Hyperlipidaemia is a metabolic disorder with a high global prevalence characterized by elevated circulating levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triacylglycerols (TG), with a reduced concentration of high-density lipoprotein cholesterol (HDL-C) in the blood (Hongli et al., 2013). Hyperlipidaemia is a primary risk factor for the development of atherosclerosis, coronary heart disease, peripheral artery diseases, and other major metabolic diseases (Watts and Fredrik, 2011). It has been reported that even 1% reduce in the serum TC levels was estimated to result in 2–3% decrease in the risk of cardiovascular disease (Catapano et al., 2000). Therefore, the reduction of circulating TC and LDL-C levels are important strategy that can be employed to prevent or slow the progression of atherosclerosis (Hansson, 2005). Normal physiological functions can be maintained by decreasing elevated serum TC to an appropriate level. Research into functional foods has identified plants and fungi with beneficial effects in certain diseases (Mélanie and Jones, 2004); for example, a novel functional diet formulation of Auricularia auricula and Chinese hawthorn (Crataegus pinnatifida) showed effective hypolipidaemic properties (Luo et al., 2009). A large number of bioactive components of foods have been reported to potentially produce hypolipidaemic effects. These findings have intensified the search for foods that lower serum lipid levels effectively with minimum side effects (Liu et al., 2014).

Grifola frondosa is a promising candidate with lipids-lowering effects that may prevent atherosclerotic disease through its high fiber, microelement, protein, and polysaccharide content (Gu et al., 2007). G. frondosa, also known as Maitake, is a basidiomycete polypore fungus belonging to the Meripilaceae family (Shin and Lee, 2014). G. frondosa is a medicinal mushroom in traditional Chinese and Japanese herbology, as well as being widely used is an edible mushroom for cooking, and dietary supplementation in Asia. This fungus has shown various biological activities, including antitumor (Kodama et al., 2003), immuno-modulatory (Gary et al., 2009), hypotensive (Talpur et al., 2002), and antioxidant effects. Recently, an antioxidant polysaccharide was purified from G. frondosa (Chen et al., 2012), and our laboratory has previously demonstrated that G. frondosa polysaccharides produced hypoglycaemic effects in a mouse model of type 2 diabetes (Xiao et al., 2011). Therefore, G. frondosa can be considered to have potential as a functional food. The hypolipidaemic effects of G. frondosa were previously reported that it reduced serum lipid levels by promoting the conversion of cholesterol to bile acid (Kubo and Nanba, 1997). Subsequent investigations (Fukushima et al., 2001) showed that G. frondosa fiber significantly reduced serum lipid levels by enhancing fecal cholesterol excretion. Another study (Mayumi et al., 2013) also revealed that G. frondosa reduced serum lipid levels. However, few studies have investigated whether G. frondosa can regulate the expression of key genes involved in lipid metabolism.

Hepatic cholesterol metabolism is an important physiological process involved in lipid metabolic disorder (Li et al., 2014). Cholesterol homeostasis is regulated by coordinated changes in cholesterol biosynthesis, absorption, catabolism, and transport of lipoprotein particles. HMGCR is a key rate-limiting enzyme for de novo cholesterol synthesis. ACAT2 is a key tissue cholesterol-esterifying enzyme that plays an key role in cholesterol absorption by catalyzing the formation of cholesterol esters from cholesterol and long-chain fatty acids in vivo in response to excess intracellular cholesterol (Lee et al., 2005). CYP7A1 is a key rate-limiting enzyme for bile acid biosynthesis from cholesterol (Hubacek and Bobkova, 2006). Approximately 95% of LDL is ApoB, which is constitutively expressed in the liver. ACC1 is a key enzymes in the synthesis of fatty acids and triacylglycerol in the liver. And few research has been carried out concerning whether the lipid-lowering effects of G. frondosa are regulated by these key genes. Therefore, this study was undertaken to assess the lipid-lowering effects of G. frondosa and its regulation mechanism on key genes involved in lipid metabolism in liver of rat, i.e., HMGCR, ACAT2, ApoB, CYP7A1, FAS, and ACC1.

Hypercholesterolemia and hypertriglyceridemia are important risk factors that act, either alone, or in combination, to accelerate the development of cardiovascular disease and the progression of atherosclerotic lesions (Hun et al., 2007). Thus, reducing serum TC and/or TG levels are important for atherosclerosis and cardiovascular disease prevention. High levels of LDL accumulate in the extracellular subendothelial space of arteries to form oxidized LDL, which is highly atherogenic and toxic to vascular cells. It is generally accepted that lowering high serum LDL levels plays an important role in the prevention and reversal of atherosclerosis (Haiping et al., 2010). Diet plays a key role in the control of cholesterol balance, and the consumption of a high-cholesterol diet is regarded as a crucial risk factor for the development of hyperlipidaemia. In our study, the rats ingesting a high-cholesterol diet developed elevated serum levels of TG, TC, and LDL-C, which were significantly reduced by the administration of G. frondosa. The results also agreed with those of previous studies that G. frondosa significantly reduced serum TG, TC, and LDL-C levels (Kubo and Nanba, 1997; Mayumi et al., 2013). These findings suggest that G. frondosa is a potentially effective lipid-lowering food. Individuals with hyperlipidemia could consider using G. frondosa as a dietary supplement for preventing atherosclerosis and cardiovascular diseases. However, these studies didn't elaborate whether the lipid-lowering effects of G. frondosa are mediated by key genes and proteins involved in lipid metabolism. Therefore, we investigated its mechanisms on the hepatic metabolic pathway of lipid by evaluating key enzyme expression. Furthermore, we elucidated changes in liver proteomic profiles using 2DE combined with MALDI-TOF-MS.

The inhibition of HMGCR expression leads to suppression of cholesterol synthesis in liver and further efficiently reduce serum cholesterol level (Lee et al., 2003). Therefore, HMGCR is a key target of several widely available cholesterol-lowering drugs. Statins, currently the most widely used hypolipidaemic drugs, reduce serum TC levels by inhibiting hepatic HMGCR expression (Lammi et al., 2015). A quantity of edible mushroom such as Pleurotus ostreatus, Cratharellus cornucopiodes, Amanita ponderosa, and particularly Lentinus edodes showed HMGCR inhibitory capacities ranging from 52 up to 76% in vitro (Gil-Ramírez et al., 2013). A. auricular significantly decreased HMGCR activity in mice liver (Chen et al., 2011). Our study show that G. frondosa potentially capable of down-regulating the mRNA expression of HMGCR and markedly reduced serum TC levels in rats. These results showed the inhibitors of HMGCR represents the most efficient way to reduce serum TC levels. Accumulation of hepatic cholesteryl esters promote potentially hyperlipidemia. Tissue-specific knockouts of ACAT2 prevent diet induced hepatic cholesteryl esters and TC accumulation in the liver and blood of mice (Zhang et al., 2012). Inhibition of ACAT2 expression may reduce the levels of plasma TC and accumulation of cholesterol ester on the arterial wall (Bei et al., 2012). In current study, G. frondosa reduce the accumulation of serum TC of rats by down-regulating the mRNA expression of ACAT2. Therefore, the ACAT2-specific inhibitors are effective in treating or preventing hyperlipidemia. ApoB is an essential component of LDL and inhibition of ApoB synthesis in the liver may efficiently reduce the serum LDL. Mipomersen decreases ApoB synthesis by inhibiting translation of ApoB mRNA (Thomas et al., 2013). G. frondosa decreased ApoB synthesis by reducing content of ApoB mRNA and further reduce serum LDL level. The increase of CYP7A1 expression may enhance catabolic of cholesterol and thus reduce the serum TC level. L. edodes may contribute to reducing TC, LDL, TG, hepatic fat accumulation and aortic atherosclerotic plaque formation by increasing mRNA expression of CYP7A1 in liver tissues (Yang et al., 2013). In our study, administration of G. frondosa enhanced CYP7A1 mRNA expression, resulting in increase of cholesterol conversion into bile acids.

Cholesterol homeostasis is regulated by well-balanced mechanisms controlling mRNA encoding multiple enzymes (Kenichiro et al., 2005). The concentrations of these enzymes in the present study also paralleled the observed changes in mRNA levels. Down-regulation of HMGCR, ACAT2, and ApoB mRNA expression by administering G. frondosa reduced the concentration of these enzymes and may have resulted in the decreased serum TC and LDL levels. Conversely, up-regulation of CYP7A1 mRNA expression by administering G. frondosa increased concentration of the enzyme and may promote the conversion of cholesterol to bile acid, thus reducing serum TC levels. Therefore, cholesterol-lowering effects of G. frondosa are regulated by down-regulation of HMGCR, ACAT2, and ApoB and up-regulation of CYP7A1 levels, which inhibited the synthesis, absorption and transport of cholesterol, and enhanced its catabolism.

Accumulation of TG in hepatocytes is associated with metabolic disorder (Fatiha et al., 2013). ACC1 is a key rate-limiting enzyme in synthesis of fatty acids and TG in the liver. ACC1 catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, while FAS catalyzes palmitate formation (Menendez et al., 2009). The inhibition of ACC1 expression will limit TG de novo synthesis and further reduce serum TG levels. Gemfibrozil, currently widely used hypotriglyceridemic drugs, reduce serum TG levels by inhibiting hepatic ACC1 expression. We found significant reductions in the mRNA expression levels and concentration of FAS and ACC1 in rats treated with G. frondosa, as well as lower serum TG levels. Consequently, G. frondosa may inhibit FAS and ACC1 expression to produce beneficial effects in hypertriglyceridaemia, thus reducing serum TG and risk of cardiovascular disease.

To further investigate the influence of G. frondosa on liver lipid metabolism, we employed proteomic tools (2-DE and MALDI-TOF-MS) to globally identify proteins that were differentially expressed in the livers of rats administered with G. frondosa compared with untreated hyperlipidemic rats. Our proteomic data revealed that 20 proteins were differentially expressed between the two groups. Interestingly, HMGCS2 is relevant synthesis of cholesterol and condenses acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMGC), which is the substrate for HMGCR. HMGCR is a key rate-limiting enzyme in cholesterol synthesis and catalyzes the production of mevalonic acid from HMGC. Down-regulation of HMGCS2 protein expression (Table 3) may inhibit cholesterol biosynthesis and further decrease serum TC levels. The marked reduction in serum TC levels observed in rats administered G. frondosa could be explained by the down-regulation of HMGCS2 protein expression. Among the 20 identified proteins, ACSL1 and AMACR are of interest with respect to fatty acid metabolism. ACSL1 catalyzes the conversion of free fatty acids to acyl–CoA and the reaction of acyl–CoA with diacylglycerol to form TG. Similar investigations (Ying-Ling et al., 2014) showed that toll-like receptor agonists promote prolonged TG storage in murine and human macrophages by enhancing fatty acid uptake and increasing TG synthesis, with an associated persistent increases in ACSL1. Therefore, down-regulation of ACSL1 protein expression could inhibit fatty acid biosynthesis. The other protein, AMACR, is required for oxidation of fatty acids (Mobley et al., 2003), and it's up-regulation could enhance catabolism of fatty acids. The inhibition of ACSL1, required for fatty acid biosynthesis, and the increase in AMACR, involved in fatty acid catabolism, could therefore result in decreased fatty acid content and further decrease serum TG levels in rats treated with G. frondosa.

High levels of LDL are present in the vessel walls, and LDL undergoes oxidative attack by reactive oxygen species (ROS) to generate oxidized LDL (oxLDL). Endothelial cell dysfunction and the accumulation of oxLDL in the vessel wall are early events in atherogenesis, resulting in the recruitment of circulating monocytes, their adhesion to the endothelium, their subsequent differentiation into macrophages, and the accumulation of oxLDL to form foam cells. Therefore, oxLDL plays a pivotal role in the initiation and promotion of atherosclerosis (Augusti et al., 2012), and lowering oxLDL levels represents an important strategy for the treatment and prevention of atherosclerosis. SOD and Prdx-1 both are member of a ubiquitous family of antioxidant enzymes that reduces oxLDL levels and early atherosclerosis. SOD destroys radicals that are toxic to biological systems. Tea polyphenols may confer anti-atherosclerotic effects by inhibiting oxidation of LDL by SOD (Uto-Kondo et al., 2013). The elevated Prdx-1 level associated with T-cell death-associated gene 51 deficiency contributes to increased resistance to oxidative stress, leading to reduce atherosclerotic lesion growth in mice (Hossain et al., 2013). HPX has a strong binding capacity to haem and thus can transport free toxic haem. HPX also exhibits anti-oxidant, immune regulation, and organic protection effects. When bound to HPX, haem significantly reduced oxygen free radical levels and decreased the catalytic activity of lipid peroxidation (oxLDL) (Grinberg et al., 1999). Therefore, up-regulation of SOD1, Prdx-1, and HPX expression can increase resistance to oxLDL formation, thereby reducing atherosclerotic lesion progression. CPPED1 dephosphorylates AKT (protein kinase B) family kinases, specifically at Ser-473. Studies (Vaittinen et al., 2013) demonstrated that knockdown of CPPED1 expression enhances insulin-stimulated glucose uptake in mature adipocytes, leading to improved glucose metabolism via adiponectin-mediated mechanisms. The results (Walter, 2006) indicated that inhibitors of CPPED1 may be useful for the treatment or prevention of arteriosclerotic diseases. These results showed down-regulation of CPPED1 expression prevent arteriosclerotic disease. In response to free radicals and oxidative stress, cells produce high levels of HSP60. Studies (Yu et al., 2013) showed that Porphyromonas gingivalis challenge increased the expression of oxidative stress-related mediators, such as CCL2 and HSP60. In contrast, consumption of green tea epigallocatechin-3-gallate decreased atherosclerotic lesions and oxidative stress-related mediators in atherosclerotic mice induced by P. gingivalis. Overall, down-regulation of CPPED1, and HSP60 reduces atherosclerotic lesion progression. To summarize, we showed that G. frondosa produced anti-atherosclerotic effects by inhibiting LDLox formation via a reduction CPPED1 and HSP60 levels and increased levels of SOD1, Prdx-1, and HPX in rat liver.

Eight of the 20 proteins identified were relevant to lipid metabolism. The remaining 12 proteins are implicated in other metabolic pathways, MAT1A, GCLM, BHMT, CPS1, and SARDH are relevant to amino acid metabolism. MAT1A form S-adenosylmethionine (SAM), which is precursor of glutathione (GSH). GCLM is an important enzyme in synthesis of GSH, which has antioxidation effect. Therefore, MAT1A and GCLM indirectly involved in lipid peroxidation. ACTG1, KRT18, and TPM3 keep cytoskeleton stability to provide support frame for lipid metabolism. CTSB and PSMB9 degrade excess lipoprotein in cell. ATP5a1 and Cyb5a provide energy for lipid metabolism.

G. frondosa exerted hypolipidaemic and anti-atherosclerotic effects in this study. Figure 5 shows the hypolipidaemic effects of G. frondosa through down-regulation of HMGCR, HMGCS2, ACAT2, and ApoB and up-regulation of CYP7A1 expression levels of mRNA or proteins involved in cholesterol biosynthesis, absorption, catabolism. Furthermore, G. frondosa down-regulated FAS, ACC1, and ACSL1 and up-regulated AMACR expression levels of mRNA or proteins involved in triacylglycerol biosynthesis and catabolism. The anti-atherosclerotic effects of G. frondosa can function through the inhibition of LDL oxidation by down-regulated HSP60 and CPPED1 and up-regulated Prdx-1, SOD1, and HPX expression levels in rat liver. These findings indicate that G. frondosa has significant potential as a functional food to treat or prevent hyperlipidaemia and atherosclerosis.

YD and QW designed the experiments; YD, CX, XL, and HH performed the experiments; YD and YX performed data analysis; LL provided scientific expertise; YD wrote the manuscript; QW edited the manuscript.