Open Access

DFMG attenuates the activation of macrophages induced by co‑culture with LPC‑injured HUVE‑12 cells via the TLR4/MyD88/NF‑κB signaling pathway

  • Authors:
    • Li Cong
    • Shuting Yang
    • Yong Zhang
    • Jianguo Cao
    • Xiaohua Fu
  • View Affiliations

  • Published online on: February 23, 2018     https://doi.org/10.3892/ijmm.2018.3511
  • Pages: 2619-2628
  • Copyright: © Cong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

7‑difluoromethoxy‑5,4'‑dimethoxy‑genistein (DFMG) is a novel active chemical entity, which modulates the function and signal transduction of endothelial cells and macrophages (MPs), and is essential in the prevention of atherosclerosis. In the present study, the activity and molecular mechanism of DFMG on MPs was investigated using a Transwell assay to construct a non‑contact co‑culture model. Human umbilical vein endothelial cells (HUVE‑12), which were incubated with lysophosphatidylcholine (LPC), were seeded in the upper chambers, whereas PMA‑induced MPs were grown in the lower chambers. The generation of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH) were measured using the corresponding assay kits. The proliferation and migration were assessed using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide and wound healing assays, respectively. Foam cell formation was examined using oil red O staining and a total cholesterol assay. The protein expression levels of Toll‑like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)‑κB p65 were detected by western immunoblotting. The secretion of interleukin (IL)‑1β was examined using an enzyme‑linked immunosorbent assay. It was found that LPC significantly increased the generation of ROS and the release of LDH in HUVE‑12 cells. The LPC‑injured HUVE‑12 cells activated MPs under co‑culture conditions and this process was inhibited by DFMG treatment. LPC upregulated the expression levels of TLR4, MyD88 and NF‑κB p65, and the secretion of IL‑1β in the supernatant of the co‑cultured HUVE‑12 cells and MPs. These effects were reversed by the application of DFMG. Furthermore, CLI‑095 and IL‑1Ra suppressed the activation of MPs that was induced by co‑culture with injured HUVE‑12 cells. These effects were further enhanced by co‑treatment with DFMG, and DFMG exhibited synergistic effects with a TLR4‑specific inhibitor. Take together, these findings revealed that DFMG attenuated the activation of MP induced by co‑culture with LPC‑injured HUVE‑12 cells. This process was mediated via inhibition of the TLR4/MyD88/NF‑κB signaling pathway in HUVE‑12 cells.

Introduction

Atherosclerosis (AS) is as a chronic inflammatory disease that exhibits severe complications, including myocardial infarction and ischemic stroke, which are considered two of the main causes of mortality worldwide (1). Macrophages (MPs) are the most abundant inflammatory cell to invade atherosclerotic lesions, and are essential during the atherogenic process (1,2). During initiation, inflammatory cytokines produced by MPs stimulate the generation of endothelial adhesion molecules, proteases and other mediators that enter the systemic circulation in soluble forms (3). Furthermore, MPs internalize modified lipoproteins in order to produce foam cells, which are considered a hallmark event in the formation of early atherosclerotic lesions (4). During the advanced stage of the disease, MPs contribute to the plaque morphology, affecting the fibrous cap and necrotic core, which increase the pro-inflammatory responses and the apoptotic signals (5). Therefore, strategies focusing on the inhibition of MP activation may be beneficial for the prevention and treatment of AS.

AS is a multiphase process characterized by activation of the endothelium with the secretion of monocytic maturation factors (6). AS is initiated by endothelial injury due to oxidative stress associated with several cardiovascular risk factors (7). Endothelial cell dysfunction (ECD) manifested in lesion-prone areas of arterial vasculature recruits circulating monocytes from the blood into the tunica intima, which eventually differentiate into MPs (8-10). In turn, monocyte-derived MPs trigger an inflammatory cascade, including the production of pro-inflammatory cytokines, interleukin (IL)-1β, IL-12, tumor necrosis factor-α (TNF-α), and chemokines, including monocyte chemoattractant protein-1 (MCP-1). These mediators attract additional monocytes, which adhere to the endothelial cells (ECs) (1,11). In previous years, the interaction between MPs and ECs has been considered a significant feature of a majority of pathophysiological conditions (12). However, the precise signaling pathways between them remain to be fully elucidated.

Toll-like receptor 4 (TLR4) is expressed in MPs, ECs and smooth muscle cells. The receptor is mediated by myeloid differentiation factor 88 (MyD88)-dependent and -independent pathways (13,14). Nuclear factor-κB (NF-κB) is a transcription factor, which localizes to the cytoplasm and mainly exists as a heterodimer consisting of the p50 and p65 subunits (15). NF-κB signaling upregulates the expression levels of lectin-like oxidized low-density lipoprotein receptor 1, vascular adhesion molecule 1 and MCP-1 in ECs induced by oxidized low-density lipoprotein (ox-LDL) in vitro (16). The TLR4/MyD88/NF-κB signaling pathway is the main signaling pathway to be activated during TLR4-mediated inflammation (17). Activated TLR4 signaling upregulates the protein expression levels of TLR4 pathway-associated mediators, including upstream (TLR4, MyD88 and NF-κB) and downstream (IL-1β, IL-6 and TNF-α) factors (18). IL-1β belongs to the IL-1 cytokine family together with IL-1α, IL-1Ra, IL-18 and IL-33 (19,20). The generation of mature IL-1β is tightly controlled by a diverse class of cytosolic protein complexes, inflammasome components, and the K+ efflux (19,21,22). However, the exact mechanism by which the activation of TLR4 affects the interaction between ECs and MPs, during the oxidative damage of ECs remains to be elucidated.

Currently, the management of AS aims to reduce inflammation and adjust dyslipidemia. Therefore, research efforts have focused on the development of several natural and synthetic agents in order to attenuate inflammation at the molecular level. Resveratrol inhibits the IL-1β-induced expression of matrix metalloproteinase-13, IL-6 and TNF-α via the TLR4/MyD88-dependent signaling cascades (23,24), and it further attenuates neuronal autophagy and inflammatory injury in experimental traumatic brain injury (25). Diallyl trisulfide exerts anti-inflammatory effects via suppressing the TLR4/NF-κB signaling pathway in lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages and isch-emic stroke-induced inflammation (26,27). In addition, (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid inhibited LPS-induced NF-κB and p38 mitogen-activated protein kinase signaling pathways, and reduced the secretion of IL-1β, TNF-α and IL-10 from peritoneal cells in vitro and ex vivo (28,29). However, there are several disadvantages limiting the clinical application of these agents, including side effects and poor oral bioavailability (30). 7-difluoromethoxy-5,4′-dimethoxy-genistein (DFMG) is a novel active chemical entity, which is synthesized by the precursor genistein (GEN), and exhibits optimal fat-solubility, antioxidant and anti-inflammatory activities (31,32). DFMG is involved in the regulation of different inflammatory cytokines, including E-selectin, inter-cellular adhesion molecule 1 (ICAM-1), IL-6 and TNF-α (33). In addition, DFMG interacts with the inhibition of different signaling pathway activations, including mitochondrial apoptosis and TLR4 signaling (33-35).

The activation of MPs has been shown to be the critical step in the development of chronic inflammation. Therefore, the mechanism involving the activation of MPs is important for the development of preventive and therapeutic strategies for AS. The aim of the present study was to: i) identify the function of injured ECs with regard to the activation of MPs in a non-contact co-culture model and ii) to investigate the effect and molecular mechanism of DFMG treatment on the activation of MPs induced by co-culture with injured ECs.

Materials and methods

Reagents and antibodies

Phorbol 12-myristate 13-acetate (PMA), lysophosphatidylcholine (LPC) and oil red O powder were purchased from Sigma-Aldrich; EMD Millipore (Billerica, MA, USA). DFMG (purity >99%) was synthesized as previously reported (31) and dissolved in dimethyl sulfoxide (DMSO; Ameresco, Inc., Framingham, MA, USA). This compound was subsequently sterilized by filtration. CLI-095 was obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The IL-1 receptor antagonist (IL-1Ra) was purchased from Bioss (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was procured from Beyotime Institute of Biotechnology (Shanghai, China). Hematoxylin staining reagents were obtained from Servicebio Technology Co., Ltd. (Wuhan, China). Anti-TLR4 and NF-κB p65 antibodies were provided by ProteinTech Group, Inc. (Chicago, IL, USA, cat. nos. 19811-1-AP and 10745-1-AP). Anti-MyD88 and GAPDH antibodies were provided by Abgent, Inc. (San Diego, CA, USA cat. nos. ABO10975 and AM1020b). Goat anti-rabbit IgG and goat anti-mouse IgG were provided by ComWin Biotech Co., Ltd. (Beijing, China, cat. nos. CW0156S and CW0102S).

Cell culture and treatment

Human umbilical vein ECs (HUVE-12) and human acute monocytic leukemia cells (THP-1) were purchased from the China Center for Type Culture Collection (Wuhan, China) and Institute of Biochemistry and Cell Biology (Shanghai, China), respectively. The two cell lines were incubated in a humidified incubator at 37°C with 5% CO2 and were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The THP-1 cells (1×106 cells/ml) were seeded in 6-well plates and activated with 200 nM of PMA for 24 h, which allowed them to transform into MPs. All cell culture reagents were purchased from Biological Industries (Beit Haemek, Israel).

Reactive oxygen species (ROS) assay

The HUVE-12 cells were treated at 37°C with various concentrations of LPC (10, 20, 30, 40 and 50 µM). Following incubation for 24 h, fresh medium with 10% FBS was used and the cells were cultured for another 24 h. Subsequently, ROS was determined using a ROS assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Fluorescence intensity was detected at excitation and emission wavelengths of 488 and 525 nm, respectively. Hematoxylin staining was used to quantify the number of cells under a light microscope (CX41RF, Olympus, Tokyo, Japan). The fluorescence intensity of each cell was obtained from the ratio of total fluorescence intensity to the total cell count.

Lactate dehydrogenase (LDH) assay

The HUVE-12 cells were incubated with various concentrations of LPC and subsequently treated as described in the aforementioned protocol. Subsequently, the supernatants were collected in order to determine the concentration of LDH using the LDH cytotoxicity assay kit (Jiancheng Bioengineering Institute, Nanjing, China). The optical density (OD) was measured at 450 nm with a microplate reader (Elx800; BioTek Instruments, Inc., Winooski, VT, USA). The concentration of LDH was calculated according to the manufacturer's protocol.

Establishment of the co-culture model

The HUVE-12 cells, which had been stimulated by LPC were co-cultured with MPs in a Transwell chamber consisting of a 10-µm thick porous membrane with 0.4-µm pores in the culture inserts (Corning Incorporated, Corning, NY, USA). This methodology was used to construct an indirect co-culture system. Briefly, the MPs were seeded in the wells of a 6-well (1×105 cells/well) and/or a 24-well (2.5×104 cells/well) plate (lower layer) at 37°C prior to co-culture with the HUVE-12 cells. The HUVE-12 cells were individually grown in the upper layer of the Transwell under the following treatment conditions: Pretreatment with LPC for 24 h, and/or pretreatment with DFMG for 1 h, followed by CLI-095 and IL-1Ra for 24 h, and subsequent treatment with LPC for another 24 h. The HUVE-12 cells in fresh medium containing the inserts were added to the upper chamber of the Transwell system, with the MPs located at the bottom.

Proliferation assay

The MTT assay was used to detect the viability of the cells. The MPs were co-cultured with the HUVE-12 cells, which had been pretreated for 24 h in 24-well plates, and 50 µl of MTT solution (MTT powder dissolved in PBS, 5 mg/ml) was added to each well. The plates were incubated at 37°C for 4 h. Following removal of the medium, 750 µl of DMSO was added to each well for 10 min in order to fully dissolve the formazan crystals. The OD was measured on a microplate reader (BioTek Instruments, Inc.) at 490 nm. The viability of the cells was assessed in terms of OD values.

Migration assay

A wound healing assay was used to analyze the migratory activity of the cells. Prior to co-culture, the MPs were seeded in 6-well plates and scratched with a pipette tip (200-µl), followed by rinsing with PBS and incubation in RPMI-1640 medium in the absence of FBS. Images were captured at the 0 h time point. The pretreated HUVE-12 cells containing inserts were added to the upper chamber of the Transwell system and incubated at 37°C. After 24 h, an additional image (24 h) was captured in the same position. The cell-free area was quantified using Adobe Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA, USA).

Oil red O staining

The oil red O working solution was prepared by diluting the stock solution (0.05 g of oil red O powder dissolved in 10 ml of isopropanol) with distilled water (3:2), following which the solution was filtered. The MPs were co-cultured with HUVE-12 cells for 24 h and fixed in 4% paraformaldehyde for 15 min in order to prepare the samples for oil red O staining. The samples were washed with distilled water twice and were incubated with filtered oil red O working solution at room temperature for 10 min. Subsequently, the samples were washed twice with distilled water, and the cells were observed for image capture under an optical microscope (CX41RF, Olympus, Tokyo, Japan). The cells containing oil red O-positive fat droplets were considered as foam cells.

Assessment of total cholesterol (TC)

The quantification of TC was performed according to the protocols provided by the manufacturer (Solarbio, Beijing, China). The MPs were co-cultured with HUVE-12 cells for 24 h, and the cells were collected and dissolved in isopropanol (1 ml/4×104 cells), followed by ultrasound treatment for 1 min. The samples were centrifuged at 4°C at 8,000 × g for 10 min. The OD of the supernatants was detected at 500 nm using a spectrophotometer. The concentration of TC was calculated according to the manufacturer's protocol.

Western blot analysis

The pretreated HUVE-12 cells, which were co-cultured with MPs for 24 h, were digested and lysed in radioimmunoprecipitation assay buffer containing 1% phenylmethyl sulfonyl fluoride. The samples were centrifuged at 12,000 × g at 4°C for 10 min. Protein quantitation was performed using the BCA protein assay kit (Solabio). A total of 25 µg of each protein lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Solarbio), followed by transfer onto polyvinylidene difluoride membranes (EMD Millipore), which were blocked with 5% non-fat milk in PBST (PBS containing 0.05% Tween-20) at room temperature for 1 h. The membranes were incubated with antibodies against TLR4 (1:1,000), MyD88 (1:10,000), NF-κB p65 (1:2,500) and GAPDH (1:1,000) on a shaking platform at 4°C overnight. Subsequently, the membranes were washed with PBST and incubated with goat anti-rabbit antibody (1:10,000) and goat anti-mouse antibody (1:10,000) for 1 h at room temperature, followed by washing in PBST three times. Finally, the protein bands were visualized using enhanced chemiluminescence. The results were analyzed using densitometry with GelPro 3.2 software (Media Cybernetics, Inc., Rockville, MD, USA).

Enzyme-linked immunosorbent assay (ELISA)

Following co-culture for 24 h, the concentration of IL-1β in the supernatants was measured using a human IL-1β ELISA kit (MultiSciences Biotech Co., Ltd., Hangzhou, China) according to the manufacturer's protocol. The standards and/or samples were incubated in the wells at 37°C for 1.5 h. Subsequently, the wells were washed and primary antibody was added, covered with an adhesive strip and incubated at 37°C for 1 h. Following washing of the unbound biotinylated antibody, streptavidin-HRP was added for incubation at 37°C for 30 min. A cycle of five washing steps (with PBST for 30 sec each time) was performed and substrate solution was added to the samples. Following incubation at dark for 30 min, the stop solution was added. The OD was detected at 450 nm on a microplate reader (BioTek Instruments, Inc.), and the concentration of IL-1β was calculated according to the manufacturer's protocol.

Statistical analysis

All experiments were performed independently at least three times, and the values are expressed as the mean ± standard deviation. SPSS 20.0 software (IBM SPSS, Armonk, NY, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) were used for statistical analysis. Student's paired t-test was used for the comparison of two samples, and one-way analysis of variance was used for multiple group comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

Establishment of the injured HUVE-12 cell-MP co-culture model

Initially, HUVE-12 cells were incubated with different concentrations (10, 20, 30, 40 and 50 µM) of LPC for 24 h, and the production of ROS and LDH were detected. The results indicated that LPC damaged the HUVE-12 cells in a concentration-dependent manner. LPC significantly promoted the generation of ROS at a concentration range of 20-50 µM (Fig. 1A and B). In terms of LDH activity, the effective concentration range of LPC was estimated to be between 30 and 50 µM (Fig. 1C). Therefore, 30 µM of LPC was selected in order to injure the HUVE-12 cells.

Subsequently, the capacity of MPs to proliferate, migrate and develop into foam cells following co-culture with injured HUVE-12 cells was detected. The HUVE-12 cells were incubated with LPC (30 µM) for 24 h and co-cultured with MPs in a Transwell system for another 24 h. A significant increase in the proliferation and migration of MPs was observed, which was caused by co-culture with injured HUVE-12 cells (Fig. 1D–F). In addition, co-culture with injured HUVE-12 cells resulted in abundant cytoplasmic lipid droplet accumulation in the MPs, as demonstrated by oil red O staining (Fig. 1G) and TC (Fig. 1H). Consequently, 30 µM of LPC was selected as the optimum concentration in order to construct the LPC-injured HUVE-12 cell-MP co-culture model.

LPC promotes the activation of TLR4/MyD88/NFκ-B signaling pathway in HUVE-12 cells co-cultured with MPs

It has been shown that the TLR4/MyD88/NF-κB transduction pathway is an important event in inflammation and in the subsequent secretion of IL-1β (36). Consequently, the change in the expression levels of TLR4 and its downstream signaling molecules was monitored in LPC-injured HUVE-12 cells. The supernatants collected from the MPs following co-culture with HUVE-12 cells for 24 h were obtained. It was noted that the expression levels of TLR4, MyD88 and NF-κB p65 in HUVE-12 cells were significantly upregulated (Fig. 2A and B) and the secretion of IL-1β in the co-culture supernatant was effectively increased (Fig. 2C). Taken together, these results suggested that LPC promoted the activation of the TLR4/MyD88/NF-κB signaling pathway in HUVE-12 cells. This pathway is essential in the cross-talk between LPC-injured-HUVE-12 cells and MPs.

DFMG inhibits the activation of MPs induced by co-culture with injured HUVE-12 cells

To determine whether DFMG was able to attenuate the activation of MPs induced by co-culture with LPC-oxidative damaged HUVE-12 cells, the HUVE-12 cells were treated with various concentrations (0.3, 1.0 and 3.0 µM) of DFMG for 1 h prior to LPC (30 µM) treatment. DFMG effectively inhibited the proliferation of MPs at a concentration range of 0.3–3.0 µM (Fig. 3A), and treatment at concentrations of 1.0–3.0 µM DFMG effectively suppressed the migration (Fig. 3B and C) and the formation of foam cells (Fig. 3D and E) in a dose-dependent manner.

DFMG inhibits the TLR4/MyD88/NF-κB signaling pathway in HUVE-12 cells

As mentioned above, activation of the TLR4/MyD88/NF-κB signaling pathway in HUVE-12 cells was associated with the cross-talk between HUVE-12 cells and MPs. The molecular mechanism underlying the effect of DFMG, in relation to the activation of MPs induced by co-culture with injured HUVE-12 cells, was examined by investigating the expression levels of TLR4, MyD88 and NF-κB p65 in the HUVE-12 cells co-cultured with MPs. In addition, the concentration of IL-1β in the co-culture supernatant was monitored using ELISA. The data indicated that DFMG (1.0 and/or 3.0 µM) effectively reduced the expression levels of TLR4, MyD88 and NF-κB p65 (Fig. 4A–D) and the secretion of IL-1β (Fig. 4E). These observations suggested that DFMG attenuated the activation of MPs induced by co-culture with injured HUVE-12 cells, at least partly, via inhibition of the TLR4/MyD88/NF-κB signaling pathway in the LPC-injured HUVE-12 cells. Accordingly, 3.0 µM was selected as the optimum concentration of DFMG to be used for the following experiments.

TLR4 signaling pathway is involved in the activation of MPs induced by co-culture with injured HUVE-12 cells

To further confirm the effect of the TLR4 signaling pathway on the activation of MPs induced by co-culture with injured HUVE-12 cells, the HUVE-12 cells were pre-incubated with DFMG (3.0 µM) for 1 h, and CLI-095 (5 µg/ml) or IL-1Ra (10 µg/ml) were added for 24 h. Subsequently, LPC (30 µM) was added and incubated for another 24 h, followed by co-culture with MPs for 24 h. The results indicated that DFMG, CLI-095 and IL-1Ra significantly decreased the proliferation and migration of MPs (Fig. 5A–C) and the formation of foam cells (Fig. 5D and E), which were induced by co-culture with injured HUVE-12 cells. In addition, DFMG with CLI-095 or IL-1Ra exhibited synergistic effects in promoting the stability of MPs. These data indicated that the inhibition of TLR4 or its downstream targets in HUVE-12 cells effectively attenuated the activation of MPs induced by co-culture with LPC-injured HUVE-12 cells. The results also confirmed that the TLR4 signaling pathway in LPC-injured HUVE-12 cells was associated with the activation of MPs induced by co-culture.

DFMG attenuates activation of the TLR4/MyD88/NF-κB signaling pathway in LPC-injured HUVE-12 cells co-cultured with MPs

DFMG (3.0 µM), CLI-095 (5 µg/ml), IL-1Ra (10 µg/ml) and LPC (30 µM) were incubated with HUVE-12 cells, as described above, in order to investigate the molecular mechanism underlying the effect of DFMG on the inhibition of the activation of MPs induced by co-culture with LPC-injured HUVE-12 cells. The results indicated that DFMG, CLI-095 and IL-1Ra significantly downregulated the expression levels of TLR4, MyD88 and NF-κB p65 (Fig. 6A–D) in the HUVE-12 cells and decreased the secretion of IL-1β (Fig. 6E) in the co-culture supernatant. Furthermore, DFMG and CLI-095 or IL-1Ra exhibited synergistic effects in the inhibition of the TLR4 signaling pathway. Consequently, DFMG suppressed activation of the TLR4/MyD88/NF-κB signaling pathway in LPC-injured HUVE-12 cells following co-culture with MPs.

Discussion

MP dysregulation is considered a risk factor for several inflammatory complications, including AS and cancer (37). The process of atherogenesis involves several types of cells, notably ECs, smooth muscle cells, monocytes and macrophages. AS initiates from the endothelium of the arterial wall (7). ECD is a vital contributor to the pathobiology of atherosclerotic cardiovascular disease (8). Previous studies have shown that ECD induces the functional changes of MPs through the mutual cross-talk between cells in the vascular microenvironment (12,38). The in vitro Transwell co-culture model can be used to examine the effects of ECD on MPs. Oxidative stress is key in the progression of AS (39). LPC is a major component of ox-LDL, which can cause AS (40). In the present study, LPC stimulated the generation of ROS and the release of LDH (cell oxidative damage index) in ECs, but promoted the activation of MPs induced by co-culture with EC. The results also indicated that LPC increased the expression levels of TLR4, MyD88 and NF-κB p65 in the ECs co-cultured with THP-1-derived MPs. LPC also increased the secretion of IL-1β in the co-culture supernatant. These findings demonstrated that LPC exhibited oxidative effects on ECs. In addition to these observations, ECD promoted the activation of MPs, which may be associated with activation of the TLR4/MyD88/NF-κB signaling pathway in ECs.

AS, the main cause of cardiovascular disease, is characterized by the accumulation of inflammatory cells in the artery wall (41). TLRs are crucial in the initiation of an innate immune response through activating the inflammatory cells (42). To confirm that the TLR4 signaling pathway was involved in the activation of macrophages induced by co-culture with LPC-injured HUVE-12 cells, specific inhibitors of the proteins and the mediators acting on this pathway were used. CLI-095, also known as TAK-242, is a novel cyclohexene derivative, which selectively suppresses TLR4 signaling, potently suppressing ligand-dependent and ligand-independent signaling of TLR4 (43,44). IL-1 belongs to the innate immune system and is important in the initiation of the immunoinflam-matory cascade reaction (45). IL-1Ra is a specific and pure antagonist of the IL-1 receptor family (46), which has the capacity to inhibit inflammasome activation and control the balance between the pro-inflammatory and anti-inflammatory response (47). As expected, treatment of LPC-injured EC with CLI-095 and IL-1Ra significantly impaired the proliferation and migration of MPs, and the formation of foam cells induced by co-culture with injured ECs. Taken together, the data demonstrated that activation of the TLR4 signaling pathway in ECs led to the proliferation, migration and lipid accumulation of MPs in the co-culture model.

GEN, a primary soy isoflavone, has received considerable attention as a protein kinase inhibitor (48). In addition, studies have demonstrated that GEN possesses atheroprotective effects via regulation of the TLR4/NF-κB signaling pathway (49,50). GEN exhibits low bioavailability and intestinal absorption in vivo, and its clinical application is limited (51). DFMG is synthesized using GEN as a precursor. DFMG exhibits improved bioavailability and absorption. Our previous studies indicated that DFMG was more efficient than GEN in reducing the risk of cardiovascular disease by the inhibition of oxidative damage, mitochondrial apoptosis and/or inhibition of the TLR4 signaling pathway (33-35). In the present study, DFMG was shown to effectively attenuate the activation of THP-1-derived MPs induced by co-culture with LPC-injured ECs. Furthermore, inhibition of the TLR4 signaling pathway using specific inhibitors (CLI-095 and IL-1Ra) increased the anti-inflammatory potential of DFMG. These observations indicated that DFMG may exert its anti-inflammatory activity through inhibition of the TLR4 signaling pathway.

In view of the aforementioned results, the expression levels of TLR4 were further assessed. In the present study, DFMG significantly downregulated the protein expression levels of the aforementioned indicators (TLR4, MyD88 and NF-κB p65) in ECs, and the levels of IL-1β in the co-culture supernatant. CLI-095 and IL-1Ra markedly reduced the activation of TLR4, which was markedly enhanced by DFMG treatment. The endogenous IL-1Ra has been shown to regulate the extent of TLR9-induced liver damage (52), whereas IL-1Ra deficiency can promote TLR4-dependent arthritis (53). The present study hypothesized that DFMG may act by upregulating the expression of endogenous IL-1ra in order to inhibit the activity of TLR4.

Considering the above findings, it was suggested that the atheroprotective effects of DFMG against the activation of MPs induced by co-culture with LPC-injured ECs was mediated via regulation of the TLR4/MyD88/NF-κB signaling pathway in ECs. However, the effects of DFMG on MPs were not examined in vivo. Consequently, the detailed effects of DFMG on the TLR4 signaling network in AS require further investigation using animal knockout models. TLR4 signaling is also crucial in other diseases, including systemic lupus erythematosus (29), non-small cell lung cancer (54), idiopathic pulmonary fibrosis (55), myocardial inflammation (56) and type 2 diabetes (57). According to the present study, it was hypothesized that DFMG may prevent and/or treat these diseases via inhibiting the TLR4 signaling pathway.

Glossary

Abbreviations

Abbreviations:

DFMG

7-difluoromethoxy-5,4′-dimethoxy-genistein

ECs

endothelial cells

MPs

macrophages

HUVE-12

human umbilical vein endothelial cell

LPC

lysophosphatidylcholine

ROS

reactive oxygen species

LDH

lactate dehydrogenase

AS

atherosclerosis

ECD

endothelial cell dysfunction

TNF-α

tumor necrosis factor-α

MCP-1

monocyte chemoattractant protein-1

TLR4

Toll-like receptor 4

MyD88

myeloid differentiation factor 88

NF-κB

nuclear factor-κB

ox-LDL

oxidized low-density lipoprotein

LPS

lipopolysaccharide

GEN

genistein

PMA

phorbol 12-myristate 13-acetate

DMSO

dimethyl sulfoxide

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

THP-1

human acute monocytic leukemia cells

RPMI

Roswell Park Memorial Institute

FBS

fetal bovine serum

TC

total cholesterol ELISA, enzyme-linked immunosorbent assay

Acknowledgments

Not applicable.

Notes

[1] Funding

Financial support was provided by the Natural Science Foundation of China (grant no. 81370382).

[2] Availability of data and materials

All data generated or analysed during this study were included in this published article.

[3] Authors' contributions

XF designed the study and prepare the manuscript. LC and SY performed the experiments. YZ analysed the data. JC revised the manuscript. All authors read and approved the final manuscript.

[4] Ethics approval and consent to participate

Not applicable.

[5] Consent for publication

Not applicable.

[6] Competing interests

The authors declare that they have no competing interests.

References

1 

Cochain C and Zernecke A: Macrophages in vascular inflammation and atherosclerosis. Pflugers Arch. 469:485–499. 2017. View Article : Google Scholar : PubMed/NCBI

2 

Gerrity RG, Naito HK, Richardson M and Schwartz CJ: Dietary induced atherogenesis in swine. Morphology of the intima in prelesion stages. Am J Pathol. 95:775–792. 1979.PubMed/NCBI

3 

Galkina E and Ley K: Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol. 27:165–197. 2009. View Article : Google Scholar

4 

Weber C and Noels H: Atherosclerosis: Current pathogenesis and therapeutic options. Nat Med. 17:1410–1422. 2011. View Article : Google Scholar : PubMed/NCBI

5 

Tabas I: Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: The importance of lesion stage and phagocytic efficiency. Arterioscler Thromb Vasc Biol. 25:2255–2264. 2005. View Article : Google Scholar : PubMed/NCBI

6 

Ilhan F and Kalkanli ST: Atherosclerosis and the role of immune cells. World J Clin Cases. 3:345–352. 2015. View Article : Google Scholar : PubMed/NCBI

7 

Husain K, Hernandez W, Ansari RA and Ferder L: Inflammation, oxidative stress and renin angiotensin system in atherosclerosis. World J Biol Chem. 6:209–217. 2015. View Article : Google Scholar : PubMed/NCBI

8 

Gimbrone MA Jr and García-Cardeña G: Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res. 118:620–636. 2016. View Article : Google Scholar : PubMed/NCBI

9 

Tabas I: Heart disease: Death-defying plaque cells. Nature. 536:32–33. 2016. View Article : Google Scholar : PubMed/NCBI

10 

Linton MF, Babaev VR, Huang J, Linton EF, Tao H and Yancey PG: Macrophage apoptosis and efferocytosis in the pathogenesis of atherosclerosis. Circ J. 80:2259–2268. 2016. View Article : Google Scholar : PubMed/NCBI

11 

Tabas I and Bornfeldt KE: Macrophage phenotype and function in different stages of atherosclerosis. Circ Res. 118:653–667. 2016. View Article : Google Scholar : PubMed/NCBI

12 

Kalucka J, Bierhansl L, Wielockx B, Carmeliet P and Eelen G: Interaction of endothelial cells with macrophages-linking molecular and metabolic signaling. Pflugers Arch. 469:473–483. 2017. View Article : Google Scholar : PubMed/NCBI

13 

Pryshchep O, Ma-Krupa W, Younge BR, Goronzy JJ and Weyand CM: Vessel-specific Toll-like receptor profiles in human medium and large arteries. Circulation. 118:1276–1284. 2008. View Article : Google Scholar : PubMed/NCBI

14 

Takeuchi O and Akira S: Pattern recognition receptors and inflammation. Cell. 140:805–820. 2010. View Article : Google Scholar : PubMed/NCBI

15 

Ghosh G, Wang VY, Huang DB and Fusco A: NF-κB regulation: Lessons from structures. Immunol Rev. 246:36–58. 2012. View Article : Google Scholar : PubMed/NCBI

16 

Feng Y, Cai ZR, Tang Y, Hu G, Lu J, He D and Wang S: TLR4/NF-κB signaling pathway-mediated and oxLDL-induced up-regulation of LOX-1, MCP-1, and VCAM-1 expressions in human umbilical vein endothelial cells. Genet Mol Res. 13:680–695. 2014. View Article : Google Scholar : PubMed/NCBI

17 

Zheng Z, Yuan R, Song M, Huo Y, Liu W, Cai X, Zou H, Chen C and Ye J: The toll-like receptor 4-mediated signaling pathway is activated following optic nerve injury in mice. Brain Res. 1489:90–97. 2012. View Article : Google Scholar : PubMed/NCBI

18 

Wang Z and Wu L: Melatonin alleviates secondary brain damage and neurobehavioral dysfunction after experimental subarachnoid hemorrhage: Possible involvement of TLR4-mediated inflammatory pathway. J Pineal Res. 55:399–408. 2013.PubMed/NCBI

19 

Palová-Jelínková L, Dáňová K, Drašarová H, Dvořák M, Funda DP, Fundová P, Kotrbová-Kozak A, Černá M, Kamanová J, Martin SF, et al: Pepsin digest of wheat gliadin fraction increases production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB signaling pathway and an NLRP3 inflammasome activation. PLoS One. 8:e624262013. View Article : Google Scholar

20 

Nicoletti F, Patti F, DiMarco R, Zaccone P, Nicoletti A, Meroni P and Reggio A: Circulating serum levels of IL-1ra in patients with relapsing remitting multiple sclerosis are normal during remission phases but significantly increased either during exacerbations or in response to IFN-beta treatment. Cytokine. 8:395–400. 1996. View Article : Google Scholar : PubMed/NCBI

21 

Netea MG, Simon A, van de Veerdonk F, Kullberg BJ, Van der Meer JW and Joosten LA: IL-1beta processing in host defense: Beyond the inflammasomes. PLoS Pathog. 6:e10006612010. View Article : Google Scholar : PubMed/NCBI

22 

Piccini A, Carta S, Tassi S, Lasiglié D, Fossati G and Rubartelli A: ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1beta and IL-18 secretion in an autocrine way. Proc Natl Acad Sci USA. 105:8067–8072. 2008. View Article : Google Scholar : PubMed/NCBI

23 

Gu H, Jiao Y, Yu X, Li X, Wang W, Ding L and Liu L: Resveratrol inhibits the IL-1β-induced expression of MMP-13 and IL-6 in human articular chondrocytes via TLR4/MyD88-dependent and -independent signaling cascades. Int J Mol Med. 2017.Epub ahead of print. View Article : Google Scholar

24 

Liu L, Gu H, Liu H, Jiao Y, Li K, Zhao Y, An L and Yang J: Protective effect of resveratrol against IL-1β-induced inflammatory response on human osteoarthritic chondrocytes partly via the TLR4/MyD88/NF-κB signaling pathway: An 'in vitro study'. Int J Mol Sci. 15:6925–6940. 2014. View Article : Google Scholar : PubMed/NCBI

25 

Feng Y, Cui Y, Gao JL, Li MH, Li R, Jiang XH, Tian YX, Wang KJ, Cui CM and Cui JZ: Resveratrol attenuates neuronal autophagy and inflammatory injury by inhibiting the TLR4/NF-κB signaling pathway in experimental traumatic brain injury. Int J Mol Med. 37:921–390. 2016. View Article : Google Scholar : PubMed/NCBI

26 

Lee HH, Han MH, Hwang HJ, Kim GY, Moon SK, Hyun JW, Kim WJ and Choi YH: Diallyl trisulfde exerts anti-inflammatory effects in lipopolysaccharide-stimulated RAW 264.7 macrophages by suppressing the Toll-like receptor 4/nuclear factor-κB pathway. Int J Mol Med. 35:487–495. 2015. View Article : Google Scholar

27 

Zhu S, Tang S and Su F: Dioscin inhibits ischemic stroke-induced inflammation through inhibition of the TLR4/MyD88/NF-κB signaling pathway in a rat model. Mol Med Rep. 17:660–666. 2018.

28 

Stojanovic I, Cuzzocrea S, Mangano K, Mazzon E, Miljkovic D, Wang M, Donia M, Al Abed Y, Kim J, Nicoletti F, et al: In vitro, ex vivo and in vivo immunopharmacological activities of the isoxazoline compound VGX-1027: Modulation of cytokine synthesis and prevention of both organ-specific and systemic autoimmune diseases in murine models. Clin Immunol. 123:311–323. 2007. View Article : Google Scholar : PubMed/NCBI

29 

Fagone P, Muthumani K, Mangano K, Magro G, Meroni PL, Kim JJ, Sardesai NY, Weiner DB and Nicoletti F: VGX-1027 modulates genes involved in the lipopolysaccharide-induced toll-like receptor 4 activation and in a murine model of systemic lupus erythematosus. Immunology. 142:594–602. 2014. View Article : Google Scholar : PubMed/NCBI

30 

Pangeni R, Sahni JK, Ali J, Sharma S and Baboota S: Resveratrol: Review on therapeutic potential and recent advances in drug delivery. Expert Opin Drug Deliv. 11:1285–1298. 2014. View Article : Google Scholar : PubMed/NCBI

31 

Fu XH, Wang L, Zhao H, Xiang HL and Cao JG: Synthesis of genistein derivatives and determination of their protective effects against vascular endothelial cell damages caused by hydrogen peroxide. Bioorg Med Chem Lett. 18:513–517. 2008. View Article : Google Scholar

32 

Zhao H, Li C, Cao JG, Xiang HL, Yang HZ, You JL, Li CL and Fu XH: 7-Difluoromethyl-5,4′-dimethoxygenistein, a novel genistein derivative, has therapeutic effects on atherosclerosis in a rabbit model. J Cardiovasc Pharmacol. 54:412–420. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Wang L, Zheng X, Xiang HL, Fu XH and Cao JG: 7-Difluoromethyl-5,4′-dimethoxygenistein inhibits oxidative stress induced adhesion between endothelial cells and monocytes via NF-kappaB. Eur J Pharmacol. 605:31–35. 2009. View Article : Google Scholar : PubMed/NCBI

34 

Liu S, Li L, Zhang J, Huang H, Yang S, Ren C, Fu X and Zhang Y: 7-Difluoromethyl-5, 4′-dimethoxygenistein reverses LPC-induced apoptosis of HUVE-12 cells through regulating mitochondrial apoptosis pathway. Curr Signal Transd T. 9:50–58. 2014. View Article : Google Scholar

35 

Liu F, Cao JG, Li C, Tan JS and Fu XH: Protective effects of 7-difluoromethyl-5,4′-dimethoxygenistein against human aorta endothelial injury caused by lysophosphatidyl choline. Mol Cell Biochem. 363:147–155. 2012. View Article : Google Scholar

36 

Kong F, Ye B, Cao J, Cai X, Lin L, Huang S, Huang W and Huang Z: Curcumin represses NLRP3 inflammasome activation via TLR4/MyD88/NF-κB and P2X7R signaling in PMA-induced macrophages. Front Pharmacol. 7:3692016. View Article : Google Scholar

37 

Eaton KV, Yang HL, Giachelli CM and Scatena M: Engineering macrophages to control the inflammatory response and angiogenesis. Exp Cell Res. 339:300–309. 2015. View Article : Google Scholar : PubMed/NCBI

38 

Im GI: Coculture in musculoskeletal tissue regeneration. Tissue Eng Part B Rev. 20:545–554. 2014. View Article : Google Scholar : PubMed/NCBI

39 

Wang L, Huang Z, Huang W, Chen X, Shan P, Zhong P, Khan Z, Wang J, Fang Q, Liang G and Wang Y: Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress. Sci Rep. 8:459172017. View Article : Google Scholar : PubMed/NCBI

40 

Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK, Hindy G, Hólm H, Ding EL, Johnson T, et al: Plasma HDL cholesterol and risk of myocardial infarction: A mendelian randomisation study. Lancet. 380:572–580. 2012. View Article : Google Scholar : PubMed/NCBI

41 

Canfrán-Duque A, Rotllan N, Zhang X, Fernández-Fuertes M, Ramírez-Hidalgo C, Araldi E, Daimiel L, Busto R, Fernández-Hernando C and Suárez Y: Macrophage deficiency of miR-21 promotes apoptosis, plaque necrosis, and vascular inflammation during atherogenesis. EMBO Mol Med. 9:1244–1262. 2017. View Article : Google Scholar : PubMed/NCBI

42 

Szatmary Z: Molecular biology of toll-like receptors. Gen Physiol Biophys. 31:357–366. 2012. View Article : Google Scholar : PubMed/NCBI

43 

Ii M, Matsunaga N, Hazeki K, Nakamura K, Takashima K, Seya T, Hazeki O, Kitazaki T and Iizawa Y: A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl] cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling. Mol Pharmacol. 69:1288–1295. 2006. View Article : Google Scholar

44 

Kawamoto T, Ii M, Kitazaki T, Iizawa Y and Kimura H: TAK-242 selectively suppresses Toll-like receptor 4-signaling mediated by the intracellular domain. Eur J Pharmacol. 584:40–48. 2008. View Article : Google Scholar : PubMed/NCBI

45 

D ujmovic I, Mangano K, Pekmezovic T, Quattrocchi C, Mesaros S, Stojsavljevic N, Nicoletti F and Drulovic J: The analysis of IL-1 beta and its naturally occurring inhibitors in multiple sclerosis: The elevation of IL-1 receptor antagonist and IL-1 receptor type II after steroid therapy. J Neuroimmunol. 207:101–106. 2009. View Article : Google Scholar

46 

van Oosten BW, Lai M, Hodgkinson S, Barkhof F, Miller DH, Moseley IF, Thompson AJ, Rudge P, McDougall A, McLeod JG, et al: Treatment of multiple sclerosis with the monoclonal anti-CD4 antibody cM-T412: Results of a randomized, double-blind, placebo-controlled, MR-monitored phase II trial. Neurology. 49:351–357. 1997. View Article : Google Scholar : PubMed/NCBI

47 

Yoon GS, Sud S, Keswani RK, Baik J, Standiford TJ, Stringer KA and Rosania GR: Phagocytosed Clofazimine Biocrystals can modulate innate immune signaling by inhibiting TNFα and boosting IL-1RA secretion. Mol Pharm. 12:2517–2527. 2015. View Article : Google Scholar : PubMed/NCBI

48 

Jeong JW, Lee HH, Han MH, Kim GY, Kim WJ and Choi YH: Anti-inflammatory effects of genistein via suppression of the toll-like receptor 4-mediated signaling pathway in lipopoly-saccharide-stimulated BV2 microglia. Chem Biol Interact. 212:30–39. 2014. View Article : Google Scholar : PubMed/NCBI

Zhou X, Yuan L, Zhao X, Hou C, Ma W, Yu H and Xiao R: Genistein antagonizes inflammatory damage induced by β-amyloid peptide in microglia through TLR4 and NF-κB. Nutrition. 30:90–95. 2014. View Article : Google Scholar

49 

Ma W, Ding B, Yu H, Yuan L, Xi Y and Xiao R: Genistein alleviates β-amyloid-induced inflammatory damage through regulating Toll-like receptor 4/nuclear factor κB. J Med Food. 18:273–279. 2015. View Article : Google Scholar :

50 

Cave NJ, Backus RC, Marks SL and Klasing KC: The bioavailability and disposition kinetics of genistein in cats. J Vet Pharmacol Ther. 30:327–335. 2007. View Article : Google Scholar : PubMed/NCBI

51 

Petrasek J, Dolganiuc A, Csak T, Kurt-Jones EA and Szabo G: Type I interferons protect from Toll-like receptor 9-associated liver injury and regulate IL-1 receptor antagonist in mice. Gastroenterology. 140:697–708.e4. 2011. View Article : Google Scholar :

52 

Rogier R, Ederveen THA, Boekhorst J, Wopereis H, Scher JU, Manasson J, Frambach SJCM, Knol J, Garssen J, van der Kraan PM, et al: Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17 and TLR4-dependent arthritis. Microbiome. 5:632017. View Article : Google Scholar

53 

Li D, Jin Y, Sun Y, Lei J and Liu C: Knockdown of toll-like receptor 4 inhibits human NSCLC cancer cell growth and inflammatory cytokine secretion in vitro and in vivo. Int J Oncol. 45:813–821. 2014. View Article : Google Scholar : PubMed/NCBI

54 

Samara KD, Antoniou KM, Karagiannis K, Margaritopoulos G, Lasithiotaki I, Koutala E and Siafakas NM: Expression profiles of Toll-like receptors in non-small cell lung cancer and idiopathic pulmonary fbrosis. Int J Oncol. 40:1397–1404. 2012.PubMed/NCBI

55 

Yang Y, Lv J, Jiang S, Ma Z, Wang D, Hu W, Deng C, Fan C, Di S, Sun Y and Yi W: The emerging role of Toll-like receptor 4 in myocardial inflammation. Cell Death Dis. 7:e22342016. View Article : Google Scholar : PubMed/NCBI

56 

Cha JJ, Hyun YY, Lee MH, Kim JE, Nam DH, Song HK, Kang YS, Lee JE, Kim HW, Han JY and Cha DR: Renal protective effects of toll-like receptor 4 signaling blockade in type 2 diabetic mice. Endocrinology. 154:2144–2155. 2013. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

May-2018
Volume 41 Issue 5

Print ISSN: 1107-3756
Online ISSN:1791-244X

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Cong L, Yang S, Zhang Y, Cao J and Fu X: DFMG attenuates the activation of macrophages induced by co‑culture with LPC‑injured HUVE‑12 cells via the TLR4/MyD88/NF‑κB signaling pathway. Int J Mol Med 41: 2619-2628, 2018
APA
Cong, L., Yang, S., Zhang, Y., Cao, J., & Fu, X. (2018). DFMG attenuates the activation of macrophages induced by co‑culture with LPC‑injured HUVE‑12 cells via the TLR4/MyD88/NF‑κB signaling pathway. International Journal of Molecular Medicine, 41, 2619-2628. https://doi.org/10.3892/ijmm.2018.3511
MLA
Cong, L., Yang, S., Zhang, Y., Cao, J., Fu, X."DFMG attenuates the activation of macrophages induced by co‑culture with LPC‑injured HUVE‑12 cells via the TLR4/MyD88/NF‑κB signaling pathway". International Journal of Molecular Medicine 41.5 (2018): 2619-2628.
Chicago
Cong, L., Yang, S., Zhang, Y., Cao, J., Fu, X."DFMG attenuates the activation of macrophages induced by co‑culture with LPC‑injured HUVE‑12 cells via the TLR4/MyD88/NF‑κB signaling pathway". International Journal of Molecular Medicine 41, no. 5 (2018): 2619-2628. https://doi.org/10.3892/ijmm.2018.3511