Effects of Agaricus blazei Murrill polysaccharides on hyperlipidemic rats by regulation of intestinal microflora

Abstract The present research envisaged the effects of Agaricus blazei Murrill polysaccharides (ABPs) on blood lipids and its role in regulation of the intestinal microflora in hyperlipidemic rats. The acidic polysaccharide fraction of Agaricus blazei Murrill was obtained by DEAE‐cellulose ion exchange column chromatography. The sugar content of ABP was 75.1%. Compared with the model group (MG), the serum TC, TG, and LDL‐C levels decreased (p < .05 or p < .01) and the HDL‐C levels increased (p < .01) significantly in the ABP group. Expression of CYP7A1 was up‐regulated (p < .01), and that of SREBP‐1C (p < .05) was down‐regulated significantly in the liver tissue of rats in the ABP group. Additionally, the disordered hepatic lobules and the steatosis of hepatocytes were found to be significantly alleviated in the ABP group. We believe that ABP can reduce the ratio of Firmicutes/Bacteroidetes and reduce the relative abundance of Firmicutes, Ruminococcaceae_unclassified, and Ruminococcaceae, increasing the relative abundance of Proteobacteria, Clostridium_sensu_stricto, Allobaculum, Peptostreptococcaceae, Clostridiaceae_1, and Erysipelotrichaceae as targets to regulate blood lipids. The results showed ABP could regulate the dyslipidemia in rats with hyperlipidemia. The mechanism may be through the regulation of the imbalance of intestinal microflora induced by the high‐fat diet in rats, which may be one of the important ways of its intervention on the dyslipidemia induced by high‐fat diet.

attracting a good research attention. A large number of studies have shown that the natural polysaccharides presented many pharmacological activities such as antitumor, hypolipidemic, hypoglycemic, antioxidant, and immune activities, with broad application prospects Yu et al., 2013;Zhao, Qian, Yin, & Zhou, 2014).
Agaricus blazei Murrill (ABM) used in this study is a kind of medicinal and edible fungus, rich in a variety of nutrients and chemicals, such as polysaccharides, phytosterols, saponins, glycoproteins, beta-D-glucan, and other phytochemicals (Niwa, Tajiri, & Higashino, 2011). Agaricus blazei Murrill polysaccharides (ABPs), one of the main active substances in Agaricus blazei Murrill, is known to have antihyperlipidemic, antioxidant, antiradiation damage, immune, and anti-inflammatory activities (Da Silva et al., 2013). Although it has been reported in the literature that it has a role in regulating blood lipids (Wei et al., 2019), its mechanism is still unclear.
Intestinal microflora is closely related to the occurrence of hyperlipidemia. Patients with hyperlipidemia are often accompanied by the imbalance in the intestinal microflora, which aggravates the body's lipid metabolism disorder and is a vicious cycle (Shuang, Wenfei, & Haitao, 2013). In recent years, a large number of studies have shown that polysaccharides can shape the intestinal microflora to promote the growth and proliferation of the intestinal hyperlipidemia-related beneficial bacteria and inhibit those of harmful bacteria, thereby regulating and maintaining their normal physiological activities (Kaoutari, Armougom, Gordon, Raoult, & Henrissat, 2013). The products produced by intestinal microflora by degrading polysaccharides such as acetic acid, propionic acid, butyric acid, and lactic acid provide energy for the body and regulate the intestinal pH and microbial diversity, thereby playing an important role in protecting intestinal peristalsis and intestinal barrier (Jang, Ridgeway, & Kim, 2013;Okeke, Roland, & Mullin, 2014).
Whether ABP can play a hypolipidemic role through the regulation of intestinal flora needs further research.
In this study, a rat model for hyperlipidemia was established by giving a high-fat diet to rats for exploring the improvement of ABP on the hyperlipidemia in rats, and the relationship between the hypolipidemic effects of ABP from the perspective of intestinal microflora regulation was studied. The mechanism of the hypolipidemic effect of ABP was revealed, which may provide an experimental basis and theoretical basis for the development of highly processed products of Agaricus blazei Murrill.

| Materials
SPF-grade male SD rats, aged from 4 to 5 weeks and weighing 190.0 ± 2.0 g, were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. (license number was SCXK (Ji)-2016-0003). A high-fat diet was obtained from the Jilin Medical College, and the formula is shown in detail in Table 1.
During the experiments, the rats were fed with a free access to water and food, and acclimated to the environment in the laboratory for 7 days. The temperature of the animal room was 20.0°C-22.0°C, and the relative humidity was 50%-60%.

| Preparation of ABP
The fruiting bodies of ABM were crushed into pieces at room temperature, and the pieces of the fruiting bodies of ABM were prepared to obtain ABP. The fruiting bodies were sieved through 60-mesh sieve, and the impurities were removed manually.
Anhydrous ethanol was added to the powdered ABM and filtered to separate the residue, which was dried to constant weight and extracted with water. The filtrate was concentrated and subjected to alcohol precipitation using anhydrous ethanol for removal of proteins by the Sevage method (left at room temperature overnight). Subsequently, dialysis was performed for the sample with a dialysis bag (retention molecular weight was 3500D and dialyzed in distilled water for 48 hr to remove the small molecular substances). The product was freeze-dried to obtain crude ABPs, which was fractionated by chromatography on a DEAE-cellulose ion exchange column to obtain the pure ABP. The analysis of monosaccharide composition of the ABP was done by Shimadzu HPLC (Wu et al., 2014).

| Establishment of the hyperlipidemia rat model
Thirty-two SPF-grade male SD rats were randomly divided into four groups: normal control group (NG), model group (MG), positive drug group (PD), and ABP group (ABP). Rats in the NG group were fed with the general diet, and those in MG, PD, and ABP groups were fed with the high-fat diet. Rats in the PD group were daily given 8.4 mg/ kg lovastatin intragastrically once, those in ABP group were given 640 mg/kg ABP, and those in the NG and MG groups were given an equal volume of distilled water in the same way. The high-fat feeding and the administration lasted 8 weeks continuously.

| Calculations of body weight and organ index
The rats were weighed once a week during feeding, and 8 weeks later, the spleen and liver of rats were taken by dissection and washed with saline, and the wet weights of the spleen and liver were recorded for the calculation of the organ indexes as shown in Equation 1 (Khlifi et al., 2019).

| Determination of biochemical indicators
Serum preparation: After the high-fat feeding and administration for 8 weeks, the rats fasted for 12 hr with a free access to water, and then, their blood samples were collected through the abdominal aorta. The blood samples were centrifuged at 3,500 r/min for 15 min to separate the serum, and the serum samples were kept at −20°C for use. TC, TG, LDL-C, and HDL-C contents in the serum of rats were determined according to the manufacturer's instructions.

| Detection of Protein Expression by Western Blotting
One hundred milligrams of the liver tissue of each rat in all groups was added with RIPA protein lysis buffer. The liver lysis buffer solution was homogenized, and the homogenate was cracked on ice for 1 hr and then centrifuged at 12,000 r/min for 10 min at 4°C to take the supernatant. The standard curve was established using the BCA method, and the protein concentration in the liver tissue of rats in each group was measured and adjusted. The protein was separated by SDS-PAGE, in which the loading amount of each sample was 10 µl, and then transferred to a PVDF membrane. 5% skimmed milk powder blocking buffer was added onto the membrane, which was shaken at room temperature for 2 hr. After washing the membrane, the primary antibody dilution solution (CYP7A1: 1:1,000 dilution; SREBP-1C: 1:1,000 dilution; and NADPH: 1:20,000 dilution) was added onto the membrane, and the membrane was incubated at 4°C overnight. After washing the membrane, the corresponding second rabbit antibody (1:2,000) was added onto the membrane, which was incubated at room temperature for 1 hr. The membrane was washed again and was incubated in ECL luminescent liquid for the development of color; subsequently, the image was photographed and analyzed by an automatic analysis system of electrophoresis gel imaging.

| Hematoxylin-eosin (HE) staining
The liver tissue of rats was fixed in 10% formalin solution and then routinely sectioned for the preparation of slices. The slices were stained with HE, and the pathological features of the liver tissue were observed under a light microscope.

| Extraction of total DNA from fecal bacteria
The fecal samples in the rectum of rats (NG, MG, and ABP groups) were collected under aseptic condition and frozen rapidly in liquid nitrogen. The samples were stored at −80°C for use.

| PCR amplification and 16S rDNA sequencing
The V3-V4 region of the prokaryotic (bacterial and archaeal) smallsubunit (16S) rRNA gene was amplified with slightly modified versions of primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Fadrosh et al., 2014). The 5′ ends of the primers were tagged with specific barcodes per sample (1) Visceral index (%) = visceral mass (mg) animal body weight (g) × 100% and universal sequencing primers. PCR amplification was performed in a total volume of 25 µl reaction mixture containing 25 ng of template DNA, 12.5 µl PCR Premix, 2.5 µl of each primer, and PCR-grade water to adjust the volume. The PCR conditions to amplify the prokaryotic 16S fragments consisted of an initial denaturation at 98°C for 30 s; 35 cycles of denaturation at 98°C for 10 s, annealing at 54/52°C for 30 s, and extension at 72°C for 45 s; and a final extension at 72°C for 10 min.
The PCR products were confirmed with 2% agarose gel electrophoresis. Throughout the DNA extraction process, ultrapure water was used instead of a sample solution to exclude the possibility of false-positive PCR results as a negative control. The PCR products were purified by AMPure XP beads (Beckman Coulter Genomics) and quantified by Qubit (Invitrogen). The amplicon pools were prepared for sequencing, and the size and quantity of the amplicon library were assessed on Agilent 2100 Bioanalyzer (Agilent) and with the Library Quantification Kit for Illumina (Kapa Biosciences), respectively. PhiX control library (v3) (Illumina) was combined with the amplicon library (expected at 30%). The libraries were sequenced either on 300PE MiSeq runs, and one library was sequenced with both protocols using the standard Illumina sequencing primers, eliminating the need for a third (or fourth) index read. OTU abundance information was normalized using a standard of sequence number corresponding to the sample with the least sequences.

| Data analysis
Alpha diversity was applied to analyze complexity of species diversity for a sample through 4 indices, including Chao1, Shannon, Simpson, and Observed species. All these indices in our samples were calculated with QIIME (version 1.8.0). Beta diversity analysis was used to evaluate differences in samples in species complexity. Beta diversity was calculated by principal coordinates analysis (PCoA) and cluster analysis by QIIME

| Statistical methods
All values were expressed as mean ± standard deviation (x±S).
"n" was used to represent the number of samples in each group.
SPSS software (version 16.0) was used for the statistical analysis.

| Extraction and content determination of ABP
ABP was obtained by the dialysis by employing a 1000-Da dialysis bag to remove the ash content, and the yield was 4.7% relative to the raw material, of which the sugar content was 75.1%, as shown in Table 2. The monosaccharide composition was composed of 79.1% Glc and is shown in Table 3.

| Isolation and purification of ABP
ABP consisted of small amount of GlcA (2.7%), which was fractionated by ion exchange chromatography on a DEAE-cellulose column.
The acid sugar fraction was obtained with an elution containing 0.5 M NaCl solution. The yield was 47.5%, as shown in Table 4, and the monosaccharide composition is shown in Table 5.

| Effects of ABP on the body weight and organ index in rats
There was a persistent increase in the body weight of the rats in the MG group. The increasing trend of body weight of rats in the MG group was almost the same as that in the NG group, but the body weight of rats in the MG group was slightly higher than that in the NG group. On the ninth week, the body weight of rats in the MG group was significantly higher than that in the NG group (p < .05). The body weight of rats in the ABP group was significantly lower than that in the MG group (p < .01), as shown in Figure 1.
Compared with those in the NG group, the liver and spleen indexes of rats in the MG group increased significantly (p < .01). On the other hand, compared with that of the MG group, the liver index of rats in the PD and ABP groups decreased significantly (p < .05).

TA B L E 2 Composition analysis of ABP
Compared with that of the MG group, the spleen index of rats in the PD group and ABP group decreased significantly with p < .01 and p < .05, respectively, as shown in Figure 2.

| Effect of ABP on biochemical indicators
Compared with the NG group, the levels of TC, TG, and LDL-C in the serum of rats increased significantly in the MG group (p < .05 or p < .01), while the levels of HDL-C decreased significantly (p < .05 or p < .01). Compared with those in MG group, the levels of TC, TG, and LDL-C in the serum of rats decreased significantly in the ABP and PD groups (p < .05 or p < .01), while the HDL-C levels increased significantly (p < .05 or p < .01) ( Table 6).

| Effects of ABP on the protein expression of CYP7A1 and SREBP-1C
Compared with the NG group, the expression of SREBP-1C protein increased significantly in the MG group (p < .01), while the expression of CYP7A1 protein decreased significantly (p < .01). The expression of SREBP-1C protein decreased significantly (p < .05 or p < .01), and the expression of CYP7A1 protein increased significantly (p < .05) in the PD and ABP groups compared with that in the MG group ( Figure 3).

| Liver HE staining results
HE staining showed that no abnormal hepatocytes were found, the size of hepatocytes was normal, the hepatic cords were arranged neatly and orderly, and no fat vacuoles and fat infiltration were found in the hepatocytes of rats in the NG group. However, the hepatocytes with lipid-induced hepatic steatosis were significantly increased, the hepatocytes were extremely swollen and became round, the edema of hepatocytes was noted, the hepatic cords were disordered, and an obvious steatosis of hepatocytes was found in the MG group. The fat vacuoles in the hepatocytes of rats in PD and ABP groups were significantly less than those in the MG group ( Figure 4).

| Comparative study of intestinal microflora at OTU level in rats
It was found that 1,705 of 3,507 totally enriched OTUs were shared in all samples. About 48% of the OTUs were not deleted in the ABP group.
The order of species richness was as follows: ABP > NG > MG, among which the species richness in the ABP group was highest ( Figure 5).

| Alpha diversity analysis of intestinal microflora in rats
Alpha diversity is usually used to measure the species richness in the community ecology, and it is a comprehensive indicator that reflects the species richness and uniformity. Based on the statistical results of the OTU, the alpha diversity of each sample was calculated, and four diversity indexes were used to analyze the changing    that the abundance and uniformity of the ABP group were higher than those of the MG group, and the diversity of the total bacterial community in the NG group was higher (Table 7).

| Beta diversity analysis of intestinal microflora in rats
In Figure

| Comparison at the level of phylum
Nineteen bacterial floras were identified at the level of phylum in the feces of rats in NG, MG, and ABP groups (Figure 8), (p < .05) was higher than in the ABP group (Table 9).

| Comparison at the level of genus
Twenty bacterial floras were identified at the level of genus in the feces of rats in NG, MG, and ABP groups ( Figure 9, and Allobaculum (p < .01) was higher in the ABP group (Table 11).

| Comparison at the level of family
Twenty bacterial floras were identified at the level of family in the feces of rats in NG, MG, and ABP groups (Figure 10)  (p < .01), and Erysipelotrichaceae (p < .01) was higher in the ABP group (Table 13).

| D ISCUSS I ON
A hyperlipidemia rat model was established by administering the rats with a high-fat diet in our laboratory. It was found that the serum TC, TG, and LDL-C levels of rats increased significantly, the HDL-C levels decreased significantly, and the liver and spleen indexes increased significantly in the MG group. Serum TC, TG, LDL-C, and HDL-C levels are the main indicators to reflect the body's lipid metabolism (Dechesne, Musovic, Palomo, Diwan, & Smets, 2016). Increased TC and LDL-C are one of the main causes of hyperlipidemia. High-fat diet can cause the enlargement of liver, while elevated liver and spleen indexes reflect the presence of hyperlipidemia to some extent (Cho et al., 2017). Our results prove that the hyperlipidemia rat model induced by high-fat diet was successful. After the administration of ABP, the serum TC, TG, and LDL-C levels of hyperlipidemic rats decreased significantly, while the HDL-C level increased significantly, and the liver and spleen indexes decreased significantly, suggesting that ABP effectively regulated the blood lipid metabolism in hyperlipidemic rats.
Long-term high-fat diet may lead to the dyslipidemia in rats, wherein the levels of TC and TG increase in the blood of the rats.
The key enzyme involved in the metabolism of TC is 7α-hydroxylase (CYP7A1), the rate-limiting enzyme for the transformation of  showed that the administration of ABP led to an increase in the   (Tang et al., 2018) showed that Bacteroides were positively correlated with the TG levels and negatively correlated with the HDL-C levels, and were the core flora in the cause of hyperlipidemia.
Clostridium_IV belongs to the order Clostridium and is known to be a beneficial gut bacterium (Clausen & Mortensen, 1995). It was found that the abundance of Clostridium_IV was significantly reduced, which was consistent with the earlier findings. In the ABP intervention group, the harmful bacteria Ruminococcaceae_ unclassified decreased, and there are reports in the literature that Ruminococcaceae is a group of gut flora known to be directly related to obesity (Arumugam et al., 2011;Daniel et al., 2014;Velagapudi et al., 2010). It was observed that the bene-

TA B L E 11
Statistics of top 20 meaningful flora in genus the order Clostridium and can decompose polysaccharides to produce short-chain fatty acids. It is also known to promote the lipopolysaccharide absorption and inhibit the adipokines induced by intestinal hunger (Clausen & Mortensen, 1995).
Allobaculum is a gram-positive bacterium whose end products are lactic acid and butyric acid, which can improve the nonalcoholic fatty liver (Greetham et al., 2004;Pitts & Van Thiel, 1986 (Guo et al., 2008) that the Bacteroides order has a negative correlation with obese body weight, which is consistent with the results of our studies at the level of phylum and genus. The abundance of harmful bacteria Ruminococcaceae in the ABP intervention group was significantly reduced, which was consistent with the results at the genus level. The abundance of probiotics increased significantly, including that of Peptostreptococcaceae, Clostridiaceae_1, and Erysipelotrichaceae. Peptostreptococcaceae is a gram-positive and anaerobic coccus, and its main function is to regulate blood lipids (Sookoian et al., 2020). Clostridiaceae_1 belongs to the order Clostridium, which is consistent with the genus-level study. Chen et al. (2018)  This study clarified the lipid-lowering activity of ABP and its correlation with the regulation of intestinal flora, thereby laying a foundation for the research on the role and mechanism of the active ingredients of Agaricus Blazei Murrill.

| CON CLUS ION
ABP has a hypolipidemic effect, which may be related to its ability to regulate the expression of key lipid metabolism-related factors CYP7A1 and SREPP-1C. The hypolipidemic effect of Agaricus blazei Murrill polysaccharides is related to its modulation and regulation of the imbalance of the intestinal microflora structure.

ACK N OWLED G M ENTS
This research was supported by the Development and Reform

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.

E TH I C A L S TATEM ENT
The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Beihua University. All of the experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (China).