Soluble dietary fiber of Lentinus edodes was according to Wu Liping’s method (12).

30 male SPF-grade KM mice (4 weeks old, body weight 20 ± 2 g, No. SCXK2019-0004) were obtained from Hunan Slake Kingda Laboratory Animal Co.

All animal experiments were conducted following the guidelines of the Experimental Animal Ethics Committee of Hunan University of Traditional Chinese Medicine (approval number: LL2023032901). Mice were housed in an environmentally controlled room maintained at a temperature of 20 ± 2°C, humidity of 40–60%, and a light–dark cycle of 12 h. After acclimatization to one week of feeding under specific pathogen-free conditions, the mice were randomly divided into 5 treatment groups (n = 6). The first group (N) was fed a normal diet (23.07% protein, 11.85% fat, 65.08 carbohydrates). The second group (F) was fed a high-fat diet (basal diet with 15 percent lard, 20 percent sucrose, 1.2 percent cholesterol, and 0.2 percent sodium cholate). The third group (FL) was fed high-fat chow, and 150 mg/kg of water-soluble dietary fiber from Lentinus edodes. The fourth group (FM) was fed high-fat chow, and 300 mg/kg of water-soluble dietary fiber from Lentinus edodes. The fifth group (FH) was fed high-fat chow, and 450 mg/kg of water-soluble dietary fiber from Lentinus edodes.

Euthanasia of mice was typically performed using methods that ensure minimal pain and distress. We used the carbon dioxide (CO₂) inhalation method, where mice were exposed to a gradual fill of CO₂ in a chamber to induce unconsciousness and death. This method was often followed by a secondary physical method to confirm death, such as cervical dislocation or decapitation. Injectable anesthetics like pentobarbital were also be administered to induce a deep state of anesthesia before death. All methods comply with ethical guidelines and regulations set by institutional and governmental bodies to ensure humane treatment.

Freshly collected liver tissues from all groups of mice were sliced and soaked in 10% formalin overnight, then gradient dehydrated and embedded in paraffin wax, after which the liver tissue blocks were cut into 6 μm thick sections and stained with hematoxylin and eosin (H&E) staining for morphological observation. The liver condition was evaluated by the balloon like change in NASH-CRN scoring standard (17).

Spleen and thymus tissues were freshly collected from each group of mice, weighed, and the corresponding organ index was calculated using the formula (18):

The serum concentrations of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Serum total bile acids (TBA), Alanine aminotransferase (ALT), Gamma-glutamyltransferase (γ-GT), Aspartate aminotransferase (AST), Acid phosphatase (ACP) contents of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in liver were analyzed using commercial kits (Konodi Bio Ltd.)

Fresh liver tissue samples of each experimental group were taken, and immediately frozen with liquid nitrogen. After the tissue sample was ground into fine powder with a precooled mortar and pestle, 100 mg of fine powder was quickly weighed. The RNA in the sample was extracted in strict accordance with the instructions of the universal RNA extraction kit. After the 5 μL RNA sample was diluted 10 times with ddH₂O, the OD260 value of RNA was determined. After the concentration of RNA in the sample was unified, the RNA sample was reverse transcribed into cDNA in strict accordance with the instructions of the Takara reverse transcription kit, and stored at −20°C (19).

0.5 μL of upstream and downstream primers, 2.0 μL of cDNA sample and 2.0 μL of ddH₂O were added into the octuple, respectively. The solution was mixed and centrifuged at low speed for 30 s. 5 μL of SYBR enzyme was added into the octuple under dark conditions. The solution was mixed and centrifuged at low speed for 30 s. With GADPH as the internal reference, AMPKα, SREBP2, PPARα, CPT1A, ACS and CYP7A1 were determined by fluorescence quantitative PCR. The CQ value was automatically calculated by the system, and the relative gene expression was calculated by 2^−ΔΔ method. The base sequence and product size of the mouse derived gene primers used are shown in Table 2.

Chromatographic data were processed using the software ASTRA6.1. The CQ value of gene expression related data was automatically calculated by the built-in software, and the relative gene expression was calculated by 2^−ΔΔ method. All data were presented as mean ± standard deviation, and analyzed by SPSS 22.0 software. Independent sample T-test was used to analyze the differences between the two groups. p < 0.05 indicates a significant difference between the two groups, and p < 0.01 indicates a very significant difference between the two groups.

The extracted Lentinus edodes soluble dietary fiber was a light yellow powder with a soft texture and small particles (Figure 1). Preliminary physicochemical analysis of the extracted Lentinus edodes soluble dietary fiber showed that the extracted Lentinus edodes soluble dietary fiber contained saccharides and alditric acid, whereas there was no starch and amino acid (Table 3).

The molecular weight distribution of the soluble dietary fiber, In the molecular weight analysis chart (Figure 2), the red line represents the multi-angle laser light scattering signal (i.e., LS, in V), and the intensity of scattered light is proportional to the molecular size and molecular weight of the substance; the blue line represents the difference signal (i.e., RI, in RIU). The response value depends on the change of the outflow refractive index after the column and is related to the type, concentration and molecular weight; the black line is the molecular weight fitted by the two signals. Where the inorganic salt phase around 37 min is the solvent peak of the mobile phase. The molecular weight distribution is shown in Table 4. After analysis, the average molecular weight of soluble dietary fiber of Lentinus edodes was 8.356 kDa, the peak molecular weight Mp is 13.124 kDa, the weight Mw is 17.019 kDa, and Z Mz is 36.781 kDa.

The chromatographic qualitative was mainly based on the retention time of the target compounds on the analytical column. For elution conditions (gas/liquid), different compounds on the retention ability of analytical column is different, there are many differences, column is mainly through analytical column separation of different compounds, reuse ultraviolet detector, evaporation light detector, differential detector, electrochemical detector, conductance detector, generally can be through the peak time to determine the target compound. Figure 3A shows the ion chromatogram of the standard sample, and Figure 3B shows the ion chromatogram of the extracted mushroom soluble dietary fiber. After comparison, it was found that the monosaccharides in the soluble dietary fiber of the extracted Lentinus edodes were mainly galactose, glucose and mannose.

Using a scanning electron microscope (SEM) on the surface microstructure of mushroom soluble dietary fiber, SEM photos of different magniations of mushroom soluble dietary fiber are shown in Figure 4, at 500× and 1,000×, the soluble dietary fiber of Lentinus edodes showed an irregular network shape (Figures 4A,B), higher power with a clearer display as shown in panels Figures 4C,D, the sample surface is uneven, an irregular geometric shape, with a distinct fibrous structure, tightly connected, with a small gap.

The soluble dietary fiber of Lentinus edodes and potassium bromide were pressed into bromide and placed into the Fourier infrared spectrophotometer to determine the infrared absorption spectroscopy at 400–4,000 cm⁻¹ (Figure 5). The scan spectrum is shown in Figure 6. According to the atlas, the characteristic peak frequency area at 3,353.03 cm⁻¹ in the range of 4,000–650 cm⁻¹ is the absorption peak generated by the stretching vibration of sugar intra-molecular or intermolecular O–H, At 2,921.68 cm⁻¹ is the absorption peak generated by the C–H expansion vibration, at 1,393.37 cm⁻¹ is the C=O expansion vibration peak, 1,151.61 cm⁻¹ for C–O–C expansion vibration, 1,012.72 cm⁻¹ is the telescopic vibration of the C–O, 711.87 cm⁻¹ for the absorption peak generated by the β-substitution, Show that the presence of the β-glycosidic bond in the sample. This infrared map verified the presence of alcoic acid in the soluble dietary fiber of Lentinus edodes, the monosaccharides are mainly connected by β-glycosidic bonds.

Before the start of the experiment, the fur of each group of mice was white, smooth, and shiny, the skin was light pink, and the reaction was sensitive. The feces were oval shaped and moderately moist and soft before the experiment. As the experiment progressed, the fur of Group F mice gradually became disheveled, withered, yellow and dull, their skin appeared slightly dull and pale, their bodies were obese, and their feces were wet and oily. The fur of mice in the FL group, FM group, and FH group, which added soluble dietary fiber from Lentinus edodes, showed varying degrees of disorder, with the disorder degree being FH > FL > FM, but none of them lost their luster. Striped fibers were clearly visible in the feces, and the feces were dry and hard, with a lower water content than the normal group. The body size of mice in each group was F > FL > FH > FM > =N (Figure 6A).

Weigh the mice at designated locations every 4 days and record their weight changes. The trend of weight changes in each group of mice is shown in Figure 6B. After adaptive feeding, there was no significant difference in the initial weight of mice in each group (day 0). As the experimental days increased, the weight of mice in group F rapidly increased. On day 28, the weight reached 48.87 ± 3.08 g, which was significantly different from group N and FM (p < 0.01). On the 4th, 8th, 12th, 16th, 20th, 24th, and 28th days of the experiment, compared with the beginning of the experiment, the weight growth rate is shown in Figure 6C. At the end of the experiment, compared with the 0th day, the weight growth rate of each group is F > FL > FH > FM > N, which is consistent with the observed body size. There is a significant difference between the F group and the N and FM groups (p < 0.01), and there is also a significant difference between the FL and FH groups and the N and FM groups (p < 0.01). Indicating that soluble dietary fiber from Lentinus edodes can to some extent slow down weight gain caused by high-fat diets.

Organ coefficient, also known as organ to body ratio, is the ratio of the weight of a certain organ in an experimental animal to its body weight. The ratio of various organs to body weight is relatively constant under normal circumstances. When organs undergo pathological changes, the weight of the damaged organs changes, and the organ coefficient also changes accordingly. From Figure 7, it can be seen that there is no significant difference in the spleen coefficient and thymus coefficient among the experimental groups, indicating that there are no abnormalities in the immune organs spleen and thymus. However, there is a significant difference in the liver coefficient between group F and group N (p < 0.01), indicating that the liver of mice fed a high-fat diet for a long time has lipid accumulation, which may have caused lesions. Although the liver coefficients of the FL group, FM group, and FH group slightly increased, there was no significant difference compared to the N group, indicating that there were no organic changes in the liver tissue.

It can be seen from Figures 9C–F that the high-fat diet led to the decrease of liver SOD, GSH and cat levels compared with group N (p < 0.01), the transformation ability of SOD, GSH and cat was weakened, and the MDA level was significantly increased compared with group N (p < 0.01), the damage of hepatocyte membrane was aggravated. The levels of SOD, GSH, cat and MDA in the liver of FL group, FM group and FH group added with Lentinus edodes soluble dietary fiber were all decreased, and there was no difference between FM group and FH group and N group (p > 0.05), indicating that a certain amount of Lentinus edodes soluble dietary fiber can effectively improve the oxidative stress caused by high-fat diet.

Liver is an important organ involved in lipid metabolism, in which there are many enzymes and transcription factors related to lipid metabolism. The effect of Lentinus edodes soluble dietary fiber on the expression of genes related to liver lipid metabolism in mice fed with high fat diet is shown in Figure 10. The high-fat diet led to a decrease in the expression of PPAR-α, ACS and CPT1a genes in the liver of mice, and an increase in the expression of AMPKα and SREBP-2 genes, with no significant effect on the gene expression of CYP7A1. Lentinus edodes soluble dietary fiber intervened in high-fat diet, increased the expression of PPAR-α, ACS, CPT1A and CYP7A1 genes in the liver of mice, and significantly decreased the expression of AMPKα and SREBP-2.

Monosaccharide composition and glycosidic bonds of molecules have important effects on the function of soluble dietary fiber. Studies have shown that the soluble dietary fiber in fungi is mainly composed of galactose, glucose and mannitol in different proportions (21). Studies have also shown that arabinose, xylose, fucose, ribose, rhamnose, etc. are also detected in the soluble dietary fiber in fungi (10, 22, 23). The soluble dietary fiber extracted from Lentinus edodes in this study is mainly composed of galactose, glucose and mannitol, which is mainly connected by β-glycosidic bonds. Schulthess and other researchers have shown that glucose and mannitol can be used by anaerobic bacteria in the intestine to metabolize a large amount of acetic acid, which is conducive to reducing the proportion of pathogenic bacteria and maintaining the integrity of the intestinal barrier. Galactose, glucose and mannitol in Lentinus edodes soluble dietary fiber have a good regulatory effect on intestinal flora, can affect the secretion of intestinal SCFAs, and maintain the integrity of intestinal barrier. Compared with α-glycosidic bond, β-glycosidic bond is more likely to form a vertical conformation. Studies have shown that dextran linked by β-(1,4) glycosidic bond is more prone to intramolecular aggregation, resulting in reduced solubility, while β-(1,3) bond in glycosidic bond can reduce the polymerization, making dextran in a dissolved state in the body, which is more likely to be used and fermented by intestinal microorganisms (24). It has been reported that the main chain of lipid-lowering polysaccharides from edible fungi (Pleurotus ostreatus and Pleurotus ostreatus) is β-(1,3)-glucan, and contains a certain amount of β-(1,3)-glucose side chain. The glycosidic bond of Lentinus edodes soluble dietary fiber is β-glycosidic bond. The related structure may play the physical role of adsorbing lipids or bile salts in vivo, regulating intestinal flora and increasing the content of intestinal SCFAs.

The molecular weight (Mw) also plays an important role in the efficacy of soluble dietary fiber. When the molecular weight of soluble dietary fiber is too large, it will be difficult for organisms to absorb and utilize because the molecular weight of the substance is too large to pass through the cell membrane. When the molecular weight of soluble dietary fiber is too low, it will be difficult to form active substances, resulting in the reduction of physiological activity. The weight average molecular weight of soluble dietary fiber from Lentinus edodes is 17.019 kDa, and its molecular weight is uniform. Studies have shown that (25–28), the biological activity of polysaccharides is negatively correlated with the relative molecular weight. Some researchers isolated five kinds of Polysaccharides from Lycium barbarum, of which IBPA 4 with a molecular weight of 10.2 kDa showed good anti-cancer activity, while ibp-p 8 with a molecular weight of 6.5 × 103 kDa did not show anti-cancer activity. It may be due to the difference in the three-dimensional structure corresponding to the relative size of molecular weight, resulting in the difference in physiological activity.

The liver plays an important role in lipid synthesis, metabolism, and transport processes (29). When the balance between energy intake and consumption is broken, the lipid metabolism of the liver is abnormal, and the lipids in the liver cells begin to accumulate (30, 31). According to the data released by the China Center for Disease Control and prevention, the overweight and obesity rates of all age groups in China are increasing rapidly. At present, about 19% of Chinese adolescents aged 6–17 are overweight and obese, while the proportion among adults is more than 50%, higher than the international level (32). A large of studies (33–35) have found that dietary fiber can reduce fat and weight, prevent obesity, improve insulin resistance, reduce the absorption of food fat, inhibit the synthesis of cholesterol in the liver, and reduce the accumulation of fat in the liver. Long term high-fat diet made the weight and liver coefficient of mice in Group f the highest among the five experimental groups. After the experiment, it was found that the isolated liver of mice fed with high-fat diet was significantly white. H&E staining showed that the liver cells were disordered, and the levels of ALT and AST in the liver were significantly increased. Further detection of the lipid level in the liver of mice fed with high-fat diet showed that the levels of FFA, TG and TC in the liver were significantly increased. All of the above indicate that high-fat diet leads to liver damage and lipid metabolism disorder. However, the liver of mice fed with high-fat diet supplemented with Lentinus edodes soluble dietary fiber was still bright red, and the levels of alt, AST, FFA, TG and TC in the liver were all corrected. The lipid accumulation in the liver was reduced, and the liver lipid was significantly reduced, which confirmed that Lentinus edodes soluble dietary fiber had a better lipid-lowering effect. Through the comprehensive analysis of the general state, weight gain, organ changes, liver injury, lipid metabolism and oxidative stress reaction of mice fed with high-fat diet supplemented with different doses of Lentinus edodes soluble dietary fiber, it was found that daily supplementation of 300 mg/kg Lentinus edodes soluble dietary fiber could achieve the effect of lipid-lowering and liver protection.

Liver lipid metabolism disorder is usually manifested as dyslipidemia (36). The levels of TG and TC in mice fed with high-fat diet were significantly abnormal, indicating that high-fat diet led to dyslipidemia in mice. TG is the energy source of muscle and adipose tissue and one of the most critical plasma lipids in clinic. Plasma TG comes from food and liver synthesis (37–39). Fatty acids are absorbed by cell fatty acid transporters (40), and TG is synthesized by diacylglycerol o-acyltransferase and other steps (41). Because the size of chylomicrons (CM) rich in TG limits the penetration of the arterial wall, the increase of TG indirectly promotes atherosclerosis mechanisms such as inflammation, thrombosis and abnormal cell proliferation (42). Cholesterol in the blood is mainly transported by apolipoprotein binding to lipoprotein to form corresponding lipoprotein cholesterol. Low density lipoprotein plays an important role in cholesterol transport (43). After cholesterol is synthesized in the liver, it combines with LDL apolipoprotein to produce LDL-C into the blood. LDL-C is transported to the lysosome through receptor-mediated endocytosis (44). LDL-C enters the inner wall of blood vessels. After the vascular endothelial layer is modified and oxidized to oxidized low density lipoprotein cholesterol, it is engulfed by macrophages to form foam cells (45). Foam cells continue to increase and fuse, forming the lipid core of atherosclerotic plaque. The liver is the largest metabolic organ in the body. GSH is an important antioxidant in the body and can eliminate ROS (46). Therefore, the change of GSH content can be regarded as a sign of oxidative stress imbalance. When the organs are in a state of overload metabolism, a large number of ROS will be produced, accompanied by the decrease of GSH content in the liver. SOD, GSH and cat play an important biological role in organisms (47). They can transform reactive oxygen species into harmless substances and protect cells from oxidative damage through their cooperative effect. At the same time, they can also be used as indicators of the degree of oxidative stress in organisms, reflecting the health status of organisms (48). The content of MDA is an indicator of cell membrane peroxidation. The levels of GSH, SOD and cat in the liver of mice fed with high-fat diet were significantly decreased, while the level of MDA increased, while the levels of GSH, SOD, cat and MDA in the experimental group supplemented with medium and high doses of Lentinus edodes soluble dietary fiber were all corrected.

Liver is an important organ involved in lipid metabolism, in which there are many enzymes and transcription factors related to lipid metabolism. Peroxisome proliferator activated receptor α (PPARα) is a ligand activated nuclear transcription factor in the nuclear receptor superfamily (49). It can affect lipid metabolism by regulating the expression of acyl CoA oxidase (acox) and cytochrome P4504A (CYP4A), and also plays an important role in blood glucose homeostasis and insulin resistance. Acyl CoA synthase (ACS) is a large and diverse enzyme family, which can be divided into different types according to the length of fatty acid substrate chain (50). In the process of fatty acid metabolism, ACS catalyzes the occurrence of initial reaction to synthesize fatty acid CoA, thus activating fatty acid oxidation in cells. Carnitine palmityl transferase 1α (CPT1α) is a member of the carnitine palmityl transferase-1 (CPT1) family. The enzyme family is located on the outer membrane of mitochondria. It catalyzes the synthesis of fatty carnitine from fatty CoA and carnitine, and mediates the transfer of long-chain fatty CoA into the mitochondria for oxidation. The entry of fatty acid CoA into mitochondria is the rate limiting step of fatty acid β-oxidation. Therefore, CPT1α is the rate limiting enzyme of fatty acid β-oxidation. High fat diet reduced the mRNA expression of PPARα, ACS and CPT1α genes in the liver of mice, and different doses of Lentinus edodes soluble dietary fiber intervention could reduce this change (51). AMPKα, the catalytic subunit of AMPK known as “energy sensors,” plays a key role in maintaining the energy balance of AMPK and the dynamic balance of lipid metabolism. SREBP-2 is a member of the sterol regulatory element-binding protein family that mainly regulates gene expression related to cholesterol synthesis and uptake (52), therefore, downregulation of hepatic SBREP-2 expression reduces plasma cholesterol synthesis. Different doses of polysaccharide intervention significantly downregulated the mRNA expression of AMPKα and SREBP-2 genes, indicating that the mushroom soluble dietary fiber has a strong ability to inhibit de novo cholesterol synthesis (53). Liver X receptor α (LXRα) is the main nuclear receptor regulating cholesterol metabolism. It regulates the expression of cholesterol metabolism-related genes, such as SREBP-1c, FAS, CYP7A1, Acetyl-CoA carboxylase (ACC), Stearyl-CoA desaturase 1 (SCD 1), and promotes the liver lipid generation (54). Among them, CYP7A1 is the rate-limiting enzyme that catalyzes the synthesis of bile acids by cholesterol from the classical pathway (55). The cholesterol in the body is removed by the formation of bile acids under the action of CYP7A1, which plays an important role in maintaining the dynamic balance of cholesterol and bile acid levels (56, 57). The Lentinus edodes soluble dietary fiber intervention significantly upregulated CYP7A1 gene expression, indicating it could effectively promote the conversion of cholesterol into bile acid. The above results show that the soluble dietary fiber of mushroom can promote the conversion of cholesterol into bile acid, and effectively reduce the total cholesterol content of the body (Figure 11).

In summary, in the case of high-fat diet, supplementation of 300 mg/kg of Lentinus edodes soluble dietary fiber daily, which can achieve the effect of lipid-lowering and liver protection. However, it is not clear whether the Lentinus edodes soluble dietary fiber has an impact on the intestinal microecology, and whether the Lentinus edodes soluble dietary fiber further affects the lipid metabolism of the liver by affecting the “gut liver” axis, which will be the focus of our follow-up research.