Green tea polyphenol treatment attenuates atherosclerosis in high-fat diet-fed apolipoprotein E-knockout mice via alleviating dyslipidemia and up-regulating autophagy

<div><p>Background: Green tea polyphenol (GTP) is a polyphenol source from green tea that has drawn wide attention owing to epidemiological evidence of its beneficial effects in the prevention of cardiovascular disease; the underlying molecular mechanisms of these effects are not well understood. This study aimed to investigate the effects of GTP treatment on autophagy regulation in the vessel wall and lipid metabolism of HFD-fed male ApoE-knockout mice. Methods: Adult male ApoE-knockout mice (n = 30) fed with a high-fat diet (HFD) were treated with either vehicle or GTP (3.2 or 6.4 g/L) administered via drinking water for 15 weeks, and C57BL/6J mice fed with standard chow diet (STD) were used as the control group. Metabolic parameters, expression of key mRNAs and proteins of hepatic lipid metabolism and autophagy in the vessel wall of mice were determined after the 15-week treatment. Results: A HFD induced atherosclerosis formation and lipid metabolism disorders as well as reduced autophagy expression in the vessel wall of ApoE-knockout mice, but GTP treatment alleviated the lipid metabolism disorders, decreased the oxLDL levels in serum, and increased the mRNA and protein expressions of hepatic PPARα and autophagy markers (LC3, Beclin1 and p62) in the vessel wall of ApoE-knockout mice. Conclusions: Our findings suggest that GTP supplementation showed marked suppression of atherogenesis through improved lipid metabolism as well as through a direct impact on oxLDL and autophagy flux in the vessel wall.</p></div

Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats

<div><p>Background</p><p>Studies have demonstrated that resveratrol (a natural polyphenol) and caloric restriction activate Sirtuin-1 (SIRT1) and induce autophagy. Furthermore, autophagy is induced by the SIRT1-FoxO signaling pathway and was recently shown to be a critical protective mechanism against non-alcoholic fatty liver disease (NAFLD) development. We aimed to compare the effects of resveratrol and caloric restriction on hepatic lipid metabolism and elucidate the mechanism by which resveratrol supplementation and caloric restriction alleviate hepatosteatosis by examining the molecular interplay between SIRT1 and autophagy.</p><p>Methods and results</p><p>Eight-week-old male Wistar rats (40) were divided into four groups: the STD group, which was fed a standard chow diet; the HFD group, which was fed a high-fat diet; HFD-RES group, which was fed a high-fat diet plus resveratrol (200 mg/kg.bw); and the HFD-CR group, which was fed a high-fat diet in portions containing 70% of the mean intake of the HFD group rats. The groups were maintained for 18 weeks. Metabolic parameters, Oil Red O and hematoxylin-eosin staining of the liver, and the mRNA and protein expression of SIRT1, autophagy markers and endoplasmic reticulum(ER) stress-associated genes in the liver were assessed after the 18-week treatment. We found that resveratrol (200 mg/kg bw) and caloric restriction (30%) partially prevented hepatic steatosis and hepatocyte ballooning, increased the expression of SIRT1 and autophagy markers while decreasing ER stress markers in the liver and alleviated lipid metabolism disorder. Moreover, caloric restriction provided superior protection against HFD-induced hepatic fatty accumulation compared with resveratrol and the effects were associated with decreased total energy intake and body weight.</p><p>Conclusion</p><p>We conclude that the SIRT1-autophagy pathway and decreased ER stress are universally required for the protective effects of moderate caloric restriction (30%) and resveratrol (a pharmacological SIRT1 activator) supplementation against HFD-induced hepatic steatosis.</p></div

Hematoxylin and eosin staining observation of rat liver tissue (original magnification: ×400, scale bars = 50 μm).

<p>(A) STD group. (B) HFD group. (C) HFD-RES group. (D) HFD-CR group. (E) Volume density of quantitation of hepatic steatosis (n = 5 per group). **, <i>P</i><0.01 compared with the STD group; ^#, <i>P</i><0.05 and ^##, <i>P</i><0.01 compared with the HFD group.</p

Oil red O staining of cross-sections of aortic roots in the hearts of ApoE^-/- mice (n = 3 per group).

<p>(A) C57BL/Control; (B) ApoE^-/-/Control; (C) ApoE^-/-/GTP-L; (D) ApoE^-/-/GTP-H (20× magnification).</p

Effects of RES and CR on the protein levels of endoplasmic reticulum stress markers in the liver (n = 3 per group).

<p>(A) Western blotting results for CHOP protein. (B) Quantitative analysis of CHOP band densities. (C) Western blotting results for GRP78 protein. (D) Quantitative analysis of GRP78 band densities. ^<b>*</b>, <i>P</i><0.05 and **, <i>P</i><0.01 compared with the STD group; ^#, <i>P</i><0.05 and ^##, <i>P</i><0.01 compared with the HFD group. Data are expressed as the mean ± SD.</p

Effects of green tea polyphenol on protein expression of p62 in aortic tissues of mice (n = 3).

<p>(A) Western blotting results for p62 and β-actin. (B) Relative protein levels of p62. Blotting with anti-β-actin was used as a protein loading control. ^aa <i>P</i><0.01 compared to C57BL/Control group; ^a <i>P</i><0.05 compared to C57BL/Control group; ^b <i>P</i><0.05 compared to ApoE^-/-/Control group. Data are expressed as the mean ± SD.</p

Hematoxylin and eosin (HE) staining of livers of mice in the four groups.

<p>(A) C57BL/Control; (B) ApoE^-/-/Control; (C) ApoE^-/-/GTP-L; (D) ApoE^-/-/GTP-H (40× magnification, Bar = 100 μm), representative liver sections from mice.</p

Oil Red O staining observation of rat liver tissue (original magnification: ×400, scale bars = 50 μm).

<p>(A) STD group. (B) HFD group. (C) HFD-RES group. (D) HFD-CR group.</p

Changes of expression autophagy relative genes, ER stress genes and SIRT1 gene in the rat liver (n = 3 per group).

<p>(A) <i>LC3</i>. (B) <i>Beclin 1</i>. (C) <i>p62</i>. (D) <i>SIRT1</i>. (E) <i>PERK</i>. (F) <i>GRP78</i>. (G) <i>CHOP</i>. ^<b>*</b>, <i>P</i><0.05 and **, <i>P</i><0.01 compared with the STD group; ^#, <i>P</i><0.05 and ^##, <i>P</i><0.01 compared with the HFD group; ^△, <i>P</i><0.05 compared with the HFD-RES group. Data are expressed as the mean ± SD.</p

Effects of green tea polyphenol on serum and hepatic lipids in mice (n = 10 per group).

<p>(A) Serum total cholesterol; (B) Serum total triglyceride; (C) Hepatic cholesterol; (D) Hepatic triglycerides; (E) Serum HDL; (F) Serum LDL; (G) Serum oxLDL. ^a <i>P</i><0.05 compared to C57BL/Control group; ^b <i>P</i><0.05 compared to ApoE^-/-/Control group; ^aa <i>P</i><0.01 compared to C57BL/Control group; ^bb <i>P</i><0.01 compared to ApoE^-/-/Control group. Data are expressed as the mean ± SD.</p