Next Article in Journal
Recent Progress of Helicene Type Hole-Transporting Materials for Perovskite Solar Cells
Next Article in Special Issue
Glutamate: Molecular Mechanisms and Signaling Pathway in Alzheimer’s Disease, a Potential Therapeutic Target
Previous Article in Journal
Contribution of Reliable Chromatographic Data in QSAR for Modelling Bisphenol Transport across the Human Placenta Barrier
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 2
10.3390/molecules28020507
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner

by
Chuan-Hsiu Liu
1,2,†,
Mei-Ying Chen
3,†,
Yueh-Hsiung Kuo
4,5,6,
Jack Cheng
3,7,
Li-Zhong Chang
3,
Meng-Shiun Chang
3,
Tsai-Ni Chuang
3,
Wen-Tsong Hsieh
4,8,
Yan-Ru Xiao
8,
Bor-Tsang Wu
9,
Wei-Yong Lin
3,7,* and
Hsin-Ping Liu
10,*
1
Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
2
School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
3
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
4
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
5
Department of Biotechnology, Asia University, Taichung 41354, Taiwan
6
Chinese Medicine Research Center, China Medical University, Taichung 40402, Taiwan
7
Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan
8
Department of Pharmacology, School of Medicine, China Medical University, Taichung 40402, Taiwan
9
Department of Senior Service Management, National Taichung University of Science and Technology, Taichung City 40343, Taiwan
10
Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2023, 28(2), 507; https://doi.org/10.3390/molecules28020507
Submission received: 7 November 2022 / Revised: 27 December 2022 / Accepted: 28 December 2022 / Published: 4 January 2023
(This article belongs to the Special Issue Discovering New Drug Targets for Neurodegenerative Disorders)
Browse Figures
Versions Notes

Abstract

:
Microglia are neuroglia in the brain with an innate immune function and participate in the progress of neurodegenerative diseases. Osthole (OST) is a coumarin derivative extracted from Cnidium monnieri and bears a microglia-antagonizing ability. However, the underlying mechanism of the antagonism is not clear. The lipopolysaccharides-induced microglial BV2 cell line and amyloid-overexpressing fruit fly were used as models to study OST treatment. We found that OST treatment is sufficient to evoke NRF2 cascade under an LPS-induced inflammatory environment, and silencing NRF2 is sufficient to abolish the process. Moreover, we found that OST is sufficient to antagonize microglial activation in both LPS-induced BV2 cells and Aβ-overexpressing fruit flies, and silencing NRF2 abolishes OST’s antagonism. Furthermore, OST treatment rescued survival, climbing, and the learning ability of Aβ-overexpressing fruit flies and relieved oxidative stress. In conclusion, we proved that OST antagonizes microglial activation induced by either LPS or Aβ and that NRF2 is necessary for OST’s antagonism.
Keywords:
osthole; NRF2; microglia

1. Introduction

Reactive oxygen species (ROS) homeostasis is critical for cell signaling, metabolism, development, and immune function [1]. ROS homeostasis disturbed by the accumulation of aggregated proteins is believed to be involved in the progression of neurodegenerative diseases (NDs), such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD) [2]. These NDs are characterized by the activation of microglia along with neurodegeneration caused by oxidative damage [3]. Microglia are resident macrophage cells in the central nervous system (CNS) and act as an innate immune defense by scavenging pathogens and damaged neurons [4]. Notably, ROS and cytokines released by the microglia might be the cause of neurodegeneration [3]. Therefore, antioxidants are candidates for drug discovery against neurodegeneration development [5], in particular targeting the mechanism of ROS homeostasis [6].
The nuclear factor erythroid 2–related factor 2 (NRF2) is a transcription factor that critically regulates ROS homeostasis. At low levels of ROS, NRF2 tightly binds with the Kelch-like ECH-associated protein 1 (KEAP1) complex and thus briefly goes to ubiquitination and degradation [7]. However, in the case of high levels of ROS, NRF2 disassociates with KEAP1 and translocates into the nucleus, and it acts as a transcription factor by binding to ARE domains and activating the expression of antioxidant proteins, including heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), superoxide dismutase type 1 (SOD1), and catalase (CAT) [8]. Furthermore, due to the pivoting role of NRF2 in ROS homeostasis, compounds activating NRF2 cascades are targets of drug discovery against neurodegeneration [9].
Osthole (OST, 7-methoxy-8-(3-methylbut-2-enyl)chromen-2-one, PubChem ID: 10228), also known as osthol, is a coumarin derivative extracted from several medicinal plants like Cnidium monnieri. In Chinese medicine, Cnidium is characterized by its taste of bitterness, its property of warmth, running through the meridian of the kidney, and its ability to destroy parasites and relieve itching, to invigorate the kidney and support yang. However, the toxicity of a high dosage should be cautioned against [10]. In modern clinical research, osthole is applied in several fields, including the protection of the cerebrum from ischemia-reperfusion injury [11], stimulation of osteoblast differentiation and bone formation [12], alleviation of hyperglycemia [13], suppression of the secretion of hepatitis B virus [14], and inhibition of the growth of hepatocellular carcinoma [15].
Previous studies have shown the beneficial activities of OST, including antitumor, anti-inflammation, and neuroprotective ones [16]. Interestingly, OST has been reported to antagonize microglial activation under an inflammatory/oxidative environment [17]; however, of several possible underlying pathway candidates, it is still unclear which one is necessary. These pathway candidates include (1) inhibiting calcium entry and elevating cGMP levels [18], (2) modulating PI3K/Akt/mTOR signaling [17], (3) suppressing Notch signaling [19], (4) down-regulating NF-κB signaling [20], (5) inhibiting NLRP3 inflammasome [21], and (6) activating NRF2 signaling [22]. These pathways cover the regulation of inflammatory response, intrinsic immunity, and ROS scavenging, and they are interrelated. Identifying the key pathway, which is necessary for OST to antagonize microglial activation, will make the role of ROS scavenging clearer and benefit rational drug design using OST derivatives in the future.
To answer the question of whether NRF2 signaling is indispensable for OST to suppress microglial activation under an inflammatory/oxidative environment, we tested whether NRF2-RNAi abolishes this reaction in lipopolysaccharide (LPS)-treated BV-2 microglial cells. We further showed the OST’s efficacy in ameliorating AD symptoms and ROS burden in an Aβ-overexpressing fruit fly model.

2. Results

2.1. OST Activated the Nrf2/HO-1 Signaling Pathway in LPS-Stimulated BV2 Cells

To test whether OST activates the Nrf2/HO-1 signaling pathway in LPS-stimulated BV2 cells, we applied OST to a well-established BV2 cell line with the stimulus of LPS. BV2 is a microglial cell line derived from C57/BL6 mice and serves as a substitute for primary microglia in many experimental settings [23]. LPS are prototypical endotoxins derived from Gram-negative bacteria’s cell walls and serve as a well-established inducer of inflammation [24]. As shown in Figure 1A, the protein expression of the Nrf2/HO-1 signaling pathway, including phosphorylated and total Nrf2, HO-1, SOD1, and CAT, was significantly increased by OST. The quantification is shown in Figure 1B–F. Furthermore, OST increased the nuclear translocation of p-Nrf2 in BV2 cells (Figure 2A,B), but not Nrf2 (Figure 2C,D), and increased the accumulation of HO-1 in the perinuclear space (Figure 2E,F). Thus, OST activated the Nrf2/HO-1 signaling pathway in BV2 cells.

2.2. Nrf2 siRNA Suppressed OST-Induced Antagonism toward Microglial Activation

Microglial activation is an innate immune response in the central nervous system, scavenging pathogens [25] or facilitating neurogenesis [26]. However, microglial activation itself is associated with neurodegenerative diseases as a pathogenic driver, which destroys neuronal circuits. Therefore, clarifying the mechanism that antagonizes microglial activation is a strategy of drug development against neurodegenerative diseases [27]. Previous studies have shown that OST antagonizes microglial activation; however, there are several underlying candidate mechanisms, as described in the introduction. To test whether OST antagonizes microglial activation through the Nrf2/HO-1 signaling pathway, microglial activation markers, such as the gene expression of Ccl2, Ccl3, Cxcl1, Ccl4, and Ccl12, were measured in LPS-stimulated BV2 cells. As shown in Figure 3A, LPS induced the gene expression of Ccl2, Ccl3, Cxcl1, Ccl4, and Ccl12, while OST treatment significantly decreased the gain of Ccl2, Ccl3, Cxcl1, and Ccl4. Moreover, by treating Nrf2 siRNA, the antagonizing ability of OST against microglial activation was largely abolished (Figure 3B). The effectiveness of Nrf2 siRNA in silencing the expression of Nrf2 was confirmed (Figure 3C).

2.3. OST Ameliorated Alzheimer’s Disease Symptoms in Drosophila Animal Model

To test whether OST antagonizes microglial activation induced by other pathogens besides LPS in other organisms besides mice, we utilized the Alzheimer’s disease (AD) Drosophila model, whose neural system harbors the over-expression of human amyloid beta (Aβ), with symptoms of shortened survival, diminished climbing ability, defects of learning ability, and a higher oxidative burden [28]. By treating the AD Drosophila model with OST, we found that markers of microglial activation, such as the gene expression of Drpr and Ced [29], decreased (Figure 3D) and that AD symptoms, including survival (Figure 4A), climbing ability (Figure 4B), and learning ability (Figure 4C), were ameliorated. Moreover, the oxidative burden was decreased (Figure 4D–F).

3. Discussion

This study found that OST treatment is sufficient to evoke an NRF2 cascade under an LPS-induced inflammatory environment and that silencing NRF2 is sufficient to abolish the process. This means that the anti-inflammatory activity of OST is largely dependent on the NRF2 cascade. Moreover, we found that OST is sufficient to antagonize microglial activation in either LPS-induced BV2 cells or Aβ-overexpressing fruit flies. Again, silencing NRF2 abolishes OST’s antagonism, which means that NRF2 is necessary for this process. Furthermore, we found that OST treatment rescued survival, climbing, and the learning ability of AD fruit flies and relieved oxidative stress.
As described in the introduction, there are five other candidate pathways besides the NRF2 cascade for OST to antagonize microglial activation [17,18,19,20,21]. Could we exclude these candidates according to the results of this study? In Figure 3A, four microglial markers responded to OST treatment, while NRF2 RNAi abolished only three of them (Figure 3B). This may indicate that although the NRF2 cascade is the major pathway for OST to antagonize microglial activation, other minor contributing pathways are there. Thus, we cannot exclude the other five candidate pathways, but the NRF2 cascade is probably the key point.
In AD research, OST exhibits multiple benefits besides antagonizing microglial activation, including (1) decreasing Aβ cytotoxicity on neural cells by mediating phosphorylation of cAMP response element-binding protein (CREB) [30], (2) inhibiting apoptosis by mediating Wnt/β-catenin signaling [31], (3) improving synaptic plasticity and cognitive function by regulating the glutamatergic neuron [32], and (4) increasing hippocampal neurogenesis by upregulating Bdnf in AD mice [33]. Therefore, we could not exclude these factors and jump to the conclusion that OST improves AD by antagonizing microglial activation via the NRF2 cascade or by decreasing oxidative stress.
The KEAP1-NRF2 system is an evolutionarily conserved mechanism that controls not only the transcription of anti-oxidant, anti-inflammatory, and detoxifying proteins but also organism development [1]. For example, the KEAP1-knockout mice exhibit growth retardation, skin abnormalities, and early death after birth [34], and Cnc, the NRF2 homolog in Drosophila, controls development and head segment formation [35]. Therefore, the KEAP1-NRF2 system maintains a balance between excessive ROS scavenging and ROS-dependent signal transduction. In this sense, the therapeutic modulation of KEAP1-NRF2, such as OST treatment, must be carefully administered to attenuate inflammation-induced damage while leaving ROS-dependent signal transduction undisturbed. This may be a future challenge for the clinical application of OST to neurodegenerative diseases.
In conclusion, we proved that OST antagonizes microglial activation induced by either LPS or Aβ and that NRF2 is necessary for OST’s antagonism.

4. Materials and Methods

4.1. Osthole (OST)

The Cnidium monnieri source was identical to [36], and the purification method is described in [37], with a purity >98% as determined by HPLC, as described before [38]. OST forms a crystal of a light yellow color at room temperature. The stock solution was prepared at a concentration of 6 mg/mL in EtOH before use. For lifespan, climbing, learning ability, and gene expression assays, the final concentration of OST in the fruit fly medium was 6 μg/mL. For the BV2 cell culture, the OST concentration was 40 μM.

4.2. Western Blots

The western blotting assay detected specific proteins described previously [39]. In brief, cells were treated with OST 1 h before LPS. The proteins were extracted through PRO-PREP™ and separated by 8–12% SDS-PAGE. The proteins were transferred from the gel to the polyvinyl vinylidene fluoride (PVDF) membranes (Millipore Co., Billerica, MA, USA) and blocked with 5% BSA. Then, they were sampled with primary antibodies overnight and incubated with horseradish peroxidase (HRP) secondary conjugated antibody. The antibody detection response was performed using ECL. The antibody images were captured with the ImageQuant™ Las 4000 Mini Biomolecular Imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The antibodies used were anti-Nrf2 (ab62352, Abcam), anti-phospho-Nrf2 (Ser40) (PA5-67520, Thermo Fisher, Waltham, MA, USA), anti-HO-1 (#5853, Cell Signaling, Danvers, MA, USA), anti-SOD1 (#2770, Cell Signaling), anti-Catalase (#14097, Cell Signaling), anti-IL1β (MA5-23691, Thermo Fisher), and anti-TNFα (ab183218, Thermo Fisher).

4.3. Immunofluorescence (IF) Assay

An IF assay has been used to visualize antibody specificity with fluorescent dyes in cells. Therefore, it makes it possible to visualize the distribution of the target protein through the sample of a fluorescence microscope, as described previously [40]. In brief, cells were incubated in a confocal laser dish (500 cells/dish) for 16 h and treated with OST before being incubated with 100 ng/mL of LPS. The cells were fixed in 4% paraformaldehyde and permeated with Triton X 100 at 0.25% in phosphate-buffered physiological serum (PBS). They were incubated for 1 h in 5% of PBS-BSA to block nonspecific binding. Moreover, cells were incubated with primary antibodies overnight at 4 °C and then complemented with a secondary antibody labeled with an IgG Alexa Fluor 488 and Alexa Fluor 594 reagent. They subsequently contaminated the nuclei with DAPI gel (1 μg/mL) in 1% BSA for 20 min at 37 °C in the dark. The IF staining images were visualized using a confocal SP2/SP8X spectral microscope (Leica Microsystems, Wetzlar, Germany).

4.4. Nrf2 siRNA Transfection

A Nrf2 siRNA transfection assay was used to investigate the antioxidant activity Nrf2, as described above [40]. In short, BV2 microglial cells were cultured in 6-well plaques (2105 cells/wells). Transfecting the DNA fragment encoding Nrf2 siRNA or Nrf2-negative siRNA control was carried out using LipofectamineTM 3000 (Invitrogen, Waltham, MA, USA). Mouse siRNA Oligo Duplex was used for the transfection of small interference RNAs (siRNAs). Nrf2 siRNA to knock down endogenous Nrf2 following the manufacturer protocol (Invitrogen). After 24 h, the transfected cells were exposed to OST (40 μM) and LPS for 24 h, followed by western blot and further analysis.

4.5. SOD/Catalase Activity

The SOD activity was detected with a Superoxide Dismutase Assay Kit (706002, Cayman), and the Catalase activity was detected with a Catalase Assay Kit (707002, Cayman), following the manufacture’s manual. The total protein was extracted from 50 flies for one replicate, and four replicates for OST treatment and nontreatment were collected, respectively. Protein was quantified with bovine serum albumin (BSA, New England BioLabs, Ipswich, MA, USA). The input quantity for SOD and Catalase activity was 0.125 μg and 10 μg, and the absorbance was measured at 450 nm and 540 nm, respectively.

4.6. Lipid Hydroperoxide (LPO) Assay

For one replicate, 100 flies were collected for the measurement of the total lipid hydroperoxide using BIOXYTECHÒ LPO-586 (OxisResearch), following the manufacture’s manual, except that the absorbance was measured at 595 nm. Three replicates were assayed.

4.7. Quantitative PCR

For Drpr and CED detection, the total RNA was extracted from 100 heads of flies for one replicate with an RNeasy Mini kit (Qiagen, Hilden, Germany), as previously described [41], and then used for synthesizing cDNA with a VersoTM cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). RNA expression levels of the investigated genes were quantified by real-time PCR (Applied Biosystems 7700) with the Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The qPCR readouts were normalized to the relative amount of gapdh. Four independent measurements were performed.
For expression profiling of SOD/catalase genes, the total RNA was extracted from 50 flies for one replicate with PureZOL (Bio-Rad, Hercules, CA, USA), following the manufacturer’s manual. The reagents for reverse transcription and qPCR were identical to the previous section. The qPCR readouts were normalized to the relative amount of Rpl32.
For expression profiling of BV2 cells, the total RNA was extracted from cell lysates with PureZOL (Bio-Rad), following the manufacturer’s manual. The qPCR readouts were normalized to the relative amount of Actin. The primers used are listed in Supplementary Table S1.

4.8. Fly Stock Maintenance, Lifespan and Antigeotaxis Assays

The first familial offspring of the cross of the pan-neural Gal4 driver elav-GAL4c155 strain (Bloomington Drosophila Stock Center ID: 458) with UAS-Aβ42H29.3 [28] was utilized as the Alzheimer’s disease model. Only male flies were used. The flies were maintained in cornmeal standard media at 25 °C under a 12 h light-dark cycle. In a lifespan analysis, about twenty flies of the AD model were raised in a food vial with 6 μg/mL of OST, and four vials were prepared for each treatment. Food vials were replaced every 2 to 3 days, and dead flies were counted at that time. The percentage of survival is defined as the number of flies alive divided by the total number of flies at the beginning of the test. In the antigeotaxis assay to measure the climbing activity of Drosophila, flies were transferred into a new food vial just before measurement. The number of flies climbing more than 5 cm above the bottom in 18 s after being bounced to the bottom of vertically standing vials was counted. The percentage of climbing ability is defined as the number of climbing flies divided by the total number of flies at the beginning of the test.

4.9. Drosophila Learning and Memory Platform (T-Maze)

To observe Drosophilae’s learning and memory, about 100 flies were first placed in the upper cupper chamber, which can be supplied with electric voltage and deliver an electric shock to flies. Flies were first delivered with one odor, 4-methylcyclohexanol (MCH, 98%, Sigma Aldrich), along with electric shock waves at 80 V (Grass S88 stimulator, A-M Systems). Then, flies were delivered with another odor, 3-octanol (OCT, 97%, Sigma Aldrich), without electric shock waves. After training, the flies were moved to the space below, whose two open sides were supplied with either odor one or odor two, respectively. By observing the moving direction of flies, the bias of odor two over odor one can be estimated with the Performance Index (PI), which is defined as the difference of the number of flies between two odors divided by the total number of flies. The PI represents Drosophilae’s ability for learning and memory [42]. In this study, the time interval between the training session and the test session was 45 sec. The number of replicates of the learning assay was 14 and 6 for AD without treatment and for AD with 6 μg/mL of OST treatment for 14 days, respectively.

4.10. Statistical Analysis

The significance of the difference between the survival curves and climbing curves of OST-treated and control groups was judged by the log-rank (Mantel–Cox) test and Gehan–Breslow–Wilcoxon test. The significance of the difference between the PI of learning or memory of OST-treated and control groups was judged by Student’s t-test. Differences between LPS-treated and control groups were considered statistically significant at a level where the p-value was below 0.05. Statistical significance used the two-part Student t-test or One-Way Variance Analysis (ANOVA), as described previously [43], to determine statistical significance using the SPSS17.0 software system (IBM, Chicago, IL, USA). Differences between LPS-treated and control groups were considered statistically significant at a level where the p-value was below 0.05.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28020507/s1, Table S1: Primers used in this study.

Author Contributions

Conceptualization, W.-Y.L. and H.-P.L.; investigation, C.-H.L., M.-Y.C., J.C., L.-Z.C., M.-S.C., T.-N.C., W.-T.H., Y.-R.X. and B.-T.W.; resources, Y.-H.K.; data curation, W.-T.H. and W.-Y.L.; writing—original draft preparation, J.C. and W.-T.H.; writing—review and editing, W.-Y.L. and H.-P.L.; visualization, J.C. and W.-T.H.; supervision, W.-Y.L. and H.-P.L.; project administration, W.-Y.L. and H.-P.L.; funding acquisition, W.-Y.L. and H.-P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science and Technology Council, Taiwan (MOST 108-2320-B-039-031-MY3, MOST 111-2320-B-039-032, MOST 111-2314-B-039-017-MY3), and China Medical University & Hospital (CMU108-MF-61, CMU109-MF-110, CMU110-MF-92, CMU110-MF-96, CMU111-MF-65, DMR-111-147).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available in FigShare at https://figshare.com/articles/figure/OST_NRF2_molecules/21780383 (accessed on 26 December 2022).

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compound osthole is available from the authors.

References

  1. D’Autréaux, B.; Toledano, M.B. ROS as signalling molecules: Mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell. Biol. 2007, 8, 813–824. [Google Scholar] [CrossRef] [PubMed]
  2. Barnham, K.J.; Masters, C.L.; Bush, A.I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discov. 2004, 3, 205–214. [Google Scholar] [CrossRef] [PubMed]
  3. Simpson, D.S.; Oliver, P.L. ROS generation in microglia: Understanding oxidative stress and inflammation in neurodegenerative disease. Antioxidants 2020, 9, 743. [Google Scholar] [CrossRef] [PubMed]
  4. Gehrmann, J.; Matsumoto, Y.; Kreutzberg, G.W. Microglia: Intrinsic immuneffector cell of the brain. Brain Res. Rev. 1995, 20, 269–287. [Google Scholar] [CrossRef] [PubMed]
  5. Lee, K.H.; Cha, M.; Lee, B.H. Neuroprotective effect of antioxidants in the brain. Int. J. Mol. Sci. 2020, 21, 7152. [Google Scholar] [CrossRef]
  6. Teixeira, J.P.; de Castro, A.A.; Soares, F.V.; da Cunha, E.F.; Ramalho, T.C. Future therapeutic perspectives into the Alzheimer’s disease targeting the oxidative stress hypothesis. Molecules 2019, 24, 4410. [Google Scholar] [CrossRef] [Green Version]
  7. Ma, Q. Role of nrf2 in oxidative stress and toxicity. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 401. [Google Scholar] [CrossRef] [Green Version]
  8. Tonelli, C.; Chio, I.I.C.; Tuveson, D.A. Transcriptional regulation by Nrf2. Antioxid. Redox. Signal. 2018, 29, 1727–1745. [Google Scholar] [CrossRef] [Green Version]
  9. Brandes, M.S.; Gray, N.E. NRF2 as a therapeutic target in neurodegenerative diseases. ASN Neuro 2020, 12, 1759091419899782. [Google Scholar] [CrossRef]
  10. Li, Y.M.; Jia, M.; Li, H.Q.; Zhang, N.D.; Wen, X.; Rahman, K.; Zhang, Q.Y.; Qin, L.P. Cnidium monnieri: A Review of Traditional Uses, Phytochemical and Ethnopharmacological Properties. Am. J. Chin. Med. 2015, 43, 835–877. [Google Scholar] [CrossRef]
  11. Dong, W.P.; Zhang, Z.; Liu, Z.J.; Liu, H.; Wang, X.Y.; Bi, S.H.; Wang, X.W.; Ma, T.; Zhang, W.D. Protective effects of osthole, a natural derivative of coumarin, against intestinal ischemia-reperfusion injury in mice. Int. J. Mol. Med. 2013, 31, 1367–1374. [Google Scholar] [CrossRef]
  12. Tang, D.Z.; Hou, W.; Zhou, Q.A.; Zhang, M.J.; Holz, J.; Sheu, T.J.; Li, T.F.; Cheng, S.D.; Shi, Q.; Harris, S.E.; et al. Osthole Stimulates Osteoblast Differentiation and Bone Formation by Activation of beta-Catenin-BMP Signaling. J. Bone Min. Res. 2010, 25, 1234–1245. [Google Scholar] [CrossRef] [Green Version]
  13. Liang, H.J.; Suk, F.M.; Wang, C.K.; Hung, L.F.; Liu, D.Z.; Chen, N.Q.; Chen, Y.C.; Chang, C.C.; Liang, Y.C. Osthole, a potential antidiabetic agent, alleviates hyperglycemia in db/db mice. Chem. Biol. Interact. 2009, 181, 309–315. [Google Scholar] [CrossRef]
  14. Huang, R.L.; Chen, C.C.; Huang, Y.L.; Hsieh, D.J.; Hu, C.P.; Chen, C.F.; Chang, C.M. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro. Hepatology 1996, 24, 508–515. [Google Scholar] [CrossRef]
  15. Zhang, L.R.; Jiang, G.R.; Yao, F.; He, Y.; Liang, G.Q.; Zhang, Y.S.; Hu, B.; Wu, Y.; Li, Y.S.; Liu, H.Y. Growth Inhibition and Apoptosis Induced by Osthole, A Natural Coumarin, in Hepatocellular Carcinoma. PLoS ONE 2012, 7, e37865. [Google Scholar] [CrossRef]
  16. Zhang, Z.-R.; Leung, W.N.; Cheung, H.Y.; Chan, C.W. Osthole: A review on its bioactivities, pharmacological properties, and potential as alternative medicine. Evid. Based Complement. Altern. Med. 2015, 2015, 1. [Google Scholar] [CrossRef] [Green Version]
  17. Du, M.; Sun, Z.; Lu, Y.; Li, Y.-Z.; Xu, H.-R.; Zeng, C.-Q. Osthole inhibits proliferation and induces apoptosis in BV-2 microglia cells in kainic acid-induced epilepsy via modulating PI3K/AKt/mTOR signalling way. Pharm. Biol. 2019, 57, 238–244. [Google Scholar] [CrossRef]
  18. Liu, J.; Zhang, W.; Zhou, L.; Wang, X.; Lian, Q. Anti-inflammatory effect and mechanism of osthole in rats. Zhong Yao Cai Zhongyaocai J. Chin. Med. Mater. 2005, 28, 1002–1006. [Google Scholar]
  19. Wang, Y.; Zhou, Y.; Wang, X.; Zhen, F.; Chen, R.; Geng, D.; Yao, R. Osthole alleviates MPTP-induced Parkinson’s disease mice by suppressing Notch signaling pathway. Int. J. Neurosci. 2019, 129, 833–841. [Google Scholar] [CrossRef]
  20. Kong, L.; Yao, Y.; Xia, Y.; Liang, X.; Ni, Y.; Yang, J. Osthole alleviates inflammation by down-regulating NF-κB signaling pathway in traumatic brain injury. Immunopharmacol. Immunotoxicol. 2019, 41, 349–360. [Google Scholar] [CrossRef]
  21. Liu, Y.; Chen, X.; Gong, Q.; Shi, J.; Li, F. Osthole improves cognitive function of vascular dementia rats: Reducing Aβ deposition via inhibition NLRP3 inflammasome. Biol. Pharm. Bull. 2020, 43, 1315–1323. [Google Scholar] [CrossRef] [PubMed]
  22. Bao, Y.; Meng, X.; Liu, F.; Wang, F.; Yang, J.; Wang, H.; Xie, G. Protective effects of osthole against inflammation induced by lipopolysaccharide in BV2 cells. Mol. Med. Rep. 2018, 17, 4561–4566. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Henn, A.; Lund, S.; Hedtjärn, M.; Schrattenholz, A.; Pörzgen, P.; Leist, M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX: Altern. Anim. Exp. 2009, 26, 83–94. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Raetz, C.R.; Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 2002, 71, 635. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Glezer, I.; Simard, A.; Rivest, S. Neuroprotective role of the innate immune system by microglia. Neuroscience 2007, 147, 867–883. [Google Scholar] [CrossRef]
  26. Ekdahl, C.; Kokaia, Z.; Lindvall, O. Brain inflammation and adult neurogenesis: The dual role of microglia. Neuroscience 2009, 158, 1021–1029. [Google Scholar] [CrossRef]
  27. Mandrekar-Colucci, S.; Landreth, G.E. Microglia and inflammation in Alzheimer’s disease. CNS Neurol. Disord. Drug Targets 2010, 9, 156–167. [Google Scholar] [CrossRef]
  28. Finelli, A.; Kelkar, A.; Song, H.-J.; Yang, H.; Konsolaki, M. A model for studying Alzheimer’s Aβ42-induced toxicity in Drosophila melanogaster. Mol. Cell. Neurosci. 2004, 26, 365–375. [Google Scholar] [CrossRef]
  29. MacDonald, J.M.; Beach, M.G.; Porpiglia, E.; Sheehan, A.E.; Watts, R.J.; Freeman, M.R. The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 2006, 50, 869–881. [Google Scholar] [CrossRef] [Green Version]
  30. Hu, Y.; Wen, Q.P.; Liang, W.B.; Kang, T.G.; Ren, L.; Zhang, N.; Zhao, D.; Sun, D.; Yang, J.X. Osthole Reverses Beta-Amyloid Peptide Cytotoxicity on Neural Cells by Enhancing Cyclic AMP Response Element-Binding Protein Phosphorylation. Biol. Pharm. Bull. 2013, 36, 1950–1958. [Google Scholar] [CrossRef] [Green Version]
  31. Yao, Y.J.; Gao, Z.; Liang, W.B.; Kong, L.; Jiao, Y.A.; Li, S.H.; Tao, Z.Y.; Yan, Y.H.; Yang, J.X. Osthole promotes neuronal differentiation and inhibits apoptosis via Wnt/beta-catenin signaling in an Alzheimer’s disease model. Toxicol. Appl. Pharm. 2015, 289, 474–481. [Google Scholar] [CrossRef]
  32. Dong, X.H.; Zhang, D.S.; Zhang, L.; Li, W.; Meng, X.Y. Osthole improves synaptic plasticity in the hippocampus and cognitive function of Alzheimer’s disease rats via regulating glutamate. Neural. Regen. Res. 2012, 7, 2325–2332. [Google Scholar] [CrossRef]
  33. Liu, H.; Xue, X.H.; Shi, H.J.; Qi, L.F.; Gong, D.R. Osthole Upregulates BDNF to Enhance Adult Hippocampal Neurogenesis in APP/PS1 Transgenic Mice. Biol. Pharm. Bull. 2015, 38, 1439–1449. [Google Scholar] [CrossRef] [Green Version]
  34. Wakabayashi, N.; Itoh, K.; Wakabayashi, J.; Motohashi, H.; Noda, S.; Takahashi, S.; Imakado, S.; Kotsuji, T.; Otsuka, F.; Roop, D.R. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat. Genet. 2003, 35, 238–245. [Google Scholar] [CrossRef]
  35. Mohler, J.; Mahaffey, J.W.; Deutsch, E.; Vani, K. Control of Drosophila head segment identity by the bZIP homeotic gene cnc. Development 1995, 121, 237–247. [Google Scholar] [CrossRef]
  36. Chang, C.-I.; Hu, W.-C.; Shen, C.-P.; Hsu, B.-D.; Lin, W.-Y.; Sung, P.-J.; Wang, W.-H.; Wu, J.-B.; Kuo, Y.-H. 8-Alkylcoumarins from the fruits of Cnidium monnieri protect against hydrogen peroxide induced oxidative stress damage. Int. J. Mol. Sci. 2014, 15, 4608–4618. [Google Scholar] [CrossRef] [Green Version]
  37. Liao, P.-C.; Chien, S.-C.; Ho, C.-L.; Wang, E.I.-C.; Lee, S.-C.; Kuo, Y.-H.; Jeyashoke, N.; Chen, J.; Dong, W.-C.; Chao, L.K. Osthole regulates inflammatory mediator expression through modulating NF-κB, mitogen-activated protein kinases, protein kinase C, and reactive oxygen species. J. Agric. Food Chem. 2010, 58, 10445–10451. [Google Scholar] [CrossRef]
  38. Leung, Y.-M.; Kuo, Y.-H.; Chao, C.-C.; Tsou, Y.-H.; Chou, C.-H.; Lin, C.-H.; Wong, K.-L. Osthol is a use-dependent blocker of voltage-gated Na+ channels in mouse neuroblastoma N2A cells. Planta Med. 2010, 76, 34–40. [Google Scholar] [CrossRef]
  39. Huang, Y.-P.; Chen, D.-R.; Lin, W.-J.; Lin, Y.-H.; Chen, J.-Y.; Kuo, Y.-H.; Chung, J.-G.; Hsia, T.-C.; Hsieh, W.-T. Ergosta-7, 9 (11), 22-trien-3β-ol Attenuates Inflammatory Responses via Inhibiting MAPK/AP-1 Induced IL-6/JAK/STAT Pathways and Activating Nrf2/HO-1 Signaling in LPS-Stimulated Macrophage-like Cells. Antioxidants 2021, 10, 1430. [Google Scholar] [CrossRef]
  40. Velagapudi, R.; El-Bakoush, A.; Olajide, O.A. Activation of Nrf2 pathway contributes to neuroprotection by the dietary flavonoid tiliroside. Mol. Neurobiol. 2018, 55, 8103–8123. [Google Scholar] [CrossRef] [Green Version]
  41. Bae, J.H.; Yang, M.J.; Jeong, S.-h.; Kim, J.; Hong, S.P.; Kim, J.W.; Kim, Y.H.; Koh, G.Y. Gatekeeping role of Nf2/Merlin in vascular tip EC induction through suppression of VEGFR2 internalization. Sci. Adv. 2022, 8, eabn2611. [Google Scholar] [CrossRef] [PubMed]
  42. Waddell, S.; Quinn, W.G. Flies, genes, and learning. Annu. Rev. Neurosci. 2001, 24, 1283–1309. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Adhikari, N.; Shim, W.-S. Caffeic acid phenethyl ester inhibits pseudo-allergic reactions via inhibition of MRGPRX2/MrgprB2-dependent mast cell degranulation. Arch. Pharmacal. Res. 2022, 45, 644–657. [Google Scholar] [CrossRef] [PubMed]
Molecules 28 00507 g001 550
Figure 1. OST activated the Nrf2/HO-1 signaling pathway, but it was reversed by the Nrf2 siRNA transfection in BV2 cells. (A) Mock (control) or Nrf2 siRNA were transfected into BV2 cells. Cells were treated with 40 μM OST for 1 h before being stimulated with LPS for 24 h. The protein expressions were analyzed with a western blot assay. The expression of related proteins, including (B) p-Nrf2, (C) Nrf2, (D) HO-1, (E) SOD1, and (F) CAT were shown individually. All results were expressed as folds mean ± SEM compared with β-actin of three independent experiments (n = 3). # p < 0.05, ## p < 0.01 ### p < 0.001 compared with the control (mock) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the counterpart of the mock-treated group.
Figure 1. OST activated the Nrf2/HO-1 signaling pathway, but it was reversed by the Nrf2 siRNA transfection in BV2 cells. (A) Mock (control) or Nrf2 siRNA were transfected into BV2 cells. Cells were treated with 40 μM OST for 1 h before being stimulated with LPS for 24 h. The protein expressions were analyzed with a western blot assay. The expression of related proteins, including (B) p-Nrf2, (C) Nrf2, (D) HO-1, (E) SOD1, and (F) CAT were shown individually. All results were expressed as folds mean ± SEM compared with β-actin of three independent experiments (n = 3). # p < 0.05, ## p < 0.01 ### p < 0.001 compared with the control (mock) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the counterpart of the mock-treated group.
Molecules 28 00507 g001
Molecules 28 00507 g002 550
Figure 2. Illustrative images of immunofluorescence staining showed the effect of OST on the expression of p-Nrf2 in LPS-stimulated (A) BV2 cells and (B) Nrf2 siRNA-treated BV2 cells. The expression of Nrf2 in LPS-stimulated (C) BV2 cells and (D) Nrf2 siRNA-treated BV2 cells. The expression of HO-1 in LPS-stimulated (E) BV2 cells and (F) Nrf2 siRNA-treated BV2 cells.
Figure 2. Illustrative images of immunofluorescence staining showed the effect of OST on the expression of p-Nrf2 in LPS-stimulated (A) BV2 cells and (B) Nrf2 siRNA-treated BV2 cells. The expression of Nrf2 in LPS-stimulated (C) BV2 cells and (D) Nrf2 siRNA-treated BV2 cells. The expression of HO-1 in LPS-stimulated (E) BV2 cells and (F) Nrf2 siRNA-treated BV2 cells.
Molecules 28 00507 g002
Molecules 28 00507 g003 550
Figure 3. Nrf2 siRNA suppressed OST-induced antagonism toward microglial activation provoked by LPS. (A) OST antagonized LPS-induced expression of microglial activation markers in BV2 cells, while (B) this phenomenon was suppressed by Nrf2 siRNA. (C) Nrf2 siRNA effectively silenced NRF2 expression. (D) OST antagonized amyloid-beta (Aβ)-induced expression of microglial activation markers in Alzheimer’s disease model of a fruit fly. ** and *** denote p < 0.01 and 0.001 of Student’s t-test compared to CTRL group, respectively. #, ##, and ### denote p < 0.05, 0.01, and 0.001 of Student’s t-test compared to LPS-treated BV2 cells in (A), (B), or to AD flies in (D), respectively. The error bar stands for the standard error of the mean. n.s. stands for non-significant.
Figure 3. Nrf2 siRNA suppressed OST-induced antagonism toward microglial activation provoked by LPS. (A) OST antagonized LPS-induced expression of microglial activation markers in BV2 cells, while (B) this phenomenon was suppressed by Nrf2 siRNA. (C) Nrf2 siRNA effectively silenced NRF2 expression. (D) OST antagonized amyloid-beta (Aβ)-induced expression of microglial activation markers in Alzheimer’s disease model of a fruit fly. ** and *** denote p < 0.01 and 0.001 of Student’s t-test compared to CTRL group, respectively. #, ##, and ### denote p < 0.05, 0.01, and 0.001 of Student’s t-test compared to LPS-treated BV2 cells in (A), (B), or to AD flies in (D), respectively. The error bar stands for the standard error of the mean. n.s. stands for non-significant.
Molecules 28 00507 g003
Molecules 28 00507 g004 550
Figure 4. OST ameliorated Alzheimer’s disease (AD) symptoms and lowered oxidative stress in the Drosophila animal model. (A) Survival, (B) climbing ability, (C) learning ability, (D) superoxide dismutase (SOD) activity, (E) catalase activity, and (F) total lipid peroxidation (LPO). In the figures, “AD” stands for the AD Drosophila model with a pan-neuronal expression of amyloid beta (Aβ), while “AD + OST” stands for the OST-treated AD Drosophila model. * and ** denote p < 0.05 and 0.01 of Student’s t-test, respectively. The error bar stands for the standard error of the mean.
Figure 4. OST ameliorated Alzheimer’s disease (AD) symptoms and lowered oxidative stress in the Drosophila animal model. (A) Survival, (B) climbing ability, (C) learning ability, (D) superoxide dismutase (SOD) activity, (E) catalase activity, and (F) total lipid peroxidation (LPO). In the figures, “AD” stands for the AD Drosophila model with a pan-neuronal expression of amyloid beta (Aβ), while “AD + OST” stands for the OST-treated AD Drosophila model. * and ** denote p < 0.05 and 0.01 of Student’s t-test, respectively. The error bar stands for the standard error of the mean.
Molecules 28 00507 g004
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Liu, C.-H.; Chen, M.-Y.; Kuo, Y.-H.; Cheng, J.; Chang, L.-Z.; Chang, M.-S.; Chuang, T.-N.; Hsieh, W.-T.; Xiao, Y.-R.; Wu, B.-T.; et al. Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner. Molecules 2023, 28, 507. https://doi.org/10.3390/molecules28020507

AMA Style

Liu C-H, Chen M-Y, Kuo Y-H, Cheng J, Chang L-Z, Chang M-S, Chuang T-N, Hsieh W-T, Xiao Y-R, Wu B-T, et al. Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner. Molecules. 2023; 28(2):507. https://doi.org/10.3390/molecules28020507

Chicago/Turabian Style

Liu, Chuan-Hsiu, Mei-Ying Chen, Yueh-Hsiung Kuo, Jack Cheng, Li-Zhong Chang, Meng-Shiun Chang, Tsai-Ni Chuang, Wen-Tsong Hsieh, Yan-Ru Xiao, Bor-Tsang Wu, and et al. 2023. "Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner" Molecules 28, no. 2: 507. https://doi.org/10.3390/molecules28020507

APA Style

Liu, C. -H., Chen, M. -Y., Kuo, Y. -H., Cheng, J., Chang, L. -Z., Chang, M. -S., Chuang, T. -N., Hsieh, W. -T., Xiao, Y. -R., Wu, B. -T., Lin, W. -Y., & Liu, H. -P. (2023). Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner. Molecules, 28(2), 507. https://doi.org/10.3390/molecules28020507

Article Metrics

Back to TopTop