Myristica fragrans promotes ABCA1 expression and cholesterol efflux in THP-1-derived macrophages

Abstract

Myristica fragrans is a traditional herbal medicine and has been shown to alleviate the development of atherosclerosis. However, the anti-atherogenic mechanisms of M. fragrans are still to be addressed. In this study, we explored the effect of M. fragrans on lipid metabolism and inflammation and its mechanisms in THP-1-derived macrophages. The quantitative polymerase chain reaction and western blot analysis results showed that M. fragrans promotes cholesterol efflux from THP-1-derived macrophages and reduces intracellular total cholesterol, cholesterol ester, and free cholesterol contents in a dose- and a time-dependent manner. Further study found that liver X receptor alpha (LXRα) antagonist GGPP significantly blocked the upregulation of ABCA1 expression with M. fragrans treatment. In addition, chromatin immunoprecipitation assay confirmed that GATA binding protein 3 (GATA3) can bind to the LXRα promoter, and inhibition of GATA3 led to the downregulation of LXRα and ATP-binding cassette subfamily A member 1 expression. Furthermore, M. fragrans reduced lipid accumulation, followed by decreasing tumor necrosis factor-α, interleukin (IL)-6, and IL-1β and increasing IL-10 produced by THP-1-derived macrophages. Therefore, M. fragrans is identified as a valuable therapeutic medicine for atherosclerotic cardiovascular disease.

Myristica fragrans, GATA3, LXRα, ABCA1, cholesterol efflux

Introduction

Atherosclerosis is one of the main causes of cardiovascular disease, the number one inducement of death worldwide. It is well known that dyslipidemia is the major etiology of atherosclerosis and vascular inflammation [1,2]. ATP-binding cassette subfamily A member 1 (ABCA1), a cholesterol transporter, is localized in cytoplasmic membrane to mediate intracellular cholesterol efflux to apolipoprotein A-I (apoA-I) [3,4]. Liver X receptor (LXR) consists of α and β subtypes and belongs to nuclear receptor family. Both LXRα and LXRβ are widely recognized as the enhancers for elevating the expression of ABCA1, therefore accelerating cholesterol efflux from the peripheral cells [5,6]. Our previous studies focused on macrophage cholesterol efflux and found that the upregulation of ABCA1 expression is beneficial for accelerating cholesterol efflux [7–12]. Therefore, efficient removal of excess lipids is required for the prevention of atherosclerotic lesion. This may be a promising strategy for protecting against atherosclerosis.

Medicinal plants have played and continue to play an important role in the cardiovascular disease protection [13–15]. Myristica fragrans has been found to improve insulin resistance and hyperlipidemia [16–18]. Further studies also demonstrated an evident anti-atherogenic effect of M. fragrans on rabbits fed with Western diets [19]. However, whether the regulatory effect of M. fragrans on lipid metabolism is relevant to cholesterol efflux has not been well determined.

GATA binding protein 3 (GATA3) commonly acts as a transcription factor, and it is also involved in cardiovascular system development and coronary heart disease progression [20–22]. Although the role of GATA3 in atherogenesis is not well addressed, the regulatory role of GATA3 in macrophage polarization has been revealed [23,24]. These data indicated the possible effect of GATA3 on macrophage lipid metabolism. We speculated that M. fragrans may affect atherosclerosis by targeting the regulation of GATA3/LXRα pathway.

In the present study, we aimed to explore whether M. fragrans acts as a protector for atherosclerosis through the promotion of macrophage cholesterol efflux by upregulating ABCA1 expression via activating the GATA3/LXRα pathway.

Materials and Methods

Cell culture

Human THP-1 monocytes were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (Solarbio, Beijing, China) supplemented with 0.1% nonessential amino acids, penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum (FBS). Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. The differentiation of THP-1 monocytes into macrophages was induced by an incubation with 160 nM phorbol-12-myristate acetate (PMA; Sigma Aldrich, Munich, Germany) for 24 h. THP-1-derived macrophages then underwent related treatment.

siRNA transfection

THP-1-derived macrophages were cultured in 12-well plates at 2 × 10⁶ cells/well. M. fragrans (Klamar, Shanghai, China) or 10 μM GGPP (Sigma Aldrich) were incubated with cells. GATA3 siRNA (Santa Cruz Biotechnology, Dallas, USA) were transfected into THP-1-derived macrophages with lentivirus (Genechem, Shanghai, China). After 6–12 h, the medium was switched to RPMI-1640 medium containing antibiotics and 10% FBS. Twenty-four hours later, the expressions of target proteins were detected by the quantitative real-time polymerase chain reaction (qRT-PCR) or western blot analysis. The sequences of siRNAs were as follows: GATA3 sense 5ʹ-AAUUUUGGAGUUCUAGUAGCCC-3ʹ and anti-sense 5ʹ-AUUCCCGGUUAUUUUCGGCGA-3ʹ; Scramble sense 5ʹ-AGAUAGUAUGCUCUUGAGUCCU-3ʹ and anti-sense 5ʹ-AUAUGAUCUUCGGUUCGUGCC-3ʹ.

Immunofluorescence assay

Cells were treated and fixed with paraformaldehyde, followed by incubation with 5% goat serum (Solarbio) for 30 min at room temperature after washing three times with 1× PBS. Cells were then incubated with anti-GATA3 mouse monoclonal antibody (ab18180, 1:200; Abcam, Shanghai, China) at 4°C overnight, followed by incubation with Cy3-labeled goat anti-rabbit IgG (H + L) (A0516, 1:500; Beyotime, Shanghai, China) for 2 h at room temperature in the dark. The nuclei were counterstained with DAPI (Thermo Scientific, Shanghai, China) for 10 min. Fluorescence microscopy of GATA3 in the sections was performed using an EVOS (Thermo Scientific). The mean fluorescence intensity (MFI) on the stained sections of aortic roots was measured using Image J software.

Lipid content assay by high-performance liquid chromatography (HPLC)

Lipid content in THP-1-derived macrophages was examined as previously described [10]. THP-1-derived macrophages were sonicated with the ultrasonic processor (Sonics, Newtown, USA) on ice to quantify the target proteins. Isopropanol (1 mg/ml cholesterol) was added to dissolve cholesterol and reserved at 20°C. Thereafter, 0.4 U cholesterol oxidase (Solarbio) in 100 μl 0.5% NaCl was added to each sample for the examination of free cholesterol, or 0.4 U cholesterol oxidase together with 0.4 U cholesterol esterase (Solarbio) were added to each sample for total cholesterol detection. Each sample in the centrifuge tube was incubated at 37°C for 30 min, and then the reaction was terminated by addition of 100 ml of methanol:ethanol (1:1). After cooling for 30 min, each sample was centrifuged at 1000 g for 10 min at 15°C. Subsequently, 10 μl of supernatant was applied to a 2790 Chromatographer (Waters, Milford, USA) for chromatographic analysis. Absorbance at 216 nm was monitored with cholesterol content indicated by the peak area.

Enzyme-linked immunosorbent assay (ELISA)

The level of inflammatory cytokine secretion was quantitated using ELISA kits (Boster, Pleasanton, USA) according to the manufacturer’s instructions. In brief, 100 μl sample or standard preparation was added to each well of 96-well plates. Then, 100 μl biotin-conjugated antibody was added to each well and incubated for 60 min at 37°C and then washed with 0.01 M Tris-buffered saline (TBS). And 100 ml ABC was further added to each well and incubated for another 30 min at 37°C and washed again with 0.01 M TBS. Finally, the reaction was ended after incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) for 30 min in the dark. The absorbance at 450 nm was determined using the iMark microplate reader (Bio-Rad, Hercules, USA).

Chromatin immunoprecipitation (ChIP) assay

THP-1-derived macrophages incubated with M. fragrans were cross-linked in 1% formaldehyde for 15 min at 37°C. The reaction was stopped by adding glycine solution. SDS Lysis Buffer (Beyotime) and phenyl methyl sulfonyl fluoride (PMSF) (Beyotime) were then added to the cells before sonication with an ultrasonic processor (Sonics, Newtown, USA) for 14 bursts of 4.5 s with 9-s intervals under 60 W on ice. Cell lysate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant containing sheared chromatin was kept on ice. DNA fragment sizes were measured by agarose gel electrophoresis. After that, the samples were subjected to immunoprecipitation using a ChIP assay kit (Abcam) and antibody against GATA3 (Abcam) or antibodies against IgG (Abcam). The DNA was eluted and collected for analysis by qRT-PCR using the following primers. Human ABCA1 primers: forward 5′-CTCGGTGCAGCCGAATCTAT-3′ and reverse 5′-CACTCACTCTCGCTCGCAAT-3′.

Western blot analysis

Proteins were extracted from cells and were quantified and subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Shanghai, China), and incubated with respective primary and secondary antibodies [25]. Primary antibodies included antibodies against GATA3 (ab199428, 1:1000; Abcam), Histone 3 (ab183736, 1:200; Abcam), ABCA1 (ab18180, 1:200; Abcam), scavenger receptor class B type I (SR-BI) (ab217318, 1:200; Abcam), ABCG1 (ab52617, 1:200; Abcam), and LXRα (ab176323, 1:1000; Abcam). Secondary antibodies were HRP-labeled goat anti-mouse IgG (H + L) (1:1000; Abcam) and HRP-labeled goat anti-rabbit IgG (H + L) (1:1000; Abcam). Immunoreactive bands were visualized with Tanon 5500 (Tanon, Shanghai, China) using BeyoECL Plus (Beyotime).

Real-time quantitative PCR

THP-1-derived macrophages were used for RNA extraction using a TRIzol extraction kit (Beyotime) according to the manufacturer’s instructions. Total RNAs were reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The amplification of the mixture (5 μM cDNA, 2 μM DNA polymerase, 1 μM dNTP, 2 μM forward and reverse primers, 10 μM SYBR Green, and 30 μM ddH₂O₂) was performed with the ABI PRISM 7900 sequence detection system (Applied Biosystems, Shanghai, China) and experienced 30 cycles. The temperature profile included denaturation at 94°C for 3 min, annealing at 58°C for 15 s, and elongation at 72°C for 6 min. β-Actin mRNA level was used for normalization. The formula ∆∆CT was used to calculate mRNA relative expression. The primer sequences used for the real-time PCR were as follows: human ABCA1, forward 5ʹ-GTCCTCTTTCCCGATTATCTGG-3ʹ and reverse 5ʹ-CACTCACTCTCGCTCGCAAT-3ʹ; human LXRα, forward 5ʹ-ACGGTGATGCTTCTGGAGAC-3ʹ and reverse 5ʹ-AGCAATGAGCAAGGCAAACT-3ʹ; human LXRβ, forward 5ʹ-ACAGCCAGACGCTACAACCA-3ʹ and reverse 5ʹ-GGCAATGAGCAAGGCATACTC-3ʹ; human GATA3, forward 5ʹ-TCCAAATGCGAATACCTTGCT-3ʹ and reverse 5ʹ-CCTTCTCTAGTGTTCCTGACTGGA-3ʹ.

Cholesterol efflux assay

Cholesterol efflux assay was performed as described previously [8]. Briefly, macrophages were incubated with 50 µg/ml oxidized LDL (ox-LDL) (Beyotime). After 24 h, cells were labeled with 0.5 mCi/ml [³H]-cholesterol and incubated with different concentrations of M. fragrans for 24 h or 10 µM M. fragrans for various time periods. After 24 h of incubation, cells were washed with PBS and cultured in RPMI-1640 medium containing 0.2% BSA with 25 μg/ml apoA-I or 50 μg/ml HDL for 24 h to measure ABCA1-mediated cholesterol efflux. Radioactivity in culture medium and cells were counted in a liquid scintillation counter (LSC) separately. Cholesterol efflux was calculated as the ratio of radioactivity in culture medium to the total radioactivity in culture medium and cells. The efflux activity was expressed as the rate of outflow which is calculated as follows: rate of outflow = CPM in medium/total CPM in medium and cells × 100%.

Statistical analysis

All data were presented as the mean ± SD and evaluated using one-way ANOVA with GraphPad Prism (GraphPad Software Inc.), followed by the Student’s t-test. A value of P < 0.05 was considered statistically significant.

Results

Myristica fragrans accelerates cholesterol efflux from THP-1-derived macrophages

To detect the role of M. fragrans in macrophage lipid metabolism, we examined the lipid contents in THP-1-derived macrophages treated with different concentrations of M. fragrans for 24 h or with 10 μM M. fragrans for different time periods. From the results shown in Tables 1 and 2, it is seen that M. fragrans obviously reduced intracellular total cholesterol (TC), cholesterol ester (CE), and free cholesterol (FC) in a dose- and a time-dependent manner. To explore if M. fragrans-regulated macrophage lipid deposition affects the transporter of cholesterol, the efficiency of cholesterol efflux was measured. As shown in Fig. 1A,B, M. fragrans accelerates cholesterol efflux onto apoA-I also in a dose- and a time-dependent manner, while M. fragrans has no significant effect on the efflux of intracellular cholesterol onto HDL (Supplementary Fig. S1). Collectively, these data suggested that M. fragrans reduced intracellular lipid accumulation by promoting cholesterol efflux.

Table 1.

The effect of different concentration of Myristica fragrans on lipid content in THP-1-derived macrophages

	TC (μg/mg)	CE (μg/mg)	FC (μg/mg)	CE/TC (%)
0 μM MF	586.30 ± 25.04	391.63 ± 36.45	194.67 ± 18.63	66.80 ± 3.45
2.5 μM MF	575.18 ± 18.62	387.05 ± 45.37	188.13 ± 35.51	67.29 ± 5.26
5 μM MF	559.35 ± 36.29*	375.27 ± 17.28*	184.08 ± 28.65*	67.09 ± 1.03
10 μM MF	492.51 ± 42.03*	325.28 ± 35.26*	167.23 ± 39.62*	66.05 ± 2.97
20 μM MF	481.33 ± 56.24*	316.04 ± 29.51*	165.29 ± 19.32*	65.66 ± 3.84

TC: total cholesterol, FC: free cholesterol, CE: cholesterol ester. *P < 0.05 compared with control group; n = 3.

Table 2.

The effect of 10 μM Myristica fragrans on lipid content in THP-1-derived macrophages during different time periods

	TC (μg/mg)	CE (μg/mg)	FC (μg/mg)	CE/TC (%)
0 h	621.37 + 32.84	394.62 ± 24.16	226.75 ± 18.63	63.51 ± 1.87
6 h	605.20 ± 25.86	387.27 + 42.23	217.97 ± 35.51	63.40 ± 2.06
12 h	579.45 ± 42.36*	365.16 ± 53.23*	214.29 + 28.65*	63.01 ± 3.57
24 h	532.51 ± 17.61*	331.27 ± 35.26*	195.24 ± 39.62*	62.21 ± 4.01
48 h	516.71 ± 2864*	325.04 ± 29.51*	191.67 ± 1932*	62.91 ± 5.43

TC: total cholesterol, FC: free cholesterol, CE: cholesterol ester. *P < 0.05, compared with control group; n = 3.

Figure 1.

The effect of Myristica fragrans on cholesterol efflux from THP-1-derived macrophages THP-1-derived macrophages were incubated with 50 μg/ml ox-LDL to form foam cells. Foam cells were treated with different concentrations (0, 2.5, 5, 10, and 20 μM) of M. fragrans for 24 h or 10 μM M. fragrans for different time periods (0, 6, 12, 24, and 48 h). (A,B) The efficiency of cholesterol efflux was measured by liquid scintillation counting method. Data are presented as the mean ± SD. n = 3 in each group. ^*P < 0.05 vs the control group.

Myristica fragrans upregulated ABCA1 expression in THP-1-derived macrophages

Given the evidence that ABCA1 is mainly responsible for transporting intracellular excess free cholesterol, we tested the protein and mRNA levels of ABCA1. The results showed that M. fragrans dramatically increased the mRNA and protein levels of ABCA1 in macrophages (Fig. 2A,B). In addition, M. fragrans has no obvious effect on the expressions of SR-BI and ABCG1 (Supplementary Fig. S2). Therefore, it is suggested that M. fragrans stimulated cholesterol efflux by upregulating ABCA1 expression.

Figure 2.

The effect of Myristica fragrans on ABCA1 expression in THP-1-derived macrophages THP-1-derived macrophages were incubated with 50 μg/ml ox-LDL to form foam cells. Foam cells were treated with different concentrations (0, 2.5, 5, 10, and 20 μM) of M. fragrans for 24 h or 10 μM M. fragrans for different time periods (0, 6, 12, 24, and 48 h). (A,B) The mRNA and protein levels of ABCA1 were examined by qRT-PCR and western blot analysis, respectively. Data are presented as the mean ± SD. n = 3 in each group. *P < 0.05 vs the control group.

LXRα mediated the upregulation of ABCA1 by Myristica fragrans in THP-1-derived macrophages

The LXR family has been identified as classical triggers for ABCA1 transcription, both of its family members LXRα and LXRβ are involved in the upregulation of ABCA1 expression [26,27]. To confirm the regulatory upstream of ABCA1, we examined the expressions of LXRα and LXRβ in THP-1-derived macrophages. As shown in Fig. 3A,B, an obvious increase was observed in the protein and mRNA levels of LXRα when incubated with M. fragrans. In support of this, ABCA1 expression was partly inhibited by LXRα inhibitor GGPP, although cells were also incubated with M. fragrans (Fig. 3C). Therefore, our data suggest that M. fragrans upregulates the expression of ABCA1 in vitro most likely via the upregulation of LXRα, thereby promoting cholesterol efflux.

Figure 3.

LXRα mediated the upregulation of ABCA1 by Myristica fragrans THP-1-derived macrophages were incubated with 50 μg/ml ox-LDL to form foam cells. Foam cells were treated with 10 μM M. fragrans for 24 h or/and GGPP. (A,B) The mRNA and protein levels of LXRα and LXRβ were detected by qRT-PCR and western blot analysis, respectively. (C) The mRNA and protein levels of ABCA1 were detected by qRT-PCR and western blot analysis, respectively. Data are presented as the mean ± SD. n = 3 in each group. *P < 0.05 vs control group, ^#P < 0.05 vs GGPP group.

GATA3 participates in Myristica fragrans-induced LXRα expression in THP-1-derived macrophages

Previous studies have shown that GATA3 plays an important role in macrophage activity and also implied that GATA3 is implicated in cardiovascular diseases [21,24]. The data from bioinformatics prediction revealed a potential binding site in LXRα promoter for GATA3 (Fig. 4A). Consistent with this, our ChIP assay results suggested that GATA3 binds to the LXRα promoter region (Fig. 4B). In addition, we found that M. fragrans promoted the nuclear translocation of LXRα (Supplementary Fig. S3). Furthermore, M. fragrans was found to significantly increase GATA3 expression in THP-1-derived macrophages (Fig. 4C).

Figure 4.

Myristica fragrans promoted GATA3 expression and enhanced LXRα promoter activity THP-1-derived macrophages were incubated with 50 μg/ml ox-LDL to form foam cells. Foam cells were treated with 10 μM M. fragrans for 24 h or/and GATA3. (A) Bioinformatics analysis of GATA3 binding sites in the promoter region of LXRα. (B) The ChIP analysis was used to measure the binding condition between GATA3 and LXRα promoter region. (C) The mRNA and protein levels of GATA3 were detected by qRT-PCR and western blot analysis, respectively. Data are presented as the mean ± SD. n = 3 in each group. *P < 0.05 vs control GATA3 group.

We then successfully inhibited GATA3 using its siRNA (Fig. 5A,B) and examined the expressions of LXRα and ABCA1. The results indicated that silencing of GATA3 could decrease LXRα and ABCA1 expression (Fig. 5C,D). To further identify the role of GATA3 in M. fragrans-induced cholesterol efflux, we detected the expressions of LXRα and ABCA1 under the condition of co-treatment of GATA3 siRNA with M. fragrans. As shown in Fig. 5E,F, silencing of GATA3 could essentially eliminate the enhancing effects of M. fragrans on the expressions of LXRα and ABCA1. These findings indicate that GATA3 may act as an upstream target in the M. fragrans-regulated expression of LXRα and ABCA1.

Figure 5.

Knockdown of GATA3 blocked Myristica fragrans–upregulated LXRα and ABCA1 THP-1-derived macrophages were incubated with 50 μg/ml ox-LDL to form foam cells. Foam cells were treated with 10 μM M. fragrans for 24 h or/and GATA3 siRNA. (A) The mRNA levels of GATA3 detected by qRT-PCR. (B) The contents of GATA3 analyzed by immunofluorescence staining. Scale bar: 20 μm. (C,D) The mRNA and protein levels of LXRα and ABCA1 were detected by qRT-PCR and western blot analysis, respectively. Data are presented as the mean ± SD. n = 3 in each group. *P < 0.05 vs control group, ^#P < 0.05 vs GATA3 siRNA group.

Myristica fragrans reduced pro-inflammatory cytokines secreted from THP-1-derived macrophages

On the basis of the anti-inflammatory action of M. fragrans [28], we examined the level of inflammatory cytokines in THP-1-derived macrophage supernatant. Secretion of pro-atherogenic factors including TNF-α, IL-6, and IL-1β was markedly decreased, while the anti-inflammatory cytokine IL-10 was increased by M. fragrans (Fig. 6A–D). In addition, GGPP was found to markedly inhibit the decrease of these inflammatory cytokines by M. fragrans (Supplementary Fig. A4). Thus, M. fragrans not only promotes cholesterol efflux from macrophages, but also inhibits the trigger of inflammatory response through LXRα.

Figure 6.

Myristica fragrans reduced pro-inflammatory mediators from THP-1-derived macrophages THP-1-derived macrophages were treated with 10 μM M. fragrans for 24 h or/and 50 μg/ml ox-LDL. (A–D) The levels of TNF-α, IL-1β, IL-6, and IL-10 were detected by ELISA. Data are presented as the mean ± SD. n = 3 in each group. *P < 0.05 vs control group; ^#P < 0.05 vs ox-LDL group.

Discussion

In this study, we explored the effect of M. fragrans supplement on macrophage lipid metabolism. We first uncovered the anti-atherogenic properties of M. fragrans through upregulating ABCA1 to promote cholesterol efflux. In addition, M. fragrans triggers cholesterol efflux via activating the GATA3/LXRα pathway. We also demonstrated that the anti-inflammatory function of M. fragrans is realized by alleviating the secretion of pro-inflammatory cytokines.

Myristica fragrans is closely associated with cardiovascular disease, and it has been reported that M. fragrans supplement can effectively improve dyslipidemia and reduce atherosclerotic lesion in rabbits [19]. However, the underlying molecular mechanisms of M. fragrans-regulated lipid metabolism have not been clearly described. Our data revealed for the first time that the anti-atherogenic mechanism of M. fragrans is associated with the impairment of macrophage cholesterol efflux. This new observation led us to discover a novel favorable role of M. fragrans in lipid metabolism via upregulating the expression of LXRα and ABCA1. Given the fact that SR-BI and ABCG1 also serve as transporters to mediate the efflux of cholesterol onto HDL, we detected their expressions. But we found that M. fragrans cannot affect their expressions in THP-1-derived macrophages. Similarly, M. fragrans also has no significant effect on the efficiency of intracellular cholesterol efflux onto HDL. In support of the beneficial role of M. fragrans in decreasing macrophage lipid deposition, we also demonstrated a reduction in intracellular lipid contents. Many studies have shown that the overloaded lipid is an impetus for inflammatory response [29–31]. Hong et al. [32] reported that M. fragrans inhibits the activation of inflammatory response in murine macrophages. We demonstrated here that M. fragrans decreased the secretion of pro-inflammatory mediators from macrophages. Additionally, we verified that LXRα inhibitor GGPP could inhibit the anti-inflammatory response of M. fragrans. Therefore, these findings suggest that M. fragrans improves hyperlipidemia depending on the stimulation of cholesterol transporter from macrophages. Nevertheless, the effect of M. fragrans on the development and progression of atherosclerosis needs to be reconfirmed in apoE^−/− or LDLR^−/− pro-atherogenic mice models, which may provide evidence to support the atheroprotective effect of M. fragrans.

Deficiency of macrophage-derived GATA3 directly affects the progression of cardiovascular disease [33]. George et al. [21] observed that the mRNA level of GATA3 is closely associated with the development of myocardial infarction in patients with acute coronary syndrome, whereas the role of GATA3 in atherogenesis is not clear. Here, we first demonstrated the protective role of GATA3 in atherosclerosis. GATA3 knockdown obviously reversed the promotive effects of M. fragrans on the expressions of LXRα and ABCA1 in THP-1-derived macrophages, and M. fragrans increased GATA3 expression. We further found that M. fragrans induced the accumulation of nuclear LXRα, which provides further evidence to verify that LXRα is involved in the upregulation of ABCA1 induced by M. fragrans. Although we verified that GATA3 acted as a downstream molecule to mediate M. fragrans’ action on ABCA1 expression and mediated cholesterol efflux from macrophages, it remains to be defined whether other signaling factors also mediate the reduction of ABCA1 by M. fragrans [34]. In addition, whether M. fragrans directly interacts with LXRα also needs to be further verified.

In conclusion, the current study first showed that M. fragrans upregulates GATA3 expression and then induces the binding of GATA3 to LXRα promoter, leading to increased LXRα expression and subsequent promotion of ABCA1 gene transcription. This reduces lipid accumulation and the production of inflammatory cytokines. Therefore, M. fragrans may become a novel bridge that connects lipid metabolism with inflammation during atherogenesis.

Supplementary Data

Supplementary Data is available at Acta Biochimica et Biophysica Sinica online.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (No. 81770461) and the Hunan Provincial Innovation Foundation For Postgraduate (No. CX20190768).

Conflict of Interest

The authors declare that they have no conflict of interest.

Author notes

†

Shangming Liu, Jiahui Gao and Linhao He contributed equally to this work.