BV2 microglia-like cells. To detect changes in the nucleus translocation, nuclear extracts will facilitate a quantification of any inhibition of NFĸB activation by sEHi agents.

In a last attempt to see changes in the inflammatory response of BV2 cells under the sEHi effects, we performed SH-SY5Y and BV2 co-cultures. Co-culture facilitates the immediate pass pro-inflammatory molecules released by BV2 to SH-SY5Y, facilitating the study microenvironment of the activated microglia-like cells. PI/FDA staining permitted to discriminate alive from death cells. Results showed more death cells while LPS dose increased. Interestingly, UB23 treated cells showed less cell death than the rest sEHi.

The low anti-inflammatory effects of sEHi may be due to low levels of EETs in BV2 cells. Previous studies indicate that EETs have alternative pathways of degradation such as beta oxidation or chain elongation, CYP450 oxidation, reincorporation into glycerides and other pathways (Spector & Norris, 2018). It is reported that sEHi blocks the inflammatory response in modelled sepsis prevented LPS induced death, and reduced proinflammatory cytokines chemokines and prostaglandins. But possibly treating other conditions leading to a cytokine storm such as viral infections or the severe inflammation initiated by modern immunotherapy represent more reasonable paths for agents as sEHi that can moderate the cytokine release syndrome (DeFrancesco, 2014)

6. CONCLUSIONS

Taking all together, we can conclude that:

1. The neuroblastoma SH-SY5Y cell line is a suitable neuronal model for studying sEH and sEHi.

2. The newly synthetized sEHi compounds showed a higher inhibitory potency than the

reference compound TPPU. UB23 and UB28 showed IC50 in the lower nanomolar range. EV52 could not been tested.

3. All agents tested were safe at the anticipated concentrations to be used, except for EV52 that showed cytotoxicity effect.

4. The preliminary testing of anti-inflammatory effects and mechanisms of these agents was not conclusive but unveils UB23 as the best candidate for inhibiting NFĸB and iNOS pro-inflammatory pathways.

7. BIBLIOGRAPHY

Alva, G., & Potkin, S. G. (2003). Alzheimer disease and other dementias. Clinics in Geriatric Medicine. Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., & Bistoni, F. (1990). Immortalization of murine microglial cells by a v-raf / v-myc carrying retrovirus. Journal of Neuroimmunology. Cai, Z., Hussain, M. D., & Yan, L.-J. (2014). Microglia, neuroinflammation, and beta-amyloid protein in Alzheimer’s disease. International Journal of Neuroscience. Chen, D., Whitcomb, R., MacIntyre, E., Tran, V., Do, Z. N., Sabry, J., … Webb, H. K. (2012). Pharmacokinetics and pharmacodynamics of AR9281, an inhibitor of soluble epoxide hydrolase, in single- and multiple-dose studies in healthy human subjects. Journal of Clinical Pharmacology. DeFrancesco, L. (2014). CAR-T cell therapy seeks strategies to harness cytokine storm. Nature Biotechnology. Enayetallah, A. E., French, R. A., & Grant, D. F. (2004). Distribution of soluble epoxide hydrolase, cytochrome P450 2C8, 2C9 and 2J2 in human malignant neoplasms. Journal of Molecular Histology, 37(3–4), 133–141. Esposito, E., & Cuzzocrea, S. (2010). Antiinflammatory Activity of Melatonin in Central Nervous System. Current Neuropharmacology. Fang, X., Kaduce, T. L., Weintraub, N. L., Harmon, S., Teesch, L. M., Morisseau, C., … Spector, A. A. (2001). Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Journal of Biological Chemistry, 276(18), 14867–14874. Fitzpatrick, F. A., & Soberman, R. (2001). Regulated formation of eicosanoids. Journal of Clinical Investigation. Funk, C. D. (2001). Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science. Greene, J. F., Newman, J. W., Williamson, K. C., & Hammock, B. D. (2000). Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase. Chemical Research in Toxicology, 13(4), 217–226. Guo, Y., Peng, R., Liu, Q., & Xu, D. (2016). Exercise training-induced different improvement profile of endothelial progenitor cells function in mice with or without myocardial infarction. International Journal of Cardiology, 221, 335–341. Hardy, J., & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics. Science. Heppner, F. L., Ransohoff, R. M., & Becher, B. (2015). Immune attack: The role of inflammation in Alzheimer disease. Nature Reviews Neuroscience. Horvath, R. J., Nutile-McMenemy, N., Alkaitis, M. S., & DeLeo, J. A. (2008). Differential migration, LPS-induced cytokine, chemokine, and NO expression in immortalized BV-2 and HAPI cell lines and primary microglial cultures. Journal of Neurochemistry, 107(2), 557–569. Hwang, S. H., Wecksler, A. T., Zhang, G., Morisseau, C., Nguyen, L. V., Fu, S. H., & Hammock, B. D. (2013). Synthesis and biological evaluation of sorafenib- and regorafenib-like sEH inhibitors. Bioorganic and Medicinal Chemistry Letters.

Iliff, J. J., Jia, J., Nelson, J., Goyagi, T., Klaus, J., & Alkayed, N. J. (2010). Epoxyeicosanoid signaling in CNS function and disease. Prostaglandins and Other Lipid Mediators. Inceoglu, B., Zolkowska, D., Yoo, H. J., Wagner, K. M., Yang, J., Hackett, E., … Hammock, B. D. (2013). Epoxy fatty acids and inhibition of the soluble epoxide hydrolase selectively modulate GABA mediated neurotransmission to delay onset of seizures. PLoS ONE, 8(12), 8–17. https://doi.org/10.1371/journal.pone.0080922 Konkel, A., & Schunck, W. H. (2011). Role of cytochrome P450 enzymes in the bioactivation of polyunsaturated fatty acids. Biochimica et Biophysica Acta - Proteins and Proteomics, 1814(1), 210–222. Krstic, D., & Knuesel, I. (2013). Deciphering the mechanism underlying late-onset Alzheimer disease. Nature Reviews Neurology. Kumar, A., Singh, A., & Ekavali. (2015). A review on Alzheimer’s disease pathophysiology and its management: An update. Pharmacological Reports. Morisseau, C., & Hammock, B. D. (2008). Gerry Brooks and epoxide hydrolases : four decades to a pharmaceutical, 609(July 2007), 594–609. Morisseau, C., & Hammock, B. D. (2013). Impact of Soluble Epoxide Hydrolase and Epoxyeicosanoids on Human Health. Annual Review of Pharmacology and Toxicology, 53(1), 37–58. Nelson, J. W., Young, J. M., Borkar, R. N., Woltjer, R. L., Quinn, J. F., Silbert, L. C., … Alkayed, N. J. (2014). Role of soluble epoxide hydrolase in age-related vascular cognitive decline. Prostaglandins & Other Lipid Mediators, 113–115, 30–37. Newman, J. W., Morisseau, C., & Hammock, B. D. (2005). Epoxide hydrolases: Their roles and interactions with lipid metabolism. Progress in Lipid Research. Ng, V. Y., Huang, Y., Reddy, L. M., Falck, J. R., Lin, E. T., & Kroetz, D. L. (2007). Cytochrome P450 eicosanoids are activators of peroxisome proliferator-activated receptor α. Drug Metabolism and Disposition, 35(7), 1126–1134. Node, K., Huo, Y., Ruan, X., Yang, B., Spiecker, M., Ley, K., … Liao, J. K. (1999). Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science, 285(5431), 1276–1279. Oh, Y. T., Lee, J. Y., Lee, J., Lee, J. H., Kim, J. E., Ha, J., & Kang, I. (2010). Oleamide suppresses lipopolysaccharide-induced expression of iNOS and COX-2 through inhibition of NF-κB activation in BV2 murine microglial cells. Neuroscience Letters. Pålsson-McDermott, E. M., & O’Neill, L. A. J. (2004). Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology. Patel, N. S., Paris, D., Mathura, V., Quadros, A. N., Crawford, F. C., & Mullan, M. J. (2005). Inflammatory cytokine levels correlate with amyloid load in transgenic mouse models of Alzheimer’s disease. Journal of Neuroinflammation. Perkins, N. D. (2007). Integrating cell-signalling pathways with NF-κB and IKK function. Nature Reviews Molecular Cell Biology. Prince, M., Comas-Herrera, A., Knapp, M., Guerchet, M., & Karagiannidou, M. (2016). World Alzheimer Report 2016 Improving healthcare for people living with dementia. Coverage, Quality and costs now and in the future. Alzheimer’s Disease International (ADI).

Prinz, M., & Priller, J. (2014). Microglia and brain macrophages in the molecular age: From origin to neuropsychiatric disease. Nature Reviews Neuroscience. Sarkar, P., Zaja, I., Bienengraeber, M., Rarick, K. R., Terashvili, M., Canfield, S., … Harder, D. R. (2014). Epoxyeicosatrienoic acids pretreatment improves amyloid β-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes. American Journal of Physiology. Heart and Circulatory Physiology, 306(4), H475-84. Schmelzer, K. R., Kubala, L., Newman, J. W., Kim, I.-H., Eiserich, J. P., & Hammock, B. D. (2005). Soluble epoxide hydrolase is a therapeutic target for acute inflammation. Proceedings of the National Academy of Sciences of the United States of America, 102(28), 9772–9777. Seo, M., Lee, J. H., Baek, M., Kim, M. A., Ahn, M. Y., Kim, S. H., … Hwang, J. S. (2017). A novel role for earthworm peptide Lumbricusin as a regulator of neuroinflammation. Biochemical and Biophysical Research Communications, 490(3), 1004–1010. Simpkins, A. N., Rudic, R. D., Schreihofer, D. A., Roy, S., Manhiani, M., Tsai, H. J., … Imig, J. D. (2009). Soluble epoxide inhibition is protective against cerebral ischemia via vascular and neural protection. American Journal of Pathology. Spector, A. A., & Norris, A. W. (2007). Action of epoxyeicosatrienoic acids on cellular function, 996–1012. Spector, A. A., & Norris, A. W. (2018). Action of epoxyeicosatrienoic acids on cellular function, 996–1012. Sudduth, T. L., Schmitt, F. A., Nelson, P. T., & Wilcock, D. M. (2013). Neuroinflammatory phenotype in early Alzheimer’s disease. Neurobiology of Aging. Sung, H. H., Tsai, H. J., Liu, J. Y., Morisseau, C., & Hammock, B. D. (2007). Orally bioavailable potent soluble epoxide hydrolase inhibitors. Journal of Medicinal Chemistry. Wagner, K. M., Mcreynolds, C. B., Schmidt, W. K., & Hammock, B. D. (2017). Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases. Pharmacology and Therapeutics, 180, 62–76. Wu, H. F., Chen, Y. J., Wu, S. Z., Lee, C. W., Chen, I. T., Lee, Y. C., … Lin, H. C. (2017). Soluble Epoxide Hydrolase Inhibitor and 14,15-Epoxyeicosatrienoic Acid-Facilitated Long-Term Potentiation through cAMP and CaMKII in the Hippocampus. Neural Plasticity, 2017. Zhang, W., Yang, A. L., Liao, J., Li, H., Dong, H., Chung, Y. T., … Yang, G.-Y. (2012). Soluble Epoxide Hydrolase Gene Deficiency or Inhibition Attenuates Chronic Active Inflammatory Bowel Disease in IL-10(−/−) Mice. Digestive Diseases and Sciences.