CoChat
Sign Up
Cited by 110Flavonoids in Medical ResearchPhytochemistry and Biological ActivitiesBioactive natural compounds

The DOI matches its Crossref record; 61 of the 61 references it cites were found in a registry or an index.

Genkwanin Inhibits Proinflammatory Mediators Mainly through the Regulation of miR-101/MKP-1/MAPK Pathway in LPS-Activated Macrophages

Open access
Gao, Yuan, Liu, Fen, Fang, Lei, Cai, Runlan, Zong, Chuanjie, Qi, Yun·2014·PLoS ONE
DOI 10.1371/journal.pone.0096741PMC PMC4011752
Open this paper in CoChat — free accountOpen PDF

Abstract

Genkwanin is one of the major non-glycosylated flavonoids in many herbs with anti-inflammatory activities. Although its anti-inflammatory activity in vivo has been reported, the potential molecular mechanisms remain obscure. In this study, by pharmacological and genetic approaches, we explore the anti-inflammatory effects of genkwanin in LPS-activated RAW264.7 macrophages. Genkwanin potently decreases the proinflammatory mediators, such as iNOS, TNF-α, IL-1β and IL-6, at the transcriptional and translational levels without cytotoxicity, indicating the excellent anti-inflammatory potency of genkwanin in vitro. Mechanism study shows that genkwanin significantly suppresses the p38- and JNK-mediated AP-1 signaling pathway and increases the mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression at the posttranscriptional level. We also confirmed that microRNA-101 (miR-101) is a negative regulator of MKP-1 expression. Moreover, regardless of miR-101-deficient cells or miR-101-abundant cells, the suppression effects of genkwanin on supernatant proinflammatory mediators' levels are far less than that in respective negative control cells, suggesting that genkwanin exerts anti-inflammatory effect mainly through reducing miR-101 production. However, genkwanin can't affect the level of phospho-Akt (p-Akt), indicating that the phosphorylation of Akt may be not responsible for the effect of genkwanin on miR-101 production. We conclude that genkwanin exerts its anti-inflammatory effect mainly through the regulation of the miR-101/MKP-1/MAPK pathway.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0096741

Cites

61 of 61 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Effects of genkwanin on NO production and iNOS in LPS-activated RAW264.7 macrophages. (A) Effects of genkwanin on LPS-induced NO production. Cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 24 h. After treatment, nitrite levels in the medium were measured by Griess reaction. L-NAME was used as a positive control. (B) Effects of genkwanin on iNOS enzyme activity. Cells were pretreated with LPS (10 ng/mL) for 12 h and then exposed to genkwanin at the indicated concentrations for a further 12 h without LPS. Nitrite levels in the medium were measured. L-NAME was used as a positive control. (C) Effects of genkwanin on iNOS mRNA expression. Cells were pretreated with the indicated concentrations of genkwanin for 2 h and then exposed to LPS (10 ng/mL) for 4 h. mRNA of iNOS was determined by RT-qPCR analysis. (D) Effects of genkwanin on iNOS protein levels. Cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 24 h. After treatment, cellular proteins were prepared and the iNOS protein levels were determined by Western blot analysis. Bars represent mean ±SD of three independent experiments. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone.
Figure 1 Effects of genkwanin on NO production and iNOS in LPS-activated RAW264.7 macrophages. (A) Effects of genkwanin on LPS-induced NO production. Cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 24 h. After treatment, nitrite levels in the medium were measured by Griess reaction. L-NAME was used as a positive control. (B) Effects of genkwanin on iNOS enzyme activity. Cells were pretreated with LPS (10 ng/mL) for 12 h and then exposed to genkwanin at the indicated concentrations for a further 12 h without LPS. Nitrite levels in the medium were measured. L-NAME was used as a positive control. (C) Effects of genkwanin on iNOS mRNA expression. Cells were pretreated with the indicated concentrations of genkwanin for 2 h and then exposed to LPS (10 ng/mL) for 4 h. mRNA of iNOS was determined by RT-qPCR analysis. (D) Effects of genkwanin on iNOS protein levels. Cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 24 h. After treatment, cellular proteins were prepared and the iNOS protein levels were determined by Western blot analysis. Bars represent mean ±SD of three independent experiments. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone.CC BY · PubMed Central
Figure 2 Effects of genkwanin on TNF-α,IL-1β and IL-6 in LPS-activated RAW264.7 macrophages at the transcriptional and translational levels. (A) The cells were pretreated with the indicated concentrations of genkwanin for 2 h and then exposed to LPS (10 ng/mL) for 24 h. The levels of TNF-α, IL-1β and IL-6 in the supernatant were determined by ELISA. (B) The cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 4 h. The mRNA expressions of TNF-α, IL-6 and IL-1β were determined by RT-qPCR analysis. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone. Bars represent mean ±SD of three independent experiments.
Figure 2 Effects of genkwanin on TNF-α,IL-1β and IL-6 in LPS-activated RAW264.7 macrophages at the transcriptional and translational levels. (A) The cells were pretreated with the indicated concentrations of genkwanin for 2 h and then exposed to LPS (10 ng/mL) for 24 h. The levels of TNF-α, IL-1β and IL-6 in the supernatant were determined by ELISA. (B) The cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 4 h. The mRNA expressions of TNF-α, IL-6 and IL-1β were determined by RT-qPCR analysis. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone. Bars represent mean ±SD of three independent experiments.CC BY · PubMed Central
Figure 3 Effects of genkwanin on LPS-induced AP-1 and NF-κB activities. RAW264.7 macrophages were transfected with the luciferase reporter pAP-1-TA-luc (A) and pNF-κB-TA-luc (B). 24 h after transfection, the cells were pretreated with genkwanin for 2 h and then exposed to LPS (10 ng/mL) at the indicated concentrations. After 6 h, the luciferase activity was determined. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone. Bars represent mean ±SD of three independent experiments.
Figure 3 Effects of genkwanin on LPS-induced AP-1 and NF-κB activities. RAW264.7 macrophages were transfected with the luciferase reporter pAP-1-TA-luc (A) and pNF-κB-TA-luc (B). 24 h after transfection, the cells were pretreated with genkwanin for 2 h and then exposed to LPS (10 ng/mL) at the indicated concentrations. After 6 h, the luciferase activity was determined. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone. Bars represent mean ±SD of three independent experiments.CC BY · PubMed Central
Figure 4 Effects of genkwanin on LPS-induced MAPK and MKP-1. (A) Effects of genkwanin on the LPS-induced phosphorylation of p38, JNK and ERK1/2. RAW264.7 macrophages were pretreated with genkwanin at indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. Total proteins were extracted for the Western blot analysis. (B) Effects of genkwanin on MKP-1 mRNA in LPS-activated RAW264.7 macrophages. The cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. The mRNA expression of MKP-1 was determined by RT-qPCR analysis. (C) Effects of genkwanin on MKP-1 protein level in LPS-activated RAW264.7 macrophages. Cells were pretreated with genkwanin at indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. Cell lysates were analysed by Western blot. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone. Bars represent mean ±SD of three independent experiments.
Figure 4 Effects of genkwanin on LPS-induced MAPK and MKP-1. (A) Effects of genkwanin on the LPS-induced phosphorylation of p38, JNK and ERK1/2. RAW264.7 macrophages were pretreated with genkwanin at indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. Total proteins were extracted for the Western blot analysis. (B) Effects of genkwanin on MKP-1 mRNA in LPS-activated RAW264.7 macrophages. The cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. The mRNA expression of MKP-1 was determined by RT-qPCR analysis. (C) Effects of genkwanin on MKP-1 protein level in LPS-activated RAW264.7 macrophages. Cells were pretreated with genkwanin at indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. Cell lysates were analysed by Western blot. ## p<0.01 vs. normal control group; **p<0.01 vs. LPS alone. Bars represent mean ±SD of three independent experiments.CC BY · PubMed Central
Figure 5 Genkwanin exerts anti-inflammatory effects mainly through decreasing miR-101 production. (A) Effect of genkwanin on miR-101 expression in LPS-activated RAW264.7 macrophages. The cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 90 min. miR-101 expression was determined by RT-qPCR analysis. (B) Effect of miR-101 on MKP-1 protein level. RAW264.7 macrophages were transfected with miR-101 mimic or inhibitor or their negative controls, and then stimulated with LPS (10 ng/mL) for 1 h. Cell lysates were analysed by Western blot. (C–D) Effects of genkwanin on supernatant TNF-α in LPS-stimulated RAW264.7 macrophages which have been transfected with ssRNA inhibitor (C) or dsRNA mimic (D) for mmu- miR-101a. RAW264.7 macrophages were transfected with miR-101 inhibitor or mimic or their negative controls, and then stimulated with LPS (10 ng/mL) for 24 h in the presence or absence of genkwanin (50 µM). Supernatant TNF-α was measured by ELISA. ## p<0.01 vs. resting cells transfected with miR-101 inhibitor or mimic; *p<0.05 vs. LPS-treated cells transfected with miR-101 inhibitor or mimic; ΔΔp<0.01 vs. resting cells transfected with negative controls; @@ p<0.01 vs. LPS-treated cells transfected with negative controls. (E) Effect of genkwanin on the LPS-induced p-Akt. Cells were pretreated with genkwanin at indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. Cell lysated were assayed by Western blot analysis using Akt and p-Akt antibodies. ## p<0.01 vs. normal control group. Bars represent mean ±SD of three independent experiments.
Figure 5 Genkwanin exerts anti-inflammatory effects mainly through decreasing miR-101 production. (A) Effect of genkwanin on miR-101 expression in LPS-activated RAW264.7 macrophages. The cells were pretreated with genkwanin at the indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 90 min. miR-101 expression was determined by RT-qPCR analysis. (B) Effect of miR-101 on MKP-1 protein level. RAW264.7 macrophages were transfected with miR-101 mimic or inhibitor or their negative controls, and then stimulated with LPS (10 ng/mL) for 1 h. Cell lysates were analysed by Western blot. (C–D) Effects of genkwanin on supernatant TNF-α in LPS-stimulated RAW264.7 macrophages which have been transfected with ssRNA inhibitor (C) or dsRNA mimic (D) for mmu- miR-101a. RAW264.7 macrophages were transfected with miR-101 inhibitor or mimic or their negative controls, and then stimulated with LPS (10 ng/mL) for 24 h in the presence or absence of genkwanin (50 µM). Supernatant TNF-α was measured by ELISA. ## p<0.01 vs. resting cells transfected with miR-101 inhibitor or mimic; *p<0.05 vs. LPS-treated cells transfected with miR-101 inhibitor or mimic; ΔΔp<0.01 vs. resting cells transfected with negative controls; @@ p<0.01 vs. LPS-treated cells transfected with negative controls. (E) Effect of genkwanin on the LPS-induced p-Akt. Cells were pretreated with genkwanin at indicated concentrations for 2 h and then exposed to LPS (10 ng/mL) for 1 h. Cell lysated were assayed by Western blot analysis using Akt and p-Akt antibodies. ## p<0.01 vs. normal control group. Bars represent mean ±SD of three independent experiments.CC BY · PubMed Central
Figure 6 Chemical structures of genkwanin, apigenin, acacetin, wogonin, chrysin, baicalein, luteolin and velutin.
Figure 6 Chemical structures of genkwanin, apigenin, acacetin, wogonin, chrysin, baicalein, luteolin and velutin.CC BY · PubMed Central
Figure 7 Proposed mechanism by which genkwanin exerts anti-inflammatory activity in LPS-activated RAW264.7 macrophages. The gray color indicates the targets of genkwanin.
Figure 7 Proposed mechanism by which genkwanin exerts anti-inflammatory activity in LPS-activated RAW264.7 macrophages. The gray color indicates the targets of genkwanin.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Research works printed but not linked: 1

  1. The State Commission of Chinese Pharmacopoeia (2010) Pharmacopoeia of People's Republic of China. Part I. Beijing: Chemical Industry Press. p148.

Cited by

Sorted by citations

See all 110

Related

See all

Metadata

Authors
Gao, Y., Liu, F., Fang, L., Cai, R., Zong, C., & Qi, Y.
Affiliations
Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
Corresponding author
Qi, Yun
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-05-06
Volume
9
Issue
5
Pages
e96741
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0096741
PMC
PMC4011752
OpenAlex
W2035643839
Cited by
110
References
61
Citations by year
2026: 4 · 2025: 5 · 2024: 15 · 2023: 15 · 2022: 14 · 2021: 16 · 2020: 7 · 2019: 9 · 2018: 8 · 2017: 8 · 2016: 5 · 2015: 4
FWCI
4.13
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Natural Science Foundation of China (81173645, 2014ZX09201022-006, 81274163, 2012ZX09301002-001)
  • Ministry of Science and Technology of the People's Republic of China (81274163, 2012ZX09301-002, 2014ZX09201022-006, 2012ZX09301-002-001)
Topics
  • Health Sciences › Medicine › Pharmacology (medical) › Flavonoids in Medical Research
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Phytochemistry and Biological Activities
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Drug Discovery › Bioactive natural compounds
Keywords
genkwanin, anti-inflammatory activity, macrophages, lipopolysaccharide stimulation, miR-101, MKP-1, MAPK signaling, AP-1 signaling, p38 MAPK, JNK, inflammatory cytokines, inducible nitric oxide synthase, post-transcriptional regulation, Akt
MeSH terms
Animals, Anti-Inflammatory Agents, Cell Line, Lipopolysaccharides, Macrophages, Nitric Oxide, Phosphorylation, Tumor Necrosis Factor-alpha, Signal Transduction, Interleukin-6, Up-Regulation, Oligonucleotides, Antisense, Inflammation Mediators, Mitogen-Activated Protein Kinases, MicroRNAs, Flavones, Mice, Nitric Oxide Synthase Type II, Interleukin-1beta, Dual Specificity Phosphatase 1

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review