Introduction

Atherosclerotic vascular disease is a major cause of morbidity and mortality worldwide [1]. Atherosclerosis is a chronic disease characterized by inflammation and cholesterol deposition in the arterial wall [2]. Therefore, pharmacological inhibition of the vascular inflammatory state may be a valuable strategy for improving therapeutic outcomes in atherosclerosis.

Many signal transduction pathways are involved in the proatherogenic inflammatory process. In recent years, the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response protein 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway has attracted increased attention because of its pivotal function in atherosclerosis. It has been reported that C3H/HeJ mice carry a missense mutation that affects the cytoplasmic portion of TLR4, causing its loss of function and leading to resistance to atherosclerosis [3]. In apolipoprotein E-knockout (ApoE-KO) mice, genetic deficiency of TLR4 or MyD88 was related to a considerable decrease in atherosclerotic plaque areas and serum levels of proinflammatory cytokines, even with persistent hypercholesterolemia [4]. As a primary transcription factor, NF-κB is one of the most important regulators of inflammation. It has been shown that suppression of the NF-κB signaling pathway conferred protection against atherosclerosis [5, 6].

Sargassum thunbergii is a brown algae that is widely distributed in the seas of Japan and China. Studies on S. thunbergii have focused on its bioactive compounds, such as quinone derivatives, phlorotannins, polyunsaturated fatty acids, and polysaccharides [7, 8]. Polysaccharides from S. thunbergii have been found to exhibit diverse pharmacological effects, including antioxidant, anti-inflammatory, and antiproliferative properties [9,10,11]. In our previous work, we prepared sulfated galactofucan (SWZ-4) isolated from S. thunbergii and found that SWZ-4 induced lung cancer cell senescence [12]. However, few studies have explored whether polysaccharides from S. thunbergii can attenuate atherosclerosis by suppressing inflammation.

Thus, the effect and mechanism of SWZ-4 on TLR4-mediated inflammatory responses in atherosclerotic lesions remain unclear. The present study aimed to evaluate whether SWZ-4 attenuates inflammation and atherosclerosis by inhibiting the TLR4/MyD88/NF-κB signaling pathway in ApoE-KO mice. Its effects were also evaluated in RAW246.7 cells.

Methods and Materials

Materials

Bovine serum albumin (BSA), oil red O, fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) were provided by Gibco (Grand Island, USA). The OCT compound was obtained from Tissue Tek (Sakura, CA). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St Louis, USA).

Preparation and Compositional Analysis of SWZ-4

According to previous methods, SWZ-4 was prepared in our laboratory [10, 12]. Briefly, crude polysaccharides (SWZ) were extracted and subjected to anion exchange chromatography. Dialysis, concentration, and precipitation were then performed using ethanol. The autohydrolysis and compositional analysis of SWZ-4 was performed as described in our previous study [12, 13].

Animal Experiments

All animal studies were approved by the Ethics Committee of the Zhejiang Academy of Medical Sciences (License SYXK 2019–0011). The ApoE-KO mice were randomly assigned to four groups (n = 10): control group (NC group), high-fat diet with low-dose SWZ-4 treatment (L-SWZ-4 group), high-fat diet with high-dose SWZ-4 treatment (H-SWZ-4 group), and high-fat diet alone (model group). After more than one week of adaptive feeding, forty 6-week-old male ApoE-KO mice were fed a high-fat diet (HFD) containing 21% fat and 0.2% cholesterol or a normal chow diet for 16 weeks. All mice were housed in an SPF mouse facility and injected intraperitoneally with SWZ-4 or vehicle (saline) for a total of 16 weeks. Mice in the L-SWZ-4 and H-SWZ-4 groups received 50 mg/kg and 100 mg/kg SWZ-4 every 2 days. Body weight was measured once every 4 weeks. Before blood sampling and tissue collection, all the mice were euthanized with a lethal dose of xylazine hydrochloride (10 mg/kg).

Cell Culture

The RAW264.7 murine macrophage cell line was obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% FBS and 1% penicillin streptomycin at 37 °C in humidified 5% CO2 incubators. Cells were used when between passages 5 and 20 and were plated at 1 × 105 cells per well in 96-well plates. In the NC group, cells were treated only with serum-free DMEM. After pretreatment with DMSO or SWZ-4 (0.4 or 0.8 mg/ml) for 1 h, the LPS group (DMSO + LPS) and SWZ-4 group (SWZ-4 + LPS) cells were stimulated with LPS (1 μg/ml) for 24 h under serum-starvation culture conditions. All cell experiments were performed at least three times in triplicate.

Cell Viability Assay

Cell viability was measured using a Cell Counting Kit-8 (CCK-8, Tianjing BioLite Biotech, China) assay. Briefly, RAW 264.7 cells were seeded into 96-well culture plates and incubated with various concentrations of SWZ-4 (0.4, 0.8 or 1.0 mg/ml) with or without LPS (1 μg/ml). After 24 h, CCK-8 reagent was added to each well and incubated for 3 h in an incubator. The absorbance values were detected at a wavelength of 450 nm.

Measurement of Serum Lipid Profile and Inflammatory Cytokines

Commercial kits (Nanjing Jiancheng Bioengineering Institute, China) were employed to evaluate the serum lipid profile (triglycerides [TG], total cholesterol [TC], high density lipoprotein cholesterol [HDL-C], and low density lipoprotein cholesterol [LDL-C]) in ApoE-KO mice. Serum inflammatory cytokine levels of TNF-α, IL-1, and IL-6 in the ApoE-KO mice and RAW264.7 cells were determined with enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, China). All procedures were performed strictly according to the manufacturer’s instructions.

Assessment of Atherosclerosis with Sudan IV and Oil Red O Staining

The dissected aorta from the root to the abdominal region was embedded in paraffin or frozen in Tissue-Tek OCT medium. In series, 4-mm-thick sections of the aortic valve were cut and stained with oil red O. Aortas were cut longitudinally and stained with Sudan IV solution (n = 5). Two observers measured and quantified the total surface area and the total oil red O-positive lesion area using Image-Pro Plus 6.0. The atherosclerotic lesion coverage was evaluated by the percentage of lesions in the total area.

Quantitative Real-Time PCR Analysis

mRNA expression levels were analyzed by quantitative real-time PCR (qRT-PCR) analysis using specific primers (Table 1). Briefly, total RNA was isolated by Trizol Reagent (Invitrogen, USA) and reverse transcribed into cDNA with SuperScriptTM III Reverse Transcriptase (Invitrogen, USA). The expression of GAPDH was used as the internal control. Gene expression levels were calculated using the delta-Ct method. Each sample was assayed in triplicate.

Table 1 Primer sequences for real-time RT-PCR
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Western Blot Analysis

Protein samples from mouse aortic tissue and RAW264.7 cells were harvested and homogenized with SDS buffer. Samples were placed on ice for 30 min and centrifuged at 12,000 × g for 10 min at 4 °C, and then the supernatant was collected. Total proteins were denatured by boiling at 100 °C for 10 min in 5X loading buffer containing SDS. Protein samples were subjected to SDS-PAGE and then transferred onto nitrocellulose membranes. After blocking, the blots were incubated at 4 °C overnight with primary antibody TLR-4 (1:1000, ab13556, abcam, UK), MyD88 (1:1000, ab219413, abcam, UK), p-IκBα (1:1000, 2859S, CST, USA), IκBα (1:1000, 4814S, CST, USA), p-p65 (1:1000, 3033S, CST, USA), p65 (1:1000, 8242S, CST, USA), and GAPDH (1:2000, Vazyme, Nanjing, Jiangsu, China). The corresponding HRP-conjugated secondary antibodies were added to the blots and incubated for 2 h. Protein bands were visualized using enhanced chemiluminescence detection reagents. Relative changes in protein expression were quantified using ImageJ software. All proteins were subjected to western blotting four times.

Statistical Analysis

The data are expressed as the mean ± standard deviation (S.D.). Significant differences between groups were assessed by one-way analysis of variance followed by the LSD method for multiple comparisons. All tests were performing with SPSS 13.0 software. All significance was considered at p < 0.05.

Results

Mouse Body Weight and Food Intake

As shown in Fig. 1a, the mice in the model, L-SWZ-4, and H-SWZ-4 groups presented with markedly higher body weights than those in the control group after 8 weeks. No significant differences were found in body weights among the model, L-SWZ-4, and H-SWZ-4 group mice. There were no significant differences in the average daily food intake among the groups (Fig. 1b).

Fig. 1
figure 1

Body weight and food intake in the ApoE-KO mice. (a) Body weight was measured once every 4 weeks; (b) Food intake during the experiment. Values are expressed as the mean ± SD. (n = 10). #p < 0.01 versus the control group

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SWZ-4 downregulates inflammatory cytokine expression but did not reduce the lipid profiles in the ApoE-KO mice

Activating the TLR4/MyD88/NF-κB signaling pathway leads to release different proinflammatory cytokines. The serum levels of TNF-α, IL-6, and IL-1 in the model groups were enhanced compared with those in the control and SWZ-4 groups. Compared with those of controls, the expression levels were higher in the SWZ-4 group (Fig. 2a–c). SWZ-4 relieved the inflammatory responses in the ApoE-KO mice.

Fig. 2
figure 2

Serum levels of inflammatory cytokines and lipid profiles in the ApoE-KO mice. (a) TNF-α, (b) IL-6, (c) IL-1, (d) TG, (e) TC, (f) HDL-C, and (g) LDL-C. Values are expressed as the mean ± SD. (n = 10). #p < 0.01 versus the control group, *p < 0.01 versus the model group

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To investigate the effects of SWZ-4 on lipid profiles, we measured serum lipid levels in mice. There were no significant differences in TG, TC, HDL-C, or LDL-C among the model, L-SWZ-4, and H-SWZ-4 groups after 16 weeks. Compared to the those in the controls, the serum levels of TG, TC, and LDL-C were significantly higher in the model and SWZ-4 groups (Fig. 2d–g).

SWZ-4 Inhibits TLR4/MyD88/NF-κB Signaling Pathway Activation in ApoE-KO Mouse Aortas

We further examined the effects of SWZ-4 on inflammation-related signaling pathways in ApoE-KO mouse aortas by qRT-PCR and western blotting. Compared with those in the control group, the mRNA expression levels of TLR4, MyD88, and p65 were significantly upregulated in the model and SWZ-4 groups. Compared with the model group, treatment with SWZ-4 significantly suppressed the expression of TLR4, MyD88, and p65. The western blot results revealed similar trends between TLR4/MyD88/NF-κB mRNA expression and protein levels (Fig. 3).

Fig. 3
figure 3

Effects of SWZ-4 on the TLR4/MyD88/NF-κB signaling pathway in ApoE-KO mouse aortas. (a–d) The mRNA expression of TLR4, MyD88, IκBα, and p65 was determined by qRT-PCR (n = 3). (e–i) The protein expression of TLR4, MyD88, p-IκBα, IκBα, p-p65, and p65 determined by western blotting (n = 4). Values are expressed as the mean ± SD. #p < 0.01 versus the control group, *p < 0.01 versus the model group

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SWZ-4 attenuated the progression of atherosclerosis in ApoE-KO mice

To study the effects of SWZ-4 on atherosclerosis in vivo, the extent of atherosclerosis was measured by Sudan IV and oil red O staining of aortas in ApoE-KO mice. According to Fig. 4, the atherosclerotic plaque areas in the aorta were significantly increased in the model and SWZ-4 groups compared with those in the control group. Compared with those in the model group, a significant decrease was found in aortic atherosclerotic plaque areas in the L-SWZ-4 and H-SWZ-4 groups.

Fig. 4
figure 4

SWZ-4 attenuates atherosclerotic lesions in the ApoE-KO mouse aortas. (a) Representative morphological images (oil red O) of aortic sinus sections. The scale bar is 500 μm. (b) Representative images of Sudan IV-stained en face aortas. (c) Quantification of the percentage of lesion area stained by Sudan IV in the total aorta intima area. Values are expressed as the mean ± SD (n = 5). #p < 0.01 versus the control group, *p < 0.01 versus the model group

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Cytotoxic Effects of SWZ-4 and LPS

RAW264.7 cells were cultured with SWZ-4 and LPS or medium, and their viability was determined by CCK-8 assay. SWZ-4 did not exhibit cytotoxicity against RAW 264.7 cells when administered at concentrations of 0.4 and 0.8 mg/ml with or without LPS (Fig. 5).

Fig. 5
figure 5

Effect of SWZ-4 and LPS on the viability of RAW26.47 cells. Cells were pretreated with 0.4, 0.8, and 1 mg/ml of SWZ-4 for 1 h, followed by LPS (1 μg/ml) stimulation for 24 h. Cell viability was determined by CCK-8 assay. Values are expressed as the mean ± SD (n = 3). #p < 0.01 versus the control group

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SWZ-4 Downregulates Inflammatory Cytokines and the TLR4/MyD88/NF-κB Signaling Pathway in LPS-Induced RAW 264.7 Cells

Compared with the controls shown in Fig. 6, when RAW 264.7 cells were induced by LPS, the mRNA and protein expression levels of TNF-α and IL-6 were significantly increased. Compared with those in the LPS group, the protective effects of 0.4 and 0.8 mg/ml SWZ-4 against inflammation were significant.

Fig. 6
figure 6

Effect of SWZ-4 on the mRNA expression and release of TNF-α and IL-6 in LPS-induced RAW264.7 cells. (a–b) For mRNA expression analysis, cells were pretreated with 0.4 and 0.8 mg/ml SWZ-4 and stimulated with LPS (1 μg/ml) for 6 h. The mRNA levels of TNF-α and IL-6 were measured by qRT-PCR. (c–d) After cells were treated with SWZ-4 and LPS for 24 h, the culture supernatants were collected and TNF-α and IL-6 levels were measured by ELISA. Values are expressed as the mean ± SD (n = 3). #p < 0.01 versus the control group, *p < 0.01 versus the LPS group

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We further assessed the effects of SWZ-4 on the activation of the TLR4/MyD88/NF-κB signaling pathway in LPS-induced RAW246.7 cells. Compared with that in the controls, both the LPS and SWZ-4 groups showed upregulated TLR4, MyD88, and NF-κB mRNA and protein expression. Compared with the levels in the LPS group, SWZ-4 suppressed TLR4, MyD88, and NF-κB expression at the mRNA and protein levels (Figs. 7 and 8).

Fig. 7
figure 7

Effect of SWZ-4 on LPS-induced TLR4/MyD88/NF-κB mRNA expression in RAW264.7 cells. (a) TLR4 mRNA expression. (b) MyD88 mRNA expression. (c) IκBα mRNA expression. (d) p65 mRNA expression. Values are expressed as the mean ± SD (n = 3). #p < 0.01 versus the control group, *p < 0.01 versus the LPS group

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Fig. 8
figure 8

Effect of SWZ-4 on LPS-induced TLR4/MyD88/NF-κB protein expression in RAW264.7 cells. (a–e) The protein expression of TLR4, MyD88, p-IκBα, IκBα, p-p65, and p65 determined by western blotting. Values are expressed as the mean ± SD (n = 4). #p < 0.01 versus the control group, *p < 0.01 versus the LPS group

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Discussion

The findings of the present study indicated that SWZ-4 treatment significantly ameliorated atherosclerosis in ApoE-KO mice and reduced inflammatory cytokine expression in vivo and in vitro. These results suggested that the protective effects of SWZ-4 might be mediated by the modulation of TLR4/MyD88/NF-κB signaling pathway activation.

Many studies have reported that the biological components of seaweed show a variety of activities. As a compound of S. thunbergii, SWZ-4 induced no apparent side effects and exhibited many beneficial biological functions. Li et al. found that polysaccharides inhibited macrophage foam cell formation and alleviated cellular inflammation [14]. It has also been reported that polysaccharides exhibit antiangiogenic activity against tumor migration by inhibiting VEGF/HIF-1α signaling pathway activation and inflammatory reactions 9. In addition, a study demonstrated that polysaccharides exert antioxidant effects, which are closely related to antiatherosclerosis [15,16,17]. Our previous work revealed that SWZ-4 from S. thunbergii exhibited antitumor activities, which was consistent with other studies [12, 18,19,20]. In the present study, we found that SWZ-4 exerted not only anti-inflammatory effects but also antiatherogenic effects by suppressing TLR4/MyD88/NF-κB signaling pathway activation.

In recent decades, atherosclerosis has been considered an inflammatory disease characterized by immune responses that lead to artery obstruction. Studies have shown that inflammatory reactions are pivotal in all phases of atherosclerosis and constitute the relation between cardiovascular risk factors and altered arterial biology [21, 22]. An increasing number of studies have suggested that inflammatory cytokines such as IL-6, TNF-α, and hsCRP are related to atherosclerosis [23, 24]. Reduction in inflammation can lead to reduce risk factors and atherosclerosis. Therefore, pharmaceutically reducing inflammatory responses appears to be a valid approach for attenuating atherosclerosis and reducing risk factors. In the present work, an HFD increased both the atherosclerotic plaque areas and serum levels of inflammatory markers in ApoE-KO mice. These effects were mitigated by SWZ-4 treatment in the present study.

Toll-like receptors constitute a family of surface molecules and important modulators of immune systems and inflammatory responses, including atherosclerosis and ischemia/reperfusion [25, 26]. TLR4 is the most associated with atherosclerosis and is essential for the cellular response to LPS. The expression of TLR4 has been revealed to be markedly increased in the atherosclerotic plaque macrophages. When TLR4 expression was silenced in ApoE-KO mice, the atherosclerotic plaque size and inflammatory cytokine levels were obviously decreased [27]. MyD88 is not only a crucial downstream link of TLR4 but also an important adapter protein of NF-κB [28,29,30]. NF-κB is a widely expressed nuclear transcription factor and plays an important role in the regulation of a number of inflammatory mediators [31]. Under normal conditions, NF-κB p65 is retained in the cytoplasm, and LPS stimulation markedly elevates the phosphorylation of IκBα, which allows NF-κB p65 to translocate to the nucleus, where it induces the transcription of inflammatory genes [32]. Several studies have reported that the NF-κB signaling pathway is important in atherosclerosis [33, 34]. In the present study, the expression levels of TLR4/MyD88/NF-κB in the aortas of ApoE-KO mice and LPS-induced RAW264.7 cells were significantly elevated, and the effects of SWZ-4 not only led to a decrease in inflammatory cytokine levels but also a reduction in the expression levels of TLR4/MyD88/NF-κB. The reduction in TLR4/MyD88/NF-κB signaling pathway activation and inflammatory cytokine levels due to SWZ-4 treatment was consistent with the reduction in atherosclerotic plaque area. These results revealed that SWZ-4 ameliorated macrophage-mediated inflammation and atherosclerosis, which was associated with the TLR4/MyD88/NF-κB signaling pathway.

There are several limitations in this study. First, we did not further investigate the effects of SWZ-4 on inflammation and atherosclerosis in TLR4−/− ApoE-KO mice. However, a previous study proved that inhibiting TLR4 markedly improved inflammatory responses and atherosclerosis [27]. In addition, these results did not establish that the same effects will be seen in humans. Therefore, further clinical studies are warranted to verify the efficacy and safety of SWZ-4 in humans. Second, TLR4 is expressed broadly in both endothelial and vascular smooth muscle cells in the vessel wall, and the effects of SWZ-4 on the TLR4/MyD88/NF-κB signaling pathway in different cell types were insufficient in this study. Finally, SWZ-4 may have triggered protective mechanisms in addition to the inhibition of the TLR4/MyD88/NF-κB signaling pathway. More studies are needed to further explore the relationship between SWZ-4 and atherosclerosis.

Conclusion

In conclusion, these results suggest that SWZ-4 exerts anti-inflammatory and antiatherosclerotic effects in ApoE-KO mice. The protective efficacy of SWZ-4 may be mediated by inhibition of TLR4/MyD88/NF-κB signaling pathway activation. Therefore, SWZ-4 may be a potential therapeutic target for the prevention of atherosclerosis.