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ABSTRACT Fatty liver (FLD) disease is a consequence of metabolic syndrome, which is a health problem worldwide with a phenomenal rise in prevalence. In this study, two hepatoprotective

polysaccharide-peptides were extracted from the mushroom _Auricularia polytricha_ followed by chromatographic fractionation of the extract on the ion exchanger DEAE-cellulose and gel

filtration on Sephadex-200 to yield two purified fractions: APPI and APPII. The monosaccharide compositions, FT-IR, N-terminal sequences, internal peptide sequences and molecular weights of

the two fractions were determined. Furthermore, their hepatoprotective effect on human hepatoma HepG2 cells _in vitro_ and in an animal model of fatty liver disease was evidenced by the

findings that APPI and APPII diminished lipid deposit in cells, blood and the liver, increased cellular antioxidant activity and viability, and protected the liver against injury. The

mechanistic study revealed that APPI and APPII activated the adiponectin pathway, up-regulated expression of genes controlling free fatty acid (FFA) oxidation, such as _AMPK_, _CPTl_,

_ACOX1_ and _PPARα_ genes, enhanced lipid metabolism, preserved hepatic function, promoted the antioxidant defense system and reduced lipid peroxidation. Hence the bioactive compounds of

_A_. _polytricha_ could serve as therapeutic agents in the food and pharmaceutical industries. SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURE CHARACTERIZATION OF _GRIFOLA FRONDOSA_

POLYSACCHARIDE AND ITS EFFECT ON INSULIN RESISTANCE IN HFD-FED MICE Article Open access 08 January 2025 PREPARATION AND CHARACTERIZATION OF TOBACCO POLYSACCHARIDES AND ITS MODULATION ON

HYPERLIPIDEMIA IN HIGH-FAT-DIET-INDUCED MICE Article Open access 06 November 2024 PHYTOCHEMICAL COMPOSITION AND ANTIDIABETIC, ANTI-OBESITY, ANTIOXIDANT, AND CYTOTOXIC ACTIVITIES OF

_CARTHAMUS TINCTORIUS_ SEED OIL Article Open access 28 December 2024 INTRODUCTION The liver is an important organ in charge of the metabolism of lipids, glucose, proteins, alcohol, drugs,

and chemicals1. Accumulating data suggest that excessive intake of alcohol and fat induces improper triglyceride metabolism in the hepatocytes2. When synthesis proceeds faster than

anabolism, triglycerides accumulate in the liver. The excessive lipid in the hepatocytes, exceeding 5–10% of the liver weight, is the main pathogenetic factor of fatty liver disease

(FLD)3,4. The multiplicity of etiologic factors of FLD, include genetic, dietary factors, insulin resistance, and adipokines5. FLD is a metabolic syndrome, which has become a vital health

issue with a phenomenal escalation in prevalence. Fatty liver disease comprises alcoholic fatty liver disease and nonalcoholic fatty liver disease (NAFLD). Alcohol-related liver disease and

NAFLD are common chronic hepatic derangements6. The disease affects grown-ups and children alike and is getting more and more rampant in industrialized as well as developing countries.

Cirrhosis associated with NAFLD is likely to be a priority for hepatic transplantation in the foreseeable future. The incidence of liver cancer associated with NAFLD shows a rising trend7.

Cardiometabolic disorders, dyslipidemia in particular, are often associated with pediatric NAFLD8. Approximately one-third of the Americans have NAFLD with consequent heavy economic and

societal burdens9. Treatments for NAFLD are found to be not optimal. Some of the drugs used have side effects10. Hence, it would be highly desirable to devise a management plan to ascertain

efficacious natural products for human populations at high risk of developing fatty liver disease in order to forestall or retard the exacerbation of hepatic damage at an early stage. To

combat this disease, natural products may have value11. Currently, growing attention has been drawn to the exploitation of biomedicines owing to their minimal toxicity and therapeutic

efficacy. Polysaccharide, a potential agent that meets the requirement, is found in many vegetables, fruits, edible fungi and other microorganisms. Increasing studies furnish supporting

evidence demonstrating that polysaccharides display a diversity of biological activities, encompassing antioxidant12, anti-tumor13, antidiabetic14,15, renoprotective16,17, immunomodulatory18

and hepatoprotective19,20 activities. Polysaccharides isolated from mushrooms have been extensively investigated in the medicinal arena owing to the ready availability of fermentation

technology21,22,23. Mushrooms exert a myriad of health promoting actions. Thus mushrooms have captured the attention of many researchers. Two important activities are hepatoprotective and

antihyperlipidemic activities24,25,26. Polysaccharides from mushrooms exhibit diverse activities: those with hepatoprotective activities have been isolated from _Agaricus bisporus_,

_Coprinus comatus_, _Hypsizigus marmoreus_, _Oudemansiella radicata_, _Pholiota dinghuensis_, _Pleurotus eryngii_, _Pholiota nameko_, _Pleurotus djamor_, and _Russula

vinosa_27,28,29,30,31,32,33,34,35,36,37. Polysaccharides with antihyperlipidemic activity have been isolated from _Pholiota nameko_ and _Termitomyces albuminosus_38,39. _Auricularia

polytricha_, alternatively referred to as wood ear or Jew’s ear, belonging to Auriculariaceae family, is a culinary-medicinal fungus which manifests a multitude of activities. Modern

research indicates that _A_. _polytricha_ is a kind of healthy mushroom with a high carbohydrate content (about 630 g/kg in dried fruiting bodies)40. The polysaccharides from the fruiting

bodies have multiple pharmacological functions with potential for clinical application and are devoid of toxicity and significant side effects. After oral administration a soluble

polysaccharide from _A_. _polytricha_ exhibited antihypercholesterolemic activity in rats41. An ethanolic extract of mycelial culture exhibited antioxidant and tyrosinase inhibitory

activities42. An aqueous extract of fruiting bodies demonstrated hepatoprotective effect against paracetamol in rats43. An aqueous extract was devoid of cytotoxicity toward normal kidney

NRK-52E cells44 but displayed antiproliferative effect on COLO-205 colon cancer cells45. Polysaccharides with anticancer activity in S180 sarcoma bearing mice46, antimutagenic activity

against the alkylating agent cyclophosphamide47, and antiproliferative, cell cycle arresting and apoptotic activities toward A549 human lung cancer cells48 have been isolated from _A_.

_polytricha_. A hot water extract manifested hypoglycemic effects49. Hot water extracts inhibited platelet aggregation with a mechanism independent of cyclic AMP50,51. Thermostable

constituents suppressed activity of beta secretase which liberates toxic β-amyloid peptide in the brain and protects against neurodegenerative diseases such as Alzheimer’s disease52.

Polysaccharides with different activities have been reported from _A. polytricha_41,46,47,48. Three anticancer polysaccharides AAPS-1, AAPS-2, and AAPS-3 with molecular weights of 162, 259,

and 483 kDa46 and an antimutagenic 930 kDa salt-soluble polysaccharide47 from _A_. _polytricha_ have been reported. Our previous studies indicated that polysaccharide prepared from _A_.

_polytricha_ effectively lowered the levels of total cholesterol, LDL-cholesterol and triglycerides41 in the blood circulation, indicating that the polysaccharide could regulate the

metabolism of lipids, and is exploitable for alleviation of FLD and liver injury. Nevertheless, to date, no detailed studies have been carried out on the characterization, hepatoprotective

effect and mechanism of polysaccharides or polysaccharide-peptides from _A_. _polytricha_. Thus we commenced this investigation on purification and characterization of bioactive

polysaccharide-peptides, exploring their hepatoprotective effect and mechanism with the intent to ascertain novel bioactive constituents utilizable in the food and pharmaceutical industries.

RESULTS EXTRACTION AND PURIFICATION OF APPI AND APPII The crude polysaccharide-peptide from _A_. _polytricha_ fruiting bodies was firstly resolved on DEAE-cellulose. According to the charge

difference, two peaks, D1 and D2, eluted with 0 and 0.2 mol/L NaCl solution respectively, were detected by using the phenol–sulfuric acid method (see Supplementary Fig. S1a). The two

fractions were then subjected to concentration, dialysis and gel-filtration chromatography on Superdex-200. As a result, both D1 and D2 generated only a single peak, named _A_. _polytricha_

polysaccharide-peptide I (APPI) and _A_. _polytricha_ polysaccharide-peptide II (APPII), respectively (refer to Supplementary Fig. S1b,c). The yields of APPI and APPII were approximately

16.31% and 49.46% (w/w) based on the weights of dried crude polysaccharide-peptide. High performance gel permeation chromatography (HPGPC) analysis disclosed that the weight-average (Mw),

number-average (Mn), and z average (Mz) molecular weights and polydispersity ratio (Mw/Mn) of APPI were 9.213 × 105, 5.568 × 105, 1.057 × 106 and 1.655, while those of APPII were 6.340 × 

105, 7.693 × 104, 9.547 × 105 and 8.241, respectively. CHARACTERIZATION OF APPI AND APPII APPI and APPII exhibited the same monosaccharide moieties but different molar ratios of

monosaccharides and molecular weights, similar FTIR spectra. APPI and APPII are composed of the same monosaccharide moieties, but at different ratios. Monosaccharide composition analysis

revealed mannose and xylose are the major sugars, with glucose and small amounts of arabinose and galactose (see Supplementary Fig. S2a,b). APPI was composed of Ara, Gal, Glc, Xyl and Man,

in a molar ratio of 1:4.4:15.4:38.3:46.2, while the corresponding molar ratio for APPII was 1:71.5:99.2:10:5.1. Fourier transform infrared spectroscopy (FTIR) is an important method to

predict the structures of natural macromolecules such as polysaccharide-peptides. To more precisely characterize polysaccharide-peptide fractions, the characteristic absorption of

polysaccharides was performed in the region 4000–600 cm−1 by FTIR spectrum. As shown in the FTIR spectrum of APPI (Fig. 1a), the intense broad peak at 3400.42 cm−1 was typical of hydroxyl

groups with stretching vibration53, and the peak at 2934.88 cm−1 was attributed to C-H stretching vibration54. The peak in this region is typical of sugar. The peak at 1653.05 cm−1 suggested

the existence of C=O bands55. The absorptions between 1000 and 1200 cm−1 were typical absorption peaks of the pyranose ring and hence disclosed the existence of C-O-H side groups and C-O-C

glycosidic band vibrations56, and the absorption around 880 cm−1 to 900 cm−1 was attributed to the presence of α-type glycosidic linkages57. The FT–IR spectrum of APPII is shown in Fig. 1b,

which was similar to those of APPI (Fig. 1a), but the broad absorption bands of APPII with strong intensities around 1420.96 cm−1 probably reflected deforming vibrations of the C-H bond.

Absorption peaks were detected at 1375.60 cm−1and 1250.16 cm−1, which revealed that the polysaccharide-peptide APPII possessed a carboxyl group and a sulfate radical58. The amino acid

sequences of APPI and APPII at the N-terminal were DLYEVVEGEI, and VPSSMVVVVG, respectively. Results from internal amino acid sequence analysis showed that two peptide sequences of APPI,

namely VQNVGNGVLLGFHGR and HQTSGDQVTSSTQHSFR, were strikingly similar to mannose-binding lectin from _Cordyceps militaris_. The peptide sequence GTPSSYIDNLTFPK of APPII manifested

considerable homology with immunomodulatory protein from _Flammulina velutipes_. Another peptide sequence of APPII, ELATGQNGFGYAGSSFHR, demonstrated pronounced resemblance to peptidyl-prolyl

cis-trans isomerase from _Lentinula edodes_. PROTECTIVE EFFECTS OF APPI AND APPII _IN VITRO_ HepG2 cells of the injury model were exposed to different concentrations of FFA and ethanol, and

cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that cells could retain around 60% viability when 0.5 mmol/L FFA

and 1.8% (v/v) ethanol were used to induce hepatic injury. Triglyceride (TG) deposits in the cells were revealed by the Oil-red O staining method. The protective effects of APPI and APPII

against cell injury induced by pathogenetic factors was assessed by various assays comprising the MTT test, together with assays of intracellular TG, total superoxide dismutase (SOD), and

extracellular aspartate transaminase (AST) and alanine aminotransferase (ALT) activities. It was shown that APPI and APPII were not cytotoxic to HepG2 cells in the concentration range of

0–100 μg/mL (Fig. 2a). The cells were exposed to increasing concentrations of APPI or APPII (30–60 μg/mL) for 24 h after the addition of 0.5 mmol/L FFA and 1.8% (v/v) ethanol. As shown in

Fig. 2b, FFA and ethanol treatment brought about significant toxicity to HepG2 cells, whereas treatment with APPI or APPII increased cell viability with the highest ratio of 33.47% at 40 

μg/mL and 23.23% at 50 μg/mL, respectively. The results showed that intracellular TG in APPI and APPII groups was attenuated relative to that in the model group (P < 0.05), as shown in

Table 1, which was also verified through morphological observation aided by the Oil-red O stain (see Supplementary Fig. S3). APPI and APPII elevated the activity of total SOD in HepG2 cells

considerably, by 72.72% and 58.43%, relative to the model group (Table 1). The most conspicuous effect of hepatic injury is the liberation of intracellular enzymes, like aminotransferases,

into the circulation, which ensues in an escalation of the activities of these enzymes6. Thus, activities of extracellular aminotransferases can reflect the status of liver cells, with

higher values reflecting liver damage. It was shown that the activities of extracellular alanine and aspartate aminotransferases (ALT and AST) of the model group were elevated relative to

those of the control group, implying that pathogenetic factors of FFA and ethanol inflicted damage on the cells. However, APPI and APPII mitigated this damage, and the elevated activities of

alanine and aspartate aminotransferases consequently declined (P < 0.05), as shown in Table 1. MECHANISMS OF APPI AND APPII Adiponectin is an adipocyte-derived cytokine. Adiponectin and

its receptor 2 abundant in human hepatocytes, constitute a complex, and play a central role in the pathogenesis of liver disorders. Adiponectin, which has an anti-steatotic effect on liver

cells, acts mainly through the AMPK and PPARα pathway. It promotes oxidation of FFA and suppresses gluconeogenesis, entry of FFA and _de novo_ lipid synthesis7,8. When drugs stimulate the

adipocytes, adiponectin is secreted and conjugated with Adipor 2 receptors on the surface of the hepatocytes. The complex activates _AMPK_ gene expression, induces genes including _CPTl_ and

_ACOX1_ in FFA oxidation pathway upregulation. Meanwhile, _AMPK_ induces activation of transcription factor _PPARα_ and accelerates FFA metabolism. In order to validate the mechanism of

APPI and APPII, five key genes have been selected for qRT-PCR analysis (Fig. 3). The results showed that the qRT-PCR data of these genes were consistent with results of the bioassays. For

example, _Adipor 2_ gene was significantly down-regulated in the model group, as well as _AMPK_, _CPTl_, _PPARα_ and _ACOX1_, which deactivate FFA oxidation process and accelerate FFA

accumulation in hepatocytes or hepatic tissue. It was disclosed that the TG concentration of the model group rose. It was found that _Adipor 2_ gene was up-regulated in APPI and APPII

groups, the same took place regarding _AMPK_, _CPTl_, _PPARα_ and _ACOX1_. The results indicated that APPI and APPII could stimulate adipocytes to secrete adiponectin and activate the FFA

metabolic pathway, reducing lipid accumulation. HEPATOPROTECTIVE EFFECT OF APPI AND APPII _IN VIVO_ PROTECTIVE EFFECTS OF APPI AND APPII ON HEPATIC DAMAGE FLD is the leading cause of chronic

liver disease worldwide. Unhealthy high fat diets and sedentary habits result in the high prevalence of FLD. The rats were randomized to receive either standard laboratory diet (SLD) or

high cholesterol and high fat diet (HFD) for 8 weeks. The biochemical analysis showed that HFD induced a rise in the levels of TG, total cholesterol (TC) and low-density lipoprotein

cholesterol (LDL-C), and the activities of ALT and AST in serum (see Supplementary Table S1). The HFD rats gained weight faster than those with SLD, at the ratio of 105.43%, and the liver

weights in the model groups were heavier than that in control group. AST and ALT activities in the serum have been employed as biochemical markers for hepatic damage. The heightened AST and

ALT expression levels signified the enhanced permeability and injury of hepatocytes. In the present study, APPI and APPII ameliorated the biochemical indexes. Significant decreases (P < 

0.05) in ALT and AST activities were observed in APPI and APPII-treated groups similar to the simvastatin- treated group (Table 2). It indicated that APPI and APPII could protect hepatocytes

from injury and maintain their integrity. The concentrations of TC, TG, LDL-C, and high density lipoprotein cholesterol (HDL-C) in the serum are vital parameters in the analysis of lipid

metabolism. Abnormal serum lipid indicated the risks of FLD. The TC, TG and LDL-C concentrations of serum in APPI or APPII-treated groups were reduced relative to those in the model group (P

 < 0.05). It was indicated that APPI and APPII might be useful for preventing FLD through improving lipid metabolism. HFD is associated with fat accumulation in liver. As shown in Fig. 4,

hepatic TG level was elevated significantly by 2.07 fold versus the control group. However, treatment of HFD with APPI and APPII, just like treatment with simvastatin, significantly (P <

 0.05) lowered the TG level in liver tissue compared to the model group. HISTOPATHOLOGICAL OBSERVATIONS Histopathological examination was used to evaluate the hepatoprotective effects of

APPI and APPII on HFD-elicited acute liver lesion. Figure 5b revealed that the nuclei of hepatocytes in the model group were irregular, lysis diffluence and even disappearance. The cytoplasm

exhibited a ballooning degeneration with fatty droplets. It was also observed that hepatic sinus appeared eclasis and gore, some of Kupffer cells proliferated. It suggested that the tissues

in the model group turned to hepatocyte injury and the FLD model was successfully established. However, the hepatocytes showed a regular arrangement, the cytoplasmic ballooning degeneration

was mitigated in APPI and APPII with HFD groups (Fig. 5c–f). In the APPI and APPII treated groups, lipid and hepatocyte injury were reduced relative to the model group. Hepatocyte volume

was reduced, liver lobules were distinctly delineated, and there were fewer fat droplets. These histological alterations were in agreement with the serum and liver lipid profiles shown in

Fig. 4 and Table 2. Therefore, APPI and APPII minimized lipid deposition in the liver cells of FLD rats. DISCUSSION In our earlier study a soluble polysaccharide was prepared from _A_.

_polytricha_ fruiting bodies with an extraction rate approximating 20% by using the following conditions: extraction with 28 mL water/g mushroom at 95 °C for 4 h. The polysaccharide

preparation exerted an antihypercholesterolemic action in diet-induced hypercholesterolemic rats and depressed the plasma cholesterol level by about one-third at the doses of 4.5 and 9 

mg/kg/d. In the present investigation the polysaccharide was fractionated into two polysaccharide-peptides APPI and APPII have similar monosaccharide moieties and FITR spectra but differ in

molar ratio of the monosaccharides and molecular weight. APPII also differs from APPI in exhibiting absorption around 1420.96 cm−1 probably indicating deforming vibrations of the C-H bond

and absorption at 1375.60 cm−1 and 1250.16 cm−1 signifying the presence of a carboxyl group and a sulfate radical. Although APPI and APPII possess distinct N-terminal and internal partial

amino acid sequences, both of them resemble other mushrooms in these sequences. In APPI, two of the internal partial amino acid sequences closely resemble those of _Cordyceps militaris_

mannose-binding lectin. In APPII, two of the internal partial amino acid sequences manifest pronounced similarity to those of _Flammulina velutipes_ immunomodulatory protein and _Lentinula

edodes_ peptidyl-prolyl cis-trans isomerase, respectively. _Auricularia auricular_ polysaccharides simulated hydrolysates, derived from _A_. _auricular_ which is a species very closely

similar to _A_. _polytricha_, were composed of arabinose, galactose, glucosamine, glucose, xylose and mannose with the molar ratio of 1.91: 0.67:0.23:1.00: 0.52: 2.8959. _A_. _auricular_

polysaccharide was composed of arabinose, galactose, glucosamine, glucose, rhamnose and mannose with the molar ratio of 0.93:0.91:4.32:1:37.5360. APPI isolated in the present study was

composed of arabinose, galactose, glucose, xylose and mannose in a molar ratio of 1:4.4:15.4:38.3:46.2, while the corresponding molar ratio for APPII was 1:71.5:99.2: 10: 5.1. Glucosamine

was lacking in APPI and APPII. The data showed that they were all different in the composition of the sugar moiety. The FT-IR spectrum of APPI exhibited a broad peak at 3400.42 cm−1

characteristic of hydroxyl groups with stretching vibration, and a peak at 2934.88 cm−1 due to C-H stretching vibration. The absorption peak at 1250.16 cm−1 revealed that the

polysaccharide-peptide APPII possessed a sulfate radical. In the study of Zhang _et al_.61, the FT-IR spectrum of _A_. _auricular_ polysaccharide also displayed a band in the region of

3411.1 and 3410.5 cm−1 corresponding to the hydroxyl stretching vibration of the polysaccharide and at 2923.3 and 2921.3 cm−1 corresponding to a weak C–H stretching vibration at 1258 cm−1

due to an asymmetrical S=O stretching vibration. Although there were similarities the spectra of the aforementioned polysaccharides were not identical. With regard to its effect on lipid

metabolism, an aqueous extract of _A_. _polytricha_, which was composed of phenolics, tannins and polysaccharides, was found to reduce lipid storage, inflammation, proinflammatory cytokines

and oxidative stress in rats provided with a high-fat diet containing 10% lard. Upon completion of the treatment which lasted for 12 weeks. It was observed that changes brought about by the

high-fat diet including the hepatosomatic index, serum activities of aminotransferases, triglyceride, serum levels of free fatty acid, total cholesterol, high density lipoprotein

cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, antioxidant vitamins C and E, malondialdehyde, and the proinflammatory cytokines interleukin-6 and

tumor necrosis factor-alpha, were at least partially reversed by treatment with the _A_. _polytricha_ polysaccharide-peptides62. Alterations in hepatic triglyceride, cholesterol,

malondialdehyde, antioxidant vitamins E and C, and antioxidant enzymes were also attenuated62. Intake of a _Ganoderma lucidum_ preparation enriched in polysaccharide-peptides and

triterpenoids by human subjects restored the hepatic condition to normal from the initially mild fatty liver as evidenced by abdominal ultrasonography and reduction in plasma activities of

aminotransferases63. However, it is known that phenolics and tannins also displayed similar activities64. Thus, the aforementioned actions of the aqueous extract of _A_. _polytricha_62 were

probably attributed to each of its constituents: phenolics, tannins and polysaccharides, and the action of the _Ganoderma lucidum_ preparation63 was partly due to triterpenoids65,66. In the

present investigation both APPI and APPII exhibited a lipid lowering effect _in vitro_ as well as _in vivo_. In the _in vitro_ system employing ethanol and a mixture of palmitic and oleic

acids to induce hepatotoxicity, the resulting elevated level of intracellular triglyceride and activity of extracellular aspartate and alanine aminotransferase were suppressed by APPI and

APPII, and the decreased cellular superoxide dismutase activity was upregulated by the two polysaccharide-peptides. APPI and APPII increased the viability of the hepatocytes. It appeared

that APPII was more potent in inhibiting triglyceride accumulation in the hepatocytes. However, this was not true regarding the effects on hepatocyte viability, superoxide dismutase

activity, and aminotransferases released into the culture medium. APPI had a more potent effect on _Adipor 2_, _CPTl_, and _PPARα_ whereas APPI had a higher activity on _AMPK_ and _ACOX1_.

In the _in vivo_ system, the high fat diet brought about a rise in the circulatory titers of triglyceride, total cholesterol, low density cholesterol, aspartate aminotransferase and alanine

aminotransferase but a decline in plasma high density cholesterol. APPII was more potent than APPI in its antihypercholesterolemic and antihypertriglyceridemic and LDL lowering activities.

APPI was more potent than APPII in raising plasma HDL cholesterol activity. APPI and APPII were similar in their activity in preventing hepatic triglyceride accumulation and secretion of

aspartate aminotransferase and alanine aminotransferase into the blood stream. The hepatocytes after treatment with APPI and APPII were similar in appearance under the microscope.

Polysaccharides from _A_. _auricula_ very closely similar to _A_. _polytricha_ also reduced the serum concentrations of total cholesterol and low-density lipoprotein cholesterol in mice

receiving a diet enriched in cholesterol and improved lipoprotein lipase activity and total antioxidant capacity67. A water-soluble crude polysaccharide from _A_. _auricular_ mycelia grown

under solid-state fermentation lowered serum concentrations of triglyceride, total cholesterol, and low-density lipoprotein cholesterol in a high fat diet induced hyperlipidemic mice60. _A_.

_auricular_ polysaccharides simulated hydrolysates from the dried fruiting bodies induced a decline in the serum concentrations of triglyceride and low-density lipoprotein cholesterol

without affecting high-density lipoprotein cholesterol and total cholesterol in streptozotocin -induced diabetic rats59. The alcohol extract of _A_. _auricula_-judae provided to mice on a

high fat diet reduced plasma lipids and hepatic enzymes and downregulated expression of adipogenic/lipogenic genes (PPARγ, C/EBPα, FAS) in 3T3-L1 cells68. An alcohol extract of _A_.

_auricula_ enriched in polyphenolics lowered serum total cholesterol and upregulated high-density lipoprotein cholesterol level and fecal bile acid excretion. However, it was devoid of any

action on serum low-density lipoprotein cholesterol and triglycerides and fecal neutral cholesterol excretion59. The effects of _Auricularia cornea_ polysaccharides and enzymatic-extractable

polysaccharides displayed antioxidant and reactive oxygen species scavenging activities _in vitro_ and exerted a protective action on alcohol-induced liver pathology in mice. The

polysaccharides produced their hepatoprotective effects by suppressing lipid peroxidation, facilitating alcohol metabolism, and downregulatiing the expression of inflammatory mediators and

preventing the alcohol-induced histopathological alterations69. Adiponectin is anti-inflammatory and insulin-sensitizing adipokine. Adiponectin deficiency linked with an inflammatory state

is present in obesity, severe chronic hepatitis C-related steatosis, non-alcoholic fatty liver diseases such as hepatic steatosis and non-alcoholic steatohepatitis, and cancer70,71,72,73,74.

Adiponectin reduces insulin resistance, prevents excess hepatic lipid accumulation, inhibits inflammation and fibrosis, and exerts a hepatoprotective action75,76,77. It also protects

against cerebrovascular, cardiovascular, and chronic renal diseases7,71,72,76,77,78,79,80. It is a target in the therapy of obesity, cardiovascular and inflammatory ailments, nonalcoholic

steatohepatitis, diabetes and neurodegenerative diseases7,76,78,79,80. Efforts are made to upregulate adiponectin by therapeutic medications and/or changes in lifestyle77. Mushroom extracts

exhibit a stimulatory effect on adiponectin production. An elevation of the circulatory adipokine level, decline in levels of markers associated with hepatic damage, and activities of key

enzymes catalyzing fatty acid biosynthesis, together with attenuation of liver enlargement and triglyceride accumulation, were observed in _db/db_ mice after consumption of ethanol-soluble

extract of _Panellus serotinus_81,82. Supplementation with _Agaricus blazei_ reduces insulin resistance and improves circulatory adiponectin level in patients with type 2 diabetes Murill83.

β-glucan-rich polysaccharides derived from _Pleurotus sajor-caju_ upregulated adiponectin expression84. The ethyl acetate fraction of _Hericium erinaceus_ downregulated lipopolysaccharide

-elicited decline of adiponectin mRNA in 3T3-L1 fat cells in coculture with RAW264 macrophages85. An aqueous extract of _Antrodia cinnamomea_ reversed the action of a high-fat diet on body

weight gain, inflammatory cytokines and adiponectin production in mice86. Adiponectin promotes fatty acid oxidation in skeletal muscle79. The purified polysaccharide- peptides acquired from

_A_. _polytricha_ act via genes affect fatty acid oxidation. Peroxisome proliferator-activated receptors (PPARs) are metabolic regulators of lipid and lipoprotein levels which are divided

into α, β/δ and γ subtypes. The PPAR-α agonists suppress triglyceride levels. PPAR-γ agonists demonstrate potent hypoglycemic but weaker triglyceride lowering activity. PPAR-α/δ agonists

display antihyperglycemic, and triglyceride lowering activities. They are promising for the therapy of atherogenic dyslipidemias and NAFLD87. PPARs are paramount to energy homeostasis of the

entire body and as fatty acids sensors in several human lipid metabolic diseases88. Extracts, polysaccharides and other compounds from mushrooms inhibit fat production by acting via PPAR γ

and/or PPAR α. This study is the first to reveal the action of mushroom polysaccharide peptides through PPAR α. The present report on the hepatoprotective and fatty liver alleviating

activities of purified polysaccharide-peptides acquired from _A_. _polytricha_ and elucidation of the mechanism involved represents the first of its kind on purified polysaccharide-peptides.

The _A_. _polytricha_ polysaccharide peptides are of considerable interest and promising for development into therapeutic agents in view of the array of health-enhancing activities of

adiponectin. METHODS MATERIALS AND REAGENTS _A_. _polytricha_ fruiting bodies were collected in Beijing, China, and were ground to produce a fine powder. Monosaccharides (D-mannose,

D-glucose, D-glucuronic acid, L-rhamnose, D-xylose, D-fructose, D-galacturonic acid, D-galactose and D-arabinose), DEAE-cellulose, MTT, oleic acid and palmitate were products of

Sigma-Aldrich (USA). Superdex-200 column was obtained from General Electric Company (GE, USA), and HepG2 cell line was purchased from ATCC (American Type Tissue Culture Collection).

Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin solution, penicillin and streptomycin were products of

Invitrogen (USA). The assay kits for protein content, TG, ALT, AST, and SOD were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province, China). The other

chemicals and solvents employed were of analytical reagent grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). EXTRACTION AND ISOLATION OF POLYSACCHARIDE-PEPTIDES

Crude polysaccharide-peptides were extracted from _A_. _polytricha_ following published procedures41. Briefly, the dry powder was extracted thrice, for 4 h each time, by employing 40

volumes of hot water (90 °C). The extracts were combined and concentrated in a rotary evaporator. The concentrated solution was deproteinated using Sevag reagent (chloroform and n-butanol,

4:1 vol:vol)89. Ethanol (100%) was added to the deproteinated solution which was then allowed to stand at 20 °C overnight, and the precipitated polysaccharide-peptide (APP) was obtained by

centrifugation. Subsequently, a solution of the APP in distilled water was fractionated on a DEAE-cellulose column (1 cm × 30 cm) equilibrated with distilled water. The column was eluted

sequentially with 0, 0.2 and 1 mol/L NaCl solution successively at a flow rate of 2.0 mL/min. The unadsorbed peak D1 and adsorbed peak D2, with the carbohydrate content measured with the

phenol-sulfuric acid method, were enriched in polysaccharide. The D1 and D2 fractions were collected, concentrated, and further fractionated on an FPLC-Superdex 200 10/300 column in 0.2 

mol/L NH4HCO3 (pH 8.5) buffer using an AKTA Purifier (GE Healthcare), respectively to obtain bioactive polysaccharide-peptides APPI and APPII. CHARACTERIZATION OF BIOACTIVE

POLYSACCHARIDE-PEPTIDES ANALYSIS OF MONOSACCHARIDE COMPOSITION Monosaccharide compositions of APPI and APPII were determined with a gas chromatography mass spectrometer (GC-MS). The sample

was hydrolyzed following the method of Yu _et al_.90. FT–IR ANALYSES The IR spectra of APPI and APPII were obtained by using a Fourier transform infrared spectrophotometer (Nicolet iS5 FTIR

Spectrometer, USA) within the wave number range of 4000 to 400 cm−1. The dried sample was ground with spectroscopic grade potassium bromide (KBr) powder and then pressed into 1- mm pellets.

DETERMINATION OF MOLECULAR WEIGHT The purity and molecular weights of APPI and APPII were assessed with HPGPC technique using HPLC on a TSK GMPWXL column. The samples were injected and

eluted at 0.6 mL/min with 0.1 mol/L NaNO3 and 0.05%NaN3 as the mobile phase. N-TERMINAL AND INNER AMINO ACID SEQUENCE ANALYSIS The polysaccharide-peptides band excised from the SDS-PAGE gel

was transferred to a polyvinylidenedifluoride (PVDF) membrane by Western blotting and then stained with Coomassie brilliant blue R-250. Analysis of the stained band was performed with the

automated Edman degradation method91. The polysaccharide-band of SDS-PAGE gel was recovered and dispatched to Tsinghua University (Beijing, China) for partial amino acid sequence analysis.

Sequence homology with known sequences was searched using the BLAST/NCBI database. ASSAY FOR THE PROTECTIVE EFFECTS OF APPI AND APPII ON HEPATOCYTES PREPARATION OF MODEL OF HEPATOCYTE

INJURY-INDUCED BY ETHANOL AND A MIXTURE OF PALMITIC AND OLEIC ACIDS FFA and ethanol were considered as the pathogenetic factors which induce hepatocyte injury. FFA used was a mixture of

palmitic and oleic acids, prepared according to the method described by Garcia _et al_.92. The hepatocyte injury model was established as follows. HepG2 (liver cancer) cells were cultured in

Dulbecco’s Modified eagle medium (DMEM) medium containing 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin and 100 mg/L streptomycin, at 37 °C in a humidified atmosphere of 5% (v/v)

CO2. Cells were then seeded onto 96-well plates at a concentration of 8 × 103 cells/well, and allowed to incubate for 12 h before addition of FFA and ethanol. Incubation was then conducted

for 24 h. Afterwards, the MTT assay was performed to measure cell viability. As a control, PBS instead of pathogenetic factors (FFA and ethanol) was added to the wells. PROTECTIVE EFFECTS

AND MECHANISMS OF APPI AND APPII ON HEPATOCYTES To determine hepatoprotective effects, injured HepG2 cells were treated with APPI and APPII at various concentrations for 48 h. Upon

termination of the incubation, viability of the cells was measured with the MTT assay. The optimal doses, obtained by using cell viability determination, were considered as effective

concentrations to analyze the mechanism of APPI and APPII. HepG2 cells were seeded onto 6-well plates at a concentration of 3 × 104 cells/well, for preparation of the injury models before

addition of APPI and APPII at optimal doses. Following incubation for 48 h, the cells and culture medium in all treatment groups were collected to evaluate the repairing ability.

Intracellular protein, cellular TG and the activities of SOD from cells were quantified using commercial assay kits, and the activities of ALT and AST in the culture medium were assayed with

colorimetric assay kits, all in accordance with the manufacturers’ protocols. Histological analysis of cellular lipid was determined with the Oil-red O stain method. GENE EXPRESSION

ANALYSIS To analyze the therapeutic pathways of APPI and APPII, the expression levels of lipid metabolism genes in the adiponectin pathway were determined by qRT-PCR. Extraction of total RNA

from the samples was conducted by using TRIzol reagent (Invitrogen, USA) and reversely transcribed with oligo (dT) using EasyScriptTM First-Strand cDNA Synthesis SuperMix (Transgen, China),

in accordance with the manufacturer’s protocol. qRT-PCR was performed following the protocol of the Maxima SYBR Green/ROX qRT-PCR Master Mix (Fermentas, USA) employing an ABI 7500 (Applied

Biosystems, USA). The GAPDH RNA level was used as an endogenous control for mRNAs. The qRT-PCR procedure comprised pre-denaturation at 95 °C for 5 min and 40 cycles which consisted of

exposure to 95 °C for 30 seconds, and to 60 °C for 1 min. The relative expression level was computed by the 2-ÄÄCt method. Five independent experiments were performed. PROTECTIVE EFFECTS OF

APPI AND APPII AGAINST FAT-INDUCED FATTY LIVER IN RATS EXPERIMENTAL ANIMALS AND DIETS Male Wistar rats (220–280 g) obtained from the Institute of Laboratory Animal Science, Chinese Academy

of Medical Sciences, were utilized in this study. All experimental protocols were approved by the University Safety Office and Animal Experimentation Ethics Committee at The Chinese

University of Hong Kong and China Agricultural University. All animal experiments were carried out in accordance with the approved guidelines of the Animal Care and Use Committee of The

Chinese University of Hong Kong and China Agricultural University. The normal control group was fed with SLD, while the other groups received a HFD including 2% cholesterol and 25% pig fat.

ANIMAL GROUPING AND EXPERIMENTAL DESIGN Rats were randomly allocated in seven groups with six rats in a group. The normal control group was fed SLD, while the other six groups received a HFD

including 2% cholesterol and 25% pig fat for eight weeks. The six HFD groups were treated as follows: (i) the model group (no treatment), (ii) low-dose APPI group (50 mg/kg/d), (iii)

high-dose APPI group (100 mg/kg/d), (iv) low-dose APPII group (50 mg/kg/d), (v) high-dose APPII group (100 mg/kg/d), (vi) positive group of simvastatin treatment (2 mg/kg/d). APPI, APPII and

simvastatin were administered intragastrically for 4 weeks after an 8-weeks HFD model foundation. Rats in the normal control group and model control group an equal volume of normal saline

instead. Free access to food and water was allowed. The rats were weighed weekly, and fresh food was provided daily. After the final drug treatment, the animals were sacrificed by cervical

dislocation, and blood was collected for biochemical analysis. The liver was removed and weighed. A small piece of the liver was fixed in 10% buffered formalin solution for histological

processing, and the remainder was frozen at −80 °C till biochemical analysis. HISTOLOGICAL ANALYSIS AND BIOCHEMICAL EVALUATION The hepatic tissues were preserved in 4% paraformaldehyde at 4 

°C for 24 h before embedding in paraffin for sectioning. The tissue sections were prepared and stained with hematoxylin and eosin (H&E). Hepatic histology was observed using an Olympus

microscope, IX71 (Olympus, Japan). Liver tissue was homogenized using phosphate buffer saline. Following centrifugation at 10,000 r/min for 40 min at 4 °C, the supernatant was saved for

biochemical evaluation. TC and TG levels as lipid indexes, and ALT and AST activities as hepatic injury indexes were assayed using commercial kits following the manufacturer’s instructions.

STATISTICAL ANALYSIS All experiments in all of the bioassays were conducted in triplicate. Data are shown as mean ± standard deviation (SD). Statistical analysis was conducted with one-way

analysis of variance (ANOVA) using SPSS software (SPSS 16.0 software package, USA). Data were assessed by using ANOVA and P ≤ 0.05 was considered to be statistically significant. CONCLUSION

The present report on the hepatoprotective and fatty liver alleviating activities of purified polysaccharide-peptides acquired from _A_. _polytricha_ and elucidation of the mechanism

involved represents the first of its kind on purified polysaccharide-peptides. The _A_. _polytricha_ polysaccharide peptides are of considerable interest and promising for development into a

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are grateful to grant from Collaborative Innovation Center of Beijing Academy of Agricultural and Forestry Sciences (KJCX201915) and Beijing Academy of Agriculture and Forestry Science

(KJCX20170205). AUTHOR INFORMATION Author notes * Shuang Zhao, Shuman Zhang and Weiwei Zhang contributed equally. AUTHORS AND AFFILIATIONS * Institute of Plant and Environment Protection,

Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China Shuang Zhao, Shuman Zhang, Chengbo Rong & Yu Liu * Beijing Key Laboratory of Fruits and Vegetable Storage and

Processing, Key Laboratory of Vegetable Postharvest Processing, Ministry of Agriculture, Beijing, 100097, China Shuang Zhao * Institute of Medicinal Plant Development, Chinese Academy of

Medical Sciences & Peking Union Medical College, Beijing, 100193, China Weiwei Zhang * Beijing Xicheng District Health Care Center for Mothers and Children, Beijing, 100053, China Yi Gao

* State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, 100193, China Hexiang Wang * School of Biomedical Sciences, Faculty of

Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Jack Ho Wong & Tzibun Ng Authors * Shuang Zhao View author publications You can also search for

this author inPubMed Google Scholar * Shuman Zhang View author publications You can also search for this author inPubMed Google Scholar * Weiwei Zhang View author publications You can also

search for this author inPubMed Google Scholar * Yi Gao View author publications You can also search for this author inPubMed Google Scholar * Chengbo Rong View author publications You can

also search for this author inPubMed Google Scholar * Hexiang Wang View author publications You can also search for this author inPubMed Google Scholar * Yu Liu View author publications You

can also search for this author inPubMed Google Scholar * Jack Ho Wong View author publications You can also search for this author inPubMed Google Scholar * Tzibun Ng View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.Z. designed the purification and bioactivities studies. S.Z., Y.L. and H.W. conducted the

purification and characterization studies. S.Z., S.Z., W.Z. and Y.G. conducted the bioactivities studies _in vivo_ and _in vitro_. C.R. conducted the mechanistic studies. S.Z., H.W., J.H.W.

and T.B. Ng were in charge of the writing of the manuscript. All authors edited the manuscript and provided comments. CORRESPONDING AUTHORS Correspondence to Hexiang Wang, Jack Ho Wong or

Tzibun Ng. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

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license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zhao, S., Zhang, S., Zhang, W. _et al._ First demonstration of

protective effects of purified mushroom polysaccharide-peptides against fatty liver injury and the mechanisms involved. _Sci Rep_ 9, 13725 (2019). https://doi.org/10.1038/s41598-019-49925-0

Download citation * Received: 15 January 2019 * Accepted: 31 August 2019 * Published: 23 September 2019 * DOI: https://doi.org/10.1038/s41598-019-49925-0 SHARE THIS ARTICLE Anyone you share

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