Inflammatory bowel disease (IBD) is a chronic, idiopathic inflammatory disease of the small and/or large intestine. Endothelial expression of inflammatory mediators, including cytokines and adhesion molecules, serves a critical role in the initiation and progression of IBD. The dietary flavonoid, kaempferol, has been reported to inhibit expression of inflammatory mediators; however, the underlying mechanisms require further investigation. In the present study, a novel molecular mechanism of kaempferol against IBD was identified. The potential anti-inflammatory effect of kaempferol in a cellular model of intestinal inflammation was assessed using lipopolysaccharide (LPS)-induced rat intestinal microvascular endothelial cells (RIMVECs), and an underlying key molecular mechanism was identified. RIMVECs were pretreated with kaempferol of various concentrations (12.5, 25 and 50 µM) followed by LPS (10 µg/ml) stimulation. ELISA was used to examine the protein levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the supernatant. Protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) p65, inhibitor of NF-κB, mitogen-activated protein kinase p38 and signal transducer and activator of transcription (STAT) in cells were measured by western blotting. Kaempferol significantly reduced the overproduction of TNF-α, IL-1β, interleukin-6, ICAM-1 and VCAM-1 induced by LPS, indicating the negative regulation of kaempferol in TLR4, NF-κB and STAT signaling underlying intestinal inflammation. The present results provide support for the potential use of kaempferol as an effective therapeutic agent for IBD treatment.
Inflammatory bowel disease (IBD) primarily comprises two principal conditions, Crohn's disease and ulcerative colitis (UC), characterized by chronic gastrointestinal inflammation with alternating periods of relapse and remission (
The etiology of IBD is not yet completely understood; it is believed that a complex interaction among genetic, immunological, metabolic, vascular, microbial and social factors leads to dysregulated and persistent inflammation (
Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), a flavonoid widely distributed in fruits, vegetables and plant-based foods, has been reported to possess anti-inflammatory, anti-diabetic, anti-hypertensive, anti-depressant and anti-ulcerative properties by acting on various cellular pathways (
In order to further investigate the beneficial effect of kaempferol and the possible mechanisms involved in the intestinal inflammation process, LPS-stimulated rat intestinal microvascular endothelial cells (RIMVECs) were used to establish a cell model of IBD. It was demonstrated that kaempferol may alleviate LPS-induced inflammatory mediators, including TNF-α, IL-1β, IL-6, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1), by suppressing the activation of toll-like receptor 4 (TLR4), signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB).
Kaempferol (purity ≥98%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Kaempferol was dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO was <0.1% (v/v) when kaempferol was added to the experimental cells. Cell culture reagents, namely Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Endothelial cell growth supplement (ECGS) was purchased from EMD Millipore (Billerica, MA, USA). LPS (Escherichia coli 055:B5) was provided by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Rat TNF-α (cat. no. DY510), IL-1β (cat. no. DY501), IL-6 (cat. no. DY506), ICAM-1 (cat. no. DY583) and VCAM-1 (cat. no. DY809) ELISA kits were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Antibodies for TLR4 (cat. no. ab22048), phosphorylated (p)-inhibitor of κB (I-κB) (cat. no. ab133462) and p-NF-κB p65 (cat. no. ab28856) were purchased from Abcam (Cambridge, UK). Antibodies for p-p38 (cat. no. 4511) mitogen-activated protein kinase (MAPK) and p-STAT (cat. no. 9145) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against β-actin (cat. no. AC006) was purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).
RIMVECs were isolated and cultured, as described previously (
Cell viability was measured with a CCK-8 assay. RIMVECs were seeded at a density of 1×104 cells/well in 96-well plates for 24 h. Subsequently, the cells were treated with 100 µl of kaempferol at different concentrations (200, 100, 50, 25, 12.5 and 6.25 µM) for 12 h. Following treatment, the medium was removed, and cells were cultured in 100 µl fresh DMEM containing 10% CCK-8 solution at 37°C for 2 h. Cell viability was determined by measuring the absorbance with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.
TNF-α, IL-1β, IL-6, ICAM-1, and VCAM-1 protein expression levels in culture supernatant were measured by ELISA, according to the manufacturer's protocol. RIMVECs were seeded in 12-well plates (1×105 cells/well). At 90% confluency, the cells were washed with PBS prior to the addition of medium with different concentrations of kaempferol (50, 25 and 12.5 µM). After 3 h, cells were washed once with PBS and subsequently stimulated with 10 µg/ml LPS (1 ml/well) for 6 h (TNF-α, IL-1β and IL-6) or 12 h (ICAM-1 and VCAM-1) prior to the collection of the supernatant and measurement of inflammatory factors.
At the end of the incubation period, cells were lysed in radioimmunoprecipitation lysis buffer (2% SDS; 10% glycerol; 62.5 mM Tris-HCl buffer; pH 6.8) on ice. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Samples with equal quantities of total protein (20 µg) were loaded and separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Pierce; Thermo Fisher Scientific, Inc.). The membranes were blocked in 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) for 1 h at room temperature, and were immunoblotted with the specific primary antibodies against TLR4 (1:800), p-NF-κB p65 (1:1,000), p-I-κB (1:1,000), p-p38 (1:800), p-STAT (1:800) and β-actin (1:2,000) overnight at 4°C. Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; Cell Signaling Technology, Inc.; cat. no. SC3901) for 1 h at room temperature. Subsequently, the blots were visualized with an enhanced chemiluminescence immunoblotting detection system (Beyotime Institute of Biotechnology) and quantified using ImageJ (v1.51; National Institutes of Health, Bethesda, MD, USA).
Data are expressed as mean ± standard deviation, and analyzed by one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons. All analyses were performed by GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
The chemical structure of kaempferol is presented in
Pretreatment for 3 h with kaempferol at different concentrations (12.5, 25 and 50 µM) was followed by treatment with LPS (10 µg/ml) stimulation for 6 h. As presented in
As presented in
The NF-κB pathway serves a critical role in the secretion of cytokines (
Activation of the TLR4 signaling pathway may directly lead to the activation of NF-κB to regulate the production of cytokines (
The p38 MAPK signaling pathway additionally serves an important role in inflammatory response. In response to LPS, the level of p-p38 was significantly upregulated in the model group compared with the control group (
The STAT pathway is another important inflammatory signaling pathway. As demonstrated in
IBD is a chronic inflammatory disease of intestinal mucosa and submucosa, characterized by disruption of the intestinal epithelial barrier, increased production of inflammatory mediators and excessive tissue injury (
Inflammatory mediators, including cytokines, adhesion proteins and chemokines, have an important role in the pathogenesis of IBD. TNF-α is the earliest and primary endogenous mediator in the inflammatory process. It has a pro-inflammatory effect through an increased production of IL-1β, IL-6, adhesion molecules and pro-coagulant factors (
In IBD, activation of microvascular endothelial cells and adhesion of circulating immune cells is required for the initiation and maintenance of inflammation (
NF-κB has an essential role in regulating the expression of inflammatory cytokines and adhesion molecules (
TLRs are pattern recognition receptors that ‘sense’ microbes and alert the immune system through activation of transcription factors (including NF-κB) and the secretion of inflammatory mediators (
The MAPK signaling pathways, including extracellular signal-regulated kinase 1 and 2, c-jun N-terminal kinase and p38, are closely associated with the expression of inflammatory mediators and oxidative damage (
The STAT pathway is another crucial signaling cascade involved in IBD. It is well documented that the STAT pathway is not only associated with the inflammatory process in IBD; however, is additionally associated with further serious conditions, including colorectal cancer development (
In conclusion, it was demonstrated that kaempferol significantly inhibits the LPS-induced expression of the inflammatory mediators TNF-α, IL-1β, IL-6, ICAM-1 and VCAM-1 by inhibiting TLR4, NF-κB and STAT signaling, but not by activating p38 MAPK signaling in RIMVECs. The present results provide novel insight for the inflammatory effect of kaempferol in endothelial cells. It suggests that kaempferol may be an effective therapeutic agent for treatment of IBD, and that fruits and herbs rich in kaempferol may be incorporated into potential health care products for patients with IBD.
Not applicable.
The present study was funded by the National Natural Science Foundation of China (grant no. 31472228).
All data generated or analyzed during this study are included in this published article.
YB designed and participated in all of the experiments, and was a major contributor in writing the manuscript. PL, JZ and YH were in charge of culturing the primary cells. YF and SZ performed the western blotting. ZL revised the manuscript, and was a major contributor in designing the experiments. All authors read and approved the final manuscript.
The present study was approved by the China Agriculture University Institutional Animal Care and Use Committee (approval no. CAU20160031201).
Not applicable.
The authors declare that they have no competing interests.
Effects of kaempferol on cell viability of rat intestinal microvascular endothelial cells. (A) Chemical structure of kaempferol. (B) Cells were incubated with different concentrations of kaempferol (200, 100, 50, 25, 12.5 and 6.25 µM) for 12 h. Cell viability was determined by a Cell Counting Kit-8 assay. Data are presented as the mean ± standard deviation. n=6. **P<0.01 vs. control cells. OD, optical density.
Kaempferol inhibits cytokine production induced by LPS in rat intestinal microvascular endothelial cells. Cells were treated with three different concentrations of kaempferol (50, 25 and 12.5 µM) for 3 h and subsequently stimulated with LPS (10 µg/ml). After 6 h, (A) TNF-α, (B) IL-1β and (C) IL-6 protein secretion in the culture supernatant was quantified using an ELISA kit. Data are presented as the mean ± standard deviation. n=4. ##P<0.01 vs. control cells; **P<0.01 vs. cells stimulated with LPS. LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL, interleukin.
Kaempferol inhibits the production of adhesion protein induced by LPS in RIMVECs. RIMVECs were treated with three different concentrations of kaempferol (50, 25 and 12.5 µM) for 3 h and subsequently stimulated with LPS (10 µg/ml). After 12 h, (A) ICAM-1 and (B) VCAM-1 expression level in the culture supernatant was quantified with an ELISA kit. Data from individual experiments are presented as the mean ± standard deviation. n=4. ##P<0.01 vs. control cells; *P<0.05, **P<0.01 vs. cells stimulated with LPS. RIMVECs, rat intestinal microvascular endothelial cells; ICAM-1, intercellular adhesion molecule 1; LPS, lipopolysaccharide; VCAM-1, vascular cell adhesion molecule 1.
Kaempferol inhibits activation of NF-κB induced by LPS in rat intestinal microvascular endothelial cells. Cells were treated with various concentrations (50, 25 and 12.5 µM) of kaempferol for 3 h followed by treatment with LPS (10 µg/ml). (A) After 3 h, p-NF-κB p65, NF-κB p65, p-I-κB and I-κB protein expression levels were analyzed by western blotting. (B) p-NF-κB p65/NF-κB and (C) p-I-κB/I-κB ratios were determined by densitometry. Data are presented as the mean ± standard deviation. n=3. ##P<0.01 vs. control cells; **P<0.01 vs. cells stimulated by LPS. p-, phosphorylated; NF-κB, nuclear factor-κB; I-κB, inhibitor of κB; LPS, lipopolysaccharide.
Kaempferol inhibits expression of TLR4 induced by LPS in RIMVECs. RIMVECs were pretreated with kaempferol (50 µM) and subsequently stimulated with LPS (10 µg/ml) for 3 h. TLR4 protein expression levels were determined by (A) western blotting and (B) densitometry analysis with β-actin as the loading control were performed. Data are presented as the mean ± standard deviation. n=3. ##P<0.01 vs. control cells; **P<0.01 vs. cells stimulated by LPS. RIMVECs, rat intestinal microvascular endothelial cells; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.
Kaempferol does not affect the phosphorylation of p38 mitogen-activated protein kinase induced by LPS in RIMVECs. RIMVECs were pretreated with kaempferol (50 µM) and subsequently stimulated with LPS (10 µg/ml) for 3 h. p-p38 expression was determined by (A) western blotting and (B) densitometry analysis with β-actin as the loading control. Data are presented as the mean ± standard deviation. n=3. ##P<0.01 vs. control cells. RIMVECs, rat intestinal microvascular endothelial cells; LPS, lipopolysaccharide; p-, phosphorylated.
Kaempferol decreases the phosphorylation of STAT induced by LPS in RIMVECs. RIMVECs were pretreated with kaempferol (50 µM) and subsequently stimulated with LPS (10 µg/ml) for 3 h. p-STAT was measured by (A) western blotting and (B) densitometry analysis with β-actin as the loading control. Data are presented as the mean ± standard deviation. n=3. ##P<0.01 vs. control cells; *P<0.05 vs. cells stimulated by LPS. RIMVECs, rat intestinal microvascular endothelial cells; p-, phosphorylated; STAT, signal transducer and activator of transcription; LPS, lipopolysaccharide.