Folic acid delays development of atherosclerosis in low‐density lipoprotein receptor‐deficient mice

Abstract Many studies support the cardioprotective effects of folic acid (FA). We aimed to evaluate the utility of FA supplementation in preventing the development of atherosclerotic in low‐density lipoprotein receptor‐deficient (LDLR−/−) mice and to elucidate the molecular processes underlying this effect. LDLR−/− mice were randomly distributed into four groups: control group, HF group, HF + FA group and the HF + RAPA group. vascular smooth muscle cells (VSMCs) were divided into the following four groups: control group, PDGF group, PDGF + FA group and PDGF + FA + RAPA group. Blood lipid levels, oxidative stress and inflammatory cytokines were measured. Atherosclerosis severity was evaluated with oil red O staining. Haematoxylin and eosin (H&E) staining was used to assess atherosclerosis progression. Immunohistochemical staining was performed with antismooth muscle α‐actin (α‐SMA) antibodies and anti‐osteopontin (OPN) antibodies that demonstrate VSMC dedifferentiation. The protein expression of α‐SMA, OPN and mechanistic target of rapamycin (mTOR)/p70S6K signalling was detected by Western blot analysis. FA and rapamycin reduced serum levels of total cholesterol, triacylglycerol, LDL, inhibiting oxidative stress and the inflammatory response. Oil red O and H&E staining demonstrated that FA and rapamycin inhibited atherosclerosis. FA and rapamycin treatment inhibited VSMC dedifferentiation in vitro and in vivo, and FA and rapamycin attenuated the mTOR/p70S6K signalling pathway. Our findings suggest that FA attenuates atherosclerosis development and inhibits VSMC dedifferentiation in high‐fat‐fed LDLR−/− mice by reduced lipid levels and inhibiting oxidative stress and the inflammatory response through mTOR/p70S6K signalling pathway.

the synthesis of extracellular matrix proteins. 4,5 VSMCs exhibit a contractile phenotype characterized by the expression of contractile marker such as a-SMA and synthetic phenotype characterized by the expression of synthetic marker osteopontin (OPN). 3,6 Therefore, the regulation of the VSMC phenotype may be an alternative strategy for effective atherosclerosis prevention and treatment.
Folic acid (FA) is a water-soluble vitamin B that is essential for amino acid metabolism, also naming it vitamin B9. 7 In the past decade, epidemiological studies have shown that FA supplements can prevent neural tube defects, 8 reduce the risk of megaloblastic anaemia 9 and prevent some malignancies. 10 It has also been demonstrated that FA has anti-inflammatory, 11 anti-oxidative 12 and antiapoptotic effects. 13 FA also exhibits a cardioprotective effect, 14 and it has been reported that dietary supplementation with FA can improve endothelial function. 15 Additionally, Huo et al 16 reported that FA supplementation significantly reduced the risk of stroke among hypertensive adults in China without a prior history of stroke or myocardial infarction.
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates various cellular processes including proliferation, growth, migration and differentiation. 17 It has been reported that the VSMC switch from a contractile phenotype to synthetic phenotype is associated with mTOR/p70S6K activation in atherosclerotic lesions. 18,19 However, the regulation of the mTOR/ p70S6K signalling pathway is still not fully understood. Therefore, in this study we aimed to determine the ability of FA supplementation to delay the development of atherosclerosis lesions and to analyse the effects of FA on VSMC dedifferentiation through the mTOR/ p70S6K signalling pathway in low-density lipoprotein receptor-deficient (LDLRÀ/À) mice.

| Animal models
Twenty 6-week-old male homozygous LDLRÀ/À mice on C57BL6/J background were purchased from the model animal research centre of Nanjing University (Nanjing, China). Mice were feeding in Shaoxing City People's Hospital experimental animal centre. Following adaptation to their environment for 1 week, the LDLRÀ/À mice were randomized into four dietary groups as follows: mice fed with a standard diet (NC group), mice fed a high-fat diet (20% fat, 20% sugar and 1.25% cholesterol) (HF group), mice fed a high-fat diet with FA supplementation (75 ug/kg/d 20 ) (HF + FA group) and mice fed a high-fat diet with rapamycin (10 mg/kg 21 ) (HF + RAPA group).
Folic acid was once-daily oral gavage for 16 weeks; the control and high-fat groups received saline by oral gavage. The rapamycin group received intraperitoneal injections. The protocol was approved by the ethical committee for animal research of the Shaoxing City People's Hospital. After 16 weeks of treatment, the mice were fasted overnight and killed. Serum was collected, centrifuged at 1200 g for 5 minutes and harvested for determination of serum lipid levels. The heart and aorta were removed and perfused with phosphate-buffered saline and then for atherosclerotic lesion evaluation, haematoxylin and eosin staining was performed. The mouse aortic root which connects with the heart was isolated and fixed with 4% paraformaldehyde for 12 hour, embedded in paraffin and cut into 5lm serial sections. Two aortas were subjected to Western blotting, and the other three were treated with oil red O staining to detect atherosclerosis lesions.

| Serum biochemical determinations
An automatic biochemistry analyser (Olympus AU2700, Japan) was used to measure serum concentrations of total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C).
The level of low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula. 22 2.5 | Oxidative stress measurement and inflammatory cytokine detection interleukin-1b and interleukin-6 were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol.

| Determination of atherosclerotic plaques in the isolated aorta
After the mice were killed by euthanasia with an intraperitoneal injection of pentobarbital sodium (45 mg/kg), the aorta was harvested and stained with oil red O to assess for the presence atherosclerotic plaques. In brief, stained aortas were photographed using a digital camera (Olympus BX53, Tokyo, Japan). The area occupied by plaques was measured with DP Manager/Controller software (Mitani Co., Tokyo, Japan), and data were analysed with WIN Roof software (Ver. 5.8.1, Mitani Co.). The severity of atherosclerosis was expressed as a percentage of the atherosclerotic plaque area to the total aortic surface area.

| Haematoxylin and eosin staining of mouse aortas
The mouse aortic roots were isolated and fixed with 4% paraformaldehyde for 12 hour, embedded in paraffin and cut into 5lm serial sections. Aortic sections were stained with Lillie-Mayer's H&E to evaluate atherosclerotic lesions in the aortic root. The lesion areas were quantified using Image-Pro Plus 6.0 software (Media Cybernetics).

| Immunohistochemistry analysis
The aortic roots were harvested and fixed in 4% paraformaldehyde for 12 hour. The aortic roots were sliced into 5 lm thick for morphometric analyses. Histological sections from the aorta were treated with 3% hydrogen peroxide to block endogenous peroxidase activity, and immunohistochemical staining was performed with antia-SMA and anti-OPN antibodies. The positive areas were measured in five non-overlapping fields with a DP Manager/Controller and Image-Pro Plus software.
2.9 | Western blot for a-SMA, OPN, p-mTOR, mTOR, p-p70S6K and p70S6K Aortas and VSMCs were homogenized in ice-cold cell lysis buffer plus protease inhibitor cocktail. Protein expression in the aortas and in VSMCs was detected by Western blot analysis using the primary antibodies for a-SMA, OPN, mTOR, p-mTOR, p70S6K and p-p70S6K. Each sample was harvested and separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes. The membrane was blocked in 5% skim milk for 30 minutes, followed by incubation with the primary antibody overnight at 4°C. The samples were then further incubated with a secondary antibody for 2 hour at room temperature. The bands were detected using an enhanced chemiluminescence reagent, and protein levels were determined by normalization to GAPDH.

| Statistical analysis
At least three independent measurements were performed for all assays. All data were reported as the mean AE standard error (SE).
Parameters were evaluated by one-way ANOVA with least significant difference (LSD) post hoc multiple comparison tests. Differences were considered to be significant at P < .05.  Table 1. These findings suggest that FA provided important beneficial cardiovascular protective effects in LDLRÀ/À mice fed a high-fat diet. In addition, compared with the HF group, the weight of the HF + FA group and HF + RAPA group did not decrease, suggesting that the decrease in blood lipid level in FA and RAPA was independent of the weight.

| Folic acid supplementation decreased oxidative stress and inflammation
The effects of folic acid supplementation on markers of oxidative stress and inflammation were analysed. In the HF + FA and HF + RAPA groups, the levels of SOD and GSH-Px were significantly higher than those seen in the HF group ( Figure 1A, B and C). However, MDA levels, which are a marker of oxidative damage, were decreased in the HF + FA and HF + RAPA groups. Moreover, in the HF + FA and HF + RAPA groups, IL-6, IL-1b and TNF-a levels were reduced compared with the levels seen in the HF group ( Figure 1D, E and F). These results demonstrate that FA supplementation suppressed oxidative stress and inflammation in the high-fat-fed LDLRÀ/À mice.

| Folic acid supplementation suppressed atherosclerosis plaque progression
Atherosclerotic lesions were stained using oil red O stain to determine the area of atherosclerosis in each study group. As shown in Figure 2A

| DISCUSSION
In this study, we demonstrated that FA supplementation reduced blood lipid levels and markers of oxidative stress and inflammation in LDLRÀ/À mice. In addition, FA supplementation decreased the area of atherosclerotic lesions in LDLRÀ/À mice in comparison with LDLRÀ/À mice fed a high-fat diet without FA supplementation.
Additionally, we found that FA inhibited the dedifferentiation of  Therefore, we suggest that the possible effects by which FA reduces VSMC dedifferentiation are related to the modulating mTOR/ p70S6K signalling pathway effects of FA, including a reduction in lipid levels and inhibition of oxidative stress and inflammation. These mechanisms may be involved in the beneficial metabolic and cardiovascular effects of FA supplementation.
In conclusion, FA showed important beneficial cardioprotective effects in high-fat-fed LDLRÀ/À mice. This study provides evidence for the role of FA in regulating VSMC phenotypic switching, decreasing blood lipids, preventing oxidative stress and decreasing the levels of inflammatory cytokines in atherosclerotic lesions. All of these effects of FA may represent mechanisms underlying the antiatherosclerotic actions of FA observed in vivo. Our research may enhance the understanding of VSMC phenotypic switching and suggest potential therapeutic or preventive targets for the treatment of patients with cardiovascular diseases.

ACKNOWLEDG EMENTS
This study was supported by the grants from the Public Welfare project of Zhejiang Province (No. 2016C33227).

CONFLI CT OF INTEREST
All of the authors declare that they have no conflict of interests regarding the contents of this article.