Auricularia polytricha aqueous extract supplementation decreases hepatic lipid accumulation and improves antioxidative status in animal model of nonalcoholic fatty liver

Background: Amelioration effect of Auricularia polytricha water extract (AP) on hepatic injury in an animal model of NAFLD was investigated. Methods: Forty six-week-old Wistar rats were housed and thirty-two fed ten percent lard high-fat diet to induce NAFLD. After eight weeks of induction, animals were divided into five groups of eight rats each: normal control, high-fat diet, RN (reversion to a normal diet), 1× AP (normal diet plus 0.75% AP, w/w), and 2×AP (normal diet plus 1.5% AP). Animals were sacrificed four weeks later. Results: Rats receiving either 0.75% or 1.5% AP exhibited effective interruption of NAFLD progression, as evidenced by decreased lipid accumulation and elevated antioxidative status. Histological examination proved AP anti-inflammatory function and lower level of related markers for tumor necrosis factor-α and interleukin-6. Besides abundant polysaccharides against lipid accumulation, AP had a specific high level of phenolic compounds and tannins thus may be a potent anti-inflammatory and antioxidative agent. Conclusion: Findings suggest that under normal diet recovery, AP supplement may represent novel, protective material against NAFLD by attenuating inflammatory response, oxidative stress and lipid deposition.


Introduction
Non-alcoholic fatty liver disease (NAFLD) involves progressive liver damage mainly caused by high dietary intake of cholesterol and saturated fat [1]. Ludwig and colleagues coined the term upon discovering macrovesicular lipid droplets, cell necrosis, inflammation, and sinusoidal fibrosis in 20 female diabetics who were not habitual alcohol consumers [2]. Besides non-alcoholic steatohepatitis (NASH), fatty liver, fibrosis, and cirrhosis are all part of NAFLD. Insulin resistance, oxidative stress, and inflammation play key roles in NAFLD progression [3], yet diverse etiologies exist. Most widely accepted is a two-hit hypothesis [4]: [5] obesity, hyperlipidemia, and diabetes induce hepatic fat accumulation; [6] fat amassed results in lipid peroxidation in hepatic cell membranes, releasing proinflammatory cytokines and activating stellate cells. The double invasion contributes to a series of immune responses: e.g., permeable fat infiltration, inflammatory response, cell necrosis, apoptosis. In brief, two-hit hypothesis entails hepatic fat deposition and lipid peroxidation due mainly to unbalanced nutrient intake. Dietary adjustment or specific functional food supplementation, it is believed, can benefit patients by postponing or even reversing pathological progression [7].
It is well known that edible mushrooms are low in calories and rich in polysaccharides, proteins, vitamins, and minerals.
Recently, researchers have paid greater attention to the value of edible mushrooms in food therapy. The family Auriculae has two well-known mushrooms, Auricularia auricula-judae (AA) and Auricularia polytricha (AP). AP is a common edible mushroom in Taiwan. Unlike AA, AP has a thicker fruiting body with very short and fine fur on its backside. Plants of the Auriculae genus have abundant functional components:

Auricularia polytricha aqueous extract supplementation decreases hepatic lipid accumulation and improves antioxidative status in animal model of nonalcoholic fatty liver
polysaccharides, polyphenols, tannins, etc. [8]. Polysaccharides, especially soluble ones, are primary active components of AP and AA, as discussed in previous studies [9,10]. In addition to polysaccharides, polyphenols and tannins are also important elements in the genus. Moreover, early research mostly focused on the health-promoting effects of AA, with little discussion of the function of AP.
Recently, antitumor effect, immunomodulation, and free radical scavenging of AP was investigated [11][12][13], unearthing evidence that active components of AP might have capacity to protect against the two-hit theory of NAFLD. Our laboratory analysis indicates AP aqueous extract (AP) having more active compounds and stronger radical-scavenging abilities than AA aqueous extract (Table 1) and water extraction producing less toxic and concentrated or elevated active components of AP. Consequently, we use AP as functional supplement on hepatic injury with animal model of NAFLD.

Chemicals and Reagents
For biochemical analysis

Plant Extract Preparation
A commercially cultivated strain of AP was purchased from Jhongpu Township, Chiayi, Taiwan, extraction procedure modified from that of Puttaraju and colleagues [13]. Briefly, rehydrated fruiting bodies were steeped in reverse osmosis (RO) water (5 times sample volume) at 126 ºC, high pressure (1.2kg/cm 2 ) for 30 min, and ultrasonicated for 1 h. After proper filtration (130-140 mesh), AP was spray-dried and ground to fine powder (0.4mm).

Animals and Treatment
Forty six-week-old, male Sprague-Dawley rats were purchased from the National Laboratory Animal Center (Taipei, Taiwan).
After a week of acclimation, 32 animals were fed a high-fat diet containing 88% laboratory rodent chow, 10% lard, and 2% cholesterol, to induce NAFLD [14]. The remaining eight rats were fed a laboratory rodent chow as normal controls. After eight weeks' induction, thirty-two rats were divided into groups: was read at 500 nm and 37 °C, VLDL-C calculated as total cholesterol-(HDL-C + LDL-C).

Plasma and hepatic triglyceride concentrations
Ten-microliter plasma samples were directly treated with 1 mL of working reagent (TR213, Randox), absorbance read at 500 nm after 5-min incubation at 37 °C. Hepatic tissue samples were first extracted with a proper amount of solvent (chloroform: methanol 2: 1, v/v) and Triton x-100 added. Extracts were vacuumed and reconstituted with the working reagent (TR213, Randox). Subsequent procedures were the same as those for serum samples [15].

Blood glucose and insulin levels
During the experiment, glucose meter and blood glucose test

Lipid peroxidation
Lipid peroxidation was adapted from Mihara et al.
Tetrazolium salt reacts with superoxide and needs SOD to form superoxide; it forms yellow formazan dye at 450 nm.
Absorbance represents clearance of superoxide by SOD, expressed as U/mg protein.

Glutathione reductase (GR)
A GR assay kit (catalog no. 703202, Cayman) was used for analysis. As GR catalyzes oxidized GSSG to reduced form, GSH, colored NADPH forms colorless NADP + at 340 nm. Decrease in absorbance represents GR activity, value expressed as nmol/min/mg protein.

Glutathione peroxidase (GPx)
A GPx assay kit (catalog no. 703102, Cayman) served for analysis. Basically, reduced glutathione (GSH) in the liver turned into oxidized form whenever GPx catalyzed hydrogen peroxide to water. Decreasing rate of NADPH was measured at 340 nm, whenever oxidized GSSG returned to its reduced form, value expressed as nmol/min/mg protein.

Plasma level of interleukin (IL)-6
A rat IL-6 Platinum ELISA kit (catalog no. BMS625TWO, BenderMedsystem, Austria) served for analysis. Tissue homogenates were applied to a 96-well plate pre-coated with a rat IL-6 antibody. Biotinylated conjugates and streptavidin-HRP were added to bind the first antibody. Finally, tetramethyl-benzidine was applied to form a purple-colored complex, the absorbance read at OD of 450 nm.

Biochemical data
As seen in Table 3

Hepatic lipids, antioxidative status, and CYP 4A protein expression
A high-fat diet dramatically decreased SOD, GPx, and GR activities with higher cholesterol, triglyceride and lipid peroxidation (MDA) levels. Upon reversion to normal diet, all enzyme activities improved. With further addition of 1.5% AP, GPx and GR activities even returned to normal, which saved vitamin E consumption. Serum vitamin C and E concentrations were kept under AP supplementation. Even hepatic vitamin E level might not be fully recovered by normal diet plus AP intervention, vitamin E levels in 1×AP and 2×AP groups were significantly higher than HFD group. CYP 4A is another identical biomarker for NAFLD. As seen in Figure 1, CYP 4A protein was highly expressed in all experimental groups fed the high-fat diet. This induction continued to the end of the experiment. Data presented as mean ± SEM. Superscripts represent statistically significant differences among groups. p<0.05 N: normal dietary group, HFD: high fat diet group, RN: normal diet revert group, 1×AP, and 2×AP: normal diet recovery supplemented with 0.75%(w/w) and 1.5% (w/w) AP.

Histological observation
Histological data also proved efficacy of AP at hepatoprotection.
According to Brunt and colleague's assessment method, the HFD group exhibited grade 3 fatty liver and grade 2~3 inflammation, while the normal control group belonged to grade 0 ( Fig. 2). After reverting to normal diet, hepatic tissues of animals were grade 2 fatty livers and grade 1~2 inflammation.
Improvement was even better with AP supplementation. Figure   1 shows animals in 1×AP group with grade 1~2 fatty liver and grade 0~1 inflammation versus 2×AP group with both fatty liver and inflammation grade 0~1. Masson's trichrome stain indicated fibrotic signs in hepatic tissues, but there were no fibrotic indications in livers from animals subsequently fed high-fat diet for 12 weeks (data not shown). Our study's model successfully induced visceral obesity, high plasma FFA levels, hyperlipidemia, and liver CYP4A (data not shown) and CYP2E1 expression. Dramatic changes in AST and ALT are known signatures of liver disease. One study showed that ALT changed more than AST in NAFLD patients; fibrosis was initiated when greater AST changes occurred [18]. plasma AST and ALT levels were not more obvious than those in the normal group, addition of AP made much greater progress in elevating insulin sensitivity and lipid metabolism than those with normal dietintervention, insulin resistance (HOMA-IR) was induced, stimulating FFA release and transport to the liver that caused triglyceride storage therein. The high-fat diet also caused dysregulation of cholesterol transport. Although changes in plasma AST and ALT levels were not more obvious than those in the normal group, addition of AP made much greater progress in elevating insulin sensitivity and lipid metabolism than those with normal diet.

Discussion
When we discuss health-promoting effect of AP, polysaccharides are always addressed as chief active compounds.
According to our laboratory data, the AP had 90% dietary fiber, including 74.0% soluble and 22.6% insoluble fibers; thus its action cannot be neglected. According to prior studies, about 5~10% (w/w) fiber in the diet, an average of 1~3 g fiber intake, can effectively improve symptoms of fatty liver disease by insulin regulation, antioxidation, and lipid-lowering action [19][20][21]. Effect of high fibers could also be shown as decreased food efficiency in our study. While it successfully regulates and decreases all the above-cited categories, 10% β-glucan did not decrease TNF-α expression in obese Zucker rats [22]. Compared to the normal (RN) group, extra fiber content in AP groups were no more than 0.4725 g (with average intake of 0.525 g of the AP), and lipid-lowering, glucose-homeostatic, antioxidative and also anti-inflammatory effects were seen in the study. These phenomena imply that the power of AP might emanate from fiber content and depend on its abundant polyphenols, especially tannins. Main phenols in AP were gallic acid, tannic acid, and protocatechuic acid [13]. Tannins [26,27].
Yet AP supplementation exerted its influence on lipoprotein metabolism and strongly elevated HDL-C. Higher amount of phenols in AP than in AA may elevate HDL-C by activating lipoprotein lipase activity [28]. Active components in AP may possess a CETP-inhibiting action to increase HDL-C [27], further research must clarify. Our results indicate AP as an ideal health food material for lipid regulation.
As for antioxidative status, high-fat diet for eight weeks yielded high oxidative status, as indicated by decrease in low-oxidative marker enzyme, GPx, and high induction of high-oxidative marker enzyme, catalase. Strictly normal diet failed to improve oxidative status or inflammatory biomarkers.
Mushrooms have abundant antioxidants (phenolic compounds, polysaccharides, nicotinic acid, ergosterols, triterpenes); activities of most antioxidative enzymes like GR, GPx, and SOD in the 2×AP group had recovered. High phenol compounds and tannins in AP reduced consumption of tocopherols and ascorbic acid, which remained as antioxidative nutrients in a hyperoxidative body. CYP4A joined ω-oxidation in microsomes.
Expression of CYP4A increases when peroxisomal lipid peroxidation occurs in mitochondria [29,30]. Under PPAR-α stimulation, CYP4A regulates long-chain fatty acid oxidation, and elevates hydrogen peroxide to cause cell damage and fatty liver formation. Overexpression of PPAR-α was observed in NASH patients [30]. In the present study, benefit of CYP 4A was not seen in normal-diet or AP-supplemented groups.
Further studies must clarify action of AP on microsomal fatty acid oxidation.
Clinical studies show NASH patients with phenomenally high TNF-α expression than those with other fatty liver disease [31]. TNF-α released by visceral fat accelerates NAFLD progression and activates its receptor to cause hepatic fat to accumulate. Increased oxidized FFAs induce a hepatic kinase (IKKβ) pathway, then secrete TNF-α and IL-6. Eventually, both cytokines might postpone the signaling pathway, triggering insulin resistance [32]. This portends AP effectively removing two hits in a NASH animal model. If we convert effective dosage in the study for human usage, 1.5 g/kg BW in rats might be equivalent to 0.24 g/kg BW for humans. Taking extraction rate into consideration, a 60 kg man would ingest 144 g dry AP powder, though crude powder without proper purification had lower polyphenol, tannin, and flavone levels. APE shows definite potential for new functional food ingredients.

Conclusions
This study proved supplementation with AP effectively mitigating two-hit factors of NAFLD and postponing disease progression. Future research can focus on health benefits toward metabolic syndrome, type 2 diabetes, and related metabolic disorders. Studies of active components in AP are also needed for health food development.