3′,4′-Dihydroxyflavonol Attenuates Lipopolysaccharide-Induced Neuroinflammatory Responses of Microglial Cells by Suppressing AKT–mTOR and NF-κB Pathways

Abstract

Microglia-related neuroinflammation contributes to the pathogenesis of a variety of neurodegenerative disorders such as Alzheimer’s disease. The synthetic flavonoid, 3′,4′-dihydroxyflavonol (3,3′,4′-trihydroxyflavone), has been shown to protect brain or myocardial ischemia reperfusion-induced cell death and prevent the aggregation of amyloid-β protein, a process that causes progressive neurodegeneration in Alzheimer’s disease. Here, we explored the anti-neuroinflammatory ability of 3′,4′-dihydroxyflavonol in lipopolysaccharide (LPS)-activated MG6 microglial cells. 3′,4′-Dihydroxyflavonol attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LPS-induced phosphorylation of mammalian target of rapamycin (mTOR), nuclear factor-κB (NF-κB), and protein kinase B (AKT) (which are all associated with the neuroinflammatory response in microglia) were attenuated by 3′,4′-dihydroxyflavonol treatment. Treatment with the mTOR inhibitor, rapamycin, NF-κB inhibitor, caffeic acid phenethyl ester, or AKT inhibitor, LY294002, also attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LY294002 treatment attenuated LPS-induced phosphorylation of mTOR and NF-κB in MG6 cells. Hence, our study suggests that 3′,4′-dihydroxyflavonol can attenuate the neuroinflammatory response of microglial cells by suppressing the AKT–mTOR and NF-κB pathways.

INTRODUCTION

Neuroinflammation induced by prolonged and persistent activation of brain immune cells plays a key role in the pathophysiology of neurodegenerative diseases, including Alzheimer’s disease (AD).¹⁾ Microglia are immune cells in the mammalian central nervous system (CNS) that respond to changes in the brain microenvironment to maintain its homeostasis under physiological conditions.^1,2) Excessive and uncontrolled activation of microglia causes the secretion of large amounts of inflammatory neurotoxic mediators such as tumor necrosis factor-α (TNF-α), nitric oxide (NO), and interleukin-6 (IL-6). This results in neuronal cell death and further induces excessive activation of microglia, thereby exacerbating brain inflammation.^2,3)

Mammalian target of rapamycin (mTOR) belongs to the serine/threonine protein kinase family and plays a central role in regulation of the innate immune system as well as cell growth, autophagy, and synaptic plasticity.⁴⁾ Excessive activation of mTOR is thought to be involved in a variety of human diseases, including neurological disorders, cancer and metabolic syndromes.^4,5) Much research has shown that overactivation of mTOR signaling in the CNS plays a major role in initiation and progression of the pathogenesis of AD, and correlates with neuroinflammation.^4–7) Excessive activation of protein kinase B (AKT) signaling in the CNS is also thought to be involved in neuroinflammatory disorders.⁸⁾ Indeed, activated microglial cells induce phosphorylation of AKT, which subsequently drives downstream pathways, such as mTOR or nuclear factor-κB (NF-κB), key molecules associated with neuroinflammation.^9–11)

Flavonol is one of the major classes of flavonoids in the plant kingdom, and is characterized by a hydroxyl group in the C-3 position of the C ring in the flavone skeleton (which has a C₆-C₃-C₆ configuration).¹²⁾ Several flavonols, such as fisetin (3,3′,4′,7-tetrahydroxyflavone), have been shown to exhibit beneficial effects in cancer, cardiovascular diseases, and neurodegenerative disorders, both in vivo and in vitro.^13–16) 3′,4′-Dihydroxyflavonol (3,3′,4′-trihydroxyflavone) is a synthetic flavonoid that has previously been shown to be more potent in producing vasorelaxation than the other flavonols (fisetin and quercetin) and flavones (apigenin, chrysin, and luteolin).¹⁷⁾ Other studies have shown that 3′,4′-dihydroxyflavonol protects against ischemia reperfusion injury in brain, heart, and vascular cells.^18–21) In addition, we have previously found that 3′,4′-dihydroxyflavonol prevents the aggregation of amyloid-β protein (Aβ), a process that may cause progressive neuronal degeneration in AD.²²⁾ The potency of 3′,4′-dihydroxyflavonol in inhibiting Aβ aggregation is much higher than fisetin or kaempferol (3,4′,5,7-tetrahydroxyflavone). Thus, 3′,4′-dihydroxyflavonol may have therapeutic potential in the treatment of AD. However, little is known about whether 3′,4′-dihydroxyflavonol can exert anti-neuroinflammatory effects. In this study, we found beneficial effects of 3′,4′-dihydroxyflavonol on the neuroinflammatory responses of microglial cells. Furthermore, we determined how 3′,4′-dihydroxyflavonol influences the potential mechanisms underlying these inflammatory responses.

MATERIALS AND METHODS

Cell Culture

The mouse microglial cell line, MG6 (#RBC2403), was purchased from RIKEN BioResource Research Center (Tsukuba, Japan) and cultured in Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, U.S.A.) with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, U.S.A.), 10 µg/mL insulin (Sigma-Aldrich), and 100 µM 2-mercaptoethanol (Wako, Osaka, Japan) at 37 °C in the presence of 5% CO₂.^23,24) MG6 cells were maintained in a resting or non-activated condition before treatment with lipopolysaccharide (LPS) (Wako).

Cell Viability Assay

MG6 microglial cells were seeded into 96 well plates (5 × 10⁴ cells/well), and treated for 48 h with FBS-free DMEM containing various concentrations of 3′,4′-dihydroxyflavonol (0.1, 1, 10, and 100 µM) (Indofine Chemical, Hillsborough, NJ, U.S.A.) in the absence or presence of 100 ng/mL LPS. Next, a Cell Counting Kit-8 assay (CCK8) (Dojindo, Kumamoto, Japan) was performed.²⁵⁾

Enzyme-Linked Immunosorbent Assay (ELISA)

MG6 microglial cells were seeded into 96 well plates (5 × 10⁴ cells/well), and treated with FBS-free DMEM containing 0.1–10 µM 3′,4′-dihydroxyflavonol, 100 nM rapamycin (Tocris Bioscience, Bristol, U.K.), 10 µM LY294002 (Wako), 10 µM caffeic acid phenethyl ester (CAPE) (Tocris Bioscience), and 100 ng/mL LPS for 24 h. A commercial ELISA kit (R&D Systems, Minneapolis, MN, U.S.A.) was used to measure the amount of TNF-α in the culture medium.²⁵⁾

NO Assay

MG6 microglial cells were seeded into 96 well plates (5 × 10⁴ cells/well), and treated for 48 h with FBS-free DMEM containing 0.1–10 µM 3′,4′-dihydroxyflavonol, 100 nM rapamycin, 10 µM LY294002, 10 µM CAPE, and 100 ng/mL LPS. As described previously, Griess reagent was used to measure the amount of NO in the culture medium by examination of accumulated nitrite, a NO oxidation product.^25–27)

Immunoblotting Assay

MG6 microglial cells were seeded into 48 well plates (12.5 × 10⁴ cells/well), and treated for 0.5–6 h with FBS-free DMEM containing 0.1–10 µM 3′,4′-dihydroxyflavonol, 10 µM LY294002, and 100 ng/mL LPS. Protein samples were prepared from MG6 cells, separated on 7.5–12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 2% bovine serum albumin for 2 h, and then treated with primary antibodies at 5 °C for 24 h. The primary antibodies used were: anti-phospho-mTOR (Ser2448), anti-phospho-AKT (Ser473), anti-phospho-NF-κB p65 (Ser536), anti-mTOR, anti-AKT, and anti-NF-κB p65 (Cell Signaling Technology Inc., Danvers, MA, U.S.A.). Next, membranes were treated with HRP-linked secondary antibodies (Cell Signaling Technology Inc.). Specific immunoreactive proteins were visualized by enhanced chemiluminescence and the intensities quantified using a ImageQuant LAS-4000 system (FUJIFILM, Tokyo, Japan).^25–27)

Statistical Analysis

Data are shown as mean ± standard error of the mean (S.E.M.) and were analyzed by one-way ANOVA or Kruskal–Wallis test by ranks followed by Dunnett, Tukey, or Steel–Dwass test, as appropriate.

RESULTS

Effect of 3′,4′-Dihydroxyflavonol on LPS-Induced Secretion of TNF-α and NO in MG6 Cells

To determine whether 3′,4′-dihydroxyflavonol influences the viability of MG6 cells, we performed a CCK8 assay. 3′,4′-Dihydroxyflavonol concentrations <10 µM had no effect, but 100 µM decreased cell viability (Fig. 1B). Treatment with 100 ng/mL LPS for 48 h significantly decreased the viability of MG6 cells (Fig. 1C). This result was similar to that of previous papers,^28–31) while there are many papers showing that treatment with 100 ng/mL LPS had no effect or increased the viability of microglia.³²⁾ The discrepancies might be attributed to differences in experimental conditions including LPS treatment periods (24–144 h), culture protocols or cell conditions. Because it is widely accepted that LPS at 100 ng/mL causes the secretion of large amounts of inflammatory neurotoxic mediators such as TNF-α and NO in microglia, we used this concentration of LPS to examine whether 3′,4′-dihydroxyflavonol can inhibit neuroinflammation. Cotreatment with 3′,4′-dihydroxyflavonol and 100 ng/mL LPS for 48 h did not change the LPS-induced decrease of cell viability (Fig. 1C). Thus, we chose concentrations of 0.1, 1, and 10 µM to examine the anti-neuroinflammatory effects of 3′,4′-dihydroxyflavonol. ELISA and Griess assays were used to determine whether 3′,4′-dihydroxyflavonol attenuated neuroinflammatory responses in activated microglial cells. Treatment with 100 ng/mL LPS for 24 or 48 h significantly elevated TNF-α levels or NO production in MG6 cells, respectively (Figs. 1D, E). Cotreatment with 3′,4′-dihydroxyflavonol and 100 ng/mL LPS significantly attenuated LPS-induced TNF-α and NO secretion in a concentration-dependent manner (Figs. 1D, E).

Fig. 1. 3′,4′-Dihydroxyflavonol Attenuated LPS-Induced NO and TNF-α Production in MG6 Cells

(A) The chemical structure of 3′,4′-dihydroxyflavonol. (B) Cells were treated with 0.1, 1, 10, and 100 µM 3′,4′-dihydroxyflavonol for 48 h (n = 10). Cell viability was expressed as a percentage of the control (none, 0 µM 3′,4′-dihydroxyflavonol). Dunnett’s test (F_{4, 45} = 49.147, p < 0.001). * p < 0.05 vs. control (none). (C) Cells were cotreated with 0.1, 1, 10, or 100 µM 3′,4′-dihydroxyflavonol and 100 ng/mL lipopolysaccharide (LPS) for 48 h (n = 10). Cell viability was expressed as a percentage of the control (none, 0 µM 3′,4′-dihydroxyflavonol). Tukey’s test (H = 30.464, p < 0.001). * p < 0.05 vs. control (none). (D, E) Cells were cotreated with 0.1, 1, or 10 µM 3′,4′-dihydroxyflavonol and 100 ng/mL LPS for 24 h (D, n = 7) or 48 h (E, n = 7). Tukey’s test ; (D: H = 27.441, p < 0.001; E: H = 31.969, p < 0.001). * p < 0.05 vs. control (none), ^#p < 0.05 vs. LPS.

Effect of 3′,4′-Dihydroxyflavonol on LPS-Induced Activation of mTOR in MG6 Cells

mTOR is a serine/threonine kinase that plays an important role in neuroinflammatory signaling pathways.⁵⁾ Thus, we investigated the effect of 3′,4′-dihydroxyflavonol on mTOR phosphorylation in activated microglial cells. 3′,4′-Dihydroxyflavonol alone did not induce mTOR phosphorylation in MG6 cells under non-activated states (Supplementary Fig. 1A). Treatment with 100 ng/mL LPS for 6 h significantly elevated the levels of mTOR phosphorylation in MG6 cells (Fig. 2A). In contrast, treatment with 3′,4′-dihydroxyflavonol prior to 100 ng/mL LPS significantly attenuated LPS-induced mTOR phosphorylation in a concentration-dependent manner (Fig. 2A). The inhibitory effect of 10 µM 3′,4′-dihydroxyflavonol on LPS-induced mTOR phosphorylation was similar to that with 100 nM rapamycin (a mTOR inhibitor) (Supplementary Fig. 2A). To confirm whether mTOR activation is required for LPS-induced TNF-α and NO secretion, we examined the effect of rapamycin. Cotreatment with 100 nM rapamycin and 100 ng/mL LPS for 24 or 48 h significantly attenuated LPS-induced TNF-α or NO secretion in MG6 cells, respectively (Figs. 2B, C).

Fig. 2. 3′,4′-Dihydroxyflavonol Attenuated LPS-Induced mTOR Phosphorylation in MG6 Cells

(A) Cells were pretreated for 2 h with 0.1, 1, or 10 µM 3′,4′-dihydroxyflavonol, and then treated with 100 ng/mL LPS for 6 h. Immunoblotting to determine expression levels of phosphorylated mTOR (P-mTOR) and total mTOR (n = 6). Steel–Dwass test (H = 20.898, p < 0.001). (B, C) Cells were cotreated with 100 nM rapamycin and 100 ng/mL LPS for 24 h (B, n = 5) or 48 h (C, n = 5). Tukey’s test (B: F_{2, 12} = 242.793, p < 0.001; C: F_{2, 12} = 271.802, p < 0.001). * p < 0.05 vs. control (none), ^# p < 0.05 vs. LPS.

Effect of 3′,4′-Dihydroxyflavonol on LPS-Induced Activation of AKT in MG6 Cells

Aberrant AKT signaling is implicated in the neuroinflammatory response of activated microglia.⁸⁾ Thus, we analyzed the effect of 3′,4′-dihydroxyflavonol on LPS-induced AKT phosphorylation in MG6 cells. 3′,4′-Dihydroxyflavonol alone did not induce AKT phosphorylation in MG6 cells under non-activated states (Supplementary Fig. 1B). Treatment with 100 ng/mL LPS for 0.5 h significantly elevated AKT phosphorylation levels (Fig. 3A). Treatment with 3′,4′-dihydroxyflavonol prior to 100 ng/mL LPS significantly attenuated LPS-induced AKT phosphorylation in a concentration-dependent manner (Fig. 3A). The inhibitory effect of 10 µM 3′,4′-dihydroxyflavonol on LPS-induced AKT phosphorylation was similar to that with 1 µM LY294002 (a phosphatidylinositol 3 kinase (PI3K) inhibitor) (Supplementary Fig. 2B). To confirm whether aberrant AKT activation is required for LPS-induced TNF-α and NO secretion, we examined the effect of LY294002. Cotreatment with 10 µM LY294002 and 100 ng/mL LPS for 24 or 48 h significantly attenuated LPS-induced TNF-α or NO secretion in MG6 cells, respectively (Figs. 3B, C).

Fig. 3. 3′,4′-Dihydroxyflavonol Attenuated LPS-Induced AKT Phosphorylation in MG6 Cells

(A) Cells were pretreated for 2 h with 0.1, 1, or 10 µM 3′,4′-dihydroxyflavonol, and then treated with 100 ng/mL LPS for 0.5 h. Immunoblotting to determine expression levels of phosphorylated AKT (P-AKT) and total AKT (n = 6). Steel–Dwass test (H = 15.726, p < 0.001). (B, C) Cells were cotreated with 10 µM LY294002 and 100 ng/mL LPS for 24 h (B, n = 5) or 48 h (C, n = 5). Tukey’s test (B: F_{2, 12} = 989.826, p < 0.001; C: F_{2, 12} = 1829.559, p < 0.001). * p < 0.05 vs. control (none), ^# p < 0.05 vs. LPS.

Effect of 3′,4′-Dihydroxyflavonol on LPS-Induced Activation of NF-κB in MG6 Cells

NF-κB is another signaling molecule that plays an important role in neuroinflammatory signaling pathways.^11,12) Thus, we analyzed the effect of 3′,4′-dihydroxyflavonol on LPS-induced NF-κB phosphorylation in MG6 cells. 3′,4′-Dihydroxyflavonol alone did not induce NF-κB phosphorylation in MG6 cells under non-activated states (Supplementary Fig. 1C). Treatment with 100 ng/mL LPS for 3 h significantly elevated NF-κB phosphorylation levels (Fig. 4A). Treatment with 3′,4′-dihydroxyflavonol prior to 100 ng/mL LPS significantly attenuated LPS-induced NF-κB phosphorylation in a concentration-dependent manner (Fig. 4A). The inhibitory effect of 10 µM 3′,4′-dihydroxyflavonol on LPS-induced NF-κB phosphorylation was similar to that with 1 µM CAPE (a NF-κB inhibitor) (Supplementary Fig. 2C). To confirm whether NF-κB activation is required for LPS-induced TNF-α and NO secretion, we examined the effect of CAPE. Cotreatment with 10 µM CAPE and 100 ng/mL LPS for 24 or 48 h significantly attenuated LPS-induced TNF-α or NO secretion in MG6 cells, respectively (Figs. 4B, C).

Fig. 4. 3′,4′-Dihydroxyflavonol Attenuated LPS-Induced NF-κB Phosphorylation in MG6 Cells

(A) Cells were pretreated for 2 h with 0.1, 1, or 10 µM 3′,4′-dihydroxyflavonol, and then treated with 100 ng/mL LPS for 3 h. Immunoblotting to determine expression levels of phosphorylated NF-κB (P-NF-κB) and total NF-κB (n = 6). Steel–Dwass test (H = 22.129, p < 0.001). (B, C) Cells were cotreated with 10 µM caffeic acid phenethyl ester (CAPE) and 100 ng/mL LPS for 24 h (B, n = 5) or 48 h (C, n = 5). Tukey’s test (B: F_{2, 12} = 3693.66, p < 0.001) or Steel–Dwass test (C: H = 9.563, p = 0.008). * p < 0.05 vs. control (none), ^# p < 0.05 vs. LPS.

Effect of AKT Inhibition on LPS-Induced Activation of NF-κB and mTOR in MG6 Cells

AKT is considered to be a key regulator of TNF-α and NO secretion by activation of the NF-κB and/or mTOR signaling pathways.^8–11) Thus, we analyzed the effect of the PI3K–AKT inhibitor, LY294002, on LPS-induced phosphorylation of NF-κB and mTOR in MG6 cells. Treatment with 100 ng/mL LPS significantly elevated phosphorylation levels of AKT, NF-κB, and mTOR (Fig. 5). Treatment with 10 µM LY294002 prior to 100 ng/mL LPS significantly attenuated LPS-induced phosphorylation of AKT, NF-κB, and mTOR (Fig. 5).

Fig. 5. AKT Inhibition Attenuated LPS-Induced Phosphorylation of mTOR and NF-κB in MG6 Cells

(A–C) Cells were pretreated for 2 h with 10 µM LY294002, and then treated with 100 ng/mL LPS for 0.5 h (A, n = 6), 6 h (B, n = 6), and 3 h (C, n = 6). Immunoblotting to determine expression levels of phosphorylated (P) proteins: P-AKT, P-mTOR, and P-NF-kB. Steel–Dwass test (A: H = 15.726, p < 0.001; B: H = 15.726, p < 0.001; C: H = 13.542, p = 0.001). * p < 0.05 vs. control (none), #p < 0.05 vs. LPS.

DISCUSSION

The synthetic small molecule, 3′,4′-dihydroxyflavonol, belongs to the flavonoids (a class of a large family of polyphenolic compounds), and has been shown to inhibit brain and myocardial ischemia reperfusion-induced cell death.^17–21) The protective effects of 3′,4′-dihydroxyflavonol are mainly due to its powerful antioxidant activities.^33–36) We have previously shown that 3′,4′-dihydroxyflavonol prevents aggregation of Aβ, a process that plays a key role in neuronal death in AD.²²⁾ However, the effects of 3′,4′-dihydroxyflavonol on neuroinflammation have not been reported. Here, we show that 3′,4′-dihydroxyflavonol attenuates LPS-induced TNF-α and NO secretion in MG6 cells. Because NO and TNF-α are major representative mediators of the neuroinflammation associated with neurodegenerative disorders, 3′,4′-dihydroxyflavonol has therapeutic potential to attenuate neurodegeneration.

mTOR is a serine/threonine protein kinase with a key role in several physiological processes such as autophagy, lipid metabolism, and protein synthesis.⁴⁾ Excessive activation of mTOR signaling is thought to be involved in the initiation and progression of the pathogenesis of neurological disorders associated with neuroinflammation.^4–7) In the present study, 3′,4′-dihydroxyflavonol inhibited LPS-induced mTOR phosphorylation (Fig. 2A), while the mTOR inhibitor, rapamycin, suppressed LPS-induced TNF-α and NO secretion (Figs. 2B, C). 3′,4′-Dihydroxyflavonol also inhibited LPS-induced AKT phosphorylation (Fig. 3A). Furthermore, pharmacological inhibition of PI3K–AKT signaling suppressed the release of LPS-induced inflammatory factors (Figs. 3B, C) and LPS-induced mTOR phosphorylation (Fig. 5B) in MG6 cells. Much evidence indicates that activation of the PI3K–AKT pathway upregulates downstream signaling molecules (such as mTOR or NF-κB) associated with neuroinflammatory responses in microglial cells.^8–11) Hence, our data suggest that 3′,4′-dihydroxyflavonol attenuates LPS-induced microglial neuroinflammatory responses via suppression of the AKT–mTOR pathway. Nonetheless, we cannot exclude the possibility that AKT-independent mTOR activation induced by LPS partly contributes to the production of TNF-α and/or NO in our experiments.

In the present study, it remains unclear how the inhibitory action of 3′,4′-dihydroxyflavonol on mTOR leads to these anti-neuroinflammatory effects in microglial cells. In general, impaired autophagy is thought to be involved in triggering neuroinflammation. Ye et al.³⁷⁾ reported that LPS enhances TNF-α and NO secretion in N9 microglial cells via activation of the PI3K–AKT–mTOR pathway, with LPS-induced neuroinflammation attenuated by upregulation of autophagy in vivo and in vitro. Therefore, 3′,4′-dihydroxyflavonol may recover LPS-induced impairment of autophagy in microglial cells by inhibiting the AKT–mTOR pathway. To clarify whether 3′,4′-dihydroxyflavonol inhibits neuroinflammation by upregulating autophagy, it would be useful to detect and/or quantify autophagy-related proteins such as LC3II or SQSTM1.^37,38)

NF-κB is also thought to be a key molecule in physiological and pathophysiological processes such as autophagy, apoptosis, and inflammation.^11,12) Considerable evidence indicates that phosphorylation of the AKT protein triggers phosphorylation of NF-κB and induces cytokine release in activated microglial cells.^39–41) Here, we found that 3′,4′-dihydroxyflavonol inhibited LPS-induced NF-κB phosphorylation (Fig. 4A), while the NF-κB inhibitor, CAPE, suppressed the release of LPS-induced inflammatory factors (Figs. 4B, C) in MG6 cells. Furthermore, pharmacological inhibition of PI3K–AKT signaling suppressed LPS-induced NF-κB phosphorylation (Fig. 5C). Hence, our data suggest that 3′,4′-dihydroxyflavonol also attenuates LPS-induced microglial neuroinflammatory responses via suppression of the AKT–NF-κB pathway. Nonetheless, we cannot exclude the possibility that AKT-independent NF-κB activation induced by LPS partly contributes to the production of TNF-α and/or NO in our experiments.

In the present study, we have successfully shown that 3′,4′-dihydroxyflavonol inhibits LPS-induced mTOR and NF-κB activation by suppressing PI3K–AKT signaling in MG6 cells. However, it is not yet known whether 3′,4′-dihydroxyflavonol directly suppresses AKT phosphorylation. Sun et al.⁴²⁾ reported that the structurally related flavanol, quercetin (3,3′,4′,5,7-pentahydroxyflavone), suppressed LPS-induced neuroinflammation by inhibiting PI3K activation in the hippocampus of an animal model of depression. Similarly, Xiao et al.⁴³⁾ have reported that the structurally related flavanol, fisetin (3,3′,4′,7-tetrahydroxyflavone), suppressed proliferation or migration of pancreatic cancer cells by inhibiting PI3K activation in vivo and in vitro. Therefore, as upstream proteins of AKT, PI3K and/or other kinases might be targeted by 3′,4′-dihydroxyflavonol. The molecular mechanisms underlying the anti-neuroinflammatory activity of 3′,4′-dihydroxyflavonol warrant further investigation.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (22K11814) and the Takeda Science Foundation awarded to T.A.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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