The Potential Effects of 2,3,4-Trihydroxybenzophenone on the Transient Cerebral Ischemic Stroke in Male Mice

Abstract

Acute ischemic stroke is a cerebrovascular disease associated with high mortality and severe aftereffects which is caused by the blockage of cerebral blood vessels by a thrombus or embolus. Treatments for this condition are extremely limited. Herein, we aimed to explore the potential of 2,3,4-trihydroxybenzophenone (THB), a drug that suppresses oxidative stress and neuroinflammation, to promote functional recovery through neurite outgrowth, and to identify its protective effects in a mouse model of ischemic stroke. To determine the effects of THB on neurite outgrowth, neurite-bearing cells and neurite lengths were measured in Neuro2a cells. 1,1-Diphenyl-2-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging assays were further performed to determine the antioxidant activity of THB, while lipopolysaccharide-activated BV2 cells were used to determine the anti-inflammatory effects of THB. Transient middle cerebral artery occlusion (tMCAO) was further performed in a mouse model to determine the effects of THB on ischemic stroke. THB increased neurite outgrowth in mouse neuroblastoma cell lines and exhibits antioxidant and anti-neuroinflammatory properties. In addition, THB reduced infarct volume in a concentration-dependent manner in the tMCAO model, leading to an increase in survival rate. Moreover, THB significantly suppressed microglial activation in the cortex and striatum. These results suggest that THB has potential for treating transient cerebral ischemic stroke.

INTRODUCTION

Cerebral ischemic stroke is a type of stroke that occurs when blood flow to a region of the brain is blocked or reduced, resulting in damage to brain tissue.¹⁾ This can be caused by a variety of situations, including blood clots or atherosclerosis (narrowing of the arteries due to plaque buildup). When blood flow to the brain is interrupted, brain cells begin to die owing to a lack of oxygen and nutrients.²⁾ The symptoms of cerebral ischemic stroke may vary depending on which part of the brain is affected; however, sudden weakness or numbness on one side of the body, difficulty speaking or understanding speech, sudden vision changes, severe headache, and difficulty with coordination or balance are common symptoms.³⁾ Immediate medical attention is critical for patients with cerebral ischemic stroke. Treatment options may include medication to dissolve blood clots using tissue plasminogen activators, or surgery to remove blockages.³⁾ However, excessive generation of reactive oxygen species (ROS) during the resulting reperfusion can lead to oxidative stress and neuroinflammation.⁴⁾ If the timing of reperfusion is delayed, or the brain damage caused by reperfusion cannot be controlled, a variety of sequelae may occur.⁵⁾ Although rehabilitation can help to recover lost abilities and functions, this requires considerable time and effort. As such, methods that can facilitate this process are required.

2,3,4-Trihydroxybenzophenone (THB), also known as benzophenone-6 or BP-6, is an organic compound in the benzophenones class. It is a derivative of benzophenone and is widely used in a variety of industries. THB is commonly used as a UV-absorbing agent in a variety of applications, including sunscreens, cosmetics, and plastics. This compound can absorb UV radiation and convert it into heat, thus protecting the skin or other materials from the damaging effects of UV radiation.⁶⁾ In addition to its UV-absorbing properties, several other potential health benefits of THB have also been investigated. For example, one previous study suggested that it may exert antioxidant and anti-inflammatory properties,⁷⁾ which could make it useful in the treatment of various diseases. Further, it has been shown to have neuroprotective effects in animal models of Parkinson's disease⁸⁾ and to exhibit anticancer activity in vitro.⁹⁾

In the present study, we hypothesized that an agent that protects the brain from ischemic injury and accelerates recovery by promoting the plasticity of surviving cells has potential as a treatment for ischemic stroke. To test whether THB could act as such an agent, we first confirmed the neurite outgrowth effect of THB in vitro, and assessed its protective effects against oxidative stress and inflammation, which are secondary injuries that occur following ischemic stroke. Based on these cellular experiments, we evaluated the protective effects of THB in a mouse model of ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO).

MATERIALS AND METHODS

Materials

THB (260576), 2,3,5-triphenyltetrazolium chloride (TTC, T8877), 2,2-diphenyl-1-picrylhydrazyl (DPPH, D9132), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, 10102946001), lipopolysaccharide (LPS, L2630), Griess reagent, 2′,7′-dichlorofluorescein diacetate (DCF-DA, 287810), and pyrogallol (P0381) were sourced from Sigma-Aldrich (St. Louis, MO, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) kits for prostaglandin E₂ (PGE₂, KGE004B), tumor necrosis factor-alpha (TNF-α, MTA00B), and interleukin-1beta (IL-1β, MLB00C) were purchased from R&D Systems (Minneapolis, MN, U.S.A.). Sutures were purchased for tMCAO (cat#602356PK10RE); Doccol Corporation (Sharon, MA, U.S.A.). For immunohistochemistry, we used an anti-Iba-1 antibody (Abcam, ab178847), an ABC kit (Vectastain, PK-4001), and a DAB kit (Vectastain, SK-4001).

Neuro2a Cell Culture

The Neuro2a cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, U.S.A.). Cells were maintained in Minimum Essential Medium (MEM; WelGENE Co., Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS; WelGENE Co.) at 37 °C in an environment with 5% CO₂ and 95% air humidity. Subculturing was performed when the cells reached 70–80% confluency.

Neurite Outgrowth

Neuro2a cells were seeded in 24-well plates (2 × 10⁴ cells/well) and cultured for 24 h, after which the medium was replaced, and cells were treated with varying concentrations of the drug (THB: 0.1, 0.5, 1, 5, 10 µM; retinoic acid, RA: 10 µM) for 6 h. Neurite outgrowth was further assessed by counting the neurite-bearing cells and measuring the neurite length using a phase-contrast microscopy and application software. Random fields of 100–200 cells were photographed using a Nikon Diaphot phase-contrast microscope, taking at least three photos per experimental point, and each experiment was repeated at least in triplicate. Cells with at least one neurite longer than their body were counted as neurite-bearing cells, with this population expressed as a percentage of the total cells. Neurite lengths were further measured using ImageJ software, tracing neurites longer than the cell nucleus and scaling in micrometers. The average neurite length was calculated using 200 processes per condition. Each experiment was performed thrice with three technical replicates, and the results were plotted.

ABTS and DPPH Assay

The ABTS and DPPH assays were performed to measure the antioxidant effects of THB. For the DPPH assay, a mixture of THB (0.1, 1, 10, 50, and 100 µM) and 0.1 mM DPPH was incubated at room temperature for 10 min, after which the absorbance of the mixture at 540 nm was measured. To prepare the ABTS solution, 7 mM ABTS and 2.4 mM potassium persulfate were mixed in a 1 : 1 ratio, and incubated at room temperature for 16 h protected from light. ABTS and THB (0.1, 1, 10, 50, and 100 µM) were then mixed and incubated at room temperature for 10 min, after which the absorbance of the mixture at 723 nm was measured.

BV2 Cell Culture and LPS Stimulation

BV2 microglial cells were provided by Professor Kyung-ja Kwon (Department of Pharmacology, School of Medicine, Konkuk University) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, U.S.A.) supplemented with 10% FBS and 1% penicillin–streptomycin in a humidified atmosphere with 5% CO₂ at 37 °C. The culture medium was changed every two days, and cells were sub-cultured at 80–90% confluency. To induce an inflammatory response, BV2 cells were stimulated with LPS at a final concentration of 100 ng/mL for 24 h. LPS was dissolved in phosphate-buffered saline (PBS) and added to the culture medium, while control cells were treated with an equal volume of PBS. After LPS stimulation, the cells were harvested for various assays, and the supernatant was collected for cytokine measurement using enzyme-linked immunosorbent assay (ELISA). All experiments were performed in triplicate. BV2 cells were treated with THB for 1 h prior to LPS treatment.

Cell Viability Assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT))

MTT assay was used to assess cytotoxicity. Cells were plated in a 24-well plate at a density of 2 × 10⁴ cells per well and incubated for 24 h. Subsequently, cells were treated with varying concentrations of THB (0, 1, 3, or 10 µM) with or without LPS (100 ng/mL) for 24 h. Following this drug treatment, a mixture of THB and MTT (0.5 µg/mL) in culture medium was added and incubated for 30 min. Subsequently, the MTT solution was aspirated, and DMSO was added. The absorbance was measured at 540 nm using ELISA plate reader (Bio-Rad, Hercules, CA, U.S.A.).

Measurements of Pro-inflammatory Mediators and Cytokines

The levels of nitric oxide (NO), PGE₂, TNF-α, and IL-1β in LPS-stimulated BV2 cells were quantified using ELISA. For NO measurement, the Griess reagent was added to the supernatant of LPS-stimulated BV2 cells, in accordance with the manufacturer’s instructions. The absorbance was measured at 540 nm using a microplate reader (BioTek Instruments, Winooski, VT, U.S.A.), and the concentration of NO was determined with reference to a standard curve generated using sodium nitrite (Sigma-Aldrich). For PGE₂, TNF-α, and IL-1β measurements, commercially available ELISA kits were used, following the manufacturer’s protocol. In brief, the supernatant was added to the wells of a 96-well plate coated with specific antibodies against each cytokine. After incubation and washing, a biotinylated antibody was added, followed by streptavidin-horseradish peroxidase and substrate solution. Absorbance was finally measured at 450 nm using a microplate reader, and the concentration of each cytokine was determined from a standard curve generated using recombinant cytokines (R&D Systems). All assays were performed in triplicate.

Detection of Intracellular ROS Levels

The production of ROS in LPS-stimulated BV2 cells was quantified using a DCF-DA assay. BV2 cells were seeded in 96-well plates and allowed to adhere overnight. Following treatment with LPS, the cells were incubated with 10 µM DCF-DA for 30 min at 37 °C. Cells were then washed with PBS and the fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 485 and 535 nm, respectively. All experiments were conducted in triplicate.

Animals

C57BL/6J male mice (6 weeks old) were obtained from Samtako (Osan, Korea) and allowed to adapt for 1 week with ad libitum access to food and water. Animals were raised in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals (NIH Publication No. 8023, revised 1978). All animal experiments were approved by the Institutional Animal Care and Use Committee of Dong-A University (DIACUC-approve-19-18). In both the dose–response and time-dependent studies, 28 mice were used, with seven mice used group.

tMCAO Surgery

Mice were subjected to transient focal ischemia via proximal tMCAO, and THB (0, 10, 30, or 60 mg/kg in 10% Tween 80 solution) or vehicle (10% Tween 80 soluntion) was orally administered one hour before the surgery. Before making neck skin incisions, mice were anesthetized with 2% isoflurane. The left common carotid artery (CCA) and external carotid artery (ECA) were carefully separated from the vagus nerve, and permanently tied using medical threads. At the CCA bifurcation, an intraluminal suture (medium MCAO filament (Doccol, Cat. No. 602356PK10Re) was inserted through the internal carotid artery to transiently block the middle cerebral artery. After one hour, the suture was removed and the surgical site was sutured. During the experiment, the core temperature of the mice was measured to maintain a body temperature of 37 °C through the use of a heating pad. The mice were allowed to fully recover on a heating pad, after which they were returned to their cages. Twenty four hours after the tMCAO, the ratio of surviving animals to all animals that underwent surgery in each group was measured and expressed as survival rate.

Neurological Severity Scores

The neurological severity score (NSS) was evaluated on days 1, 3, 5, 7, and 14 following tMCAO, in accordance with previously reported methods.¹⁰⁾ Neurological function was assessed using a scale from 0 to 18, where 0 represents normal function and 18 indicates the most severe impairment. The NSS incorporates assessments of motor abilities, sensory responses, reflexes, and balance. In this scoring system, a score of 1 is assigned when a task cannot be completed or when reflexes are absent, meaning that higher scores reflect greater injury severity. Statistical analysis only included NSS data from animals that survived the full 14-d period after the procedure.

TTC Staining

Brain slices were stained with TTC solution to visualize the ischemic infarct region. Twenty-four hours after tMCAO surgery, the mice were sacrificed and their brains were rinsed with chilled saline and coronally sectioned to a thickness of 1 mm in mouse brain matrix. The brain slices were then incubated in 0.5% TTC solution dissolved in saline at 37 °C for 10 min while protected from light. Following incubation, the TTC-stained brain slices were fixed with 4% paraformaldehyde overnight.

Infarct Volume Analysis

tMCAO-induced infarction was measured using ImageJ software. The infarct volume was assessed as follows: infarct volume (%) = (contralateral hemispheric volume-ipsilateral non-infarcted volume)/contralateral hemispheric volume.

Immunohistochemistry

The brain tissue samples were preserved in a storage solution containing 30% ethylene glycol and 30% glycerol in 20 mM phosphate buffer. To remove the storage solution, the tissues were rinsed thrice with PBS. Subsequently, they were treated with 1% hydrogen peroxide (H₂O₂) for 15 min to inactivate peroxidase activity. Non-specific binding sites were blocked by incubating the tissues in a solution of 3% normal goat serum and 0.3% Triton X-100 in PBS for 1 h. The primary antibody (Iba-1 antibody, Abcam, ab178847) was diluted 1 : 500 in a solution containing 2% normal goat serum and 0.3% Triton X-100 in PBS, and the tissues were incubated in this solution overnight. Following primary antibody incubation, tissues were exposed to secondary antibody solution prepared in 3% normal goat serum and 0.3% Triton X-100 in PBS, for 1 h. Avidin-biotin complex (ABC) formation was achieved by incubating the tissues with an ABC solution (Vectastain, PK-4001) for 1 h. Immunoreactivity was further visualized using the DAB kit (Vectastain, SK-4100). Between each step, the tissues were washed thrice with PBS to remove unbound reagents. The area in which Iba-1-immunopositive microglia had an amoeboid shape was set as the core region, while the area adjacent to the core region was set as the penumbra region. The area occupied by Iba-1-stained cells in the penumbra region was measured using the ImageJ software. First, images were converted to 8-bit format. The cortex and striatum area was measured using the polygon selections tool. Background was then removed, and the Iba1 positive area was measured by adjusting the threshold. Finally, the Iba1 positive area was compared to the cortex and striatum area

Statistics

Statistical analyses comprised a one-way ANOVA followed by Tukey’s test for multiple comparisons. For the LPS-induced BV2 activation study, two-way ANOVA followed by Bonferroni’s post hoc test was used. The results are presented as the mean ± standard deviation (S.D.). Statistical significance was set at p < 0.05.

RESULTS

THB Facilitates Neurite Outgrowth in Neuro2a Cells

To confirm the effect of THB on neurite outgrowth, Neuro2a mouse neuroblastoma cells were treated with THB (0, 2.5, 5, and 10 µM) (Fig. 1B), and the effects were assessed 6 h after treatment (Fig. 1A). Statistical analysis revealed that THB significantly affected the number of neurite-bearing cells (F_7,16 = 3.760, p = 0.0134, n = 3/group, one-way ANOVA followed by Tukey’s test, Figs. 1B and 1C), as well as the length of neurites (F_7,16 = 5.981, p = 0.0015, n = 3/group, one-way ANOVA followed by Tukey’s test, Figs. 1B, 1D). Specifically, 5 or 10 µM of THB significantly increased the number of neurite-bearing cells and the length of neurites. Treatment with the positive control, retinoic acid (10 µM), also significantly increased the number of neurite-bearing cells (Figs. 1B, 1C) and the length of neurites (Fig. 1D). These results suggest that THB promoted neurite outgrowth.

Fig. 1. Effect of THB on Neurite Outgrowth

Neuro2a cells were treated with THB (0.1, 0.5, 1, 2.5, 5, 10 µM) for 6 h. The number of cells with at least one neurite longer than its cell body was counted and expressed as a percentage of the total number of cells (neurite-bearing cells). The neurite length of each cell was measured by ImageJ software. A. Structure of THB. B. Representative images of neuro2a cells 6 h after THB treatment. C, D. Changes of neurite-bearing cells (C) and neurite length (D) by THB treatment. One-way ANOVA followed by Tukey’s test for post hoc test. Data are presented as mean ± S.D. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. Bar = 50 µm. RA retinoic acid, THB 2,3,4-trihydroxybenzophenone.

THB Has Anti-oxidative Effect

To assess the antioxidant effects of THB, we conducted DPPH and ABTS assays. Although both assays evaluate antioxidant capacity, they differ in that DPPH contains free radicals, whereas ABTS contains only cationic free radicals. THB exhibited concentration-dependent free radical scavenging activity in the DPPH assay, with an IC₅₀ of 13.69 µM (Figs. 2A, 2B). In the ABTS assay, THB decreased the levels of cationic free radicals in a concentration-dependent manner, with an IC₅₀ of 9.512 µM. It further exhibited an activity similar to that of pyrogallol, which was used as a positive control (Figs. 2C, 2D). These results indicated that THB exerts significant antioxidant activity.

Fig. 2. Anti-oxidative Effect of THB

A, B. DPPH radical scavenging activity (A) and IC₅₀ (B) of THB. A mixture of THB (0.1, 1, 10, 50, and 100 µM) and 0.1 mM DPPH was incubated at room temperature for 10 min, and the absorbance of the mixture was measured at 540 nm. C, D. ABTS+ radical scavenging activity (C) and IC₅₀ (D) of THB. The ABTS and THB (0.1, 1, 10, 50, and 100 µM) were mixed and incubated at room temperature for 10 min, and the absorbance of the mixture was measured at 723 nm. One-way ANOVA followed by Tukey’s test for post hoc test. Data are presented as mean ± S.D. N = 3. *** p < 0.001. THB 2,3,4-trihydroxybenzophenone, DPPH 2,2-diphenyl-1-picrylhydrazyl, ABTS 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt.

THB Has Anti-inflammatory Effect

To evaluate the anti-inflammatory effects of THB, its effects on the cell viability of LPS-activated BV2 cells were investigated. Statistical analysis revealed that THB was not cytotoxic to normal cells (F_3,8 = 0.2034, p = 0.8912, n = 3/group, one-way ANOVA followed by Tukey’s test, Fig. 3A) or to LPS-treated BV2 cells (F_4,10 = 1.990, p = 0.1721, n = 3/group, one-way ANOVA followed by Tukey’s test, Fig. 3B) in the indicated concentration ranges. Next, we examined the effects of THB on neuroinflammation. Statistical analysis revealed that THB significantly blocked the LPS-induced increase in NO (F_5,24 = 26.68, p < 0.0001, n = 5/group, one-way ANOVA followed by Tukey’s test, Fig. 3C), PGE₂ (F_5,24 = 104.2, p < 0.0001, n = 5/group, one-way ANOVA followed by Tukey’s test, Fig. 3D), TNF-α (F_5,24 = 31.11, p < 0.0001, n = 5/group, one-way ANOVA followed by Tukey’s test, Fig. 3E), IL-1β (F_5,24 = 124.2, p < 0.0001, n = 5/group, one-way ANOVA followed by Tukey’s test, Fig. 3F) and ROS (F_5,24 = 16.30, p < 0.0001, n = 5/group, one-way ANOVA followed by Tukey’s test, Fig. 3G) in BV2 cells. Two-way ANOVA comparing the vehicle + vehicle, vehicle + LPS, THB (10) + vehicle, THB (10) + LTP groups revealed significant interactions between THB and LPS for NO (F_1,16 = 34.99, p < 0.0001, two-way ANOVA followed by Bonferroni’s test), PGE₂ (F_1,16 = 111.4, p < 0.0001, two-way ANOVA followed by Bonferroni’s test), TNF-α (F_1,16 = 130.6, p < 0.0001, two-way ANOVA followed by Bonferroni’s test), IL-1β (F_1,16 = 315.9, p < 0.0001, two-way ANOVA followed by Bonferroni’s test) and ROS (F_1,16 = 38.5, p < 0.0001, two-way ANOVA followed by Bonferroni’s test). These results indicate that THB exerts anti-neuroinflammatory activity.

Fig. 3. Anti-inflammatory Effect of THB in BV2 Cells

A, B. Effect of THB (0, 1, 3, or 10 µM) on cell viability in normal BV2 (A) and LPS (100 ng/mL)-treated BV2 (B) cells. Cell viability was evaluated with MTT assay. BV2 cells were treated with THB 1 h before LPS treatment. One-way ANOVA followed by Tukey’s test for post hoc test. C. Effect of THB on LPS-induced nitric oxide generation in BV2 cells. Minocycline (Mino, 10 µM) was used as positive control. D–F. Effect of THB on LPS-induced cytokine generation including PGE₂ (D), TNF-α (D) and IL-1β (E) in BV2 cells. F. Effect of THB on LPS-induced ROS production productions in LPS-treated BV2 cell. Two way-ANOVA followed by Bonferroni’s test for post hoc test. Data are presented as mean ± S.D. N = 5. * p < 0.05. THB 2,3,4-trihydroxybenzophenone, LPS lipopolysaccharide, Mino minocycline, PGE₂ prostaglandin E₂, TNF-α tumor necrosis factor-α, IL-1β interleukin-1β. ROS reactive oxygen species.

THB Protects the Brain from Ischemia–Reperfusion Injury

THB (0, 10, 30, or 60 mg/kg) was orally administered one hour before tMCAO surgery, and the infarction volume was observed using TTC staining 24 h after tMCAO-reperfusion surgery. Statistical analysis revealed that THB significantly affected the infarction volume following tMCAO (F_3,24 = 4.948, p = 0.0082, n = 7/group, one-way ANOVA followed by Tukey’s test, Figs. 4A, 4B). Specifically, 60 mg/kg of THB reduced the infarction volume induced by tMCAO; however, no significant changes were observed at THB doses of 10 and 30 mg/kg (Figs. 4A, 4B). This reduction in infarction volume directly affected survival rate. The vehicle and THB (10 mg/kg) groups showed a low survival rate of 50% at 24 h after surgery, whereas the THB (30 or 60 mg/kg) group showed a survival rate of more than 80% (Fig. 4C). These results indicate that THB protects the brain from ischemia/reperfusion-induced injury.

Fig. 4. Protective Effect of THB against Ischemic–Reperfusion Injury

The mice were subjected to transient focal ischemia via proximal tMCAO, and THB (0, 10, 30, or 60 mg/kg in 10% tween 80 solution) or vehicle (10% tween 80 solution) was orally administered either one h before or after the surgery. A. Photographs of brain slices stained by TTC 24 h after tMCAO. B. Infarct area of THB-treated mice before tMCAO. C. Survival rates of THB-treated tMCAO mice. One-way ANOVA followed by Tukey’s test for post hoc test. Data are presented as mean ± S.D. N = 7. * p < 0.05. Bar = 2 mm. tMCAO transient middle cerebral artery occlusion THB 2,3,4-trihydroxybenzophenone.

THB Suppresses Neuroinflammation Induced by tMCAO

We have already confirmed the anti-inflammatory effects of THB in previous results. Therefore, we investigated whether THB alters neuroinflammation in the tMCAO model. Statistical analysis revealed that THB significantly affected neuroinflammation in the tMCAO model in cortex (F_3,16 = 5.520, p = 0.0085, n = 5/group, one-way ANOVA followed by Tukey’s test, Figs. 5A, 5B) and striatum (F_3,16 = 25.12, p < 0.0001, n = 5/group, one-way ANOVA followed by Tukey’s test, Figs. 5A, 5B) regions. Specifically, compared to the sham group, the tMCAO model showed increased Iba-1 immunopositive areas in both the cortex and striatum penumbra regions (Figs. 5A, 5B). THB administration (60 mg/kg) significantly reduced the Iba-1-immunopositive areas in the striatum but not in cortex. These results indicated that THB reduced neuroinflammation in the penumbral region.

Fig. 5. Effect of THB on Neuroinflammation in Ischemic–Reperfusion Injury

The Iba-1-positive area were measured in penumbra region (black box in upper image of A) of cortex and striatum. A. Photomicroscopic images of interest area (1st panel, bar = 2 mm) and Iba-1-immunopositive cells in the cortex (middle panel) and striatum (low panel). Bar = 100 µm. B. Quantitative analysis of Iba-1-imunopositive area. One-way ANOVA followed by Tukey’s test for post hoc test. Data are presented as mean ± S.D. N = 7. * p < 0.05. Ns not significant, tMCAO transient middle cerebral artery occlusion THB 2,3,4-trihydroxybenzophenone.

THB Improves Functional Deficit by tMCAO

To test whether THB improves functional deficits caused by tMCAO, NSS was measured on days 1, 3, 5, 7, and 14 after tMCAO. THB (60 mg/kg) was administered once daily, starting 1 h before tMCAO and continuing until day 14 after tMCAO (Fig. 6A). THB treatment significantly reduced NSS on days 7 (t₁₀ = 2.936, p < 0.05, n = 6/group) and 14 (t₁₀ = 3.606, p < 0.05, n = 6/group) compared to vehicle treatment (Fig. 6B). These results suggest that THB improves functional deficits caused by tMCAO.

Fig. 6. Effect of THB on Neurological Severity Score in tMCAO Model

A. Neurological severity score was measured on days 1, 3, 5, 7, and 14 after tMCAO. THB (60 mg/kg) was administered once daily, starting 1 h before tMCAO and continuing until day 14 after tMCAO. B. Measured neurological severity score on 1, 3, 5, 7, and 14 d after tMCAO. Data are presented as mean ± S.D. N = 6. * p < 0.05. tMCAO transient middle cerebral artery occlusion THB 2,3,4-trihydroxybenzophenone.

DISCUSSION

Overall, this study investigated the protective effects of THB against ischemic stroke, with results indicating that THB exhibited antioxidant, anti-inflammatory, and neurite outgrowth effects in vitro, and reduced infarct volume in a mouse model of ischemic stroke.

Cerebral infarction, caused by the occlusion of the middle cerebral artery, is the most common subtype of ischemic stroke, accounting for 50% of all cases.¹¹⁾ This condition predominantly affects the cerebral motor cortex, striatum, and limbic system,^12,13) leading to sequelae such as loss of motor function and cognitive decline.¹⁴⁾ Tissue plasminogen activator (tPA), the only U.S. Food and Drug Administration (FDA)-approved stroke treatment,¹⁵⁾ further opens blocked cerebral blood vessels by dissolving blood clots to prevent further brain damage, but does not improve the functional recovery of already damaged brain tissue. Many studies have thus far been conducted to identify methods to inhibit brain cell death following a stroke; however, no treatments meeting this objective have yet been developed, indicating that once a stroke occurs, preventing subsequent brain cell death can be difficult. Numerous studies have previously reported excellent neuroprotective activity with the administration of drugs prior to stroke; however, in clinical situations, it is not possible to administer drugs before stroke occurs. However, we can view these substances as effective against stroke in the context of reducing the incidence or severity of the condition before it occurs, similar to health supplements or vitamins. In such cases, easily accessible, side effect-free, and inexpensive low-molecular-weight compounds or natural products are most suitable.

From this perspective, the efficacy of a drug is linked to its ability to counteract oxidative stress and inflammatory cell death,^16–18) the most important mechanisms underlying brain cell death in ischemic strokes. Cerebrovascular occlusion leading to oxygen and glucose deprivation causes the excessive production of ROS through the activation of the mitochondria, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, and nitric oxide synthase.¹⁹⁾ Consequently, drugs that inhibit or eliminate ROS can protect the brain from ischemia/reperfusion injury. Edaravone, a drug developed following this hypothesis, has a powerful antioxidant effect, and is administered to prevent post-stroke disabilities in Japan.²⁰⁾ Moreover, previous reports suggest that consumption of antioxidant-rich foods or supplements may reduce the incidence of ischemic cerebrovascular disease.^21,22) Inflammatory responses play a significant role in the pathogenesis of ischemic stroke.²³⁾ Neuroinflammation is characterized by microglia activation, which leads to the production of pro-inflammatory cytokines, such as IL-1β and TNF-α, and chemokines, such as monocyte chemoattractant protein-1.²³⁾ These inflammatory mediators attract leukocytes to the site of injury, thereby exacerbating the inflammatory response, and leading to neuronal damage. Microglial activation is further triggered by the release of damage-associated and pathogen-associated molecular patterns, released by injured cells and invading microorganisms, respectively.^24,25) These molecules activate toll-like receptors on microglia, leading to the release of pro-inflammatory cytokines and chemokines.²⁶⁾ Our results showed that THB exerts radical-scavenging effects and could significantly reduce the release of pro-inflammatory cytokines from LPS-activated BV2 cells (a murine microglia cell line). Moreover, THB reduced LPS-induced ROS generation, indicating that it may suppress neuroinflammation in brain tissues during ischemic stroke. The effects of THB observed in vitro manifested as a reduction in brain tissue damage in the tMCAO model, suggesting that THB has the potential to reduce stroke severity.

Neurite outgrowth is a critical process for recovering neuronal function following ischemic stroke and depends on neurotrophic support and a favorable environment for neuronal growth.^27,28) Recently, bryostatin, an AD candidate that promotes neurite growth, was developed.²⁹⁾ This are likely due to the multiple effects of neurite growth on neurogenesis, axonal elongation, and dendritic spine formation.³⁰⁾ Neurite outgrowth is especially essential for the integration of newborn neurons into existing neural circuits.³¹⁾ Therefore, functional recovery relies on both neurite outgrowth and neurogenesis, which include processes such as axonal elongation and dendritic spine formation.³²⁾ Moreover, these processes share similar signaling pathways, including those involving BDNF and NGF. Consequently, bryostatin may facilitate functional recovery through this compensatory mechanism. Our results indicate that administration of this drug significantly increases the length and number of neurites in murine neuroblastoma cells, suggesting that THB has a positive effect on neurite outgrowth. We do not yet know the exact mechanism by which THB promotes neurite outgrowth. However, given that many previous studies have reported the neurite outgrowth-promoting effects of antioxidants, we expect that the antioxidative effect of THB may be related to its ability to promote neurite outgrowth.^33–35) Of course, while some studies have reported that ROS itself can induce signals for neurite outgrowth,^36,37) considering the contradictory results from other studies, it can be inferred that maintaining an appropriate level of ROS is important for the induction of neurite outgrowth.

As such, THB’s promotion of neurite outgrowth is highly likely contributes to the recovery of brain function by restoring brain damage in animal models of stroke. However, there are no reference to predict whether THB can penetrate the BBB. Nevertheless, in silico analysis using SwissADME (http://www.swissadme.ch/index.php) predicted that THB would not pass through the BBB. However, previous studies have reported that the BBB is disrupted in animal models of tMCAO,^38,39) indicating that substances that cannot cross the BBB in healthy animals may be able to do so in tMCAO models. Therefore, we could expect that THB will not pass the normal BBB but would be able to pass the damaged BBB in the tMCAO model. Another interpretation is that THB may have induced improvements in the microflow in the blood vessels around the brain, rather than directly affecting the brain. Indeed, in one pilot study, we found that THB was not effective when administered immediately or 1 h after surgery, but was effective when administered 1 h prior to surgery (data not shown), indicating that THB may trigger changes in blood vessels prior to entering the brain. To prove this hypothesis, further studies testing the translocation rate of THB to the brain after administration in tMCAO models, or to evaluate the effects of THB on cerebral blood flow, are required.

In conclusion, our study demonstrated that THB exerts antioxidant, anti-inflammatory, and neurite outgrowth effects in vitro, and further reduces severity in an ischemic stroke mouse model. THB scavenges free radicals, reduces ROS and proinflammatory cytokine production, and promotes neuronal growth. THB can also reduce the infarct volume following ischemic stroke. As such, THB could serve as a starting compound for the development of substances that can reduce severity of strokes. However, we still do not know the mechanism of THB on neurite outgrowth and anti-inflammatory effects. Therefore, further studies are required to explore the precise mechanism of THB.

Acknowledgments

This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1A2C1005251).

Conflict of Interest

The authors declare no conflict of interest.

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