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. 2024 Feb 16;7(4):1002–1012. doi: 10.1021/acsptsci.3c00282

Santacruzamate A Alleviates Pain and Pain-Related Adverse Emotions through the Inhibition of Microglial Activation in the Anterior Cingulate Cortex

Yan Qin †, Qingqing Liu †, Saiying Wang †, Qinhui Wang †, Yaya Du †, Jingyue Yao †, Yue Chen †, Qi Yang †, Yumei Wu ‡, Shuibing Liu ‡, Minggao Zhao †, Gaofei Wei §,*, Le Yang †,*
PMCID: PMC11019733  PMID: 38633586

Abstract

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Chronic pain is a complex disease. It seriously affects patients’ quality of life and imposes a significant economic burden on society. Santacruzamate A (SCA) is a natural product isolated from marine cyanobacteria in Panama. In this study, we first demonstrated that SCA could alleviate chronic inflammatory pain, pain-related anxiety, and depression emotions induced by complete Freund’s adjuvant in mice while inhibiting microglial activation in the anterior cingulate cortex. Moreover, SCA treatment attenuated lipopolysaccharide (LPS)-induced inflammatory response by downregulating interleukin 1β and 6 (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) levels in BV2 cells. Furthermore, we found that SCA could bind to soluble epoxide hydrolase (sEH) through molecular docking technology, and the thermal stability of sEH was enhanced after binding of SCA to the sEH protein. Meanwhile, we identified that SCA could reduce the sEH enzyme activity and inhibit sEH protein overexpression in the LPS stimulation model. The results indicated that SCA could alleviate the development of inflammation by inhibiting the enzyme activity and expression of sEH to further reduce chronic inflammatory pain. Our study suggested that SCA could be a potential drug for treating chronic inflammatory pain.

Keywords: santacruzamate A, chronic inflammatory pain, soluble epoxide hydrolase, anterior cingulate cortex

Santacruzamate A (SCA) is a natural carbamate derivative isolated from the dark brown clustered cyanobacteria.1 Recent studies have found that SCA and its synthetic analogues have immunomodulatory and cell proliferation inhibition activities2,3 and positive effects on breast cancer, liver cancer, and other diseases.4−7 SCA also plays an important role in central nervous system diseases; for example, it improves Alzheimer’s disease-like pathologies by enhancing endoplasmic reticulum stress tolerance.8 Additionally, SCA effectively affects pain disorders;9 however, the analgesic mechanism has not been fully elucidated.

Chronic pain is characterized by long-term pain and unpleasant sensations mediated by central or peripheral sensitization.10 Prolonged exposure to chronic pain leads to adverse effects such as anxiety, insomnia, depression, and premature aging, significantly affecting the quality of life and imposing enormous economic burdens on society and families.11 Currently, the clinical treatment of chronic pain mainly relies on opioid drugs, nonsteroidal anti-inflammatory drugs, and acetaminophen (paracetamol). However, these drugs have severe side effects, such as respiratory depression, addiction, constipation, and gastrointestinal damage.12−14 A recent study has reported that an inhibitor of soluble epoxide hydrolase (sEH) has a good effect on anti-inflammatory analgesia and neuropathic pain.15 Therefore, sEH will be an important new target in exploiting new drugs for chronic pain disease.

After the pain pathway is triggered, the anterior cingulate cortex (ACC) and other cortical regions, including the insular, primary somatosensory, secondary somatosensory, and prefrontal cortex, are activated.16 ACC activation is also associated with adverse emotions related to pain, such as disgust, fear, anxiety, and depression.17,18 In chronic inflammatory pain, the continuous excitatory input of ACC induces microglial activation.19

Microglia, also known as specialized macrophages in the brain, mediate inflammation in the central nervous system and play important roles in chronic neuropathic pain.20 Meanwhile, the interaction between neurons and microglia mediated by pain stimulation is important in neuroinflammation and excitatory synapse plasticity.21 During chronic pain induced by neuroinflammation, sEH exacerbates the neuroinflammatory response by degrading anti-inflammatory epoxide metabolites (epoxyeicosatrienoic acids, epoxyeicosatetraenoic acids, and epoxydocosapentaenoic acids).22 Thus, developing sEH inhibitors is important for treating chronic inflammatory and neuropathic pain, among other diseases.23

In this study, we used a mouse model for inflammatory pain to test the analgesic and pain-related negative emotion effects of SCA in vivo. We performed experiments to verify its anti-inflammatory potential in vitro and used molecular docking to determine the binding capability of SCA with sEH. We further tested the thermostability of SCA and sEH protein binding using a cellular thermal shift assay (CETSA) and the SCA inhibition of sEH protein activity using a fluorescence assay. The effects of SCA on sEH protein expression were assessed using western blotting. We also verified the effect of SCA on the synaptic plasticity of nerve cells in an inflammatory model. We hypothesize that SCA could inhibit sEH protein activity and expression, thereby suppressing inflammatory processes and alleviating inflammatory responses, chronic pain, and pain-related adverse emotions.

Results

Effect of SCA on Complete Freund’s Adjuvant (CFA)-Induced Chronic Inflammatory Pain

The injection of CFA into the hindpaw represents a valid chronic inflammatory pain model in mice.24 First, we demonstrated that SCA could cross the blood-brain barrier using ultraperformance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (Figure S1A). Subsequently, the validated analytical method was employed to study the mean plasma and brain concentration–time profiles of SCA presented (Figure S1B,C). The estimated pharmacokinetic (PK) parameters of the SCA are presented in Table S1. For 0–12 h after SCA administration, SCA concentrations in plasma and the brain were highest at 0.25 h (260.4 ± 35.5 ng/mL) and 1 h (43.4 ± 4.5 ng/mL), respectively. Meanwhile, we evaluated the effect of SCA on chronic inflammatory pain in mice. The experimental protocol is shown in Figure 1A. The mechanical pain threshold is significantly reduced in the CFA group compared with that in the control group. After the SCA treatment (2, 10, and 50 mg/kg), the mechanical pain threshold was significantly increased compared with that of the CFA group (Figure 1B). Furthermore, we found an obvious increase in paw thickness in the CFA group, and SCA reversed the increase in the CFA group (Figure 1C). These results demonstrated that SCA alleviated the chronic inflammatory pain caused by CFA injection.

Figure 1.

Figure 1

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Santacruzamate A (SCA) alleviates complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain. (A) The schedule shows the procedure of the experiment (i.g.: intragastric administration, i.h.: hypodermic injection). (B, C) Control group and CFA + SCA (2, 10, and 50 mg/kg) groups reduced mechanical threshold (B) and hindpaw thickness (C) in CFA injection mice. Each data expressed as mean ± SEM of three independent experiments (n = 6, *p < 0.05, **p < 0.01 versus control group; #p < 0.05, ##p < 0.01 versus CFA group).

SCA Alleviated Anxiolytic- and Depressant-like Behaviors Associated with CFA-Induced Chronic Inflammatory Pain

In the open field test (OFT), mice in the CFA group spent less time and traveled a shorter distance in the central area (Figure 2A,B) than those in the control group. After the SCA treatment, the times spent and distance traveled were significantly increased in the central area. However, the total distance traversed showed no significant differences between the groups (Figure 2C). In the elevated plus maze (EPM) test, mice in the CFA group spent less time in the open arms (Figure 2D) and entered the open arms less frequently (Figure 2E). After the SCA treatment, the times spent and number of entries significantly increased in the open arms. No difference was found regarding the total distance traveled either (Figure 2F). These results indicated that SCA could mitigate anxiety-like behaviors associated with CFA-induced chronic inflammatory pain.

Figure 2.

Figure 2

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SCA alleviates anxiolytic- and depressant-like behaviors associated with CFA-induced chronic inflammatory pain. (A–C) In the open field test (OFT), CFA + SCA (2, 10, and 50 mg/kg) increased the time spent (A) and the distance traveled (B) in the central area after CFA injection, and there were no significant differences in the total distance in each group (C). (D–F) In the elevated plus maze test (EPM) test, SCA effectively reversed the reduction in the time spent (D) and entry numbers (E) in the open arms after CFA injection, and there were no significant differences in the total distance in each group (F). (G, H) In the tail suspension tests (TST) and forced swim tests (FST), SCA effectively decreased immobility time after CFA injection. (I) In the sucrose preference test (SPT), SCA effectively reversed the amount of sugar water consumed by mice. Each data expressed as mean ± SEM of three independent experiments (n = 6, *p < 0.05, **p < 0.01 versus control group; #p < 0.05, ##p < 0.01 versus CFA group).

In the tail suspension and forced swim tests (TST and FST), mice in the CFA group showed increased time immobility compared to those in the control group. Treatment with SCA effectively decreased the time immobility (Figure 2G,H). In the sucrose preference test (SPT), the preference for sugar in the CFA group was significantly lower than that in the control group. After the SCA treatment, the sucrose preference was significantly increased compared with that in the CFA group (Figure 2I). These results indicated that SCA could alleviate depressive behaviors in a CFA-induced mouse model for chronic inflammatory pain.

SCA Inhibited Microglial Activation Induced by CFA in the ACC

The ACC is an important brain region related to pain and affective disorders. Microglial cells are abnormally active in the ACC when chronic inflammatory pain occurs.25 Consistently, we observed that the number of positive cells for Iba-1 (a marker for microglia) significantly increased in the CFA group; however, the activated cell numbers significantly decreased after the SCA treatment (Figure 3A). These results indicated that SCA decreased the activation of microglia induced by CFA in the ACC.

Figure 3.

Figure 3

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SCA inhibits microglial activation induced by CFA in the anterior cingulate cortex (ACC). Mouse brain sections were immunofluorescence-stained with the microglial marker Iba-1 antibody (green), and the nuclei were stained with DAPI (blue). Scale bar = 100 μm (A). After injection of CFA, SCA inhibited the activation of microglia in the ACC. Each data expressed as mean ± SEM of three independent experiments (n = 3 slices per group, *p < 0.05, **p < 0.01 versus control group; #p < 0.05 versus CFA group).

SCA Suppressed Inflammation Induced by Lipopolysaccharide (LPS) in the BV2 Cells

To further investigate the mechanism of SCA treatment for chronic inflammatory pain, we examined the effects of SCA on inflammatory factors in BV2 cells. The enzyme-linked immunosorbent assay (ELISA) showed that the levels of inflammatory factors, including interleukin 1β and 6 (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α), were significantly higher in the LPS group than in the control group, and SCA reduced the release of inflammatory factors induced by LPS (Figure 4A–C). Western blotting also identified significantly higher protein levels of IL-1β, IL-6, and TNF-α in the LPS group than in the control group, and SCA reduced the protein levels of IL-1β, IL-6, and TNF-α (Figure 4D). Therefore, these data indicated that SCA reduced the levels of inflammatory factors induced by LPS in the BV2 cells.

Figure 4.

Figure 4

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SCA inhibited inflammation induced by lipopolysaccharide (LPS) in the BV2 cells. (A–C) Enzyme-linked immunosorbent assay (ELISA) detected inflammatory factor interleukin 1β and 6 (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) in the medium supernatant. (D) Western blot analysis was performed to assess IL-1β, IL-6, and TNF-α expressions. Each data expressed as mean ± SEM of three independent experiments (n = 3, *p < 0.05, **p < 0.01 versus control group; #p < 0.05, ##p < 0.01 versus LPS group).

SCA is a Novel sEH Inhibitor

Next, we found that SCA could interact with sEH using a molecular docking analysis. SCA had a lower CDOCKER energy (−46.2 kcal/mol) than the standard sEH inhibitor TPPU (1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea) of sEH (−38.4 kcal/mol), indicating that SCA also had an affinity for sEH. This result supported the fact that SCA overlapped with the natural substrate well in the sEH binding site (Figure 5A,B). ASP335, TYR383, and TYR466 of sEH formed direct hydrogen bonds with the SCA at 3.0 Å, stabilizing the molecular structure within the binding site. We also observed that ILE363, PRO361, LEU408, MET419, and HIS524 residues formed hydrophobic cavities on the left and right sides of the SCA molecule. The benzyl group of the SCA occupied the hydrophobic cavity on the right, and the alkyl of the SCA formed a hydrophobic interaction with ILE363 and PRO361 on the left, which contributed to strengthening the interaction between SCA and sEH (Figure 5C,D). The CETSA assay also proved that SCA binds to sEH (Figure 5E). Moreover, we detected that the IC50 of SCA and TPPU (a potent inhibitor of sEH) on sEH enzyme activity were 7.0 ± 0.4 μM and 23.0 ± 2.5 nM, respectively. The inhibitory effect of TPPU on sEH was consistent with previous literature.26 Additionally, western blotting showed that SCA downregulates the sEH protein induced by LPS (Figure 5G). These results indicated that SCA could affect sEH activity.

Figure 5.

Figure 5

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SCA is a novel soluble epoxide hydrolase (sEH) inhibitor. (A) Chemical structure formula of SCA. (B) Alignment of SCA (yellow) and substrate (blue) at the binding site of the sEH (PDB code: 4OD0). (C) Interactions of SCA with ASP335, TYR383, and TYR466. (D) Diagram of the SCA interaction in the binding site of sEH. The hydrogen bond is depicted as a purple line. (E) Cellular thermal shift assay (CETSA) was used to evaluate the binding ability of the interaction between SCA (50 μM) and sEH at the thermodynamic level. (F) Inhibitory effects of SCA toward sEH. (G) The classical Western blot analysis showed that SCA significantly reversed the increase in sEH expression. Each data expressed as mean ± SEM of three independent experiments (n = 3, *p < 0.05, **p < 0.01 versus control group; #p < 0.05 versus LPS group).

SCA Modulates the Excitatory Synaptic Receptor Function

The results mentioned above showed that SCA reduced the neuroinflammatory response, and we further verified whether SCA affected the expression of neuronal cells. We cocultured PC12 cells with BV2 cells (Figure 6A) and examined the changes in the excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic (NMDA) receptors as follows: NMDA receptor 2A (GluN2A) and receptor 2B (GluN2B), glutamate A1 (GluA1), and glutamate A2 (GluA2) in PC12 cells. The protein expressions of GluN2A, GluN2B, GluA1, and GluA2 were significantly increased in PC12 cells cocultured with BV2 cells treated with LPS. Upon treatment with SCA, the protein expressions of GluN2A, GluN2B, GluA1, and GluA2 significantly decreased (Figure 6B–F). These results indicated that SCA could modulate the function of excitatory synaptic receptors.

Figure 6.

Figure 6

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SCA modulates the excitatory synaptic receptor function. (A) Flow diagram of coculturing of PC12 cells and BV2 cells. (B–F) Protein expression of NMDA receptor 2A (GluN2A) and receptor 2B (GluN2B), glutamate A1 (GluA1), and glutamate A2 (GluA2) was determined by western blot analysis. Data from three independent experiments, **p < 0.01, ***p < 0.001 versus control group, #p < 0.05 versus LPS group.

Discussion

In this study, we confirmed that SCA could pass through the blood-brain barrier and obtain the changes in the blood drug concentration and pharmacokinetic-related parameters of SCA in mice by the pharmacokinetic method. We also found that SCA alleviated CFA-induced chronic inflammatory pain, anxiety, and depressive-like behaviors in vivo. We referred to previous studies, most of which compared the activity of aspirin,27 diclofenac,28 or ibuprofen29 in the CFA-induced. For example, Cobos et al. stated30 that subcutaneous injection of morphine (0.06–0.5 mg/kg), prednisolone (0.62–5 mg/kg), celecoxib (2.5–20 mg/kg), diclofenac (1.25–10 mg/kg), ibuprofen (2.5–20 mg/kg), and naproxen (10–80 mg/kg) reversed inflammation and pain in CFA-induced mice. Similarly, in our study, SCA (2–50 mg/kg) had stable analgesic, antianxiety, and antidepressant-like behaviors after 3 days of continuous intragastric administration, which was compared to positive control doses and previous studies. Therefore, we believe that SCA has a certain potential for development. Furthermore, SCA decreased microglial activation in the ACC of mice and reduced the expression of inflammatory factors in vitro. Subsequently, we demonstrated that SCA has a moderate affinity for sEH and reduces the enzyme activity of sEH. SCA effectively inhibited excitatory synaptic transmission in the ACC. Overall, SCA alleviated chronic inflammatory pain and pain-related adverse emotions by inhibiting microglial activation in the ACC via affecting sEH.

Due to neuroplasticity in pathways and circuit coding for pain, chronic pain may be caused by peripheral sensitization, central sensitization, or the interaction between neuroimmunity and glia. The ACC is believed to play an essential role in pain and pain-related unpleasant emotions.31 Microglia are immune cells of the central nervous system. After stimulation, microglia were activated, releasing inflammatory factors that aggravate chronic pain.32 We found that microglia in the ACC of the mice were activated by CFA stimulation and subsequently inhibited by SCA. Additionally, SCA reduced IL-1β, IL-6, and TNF-α levels in an LPS-induced inflammatory model in vitro. Our results suggest that SCA significantly reverses the inflammatory response, contributing to a decrease in chronic inflammatory pain and the associated anxiety- and depression-like behavior caused by CFA injection in mice. Currently, some studies have explored the possibility of sex differences in the occurrence and development of pain.33,34 We referred to several studies using the CFA mouse model, most of which considered the differences in pain presentation between male and female mice to be small.35−37 Therefore, only male mice were used in our experiment, which is also a shortcoming of our experimental design. We will consider sex in the experimental design of subsequent studies on SCA.

sEH plays an important role in the biosynthesis and exogenous transformation of inflammatory mediators,38 which can produce proinflammatory substances through the catabolism of their corresponding glycols by epoxide fatty acids, intensifying inflammation processes.39,40 sEH is well studied and considered to be a therapeutic target for pain, inflammation, and neurodegenerative diseases.15,41−43 In this study, we demonstrated that SCA could stably bind to sEH using molecular docking analysis and CETSA. Meanwhile, we confirmed that SCA reduced the activity of the human sEH enzyme (IC50 = 7.0 ± 0.4 μM) in vitro. Additionally, SCA decreased the protein level of sEH in the LPS group. These results suggest that SCA might not be a specific inhibitor for sEH; sEH might have an independent regulatory mechanism. Thus, more studies are needed to validate the interaction between the SCA and sEH.

Currently, a consensus exists that synaptic plasticity is involved in the pain process. Triggering the pain pathway leads to increased responsiveness of neurons involved in transmitting pain signals and promoting excitatory synapses.44 Rapid excitatory synapse transmission in the pain pathway is primarily mediated by AMPA and NMDA receptors, which are activated by glutamate.45,46 In this study, we found that the expressions of GluN2A, GluN2B, GluA1, and GluA2 were significantly decreased after SCA administration. Thus, SCA could modulate the synaptic function in neurons by inhibiting microglial activation.

In conclusion, our results showed that SCA significantly alleviated chronic inflammatory pain, depression, and anxiety-like behavior. Furthermore, SCA inhibited the activation of microglia and reduced the release of inflammatory factors by acting on sEH. Clearly, SCA has the potential in treating inflammatory neuropathic diseases and provides a new idea for analgesic drug development in this study.

Materials and Methods

Materials

CFA (cat. no. F5881) and LPS (cat. no. L2630) were purchased from Sigma-Aldrich. SCA was purchased from TargetMol (cat. no. T2266; molecular weight: 278.352, formula: C15H22N2O3, purity: 99.55%, CAS no. 1477949-42-0). Human sEH protein was purchased from Cayman (cat. no. 10011669). PHOME was purchased from Medchamexpress (cat. no. HY-113862).

Animals

Male C57BL/6J mice aged 6–8 weeks (weight = 18 ± 2 g) were obtained from the experimental animal center of the Air Force Military Medical University and placed in an SPF level environment with a 12 h dark/light cycle, free access to food and water. All behavioral tests were performed during the light period on the designated day of the experiment. All experimental procedures were approved by the Air Force Military Medical University Animal Care and Use Committee. Every effort was made to minimize the number of animals used and their suffering. Mice must adapt to laboratory conditions for 5 days prior to testing (approval reference number no. KY20193145).

Plasma and Brain PK in Male Mice

Male C57BL/6J mice ranging from 8 to 10 weeks old were used as subjects. SCA was administered once via an intragastric administration (i.g.) of 10 mg/kg.

For the pharmacokinetic study,47 blood samples were collected from the eye socket of four mice at pre dose and again at approximately 0.083, 0.25, 0.5, 1, 2, 3, 6, and 12 h post dose for plasma. Immediately after blood collection, plasma samples were obtained via centrifugation at 13,000 rpm for 10 min and stored at −20 °C until LC-MS/MS analysis.48

For the brain kinetics evaluation, brain samples were collected at pre dose and again at approximately 0.5, 1, 2, 6, and 12 h post dose (each group consists of three mice). The residual blood in the brain was removed from circulation by perfusion of the saline solution into the heart. Brain samples were collected and stored at −20 °C until LC-MS/MS analysis.

The PK parameters including the mean plasma concentration, maximum plasma concentration (Cmax), the time to reach Cmax (Tmax), half-life time (T1/2), area under the curve (AUC0–t and AUC0–∞), and mean residence time (MRT) were calculated and evaluated by using the software of drug and statistics (DAS, version 2.1).

LC-MS/MS Analysis

Chromatographic separation was performed on an LC-30AD liquid chromatography instrument equipped with an XSelect HSS T3 (5.0 μm, 4.6 × 100 mm, Waters, USA) column. The solvent system consisted of acetonitrile (solvent A) and a 0.1% formic acid solution (solvent B). The gradient program was as follows: 0–0.5 min kept 90% B; 0.5–1 min 90–90% B; 1–2 min 40–40% B; 2–3 min 40–5% B; 3–5 min kept 5% B; 5–5.5 min 5–90% B; 5.5–6 min kept 90% B. The flow rate was 0.4 mL/min, and the injection volume was 5 μL. Analytes were monitored by positive ion multiple-reaction monitoring (MRM) mode on a UFLC30A + AB SCIEX API400 mass spectrometer (Shimazu, Japan). The operating parameters were as follows: collision gas, 9 psi; curtain gas, 25 psi; ion source gas 1 and ion source gas 2, 50 and 55 psi, respectively; ion spray voltage, 5500 V; and turbo spray temperature, 500 °C. Monitoring ion pairs for quantitative analysis: SCA, m/z 279.1/233.1 (+), declustering potential (DP): 55 V, collision energy (CE): 17 V.

Experimental Protocol and Treatment Schedule

The model of chronic inflammatory pain was established after mice adapted to the environment.49 Mice were injected CFA (50% in saline, 10 μL, i.h.: hypodermic injection) subcutaneously into the left hindpaw. Mechanical thresholds were measured on days 0, 1, 3, 5, 7, and 14 after CFA injection. Mice were randomly divided into five groups (each group consists of six mice): control, CFA, and CFA + SCA (2, 10, and 50 mg/kg). Starting from the day of CFA injury, mice were administered with SCA or sterile normal saline (solvent, 10 mL/kg). Mice were repeatedly given SCA or normal saline once a day for 3 days. Mechanical allodynia and swelling at the hindpaw area were examined on days d0, d1, d3, d5, d7, and d14 after CFA injection.

Measurement of Mechanical Hyperalgesia

Prior to the experiment, mice were placed in individual compartments with mesh floors and allowed to acclimate for 30 min. Mechanical sensitivity was assessed with a set of von Frey filaments (Ugo Basile, Italy, model number 37450-275) by using before (day 0) and after the CFA injection (days 1, 3, 7, and 14), and 50% threshold forces were calculated using the up–down paradigm. Positive responses included prolonged hindpaw withdrawal followed by licking or scratching.50

Measurement of Paw Edema

To evaluate the effect of SCA on inflammation, hindpaw thickness of mice was measured after CFA injection just before (day 0) and after the CFA injection at days 1, 3, 7, and 14 using a digimatic micrometer.51

Open Field Test (OFT)

The open field (JLBehv-LAM-4, Shanghai Jiliang software, China) was a square arena (30 × 30 × 30 cm3), with clear plexiglass walls and floors, and was placed in a dark lighting and fan isolation room. At the beginning of the test, each mouse was placed in the same left position in the corner of the rectangular arena and allowed to explore the arena freely for 15 min. The exploratory behavior of mice was recorded with a camera fixed on the floor, and the total travel distance and time of mice in the central area were analyzed with a video tracking system (MedAssociates, St. Albans, VT, USA). The time spent in the central arena was defined as the center of the open arena 15 × 15 cm2 area, accounting for one-quarter of the total area.52

Elevated Plus Maze Test (EPM)

The elevated plus maze test was performed on mice according to the previously published method.53 The instrument (DigBehv-EPMG, Shanghai Jiliang software) consists of four verticals + shaped plexiglass arms and two open arms (25 × 8 × 0.5 cm3) and two closed arms (25 × 8 × 12 cm3), which extend from a common central area (8 cm3) to a height of 50 cm above the ground. In each experiment, mice were initially placed in the central area of the elevated maze and allowed to explore freely for 5 min. At the same time, the video tracking system (MedAssociates) was used to record and analyze the time and number of people entering the open and closed arms.

Forced Swim Test (FST)

The mice were forced to swim for 6 min in a transparent plexiglass cylinder (20 cm in diameter, 50 cm in depth, filled with water at 23–25 °C, 25 cm in depth). The whole swimming test process was recorded by a camera, and the immobility time in the last 4 min was analyzed by any maze behavioral tracking software (Stoelting, Co., Illinois, USA).

Tail Suspension Test (TST)

The tail suspension test was carried out according to the previous method, and the tail of mice (2 cm from the tail tip) was fixed on the hook and suspended from a height of about 25 cm from the ground for 5 min.54 The whole process was recorded with a camera, and the immobility time of the mice for 6 min was analyzed by any maze behavioral tracking software (Stoelting, Co., Illinois, USA).

Sucrose Preference Test (SPT)

Each mouse was raised in a different cage, and the same amount of drinking water and 1% sucrose water was placed in every cage. The locations of drinking water and sucrose water in each cage could be random to avoid location interference. We removed food, water, and sucrose water in the cage for 15 h. The measurement time was from 07:00 to 22:00. On the first day, normal water and food were given. On the second day, the same time and method, the consumption volume of drinking water and sucrose water in each cage was recorded.55

Immunofluorescence Staining

After the behavioral test, the mice were anesthetized with sodium pentobarbital, perfused with 4% paraformaldehyde, and then transferred to 30% sucrose solution. Coronal sections of the ACC were cut on a cryostat (Leica Microsystems, 25 μm). The sections were blocked with 10% goat serum and 0.1% Triton X-100 PBS for 2 h at 4 °C. Then, the slices were cultured with goat anti-Iba-1 (ab289874, 1:100, Abcam) in a closed solution overnight at 4 °C and then cultured with mouse anti-rabbit IgG Alexa Fluor 594 (1:200, Invitrogen) at room temperature for 2 h. The nuclei were counterstained with DAPI. The slides were then covered with immu-mount gel and placed at 4 °C. A FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to photograph and analyze the stained samples.56

Cell Culture and Treatment

BV2 cells (a murine microglial cell line) and PC12 cells (a neuronal cell line) were purchased from the China Center for the Type Culture Collection (CCTCC). The cells were cultured in a complete medium (10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin in Dulbecco’s modified Eagle medium (DMEM). They were cultured in a humidified environment with 5% CO2 at 37 °C. The BV2 cells were divided into a control group, LPS, 1 μM group, and SCA (10 μM) + LPS (1 μM) group for 24 h at 37 °C. The treatment of PC12 cells induced inflammation of BV2 cells with LPS for 24 h, then replaced with a fresh culture medium, and continued to culture for 24 h. We collected the supernatant from the BV2 cells and cocultured with PC12 cells for 24 h.57 PC12 cells were divided into three groups the same as BV2 cells.

Enzyme-Linked Immunosorbent Assay (ELISA)

The BV2 cells were administrated for 24 h by LPS and the supernatant. The total protein of the inflammatory factor concentration in the supernatant was measured by an ELISA kit, which was all obtained from Thermo Fisher including interleukin (IL-1β: 88-7013, IL-6: 88-7064, and TNF- α: 88-7324).

Western Blot Analysis

The BV2 cells were collected 24 h after LPS treatment, and the PC12 cells were collected 24 h after being cultured with an inflammatory BV2 cell supernatant. Each sample was homogenized in ice-cold radio immunoprecipitation assay (RIPA) lysis buffer containing a mixture of a protease inhibitor, then homogenized, and centrifuged (20,000g) for 20 min at 4 °C. Protein extractions were prepared from the group of control, LPS, and SCA + LPS in BV2 cells or PC12 cells. The methods of western blot procedures and analyses were performed as previously.58 Primary antibodies included those against Signaling Technology (12912s; 1:1000), TNF-α (11948s; 1:1000), IL-1β (12507s; 1:1000), GAPDH (5174s; 1:1000), GluA1 (13185s; 1:1000), GluA2 (13607s; 1:1000), GluN2A (4205s; 1:1000), GluN2B (14544s; 1:1000), and EPHX2 polyclonal antibody (10833–1-AP; 1:1000, Proteintech).

Molecular Docking Analysis

The crystal complex (PDB code: 4OD0) was obtained from the Protein Data Bank (PDB).59 Discovery Studio 2021 was employed to perform molecular docking analysis. The SCA was conducted with the Prepare Ligands module and minimized using the CHARM force field to generate three conformers. Before docking, protein was also prepared using Protein Preparation, allowing the addition of hydrogen atoms and the deletion of unnecessary water. Subsequently, protein was optimized and minimized. Using the Receptor–Ligand Pharmacophore Generation module, we defined the ligand-binding site. The docking results were evaluated by the -CDOCKER energy, hydrogen bond interaction, and the binding mode pattern. The Ligand Interaction tool was used to view the interaction diagram of the ligands with residues at the active site of the target protein. All structural figures were made using PyMol 3.7.

Cellular Thermal Shift Assay (CETSA)

The cells were treated with SCA (50 μM) and DMSO for 3 h, and then, cells were collected. The two groups were divided into nine equal portions. Nine temperature gradients were set on a PCR amplification instrument: 37, 40, 43, 46, 49, 52, 55, 61, and 67 °C. Each group was heated for 3 min. After heating all samples, the samples were repeatedly freeze–thaw-cracked.60 This was followed with western blot analysis.

Assessment of the Inhibitory Activity

The IC50 value was tested with a fluorescent end point assay system.26,61 Human sEH (hsEH) was incubated with SCA or TPPU for 5 min in BisTris–HCl buffer (25 mM, pH 7.0, containing 0.1 mg/mL BSA) at 30 °C prior to the substrate of the PHOME introduction ([S] = 5 μM). The IC50 value was determined by linear regression analysis employing at least three replicated data at different concentrations in the linear range.

Statistical Analysis

The data were expressed as the mean ± SEM, and multiple groups were statistically analyzed by one-way analysis of variance (ANOVA) in GraphPad Prism 8.0 software (San Diego, California, USA). In all cases, p < 0.05 was considered statistically significant.

Glossary

ABBREVIATIONS

ACC

anterior cingulate cortex

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

CFA

complete Freund’s adjuvant

EPM

elevated plus maze

FST

forced swim test

Glu

glutamic acid

HPLC-MS/MS

high-performance liquid chromatography–tandem mass spectrometry

IL

interleukin

NMDA

N-methyl-d-aspartic

OFT

open field test

sEH

soluble epoxide hydrolase

SPT

sucrose preference test

SCA

santacruzamate A

TNF-α

tumor necrosis factor-alpha

TST

tail suspension test

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00282.

  • SCA crossing the blood-brain barrier and reaching the brain and mean plasma and brain concentration–time curves after intragastric administration, pharmacokinetic parameters of oral administration of SCA, inhibitory effects of TPPU toward sEH, and 10 conformations of CDOCKER-energy for SCA or TPPU (PDF)

Author Contributions

∥ Y.Q. and Q.L. contributed equally to this work. Y.Q. wrote the paper draft, Q.L., M.Z., G.W. and L.Y. corrected the draft, J.Y., Q.Y., Y.W., and S.L. supervised the experimentation, Y.Q., Q.L., S.W., Q.W., Y.D., and Y.C. performed the experiments. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of the work ensuring integrity and accuracy.

The study was supported by the National Natural Science Foundation of China, nos. 31800887 and 31972902, and partially by the China Postdoctoral Science Foundation, no. 2020M683750.

The authors declare no competing financial interest.

Supplementary Material

pt3c00282_si_001.pdf (217.4KB, pdf)

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pt3c00282_si_001.pdf (217.4KB, pdf)

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