Isorhamnetin, which is a flavonoid predominantly found in fruits and leaves of various plants, including
Microglia function as macrophages in the central nervous system (CNS), and serve critical roles in brain development and maintenance. Microglial dysfunction caused by hyperactivity in response to inflammatory signals is closely associated with the onset and progression of various neurodegenerative diseases by damaging brain neurons (
Recent studies have reported that various natural products, including flavonoids, which are a group of naturally occurring polyphenol compounds found in plants, possess anti-inflammatory effects by blocking the activation of microglial cells (
The BV2 immortalized murine microglial cell line was provided by Dr Il-Whan Choi (Department of Microbiology, College of Medicine, Inje University, Busan, Korea). BV2 microglia were maintained in Dulbecco’s modified Eagle’s medium (DMEM; WelGENE, Inc., Gyeongsan, Korea) containing 10% (v/v) fetal bovine serum (WelGENE, Inc.), L-glutamine (2 mM), penicillin (100 U/ml) and 100
Cell viability was measured based on the formation of blue formazan, which is metabolized from colorless MTT by mitochondrial dehydrogenases, enzymes that are only active in live cells. Briefly, BV2 cells were seeded into 96-well plates at a density of 1×104 cells/well. After 24 h of incubation, cells were treated with various concentrations (0, 50, 100 and 200
Levels of nitric oxide (NO) production were indirectly determined by measuring the stable NO catabolite, nitrite, in the medium using the Griess reaction. Briefly, BV2 cells (5×105 cells/ml) were stimulated in 24-well plates with or without various concentrations of isorhamnetin for 1 h prior to LPS (100 ng/ml) treatment for 24 h. Subsequently, the culture medium supernatant (100
BV2 cells were pretreated with various concentrations of isorhamnetin for 1 h, followed by treatment with 100 ng/ml LPS for 24 h. Total RNA was isolated from the cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer’s protocol, and RNA levels were quantified. For mRNA expression analysis, cDNA was synthesized from 1
BV2 cells were pretreated with various concentrations of isorhamnetin for 1 h, followed by treatment with 100 ng/ml LPS for 24 h. Alternatively, cells were treated with 100 ng/ml LPS for various durations. The cells were collected and cellular proteins were prepared using lysis buffer [25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride and 5 mM dithiothreitol], as described previously (
After cells (5×105 cells/ml) were pretreated with or without 200
To investigate the effects of isorhamnetin on TLR4 expression on the cell surface, BV2 cells were pretreated with or without 200
Production of intracellular ROS was monitored using 5,6-carboxy-2′,7′-dichlorofluorescin diacetate (DCF-DA; Sigma-Aldrich; Merck KGaA), which is a cell-permeable fluorogenic probe. Briefly, cells were treated with 100 ng/ml LPS for the indicated time periods, or were pretreated with 200
Statistical analysis was conducted using GraphPad Prism version 6.02 (GraphPad Software, Inc., La Jolla, CA, USA). All data were collected from at least three independent experiments and are presented as the means ± standard deviation. One-way analysis of variance with Tukey’s multiple comparison post hoc test was performed to analyze the data. P<0.05 was considered to indicate a statistically significant difference.
To determine the inhibitory effects of isorhamnetin on LPS-induced production of NO and PGE2, which are pro-inflammatory mediators, BV2 cells were pretreated with various concentrations of isorhamnetin for 1 h and were then stimulated with or without 100 ng/ml LPS for 24 h. Levels of NO and PGE2 in culture supernatants were determined using the Griess reaction assay and ELISA, respectively. As shown in
The present study aimed to determine whether the inhibitory effects of isorhamnetin on NO and PGE2 production were associated with regulation of iNOS and COX-2 expression. As shown in
The MTT assay was performed to investigate whether the inhibitory effects of isorhamnetin on production of pro-inflammatory mediators and cytokines were caused by cytotoxicity. As shown in
The present study aimed to determine whether isorhamnetin could attenuate LPS-induced activation of NF-κB in BV2 cells. Immunoblotting data using cytoplasmic and nuclear extracts revealed that pretreatment with isorhamnetin inhibited nuclear accumulation of NF-κB p65 subunits in LPS-stimulated BV2 cells. In addition, isorhamnetin attenuated the LPS-induced inhibition of total IκBα protein expression and reduced phosphorylation of IκBα (
To determine whether the anti-inflammatory effects of isorhamnetin were associated with blockade of the TLR signaling pathway, the expression levels of TLR4 and myeloid differentiation factor 88 (Myd88) were investigated (
The present study assessed whether isorhamnetin could inhibit the interaction between LPS and TLR4 on the surface of LPS-treated BV2 cells. As shown in
To further determine whether blockade of TLR4 signaling is mediated by the anti-inflammatory action of isorhamnetin, the present study examined the effects of the TLR4 antagonist, CLI-095, on isorhamnetin-induced inhibition of inflammatory mediators. As shown in
The present study also examined the effects of isorhamnetin on LPS-induced ROS production, in order to investigate the antioxidant potential of isorhamnetin. Flow cytometry using the fluorescent probe DCF-DA revealed that the levels of ROS were gradually increased following treatment with LPS, peaking at 1 h; thereafter, ROS levels were decreased (
The results of the present study demonstrated that isorhamnetin inhibited LPS-induced inflammatory signaling in BV2 microglia, a brain microglial cell line. Similar to the results of previous studies using macrophage and gingival fibroblast models (
NF-κB is a key transcription factor that increases the expression of pro-inflammatory enzymes and cytokines only if it has migrated to the nucleus. NF-κB is usually located in the cytoplasm in association with IκBα. When IκBα is phosphorylated and degraded, NF-κB is isolated and translocated to the nucleus (
Immune cells, including microglia, can recognize pathogen-associated molecular patterns through TLR pattern recognition receptors, which are expressed on the cell surface. Among various TLRs, TLR4 is known to recruit adapter molecules, including MyD88, LPS-binding protein and differentiation cluster co-receptor, when immune cells are activated by LPS (
Alongside inflammatory insults, oxidative stress is another major cause of CNS damage. Low levels of ROS serve an important role as signaling molecules that regulate the immune response to pathogens; however, overproduction of ROS contributes to neurotoxicity (
In conclusion, the present study demonstrated that isorhamnetin exerted potent anti-inflammatory effects on BV2 microglial cells. In LPS-stimulated BV2 cells, isorhamnetin was able to reduce the production of pro-inflammatory mediators and cytokines, which was associated with decreased expression of their regulatory genes via the suppression of NF-κB activity. Furthermore, isorhamnetin could block early intracellular signaling cascades by antagonizing TLR4 or suppressing ROS accumulation. Although the results of the current study may provide partial understanding of the mechanism underlying the anti-inflammatory effects of isorhamnetin, further studies are required to assess the mechanical role of isorhamnetin in various oxidative stress- and inflammation-mediated diseases.
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korean government (grant nos. 2018R1A2B2005705 and 2016R1A5A2007009).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SYK, HTP and YHC contributed to the conception and design of the experiment. SYK, CYJ, CHK, YHY and GYK performed all experiments and verified the analytical data. HMY and SHC contributed to the statistical analysis and helped interpret the results. YHC supervised the experiments in discussion with SYK, HTP, YHC, CYJ, CHK, YHY and GYK wrote the manuscript. All authors discussed the final results and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
Suppression of NO, PGE2, TNF-α and IL-1β production by ISO in LPS-stimulated BV2 microglial cells. Cells were pretreated with the indicated concentrations of ISO for 1 h prior to incubation with 100 ng/ml LPS for 24 h. Levels of (A) NO, (B) PGE2, (C) TNF-α and (D) IL-1β were detected in the culture media by Griess assay and commercial ELISA kits. Data are presented as the means ± standard deviation obtained from three independent experiments. *P<0.05 compared with the control group; #P<0.05 compared with the LPS group. IL-1β, interleukin-1β; ISO, isorhamnetin; LPS, lipopolysaccharide; NO, nitric oxide; PGE2, prostaglandin E2; TNF-α, tumor necrosis factor-α.
Inhibition of LPS-induced expression of iNOS, COX-2, TNF-α and IL-1β by ISO in BV2 microglial cells. BV2 cells were pretreated with various concentrations of ISO for 1 h followed by treatment with 100 ng/ml LPS for 24 h. (A) Total RNA was isolated and RT-PCR was performed using the indicated primers. (B) Total proteins were isolated and subjected to western blot analyses. Experiments were repeated three times and similar results were obtained. GAPDH and actin were used as the internal controls for the RT-PCR and western blot analysis, respectively. Bands were semi-quantified using ImageJ, normalized to (C) GAPDH and (D) actin and ratios were determined. Data are presented as the means ± standard deviation obtained from three independent experiments. *P<0.05 compared with the control group; #P<0.05 compared with the LPS group. COX-2, cyclooxygenase 2; IL-1β, interleukin-1β; ISO, isorham-netin; LPS, lipopolysaccharide; NO, nitric oxide; PGE2, prostaglandin E2; RT-PCR, reverse transcription-polymerase chain reaction; TNF-α, tumor necrosis factor-α.
Effects of ISO and LPS on the viability of BV2 microglial cells. Cells were (A) treated with various concentrations of ISO for 24 h or (B) were pretreated with the indicated concentrations of ISO for 1 h prior to treatment with 100 ng/ml LPS for 24 h. Cell viability was assessed by MTT assay. Results are expressed as the percentage of surviving cells over control cells. Data are presented as the means ± standard deviation of three independent experiments. ISO, isorhamnetin; LPS, lipopolysaccharide.
Inhibition of NF-κB nuclear translocation by isorhamnetin in LPS-stimulated BV2 microglial cells. (A) Cells were pretreated with 200
Effects of isorhamnetin on LPS-induced expression of TLR4 and Myd88 in BV2 microglial cells. Cells were (A) treated with 100 ng/ml LPS for the indicated duration or (B) pretreated with the indicated concentrations of isorhamnetin for 1 h prior to 100 ng/ml LPS treatment for 6 h. Total proteins were prepared for western blot analysis using anti-TLR4 and anti-Myd88 antibodies. Actin was used as an internal control. (C and D) Bands were semi-quantified using ImageJ, normalized to actin and the ratios were determined. Data are presented as the means ± standard deviation of three independent experiments. *P<0.05 compared with the control group; #P<0.05 compared with the LPS group. ISO, isorhamnetin; LPS, lipopolysaccharide; Myd88, myeloid differentiation factor 88; TLR4, Toll-like receptor 4.
LPS-induced interaction between LPS and TLR4 is attenuated by isorhamnetin in BV2 microglial cells. (A) BV2 cells were pretreated with or without 200
Effects of the TLR4 inhibitor, CLI-095, on LPS-induced production of pro-inflammatory mediators in BV2 microglia. (A and B) BV2 cells were treated with the indicated concentrations of ISO (200
LPS-induced ROS generation is inhibited by ISO in BV2 microglial cells. Cells were (A) treated with 100 ng/ml LPS for the indicated time periods or (B) pretreated with 200
List of antibodies used for western blot analysis in the present study.
Antibody | Dilution | Product no. | Species of origin | Supplier |
---|---|---|---|---|
iNOS | 1:1,000 | 610328 | Rabbit polyclonal | BD Transduction Laboratories; BD Biosciences, San Jose, CA, USA |
COX-2 | 1:500 | 160126 | Rabbit polyclonal | Cayman Chemical Company, Ann Arbor, MI, USA |
IL-1β | 1:1,000 | sc-7884 | Rabbit polyclonal | Santa Cruz Biotechnology, Inc., Dallas, TX, USA |
TNF-α | 1:1,000 | 3707S | Rabbit polyclonal | Cell Signaling Technology, Inc., Danvers, MA, USA |
NF-κB p65 | 1:1,000 | sc-71675 | Mouse monoclonal | Santa Cruz Biotechnology, Inc. |
IκBα | 1:1,000 | sc-371 | Rabbit polyclonal | Santa Cruz Biotechnology, Inc. |
p-IκBα | 1:1,000 | sc-8404 | Mouse monoclonal | Santa Cruz Biotechnology, Inc. |
TLR4 | 1:1,000 | ab53629 | Goat polyclonal | Abcam, Cambridge, MA, USA |
Myd88 | 1:1,000 | ab2064 | Rabbit polyclonal | Abcam |
Lamin B | 1:1,000 | sc-6216 | Goat polyclonal | Santa Cruz Biotechnology, Inc. |
Actin | 1:1,000 | sc-1615 | Goat polyclonal | Santa Cruz Biotechnology, Inc. |
COX-2, cyclooxygenase-2; IκB-α, inhibitor κB-α; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; Myd88, myeloid differentiation factor 88; NF-κB, nuclear factor-κB; p, phosphorylated; TNF-α, tumor necrosis factor-α; TLR4, Toll-like receptor 4.