Ferulic acid and protocatechuic acid alleviate atherosclerosis by promoting UCP1 expression to inhibit the NLRP3-IL-1β signaling pathway

Abstract

Dietary phenolic acids can combat metabolic diseases like obesity and non-alcoholic fatty liver by enhancing adipose tissue's thermogenic function. Uncoupling protein 1 (UCP1), a key thermogenic protein, is linked to atherosclerosis (AS) development. Whether dietary phenolic acids inhibit AS by boosting thermogenic function remains unknown. This study aims to identify phenolic acids that can enhance the thermogenic capacity of fat and investigate their roles and mechanisms in alleviating AS. Here, we utilized C3H10T1/2 cells and UCP1–luciferase gene knock-in mice to screen dietary phenolic acids, namely ferulic acid and protocatechuic acid, which could enhance the thermogenic capacity of the organism. Treating ApoE^−/− mice with these phenolic acids reduced aortic plaques and suppressed pro-inflammatory gene expression (il-1β, il-6, tnf-α), while simultaneously promoting thermogenic functionality in interscapular brown adipose tissue and perivascular adipose tissue. Furthermore, applying conditioned media from brown adipose cells whose thermogenic capacity was activated by the phenolic acids to foam cells substantially inhibited the NLRP3-IL-1β inflammatory pathway and suppressed foam cell formation. These studies reveal that ferulic acid and protocatechuic acid can inhibit AS, at least in part, by upregulating UCP1 in adipose tissue, thereby suppressing the NLRP3-IL-1β inflammatory pathway and inhibiting foam cell formation in AS plaques. This validates the potential therapeutic function of phenolic acid compounds selected using UCP1 as a target for treating AS. Our work provides a theoretical basis for the precise utilization of food resources rich in phenolic acid compounds.

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Introduction

Atherosclerosis (AS) is a multifaceted and highly prevalent chronic inflammatory vascular ailment. Statistics reveal that AS has attained high incidence and mortality rates worldwide, posing a significant threat to human health.¹ The development of AS is intimately linked to the imbalance in lipid metabolism and adverse immune responses. Macrophages at the arterial wall, stimulated by low-density lipoprotein cholesterol (LDL-C), differentiate into foam cells, triggering localized inflammation and the release of pro-inflammatory cytokines, leading to endothelial damage and further exacerbating AS symptoms.² Currently, the clinical treatment of AS includes taking statins and vascular reconstruction surgery. However, relying solely on pharmaceuticals and surgical interventions can give rise to adverse effects such as neurological dysfunction³ and rhabdomyolysis,⁴ with suboptimal outcomes. Consequently, the pursuit of safe and effective alternative therapies remains a prominent focus of current research.

Phenolic acids are an important class of secondary metabolites containing phenolic hydroxyl and carboxyl groups widely distributed in higher plants. They are abundant in fruits, vegetables, cereals and other plant species and have excellent antioxidant, anti-cancer and anti-inflammatory properties.⁵ Phenolic acids are classified into two main categories based on their chemical structures: hydroxybenzoic acids and hydroxycinnamic acids. Hydroxybenzoic acids encompass gallic acid, p-hydroxybenzoic acid, protocatechuic acid, and vanillic acid, while hydroxycinnamic acids include caffeic acid, ferulic acid, and p-hydroxycinnamic acid. The structure–activity relationship between these two classes of phenolic acids remains unclear. Research has indicated that a diet rich in dietary phenolic acids can ameliorate AS symptoms, including the improvement of lipid metabolism, modulation of vascular and endothelial function, enhancement of the body's antioxidative capabilities, and cholesterol clearance rate.⁶ This suggests the potential utility of dietary phenolic acids as a therapeutic avenue for AS. Although some studies have explored the ability of dietary phenolic acids to mitigate AS, due to the wide variety and complex functions of phenolic acids, there has been no in-depth exploration of their mechanisms.

In a recent study, Gu et al. discovered that uncoupling protein 1 (UCP1) within the perivascular adipose tissue (PVAT) surrounding blood vessels could reduce the expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasomes and interleukin-1β (IL-1β) in AS mice, thereby preserving vascular health through its anti-inflammatory properties.^7,8 UCP1 serves as a pivotal thermogenic protein in adipose tissue, and it is expressed in both brown adipose tissue (BAT) and PVAT, displaying a brown-like phenotype. The extent of browning in PVAT has been inversely correlated with the development of AS.⁹ Furthermore, increased expression of UCP1 can promote thermogenesis in the body, releasing energy through thermogenesis and, to some extent, reducing plasma triglyceride and cholesterol levels, thereby alleviating the progression of AS. Phenolic acids have been demonstrated to serve as effective activators of UCP1 in BAT. For instance, vanillic acid can stimulate thermogenesis and mitochondrial synthesis in BAT and inguinal white adipose tissue (iWAT),¹⁰ sinapic acid can induce browning in 3T3-L1 adipocytes via the p38 MAPK/CREB signaling pathway,¹¹ chlorogenic acid can stimulate thermogenesis in brown adipocytes by enhancing glucose uptake capability and mitochondrial function.¹² Therefore, dietary phenolic acids can be considered as activators of BAT thermogenesis, targeting UCP1 as a key mechanism to alleviate AS. However, no studies have yet linked these three aspects together.

To investigate the relationship between phenolic acid-induced activation of adipose thermogenesis and AS, we screened phenolic acids with the ability to activate BAT thermogenesis from two structurally different types of phenolic acids and verified their effects on AS. Lastly, through cell-based models, we substantiated that the activation of UCP1 expression in adipose tissue could alleviate the inflammatory response of foam cells within AS lesions. This validates the potential therapeutic function of phenolic acid compounds selected using UCP1 as a target for treating AS. Our work provides a theoretical basis for the precise utilization of food resources rich in phenolic acid compounds. It offers a reference for the development of dietary supplements and health foods aimed at improving AS.

Materials and methods

Cell culture and treatment

The C3H10T1/2 cell line was obtained from the Shanghai Institute of Biochemistry and Cell Biology. Mouse macrophage cells (RAW264.7) were procured from the Institute of Zoology, Chinese Academy of Sciences, and human umbilical vein endothelial cells (Ea.hy926) were purchased from the Institute of Basic Medical Sciences. All cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin–streptomycin and maintained at 37 °C under a 5% CO₂ atmosphere (Thermo Fisher Scientific).

When C3H10T1/2 cells reached over 90% confluence, the culture medium was replaced with induction medium (90% DMEM, 10% fetal bovine serum, 1 μg mL⁻¹ insulin, 1 nM 3,3′,5-triiodo-L-thyronine, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, and 0.125 mM indomethacin (Sigma-Aldrich)). After 2 days, the medium was switched to a differentiation medium (89% DMEM, 10% fetal bovine serum, 1% penicillin–streptomycin, 1 μg mL⁻¹ insulin, and 1 nM 3,3′,5-triiodo-L-thyronine). During the differentiation process, various concentrations of phenolic acids (0 μM–1000 μM) including gentisic acid (GA), protocatechuic acid (PCA), pyrocatechuic acid (PTA), ferulic acid (FA), o-coumaric acid (o-CA), and p-coumaric acid (p-CA) were simultaneously administered. All phenolic acids are purchased from Sigma-Aldrich.

Mouse brown adipose progenitor cells were isolated from neonatal mice. Brown adipose tissue was harvested from the scapulae, followed by the addition of DNase I (Thermo Fisher Scientific) and digestion at 37 °C for 20 minutes. The resulting cell suspension was filtered through a 100 μm cell strainer, briefly centrifuged, and then resuspended in culture medium for incubation. The culture method used was identical to that used for C3H10T1/2 cells.

RAW264.7 and Ea.hy926 cells were exposed to inducers: 50 μg mL⁻¹ oxidized low-density lipoprotein (Ox-LDL) (Shanghai yuanye Bio-Technology Co., Ltd) for the foam cell model and 500 ng mL⁻¹ lipopolysaccharides (LPS) (Beijing Solarbio Science & Technology Co., Ltd) for the endothelial cell injury model. Different experimental groups were established as follows: control group (no treatment), model group (inducers only), FA group (10 μM FA + inducers), PCA group (10 μM PCA + inducers), and FA + PCA group (5 μM FA + 5 μM PCA + inducers). Inducer concentrations were determined based on cell viability assays (Fig. S2†).

Cell viability assays

Cell activity was assessed via the CCK-8 assay (Beijing Solarbio Science & Technology Co., Ltd). Absorbance was measured at 490 nm using a microplate reader.

Oil red O staining

Cell oil red O staining. For cell oil red O staining, refer to the method of Han, X et al.¹²

Aortic plaque oil red O staining. An aortic vessel was trimmed, the surrounding fat was removed, and then the vessel was washed with PBS. Then it was immersed in 0.5% oil red O in 60% isopropanol for 10 minutes, washed for 3 minutes with 60% isopropanol and rinsed with water. Following this, it was counterstained with hematoxylin for 2 minutes, rinsed, mounted, and photographed under a microscope. Plaque regions were outlined using ImageJ and quantified using the area ratio between the aorta and stained plaque.

Determination of NO levels

The methods employed in this study followed the instructions provided by the NO assay kit (Beyotime Biotech. Inc.).

Animal experiments

8-week-old C57BL6J male mice and ApoE^−/− male mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. 8-week-old C57BL/6J background male mice with UCP1-luciferase gene knock-in were sourced from the Institute of Zoology, Chinese Academy of Sciences. Mice were housed 4 per cage under SPF conditions (12/12 h light/dark, 50 ± 15% humidity, 22 ± 2 °C). They had food and water ad libitum, and the water was changed every 3 days. Mice were euthanized by neck-breaking post-blood collection. Arterial tissues, PVAT, BAT, iWAT, and epididymal white adipose tissue (eWAT) were stored at −80 °C. Data from independent biological samples.

The UCP1–luciferase mice were divided into four groups, each comprising five individuals, with ad libitum access to food and water. The mice were fed a standard diet for growth. After an acclimatization period, the mice underwent intragastric gavage with phenolic compounds (control group: physiological saline; FA group: 50 mg kg⁻¹ day⁻¹ ferulic acid; PCA group: 50 mg kg⁻¹ day⁻¹ protocatechuic acid; FA + PCA group: 25 mg kg⁻¹ day⁻¹ ferulic acid + 25 mg kg⁻¹ day⁻¹ protocatechuic acid) for 8 weeks.

The ApoE^−/− mice were divided into five groups, each comprising six individuals, with ad libitum access to food and water. They were fed a diet containing 21% fat and 0.15% triglyceride. The specific composition of the ingredients in the feed can be seen in Table S1.† The mice were subjected to gavage with various treatments for 6 weeks (model group: physiological saline; FA group: 50 mg kg⁻¹ day⁻¹ of ferulic acid; PCA group: 50 mg kg⁻¹ day⁻¹ of protocatechuic acid; FA + PCA group: 25 mg kg⁻¹ day⁻¹ of ferulic acid + 25 mg kg⁻¹ day⁻¹ of protocatechuic acid; AT group: 10 mg kg⁻¹ day⁻¹ of atorvastatin). The control group consisted of C57BL/6J male mice fed with the standard growth diet and gavaged with physiological saline.

All animal experiment procedures were approved according to the guidelines of the Animal Ethical Committee of China Agricultural University and complied with the ARRIVE guidelines & the National Research Council's Guide for the Care and Use of Laboratory Animals.

In vivo fluorescence imaging of mice using IVIS

In the 7th week of UCP1–luciferase mouse experimentation, mice had shoulder blade hair removed. Then mice were intraperitoneally injected with D-luciferase solution (Gold Biotechnology) at 15 mg kg⁻¹. After 8 minutes, they were anesthetized with 2.5% isoflurane and moved to an imaging chamber for respiratory anesthesia. We used the IVIS system for live mouse fluorescence imaging, and the shoulder blade area of the mice in the images was quantified using Living Imaging software.

Mouse thermogenic capacity

In the 6th week, each mouse fasted and stayed at 4 °C for 5 hours, with hourly rectal temp checks. Then, the mice were laid down for infrared pics to measure brown fat activity at the scapula among treatments.

RT-qPCR

Total RNA was extracted using TRIzol™ reagent (Nanjing Nuoweizan Biotechnology Co., Ltd). Subsequently, cDNA was synthesized through reverse-transcription PCR with a high-capacity cDNA reverse transcription kit (Jiangsu CoWin Biotech). Real-time PCR was performed with a Taq Pro Universal SYBR qPCR Master Mix (Nanjing Nuoweizan Biotechnology Co., Ltd) on a LightCycler 480 (Roche). Data were normalized to the internal control β-actin and analyzed using the 2^−ΔΔCT method. Primer sequences are provided in Table S2.†

HE staining of adipose tissues

At the end of the experiments, mice organs were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed using conventional methods.

Serum biochemical index

Blood stood for 2 hours and then spun at 3000 rpm for 10 minutes. Serum extracted for assays: TGs, T-CHO, HDL-C, LDL-C, AST, ALT, and AKP measured using Nanjing Jiancheng Bioengineering Institute kits, following the manufacturer's instructions.

Enzyme-linked immunosorbent assay (ELISA) detection

The levels of IL-6, TNF-α, and IL-1β were determined following the instructions provided in the ELISA kits (Beijing Borui Technology Co. Ltd).

Western blotting

For western blotting, refer to the method of Han, X et al.¹³

Data analysis

All data are expressed as means ± SEM. Experimental data were analyzed using SPSS software. Student's t-test was employed for comparing two groups, while differences among three or more groups were assessed using one-way analysis of variance (one-way ANOVA) via Tukey's test followed by the Dunnett test. Statistical significance was considered at p < 0.05. Graphs were generated using Prism 9.0 software.

Result

FA and PCA promote thermogenesis in BAT by enhancing ucp1 expression

At present, the structure–activity relationships of two distinct classes of phenolic acids within plant organisms remain unclear. Consequently, this study selected six commonly found phenolic acids from these two classes for investigation. When cells were exposed to different phenolic acid concentrations, high amounts hindered cell growth. Using a 10 μM concentration ensured minimal impact on cell activity (Fig. S1A–F†). Lipid droplet distribution and genes linked to fat synthesis showed no impact on cell differentiation into brown adipocytes at this concentration (Fig. 1A and B, S1G and H†). Further analysis of genes related to thermogenesis revealed that FA and PCA treatments significantly enhanced ucp1 transcription (p < 0.01, Fig. 1C). The results obtained from detecting upstream genes of ucp1, namely pgc1α, prdm16, and cidea, also demonstrated that FA and PCA treatments could activate the expression of thermogenesis-related genes (p < 0.05, Fig. 1D–F).

Fig. 1 FA and PCA promote thermogenesis in BAT by enhancing ucp1 expression. Oil red O staining on C3H10T1/2 cells (A) and relative quantification of intracellular lipids (n = 4) (B). Scale bar: 50 μm. Relative mRNA expression of ucp1 (C), pgc1α (D), prdm16 (E), and cidea (F) in C3H10T1/2 cells (n = 6). (G) Fluorescence imaging of the scapular region in mice using IVIS and quantitative fluorescence imaging chart (H). (I) The temperature variation in the mouse colon at 4 °C. (J) After 5 hours of treatment at 4 °C, infrared thermography images of the back and temperature values (K). (L) Gene relative mRNA expression in BAT (n = 4 in all mice experiments). (M and N) The gene expressions of ucp1, pgc1α, and prdm16 and protein expressions of UCP1 and PGC1α in brown adipose primary cells after phenolic acid treatment (n = 4). Data are expressed as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. control. p-Value was assessed using one-way ANOVA via Tukey's test followed by the Dunnett test.

Next, we further explored the thermogenic activation effect of FA and PCA. In addition to using the two phenolic acids alone, a compound group was set up and used together after half the dose to explore whether the structural interaction could achieve a better effect at a reduced dose. We treated UCP1–luciferase gene mice with these acids. Using in vivo fluorescence imaging, luciferase expressed at the ucp1 gene locus allowed precise UCP1 level quantification. After an 8 week treatment, FA-treated mice showed a significantly heightened luciferase signal at the scapular region (p < 0.05), while PCA and combined groups trended towards an increase vs. controls (Fig. 1G and H). Infrared imaging showed higher temperatures in the scapular region of cold-exposed mice treated with FA and PCA, indicating increased heat release (p < 0.01, Fig. 1I–K). This correlated with elevated ucp1 and prdm16 gene expression in BAT (p < 0.05, Fig. 1L). Furthermore, we utilized primary brown adipose cells to investigate the effects of FA and PCA. Treatment with phenolic acids significantly increased the expression of the ucp1 and prdm16 genes in these cells (p < 0.05, Fig. 1M). At the protein level, the expression of UCP1, a key thermogenic protein, was significantly elevated in brown adipocytes compared to the control group (Fig. 1N). These findings suggest that PCA (hydroxybenzoic acid) and FA (hydroxycinnamic acid) can upregulate ucp1 in BAT, enhancing thermogenic energy expenditure.

FA and PCA inhibit macrophage foam cell formation and endothelial cell damage

In the following investigations, we investigated whether FA and PCA exhibit the ability to inhibit AS lesions. AS symptoms involves two key stages: macrophage foam cell formation and endothelial cell damage. After Ox-LDL induction, RAW264.7 cells quickly transformed into foam cells, accumulating many intracellular lipid droplets. Yet, FA, PCA, and their phenolic acid mix notably reduced intracellular lipid droplets, effectively halting foam cell formation in RAW264.7 cells (p < 0.05, Fig. 2A and B). Post Ox-LDL stimulation, macrophages showed heightened inos expression, elevating intracellular nitric oxide (NO) levels and causing cytotoxicity. FA and PCA treatments effectively lowered intracellular NO (p < 0.01, Fig. 2C and D). Subsequent RT-qPCR analysis showed increased interleukin-6 (il-6), il-1β, and tumor necrosis factor-α (tnf-α) in foam cells. Phenolic acid treatment, particularly FA and combined treatments, significantly reduced their expression (p < 0.05, Fig. 2E–G). The doses we used had no effect on the growth activity of RAW264.7 cells and endothelial cells (Fig. S2D and E†). Both FA and PCA notably inhibited macrophage foam cell formation.

Fig. 2 FA and PCA inhibit macrophage foam cell formation and endothelial cell damage (n = 3 in all cell experiments). (A–G) FA and PCA were applied to foam-forming RAW264.7 cells. (A) Oil red O staining and relative quantification of intracellular lipids (B). (C) Relative inos mRNA expression. (D) The intracellular levels of NO. (E–G) Relative mRNA expression of il-6 (E), il-1β (F), and tnf-α (G). (H–M) FA and PCA were applied to epithelial cells damaged by LPS. Relative mRNA expression of il-6 (H), il-1β (I), tnf-α (J), vcam-1 (H), icam-1 (I), and e-selection (J). Data are expressed as the means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. model. ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. control. p-Value was assessed using one-way ANOVA via Tukey's test followed by the Dunnett test.

The adhesion stimulation of foam cells triggers inflammation and damage in vascular endothelial cells, further accelerating the development of AS. The study found that FA and PCA treatments improved LPS-induced epithelial cell damage, partly reducing the gene expression of il-6, il-1β, and tnf-α in affected cells (Fig. 2H–J). Also, adhesion factors are pivotal in AS plaque formation. When treating damaged epithelial cells with FA and PCA, the gene expression of vcam-1 was significantly reduced (p < 0.05, Fig. 2K), and the gene expression of icam-1 was markedly suppressed under combined treatment (p < 0.05, Fig. 2L).

FA and PCA inhibit the development of AS in ApoE^−/− mice

Next, we used ApoE^−/− mice to verify the anti-AS effects of FA and PCA. The doses used in the experiment did not cause liver damage in mice (Fig. S3G–I†). Despite being fed a high-cholesterol diet, no notable mouse weight change was observed (Fig. 3A). Yet, compared to the control, the model group notably raised serum total cholesterol (T-CHO), triglycerides (TGs), and LDL-C. FA and PCA treatments markedly cut these, mimicking atorvastatin (AT), an AS therapeutic drug's impact (p < 0.001, Fig. 3B–D). On the other hand, high-density lipoprotein cholesterol (HDL-C) levels did not exhibit any significant differences (Fig. 3E). About atherosclerosis index calculations, FA and combined treatments displayed stronger inhibition compared to the PCA group (p < 0.05, Fig. 3F). Using oil red O staining on mouse aortas, we characterized AS plaque development based on lipid accumulation. The plaque area in the model group reached 41.2% of the entire aortic surface. Post phenolic acid treatment, the plaque area significantly decreased (p < 0.001, Fig. 3G and H). Cross-section analysis at aortic sinuses revealed an 18.9% lipid deposition in the model group. FA and PCA treatments notably reduced lipid deposition at the sinuses (p < 0.05, Fig. 3I and J).

Fig. 3 FA and PCA inhibit the development of AS in ApoE^−/− mice. Male WT and ApoE^−/− mice were fed from 9 to 22 weeks of age (n = 6 in A–F; n = 3 in G–K). (A) Body weight changes. (B–E) Serum levels of triglycerides (B), total cholesterol (C), low-density lipoprotein cholesterol (D) and high-density lipoprotein cholesterol (E). (F) AS index. (G and H) Oil red O staining (G) and quantification (H) of whole aortic plaque. (I and J) Oil red O staining (I) and quantification (J) of aortic sinus plaque. (K) Gene relative mRNA expression in aorta. Data are expressed as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. model. #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. control. p-Value was assessed using one-way ANOVA via Tukey's test followed by the Dunnett test.

We analyzed inflammatory gene levels in mouse aortic tissues. During AS lesions, pro-inflammatory factors (il-6, tnf-α, il-1β, and mcp-1) were notably upregulated (p < 0.05). Particularly, il-1β showed significant enhancement (p < 0.0001), indicating its crucial role in AS lesions. After FA and PCA treatments, compound formulation, and AT treatment, these factors significantly decreased, highlighting differences between treatment and model groups. Phenolic acids effectively inhibited pro-inflammatory factor expressions (p < 0.05). On the other hand, in the model group, aortic tissues exhibited notably increased gene expression levels of adhesion molecules (icam-1, vcam-1 and e-selection), indicating heightened interaction between aortic endothelial cells and foam cells, promoting AS plaque formation. After phenolic acid and AT treatment, icam-1 and vcam-1 gene expression significantly decreased among cells (p < 0.05). Moreover, e-selection, aiding lymphocyte migration, notably decreased with phenolic acid co-administration and AT treatment (p < 0.05, Fig. 3K). Using ApoE^−/− mice, we have further substantiated the alleviating effects of FA, PCA, and their combination on AS. Likely mediated by pro-inflammatory factor downregulation, cellular adhesion in AS plaque sites is reduced. Based on extensive results, FA and combined treatments are more effective than PCA alone.

FA and PCA enhance lipid metabolism and thermogenic function in ApoE^−/− mice

Next, we studied thermogenesis in these ApoE^−/− mice. In terms of fat weight, the drug-treated group showed decreased white adipose tissue, with less marked changes in BAT at the scapula and PVAT around the aorta (Fig. S3C–F†). For the fat morphology, post AS lesion development led to increased tissue volume and lipid droplets, while FA, PCA, or their combination notably reduced the adipocyte size (Fig. 4A), indicating boosted lipid expenditure. Simultaneously, PVAT and BAT displayed akin tissue morphology, hinting at a shared thermogenic function.

Fig. 4 FA and PCA enhance lipid metabolism and thermogenic function in ApoE^−/− mice. (A) Representative images of H&E-stained iWAT, eWAT, BAT and PVAT of the mice used in Fig. 3. Scale bar: 100 μm (n = 3 in B–E; n = 5–6 in G–I). (B–E) Gene relative mRNA expression in iWAT (B), eWAT (C), BAT (D) and PVAT (E). (F) The protein expressions of UCP1 and PGC1α in BAT. (G) The temperature variation in the mouse colon at 4 °C. (H) After 5 hours of treatment at 4 °C, infrared thermography images of the back and temperature values (I). Data are expressed as the means ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. model. #p < 0.05 vs. control. p-Value was assessed using one-way ANOVA via Tukey's test followed by the Dunnett test.

We delve into gene expression levels in various adipose tissues to understand thermogenic function. UCP1 emerges as crucial in BAT. Under specific stimuli, white adipose tissue can also induce ucp1 expression, boosting thermogenesis. In both iWAT and eWAT, PCA and the combination significantly improved ucp1 expression. No statistically significant difference was observed with FA, despite an enhancing trend. Similar trends were seen in the upregulation of cidea and prdm16 genes (p < 0.05, Fig. 4B and C). For BAT and PVAT, both FA and PCA notably enhanced ucp1 expression within tissues (p < 0.01, Fig. 4D–F). Cold stimuli response in mice's body temperature illustrates thermogenic capacity. FA and PCA treatments curbed body temperature decline under cold conditions, activating BAT's thermogenic capability in the scapular region (Fig. 4G–I). FA and PCA treatments notably impacted adipose tissue's thermogenic capacity in AS-afflicted mice, especially in the PVAT depot. UCP1 likely mediates these effects.

FA and PCA inhibit inflammatory levels in the thermogenic fat depot of AS mice

Inflammation response is a crucial characteristic in the pathogenesis of AS. In previous studies, we observed FA and PCA treatments curb the expression of il-6, tnf-α, and il-1β in aortic lesions (Fig. 3K). To delve deeper into inflammation changes in AS mice, we used ELISA to gauge inflammatory factor levels in serum, aortic and adipose tissues near blood vessels, and BAT near the scapula. In AS mice's blood, inflammatory factors spiked. FA and PCA treatments showed a strong anti-inflammatory impact (p < 0.01, Fig. 5A–C). In BAT and PVAT, FA and PCA decreased IL-6, TNF-α, and IL-1β secretion, matching aortic tissue trends (p < 0.05, Fig. 5D–L). This indicates the presence of factors inhibiting inflammation in these thermogenic adipose tissues, likely associated with promoting ucp1 expression by FA and PCA.

Fig. 5 FA and PCA inhibit inflammatory levels in AS mice. The levels of inflammatory factors in different parts of the mice used in Fig. 3 (n = 6 in all experiments). All data were obtained by ELISA. (A–C) The levels of IL-6 (A), TNF-α (B), and IL-1β (C) in the serum. (D–F) The levels of IL-6 (D), TNF-α (E), and IL-1β (F) in the aorta. (G–I) The levels of IL-6 (G), TNF-α (H), and IL-1β (I) in PVAT. (J–L) The levels of IL-6 (J), TNF-α (K), and IL-1β (L) in BAT. Data are expressed as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. model. ####p < 0.0001 vs. control. p-Value was assessed using one-way ANOVA via Tukey's test followed by the Dunnett test.

UCP1-overexpressing adipocytes inhibit the NLRP3-IL-1β inflammatory signaling pathway in foam cells

To validate our previous hypothesis and investigate whether the enhanced expression of the thermogenic essential protein UCP1 in adipose tissue could suppress AS inflammation, we co-cultured the conditioned media from forskolin-treated C3H10T1/2 cells with RAW264.7 cells (Fig. 6A). After forskolin treatment, C3H10T1/2 cells matured into brown adipocytes, showing increased levels of thermogenic genes (ucp1, pgc1a, and prdm16). Moreover, UCP1 protein secretion substantially increased (p < 0.01, Fig. 6B and C). Treating foam cells with the supernatant notably reduced their inflammatory expression. However, supernatant from forskolin-treated or normal cells alone had no discernible effects. We assessed NLPR3, Caspase1, and IL-1β expressions. Levels in foam cells exposed to the forskolin-treated C3H10T1/2 cell supernatant were notably lower compared to controls (p < 0.05, Fig. 6D and E). Furthermore, we treated brown adipose progenitor cells with FA and PCA to obtain a supernatant, which was then used to treat foamy macrophages. The results are shown in Fig. 6F and G. While direct treatment with phenolic acids does have a mitigating effect on the foaming of RAW264.7 cells, the effect is more pronounced following treatment with the cell supernatant. This suggests that certain substances secreted by brown adipocytes in response to thermogenesis stimulation can alleviate the foaming of macrophages. This suggests that UCP1 suppresses NLRP3-IL-1β, reducing inflammation in foam cells. As a result, intracellular lipid droplets decreased, curbing foam cell formation.

Fig. 6 UCP1-overexpressing adipocytes inhibits the NLRP3-IL-1β inflammatory signaling pathway in foam cells. (A) Schematic diagram of adipocyte and macrophage co-culture. (B) The expression of thermogenic genes in C3H10T1/2 cells after forskolin treatment (n = 3). (C) Western blotting analysis for C3H10T1/2 cells treated with forskolin on UCP1, PRDM16, PGC1-α and GAPDH (as a loading control). (D and E) The supernatant of C3H10T1/2 cells treated with forskolin was used to treat foamy RAW264.7 cells (n = 3). (D) Gene relative mRNA expression. (E) Western blotting analysis on NLRP3, Caspase1, IL-1β and GAPDH (as a loading control). (F and G) The supernatant of brown adipose primary cells treated with FA and PCA was used to treat foamy RAW264.7 cells. (F) Gene relative mRNA expression. (G) Western blotting analysis on NLRP3, Caspase1, IL-1β and β-actin (as a loading control). Data are expressed as the means ± SEM. *p < 0.05 and **p < 0.01 vs. model. #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. control. p-Value was assessed using one-way ANOVA via Tukey's test followed by the Dunnett test.

Discussion

The use of lipids to generate heat is an important thermogenesis pathway of adipocytes. Activation of BAT-mediated thermogenesis significantly reduces blood lipid levels. Adipose dysfunction in obesity is one of the important culprits in endothelial dysfunction, vascular inflammation, and the development of atherosclerotic plaques. Therefore, activated BAT emerges as a potential therapeutic target for AS. Phenolic acids serve as potential activators to enhance the thermogenic capacity of BAT. Currently, there is yet to be a definitive conclusion regarding the structure–activity relationship of these two classes of phenolic acids. In terms of promoting the thermogenic function of BAT, hydroxybenzoic acids such as vanillic acid and gentisic acid, as well as hydroxycinnamic acids such as chlorogenic acid and p-coumaric acid, have been demonstrated to have certain effects.^10,13,14 In this study, we discovered that hydroxybenzoic acids such as PCA and hydroxycinnamic acids like FA significantly promote BAT thermogenesis and browning of white adipose tissue. Particularly notable is their ability to enhance the expression of the thermogenic marker protein UCP1. FA and PCA enhance thermogenic capacity in ApoE^−/− mice systemically. Apart from the increased thermogenic capacity in BAT, the expression of thermogenic genes such as ucp1, cidea, and pgc1α in iWAT, eWAT, and PVAT was also upregulated at the transcriptional level. The fat cell structure in these areas changed to smaller lipid droplets and cell sizes. These changes in white fat suggest the body's heat-producing reaction.¹⁵ Heat-producing fat has significant effects on the overall metabolism. Boosting the amount or activity of this fat can ease metabolic strain and enhance energy use.¹⁶ Therefore, targeting fat's heat-producing abilities is a new approach for treating metabolic conditions like AS, obesity, and type 2 diabetes. Compounds like FA and PCA show promise in this aspect.

Activation of BAT with cold exposure and β3 adrenergic receptors reduced LDL in hyperlipidemic mice, reduced TGs and T-CHO in ApoE^−/− mice, increased energy expenditure, and alleviated AS symptoms.^17–19 Similarly, FA and PCA, screened using UCP1–luciferase gene knock-in mice, can activate BAT thermogenesis and relieve symptoms of AS. Endothelial dysfunction and vascular inflammation constitute the foundation for forming AS plaques within the arterial walls.²⁰ FA and PCA treatments inhibit macrophage-to-foam-cell transition, reducing endothelial damage and inflammation. In ApoE^−/− mice, phenolic acid treatment eases systemic lipid abnormalities, notably slashing serum TGs, T-CHO, and LDL levels. This aligns with local tissue changes: smaller plaques in the aortic wall and less lipid accumulation in the aortic sinus.

PVAT also plays a pivotal role in the development of AS. Disruption of PVAT function leads to direct initiation of endothelial dysfunction and vascular inflammation from outside inward due to its proximity to blood vessels and lack of an adventitial fascial boundary. This dysfunction has a direct local impact on the progression of AS. As a crucial type of adipose tissue within the organism, PVAT resembles BAT more than WAT. At the genetic level, one study revealed that only 228 genes (0.79%) displayed significant differences between thoracic PVAT and scapular BAT.²¹ Proteomic analysis aligns with PVAT's likeness to BAT.²² Hematoxylin and eosin staining showed PVAT adipocytes resembling BAT with multilocular appearance and round nuclei. FA and PCA treatments had the same effect on thermogenic genes in PVAT and BAT. The enhanced expression of ucp1, cidea, and prdm16 suggests that PVAT, akin to BAT, might be a crucial site for phenolic acid-mediated thermogenesis regulation. Furthermore, the inflammatory response of adipocytes is critical in AS and cannot be overlooked. In ApoE^−/− mice, the levels of inflammatory factors IL-6, TNF-α, and IL-1β significantly increased in adipose tissue, accompanied by abnormal enlargement of adipocytes and increased lipid droplets. In AS mice treated with phenolic acids, symptom alleviation was accompanied by a significant reduction in the levels of IL-6, TNF-α, and IL-1β in both BAT and PVAT. The “anti-inflammatory factors” released by thermogenic adipose tissues influenced vascular health. Therefore, enhancing thermogenic capacity and suppressing inflammatory responses in adipose tissues emerge as crucial focal points in alleviating AS.

The localized inflammatory storm within the organism constitutes a crucial characteristic of AS. The intake of phenolic acids proves beneficial in suppressing inflammation.²³ In ApoE^−/− mice, a significant upregulation of il-6, tnf-α, and il-1β gene expression was detected in both local and systemic inflammatory responses. This further stimulated endothelial cells to express adhesion molecules such as VCAM-1 and ICAM-1, mediating the adhesion of lymphocytes and monocytes. While phenolic acids show anti-inflammatory properties against AS-induced inflammation, their exact mechanism remains elusive. PVAT emerges as a potential pivotal factor. PVAT's shift to WAT seems to promote AS advancement in ApoE^−/− mice via IL-1β production.²⁴ The culture supernatant from brown adipocytes containing UCP1 treats foam cells, mimicking activated adipose tissue's effect on arterial plaque. Caspase-1 levels in foam cells decrease significantly, inhibiting NLRP3 inflammasome activation and reducing IL-1β expression. IL-1β overproduction is linked to various inflammatory diseases. Gu et al. showed that UCP1 knockout alters the mitochondrial membrane potential, inducing superoxide generation, NLRP3 inflammasome activation, and increased Caspase-1 and IL-1β syntheses.⁷ IL-1β is abundantly present in human AS plaques; however, even in patients, its circulating levels remain below the detection threshold. This suggests that IL-1β is primarily produced and acts locally within the lesion site.²⁵ Our current research highlights UCP1's inhibition of vascular IL-1β, showing how phenolic acids, fostering adipose thermogenesis, reduce AS by curbing inflammation.

In our study, a treatment dose of 50 mg kg⁻¹ day⁻¹ was used in mice to alleviate symptoms of AS. Several studies investigating the health benefits of FA and PCA in mice have also employed doses ranging from 30 mg kg⁻¹ day⁻¹ to 75 mg kg⁻¹ day⁻¹, which are achievable through the human diet.^26–28 Based on the body surface area conversion method, the 50 mg kg⁻¹ day⁻¹ dose in mice is approximately equivalent to 350–400 mg day⁻¹ for an adult. Foods rich in FA and PCA that can be incorporated into daily diets include maize (FA: 170.64 mg per 100 g), rye (FA: 91.49 mg per 100 g), wheat (FA: 64.68 mg per 100 g), cocoa (PCA: 40.00 mg per 100 g), and olives (PCA: 58.50 mg per 100 g). Furthermore, certain foods high in cyanidin-3-O-glucoside, such as black elderberry (794.13 mg per 100 g) and blackberry (138.72 mg per 100 g), can also degrade into PCA and FA in the body.²⁹ Therefore, the doses we selected are safe and attainable through dietary sources. The combination of two natural products has long been of interest. The co-administration of FA with p-coumaric acid and FA with piperine can achieve a synergistic effect.^28,30 Our study also found that FA and PCA can maintain their health benefits even when the doses are halved. Reducing the dosage allows for easier intake of sufficient amounts of phenolic acids from the daily diet while ensuring the richness and diversity of everyday meals. This has significant implications for guiding daily dietary choices.

Conclusions

Overall, our study found that ferulic acid and protocatechuic acid can inhibit the development of AS, which may be related to these phenolic acids to promote thermogenesis of fat tissue. However, our research can delve even deeper in the future, such as employing UCP1–KO animal models to further validate the pivotal role of UCP1 in the inhibition of AS by FA and PCA. In general, our work links dietary phenolic acids to thermogenesis-related energy metabolism and atherosclerotic disease, providing a theoretical basis for precisely utilizing food resources rich in FA and PCA. It also validates the potential therapeutic function of phenolic acid compounds selected using UCP1 as a target for treating AS, which offers novel perspectives for screening drugs aimed at inhibiting AS.

Abbreviations

AS	Atherosclerosis
LPS	lipopolysaccharides
UCP1	Uncoupling protein 1
PVAT	Perivascular adipose tissue
BAT	Brown adipose tissue
iWAT	Inguinal white adipose tissue
eWAT	Epididymal white adipose tissue
Ox-LDL	Oxidized low-density lipoprotein
NO	Nitric oxide
TGs	Triglycerides
T-CHO	Total cholesterol
HDL-C	High-density lipoprotein cholesterol
LDL-C	Low-density lipoprotein cholesterol
AT	Atorvastatin
IL-6	Interleukin-6
IL-1β	Interleukin-1β
TNF-α	Tumor necrosis factor-alpha
NLRP3	Nucleotide-binding oligomerization domain-like receptor protein 3
GA	Gentisic acid
PCA	Protocatechuic acid
PTA	Pyrocatechuic acid
FA	Ferulic acid
o-PA	o-Coumaric acid
p-CA	p-Coumaric acid

Author contributions

Kexin Hong: conceptualization, investigation, formal analysis, data curation, writing – original draft, methodology, and visualization. Jiting Wang: methodology, investigation, data curation, and formal analysis. Xiping Kang: investigation and methodology. Huimin Xue: investigation and methodology. Yunxiao Gao: investigation and methodology. Heming Liang: investigation. Weidong Huang: conceptualization, resources. Jicheng Zhan: conceptualization, resources. Yilin You: conceptualization, writing – review & editing, resources, and funding acquisition.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the General Program of National Natural Science Foundation of China [32272306] to Yilin You.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo02955k

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