Ganoderma lucidum polysaccharide ameliorates cholesterol gallstone formation by modulating cholesterol and bile acid metabolism in an FXR-dependent manner

Background Cholesterol gallstone (CG) disease is a worldwide common disease characterized by cholesterol supersaturation in gallbladder bile. Ganoderma lucidum polysaccharide (GLP) has been shown to possess various beneficial effects against metabolic disorders. However, the role and underlying mechanism of GLP in CG formation are still unknown. This study aimed to determine the role of GLP in ameliorating lithogenic diet (LD)-induced CG formation. Methods Mice were fed either a normal chow diet, a LD, or LD supplemented with GLP. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of genes involved in cholesterol and bile acid (BA) metabolism. The BA concentrations in the ileum were quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The microbiota in cecal contents were characterized using 16S ribosomal RNA (16S rRNA) gene sequencing. Results GLP effectively alleviated CG formation induced by LD. Specifically, GLP reduced the total cholesterol (TC) levels, increased the total BA levels, and decreased the cholesterol saturation index (CSI) in gallbladder bile. The protective effect of GLP was attributed to the inhibition of farnesoid X receptor (FXR) signaling, increased hepatic BA synthesis and decreased hepatic cholesterol synthesis and secretion. GLP also altered the BA composition in the ileum, reducing FXR-agonistic BAs and increasing FXR-antagonistic BAs, which may contribute to the inhibition of intestinal FXR signaling. Additionally, GLP improved dysbiosis of the intestinal flora and reduced the serum levels of hydrogen sulfide (H2S), a bacterial metabolite that can induce hepatic FXR, thereby inhibiting hepatic FXR signaling. Moreover, the protective effect of GLP against CG formation could be reversed by both the global and gut-restricted FXR agonists. Conclusions Taken together, GLP ameliorates CG formation by regulating cholesterol and BA metabolism in an FXR-dependent manner. Our study demonstrates that GLP may be a potential strategy for the prevention against CG disease. Supplementary Information The online version contains supplementary material available at 10.1186/s13020-024-00889-y.


Background
Cholelithiasis is a common and frequently-occurring disease worldwide.Cholesterol gallstones (CGs) account for almost 90% of the gallstones found during cholecystectomy [1].Cholecystectomy is the definitive treatment but leads to complications that seriously threaten patient's health and quality of life [2].Ursodeoxycholic acid (UDCA) is a first-line drug for the treatment of CGs in the gallbladder, but it also presents the disadvantages of long treatment duration and high recurrence rate [3].Therefore, more attention should be paid to the prevention of CG disease, and it is necessary to look for new methods to prevent CG disease.
As a BA sensor, the farnesoid X receptor (FXR), a nuclear receptor mainly expressed in the intestine and liver, plays a crucial role in regulating cholesterol and BA metabolism [12].Activation of FXR is the primary mechanism in inhibiting BA synthesis by directly inducing target genes, including intestinal fibroblast growth factor 15 (FGF15) and hepatic small heterodimer partner (SHP), which in turn reduces the expression of genes encoding BA synthetic enzymes [13].BAs are the endogenous ligands of FXR.Cholic acid (CA) and deoxycholic acid (DCA) are FXR agonists, while β-muricholic acid (β-MCA) is a FXR antagonist [14,15].Furthermore, the bacterial metabolite hydrogen sulfide (H 2 S) is shown to activate hepatic FXR [16].
Ganoderma lucidum is a traditional Chinese medicine and has been reported to possess various pharmacological effects such as anticancer, hypoglycemic, immunomodulatory and antioxidant effects [17][18][19].Ganoderma lucidum polysaccharide (GLP) is a watersoluble polysaccharide and one of the main bioactive components extracted from Ganoderma lucidum.Several studies reported that GLP could modulate the gut microbiota composition, which might be related to the prevention and treatment of diabetes and obesity [18,20].However, it is unclear whether GLP can prevent CG formation.
This study aimed to explore the impact and potential mechanism of GLP on lithogenic diet (LD)-induced CG formation.Our findings indicated that GLP effectively attenuated CG formation and cholesterol supersaturation of gallbladder bile in mice.We found that GLP decreased the levels of ileal CA and DCA, increased the levels of ileal β-MCA, ameliorated gut dysbiosis, and lowered serum H 2 S levels, which collectively inhibited LD-induced activation of intestinal and hepatic FXR signaling, thereby promoting hepatic BA synthesis and increasing cholesterol depletion.Notably, GLP also suppressed hepatic cholesterol synthesis and secretion.The protective effect of GLP against CG formation was reversed by both global and gut-restricted FXR agonists.Our study may provide a scientific basis for using GLP as a promising strategy for preventing CG disease.

Animal study
All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (approval number: 2021-0063).C57BL/6J mice (male, 6 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd.(Shanghai, China).All the mice were maintained under 12-h light/dark cycles at 20-22 °C and 45 ± 5% humidity, and had access to food and water ad libitum.Mice were allowed to acclimate for 1 week before experiments.The normal chow diet (LAD 0011) and LD (TP 28900) were both purchased from Trophic Diet (Nantong, China).The LD contained 15% fat, 1.25% cholesterol, and 0.5% CA.GLP was purchased from Ci Yuan Biotechnology Co., Ltd.(Shanxi, China) and the purity of polysaccharide from Ganoderma lucidum was 98%.The LD pellets were first broken to the powder form, next, GLP was added in the LD powder to the finial concentration of 2% or 4%, and then mixed well and reshaped them from powder to pellets in the sterile condition.Fexaramine was purchased from Biochempartner Co., Ltd.(Shanghai, China) and GW4064 was purchased from Selleck (Houston, TX, USA).
In the GLP intervention study, 40 mice were randomly divided into 4 groups (n = 10 per group and n = 5 per cage) and fed with (i) a normal chow diet (ND group); (ii) a lithogenic diet (LD group); (iii) a LD supplemented with low-dose GLP (2%; GLPL group); and (iv) a LD supplemented with high-dose GLP (4%; GLPH group), all for 6 weeks.The body weights of mice were recorded once a week.
At the end of experiments, mice were fasted overnight before being euthanized.Blood samples were collected and centrifuged to obtain the serum sample.Tissues including gallbladder, liver, ileum and cecum contents were obtained and then stored at − 80 °C for further analysis.

Analyses of biliary, serum and hepatic lipids and calculation of cholesterol saturation index (CSI)
The levels of total cholesterol (TC) and total BAs in gallbladder bile and levels of TC, total triglyceride (TG) and total BAs in serum were measured using commercially available kits (Jiancheng Bioengineering Institute, Nanjing, China).The level of total PLs in gallbladder bile was measured according to the manufacturer's instruction of the kit (Wako Pure Chemicals, Osaka, Japan).The CSI of gallbladder bile was calculated according to Carey's critical table [25].Hepatic lipids were extracted by the Folch method as previously described [26].The hepatic levels of TC, TG and total BAs were measured using kits from Jiancheng Bioengineering Institute, as described.

Histology of liver tissue
The liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4-µm slices and then stained with hematoxylin and eosin (H&E) for histological analysis.Hepatic lipid accumulation was measured by Oil Red O staining as previously described [27].The positively stained area was quantified by ImageJ software and results were expressed as a percentage of the total area of a high-power field.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA in liver and ileum tissues was extracted using TRIzol (Invitrogen, CA, USA).The quantity and purity of RNA were determined by a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).Total RNA was reverse transcribed using the PrimeScript ™ RT reagent kit (Takara Biotechnology, Japan), and then the SYBR ® Premix Ex Taq ™ reagent kit (Takara Biotechnology, Japan) was used to perform qPCR.The mRNA levels of target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2 −ΔΔCt method and the relative expression levels were shown as fold changes relative to the control group.The sequences of primers are listed in Table 1.

BA analysis
BA concentrations in the ileum tissues were measured using negative electrospray liquid chromatographytandem mass spectrometry (LC-MS/MS).The 47 kinds of BA standards were weighed accurately and prepared as standard solutions through serial dilution using methanol.The ileum samples were weighed precisely (25 ± 5 mg), homogenized in 50 μL of internal standard working solution and 350 μL of extraction mixture (methanol/water, 4:1), and centrifuged at 13,000×g at 4 °C for 15 min.The supernatant was dried with nitrogen, re-dissolved with 100 μL of 50% acetonitrile, vortexmixed for 30 s, and centrifuged at 13,000×g at 4 °C for 15 min.The supernatant was then ready for LC-MS/MS.Chromatographic separation was achieved on an Acquity ultraperformance liquid chromatography (UPLC) BEH C18 column (internal dimensions: 150 mm × 2.1 mm, 1.7 µm; Waters).The temperature of the column was set to 50 °C.The injection volume was set at 5 μL.The mobile phase consisted of 0.025% formic acid and 10 mM ammonium acetate in water (solvent A), and acetonitrile/ methanol (9:1) solution (solvent B).The flow rate was set at 0.4 mL/min.The following gradient pattern was used for the separation of BAs: 0-0.5 min, 5-10% B; 0.

16S ribosomal RNA (16S rRNA) gene sequencing
Total bacterial genomic DNA from cecal contents was extracted using QIAamp DNA Stool Kit (Qiagen, Germany) according to the manufacturer's guidelines.The V3-V4 regions of the bacterial 16S rRNA genes were amplified with specific primers by PCR.Amplicons were extracted from 2% agarose gel, purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), then quantified by QuantiFluor ™ -ST (Promega, USA) and sequenced on the Illumina MiSeq platform (Illumina, San Diego, USA).
The raw Illumina fasta files were demultiplexed, quality filtered, and analyzed using the Quantitative Insights into Microbial Ecology (QIIME, v1.9.1) platform with the following criteria: (i) reads were truncated at any site receiving a quality score of < 20, with the truncated reads shorter than 50 bp being discarded; (ii) only sequences that overlap longer than 10 bp were assembled according to their overlapped sequence, and reads that could not be  Sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs) according to sequences analysis using UPARSE v7.1 [28].The taxonomy of each OTU representative sequence was analyzed using the Ribosomal Database Project (RDP) Classifier v2.2 against the 16S rRNA gene database using a confidence threshold of 0.7 [29].Alpha diversity index including Ace, Chao, Shannon, Sobs, and beta diversity index (principal coordinate analysis (PCoA)) were calculated with QIIME.

Serum H 2 S and FGF15 detection
The level of serum H 2 S was determined by spectrophotometry as previously described using NaHS (2.5-200 μM) as a standard [30].The level of serum FGF15 was measured using a sandwich ELISA Kit (Bioswamp, Wuhan, China) according to the manufacturer's instruction.

Statistical analysis
Statistical analysis was undertaken using GraphPad Prism v7.0 (La Jolla, CA, USA).All data were presented as the mean ± standard error of the mean (SEM).For data that showed a normal distribution, significant differences were identified by one-way analysis of variance (ANOVA) for multiple comparisons.For data with a skewed distribution, significant differences were identified by Kruskal-Wallis test for multiple comparisons.Statistical significance was indicated by p values < 0.05.All the bar plots were generated with GraphPad Prism v7.0.

GLP attenuated CG formation and metabolic disorders in LD-fed mice
LD-induced CG model in mice was used to investigate the impact of GLP on CG formation.The gallbladders of mice in the ND group did not have CG formation and were filled with clear bile.In contrast to the ND group, CGs formed in 90% (9/10) of the mice in the LD group.With GLP supplementation, the incidence of CG formation was significantly reduced to 50% (5/10) and 30% (3/10) in the GLPL group and GLPH group respectively (Fig. 1A, B).The grading criteria described previously was adopted to grade CGs [31].As shown in Fig. 1C, GLP remarkably decreased the grade of CGs in LD-fed mice in a dose-related manner.There were no obvious differences in body weight gain among the four groups (Additional file 1: Fig. S4A).We further analyzed lipid composition in gallbladder bile.Compared with the ND group, biliary TC level and CSI were increased markedly in the LD group, whereas GLP supplementation noticeably reduced biliary TC level and CSI in a dose-dependent manner.GLP also significantly increased total biliary BA level.However, total biliary PL level was not changed significantly by GLP supplementation (Fig. 1D, E and Additional file 1: Fig. S4B).These results demonstrated that GLP could attenuate LD-induced CG formation and biliary cholesterol supersaturation.
Metabolic syndrome (MS) has been demonstrated to be associated with the incidence of gallstones [32].As shown in Fig. 1F, G, LD-fed mice exhibited obvious accumulation of lipid droplets in the liver, whereas GLP alleviated hepatic steatosis and lipid deposition dosedependently.Furthermore, GLP significantly decreased TC and TG levels both in the liver and serum, which were increased by LD consumption (Fig. 1H, I).These data indicated that GLP supplementation could improve LD-induced metabolic disorders.

GLP inhibited intestinal FXR-FGF15 and hepatic FXR-SHP signaling
It is widely known that the FXR signaling pathway plays a crucial role in regulating hepatic metabolism and is closely associated with CG formation.Thus, to further determine whether this pathway is involved in the beneficial effect of GLP on CG formation, FXR and its downstream molecules in both ileum and liver were detected.As shown in Fig. 2A, B, the expression of FGF15 was found to be increased in distal ileum of LD-fed mice at both the mRNA and protein levels, while GLP supplementation significantly decreased both the mRNA and protein levels of FGF15.Consistent with these findings, serum FGF15 level was higher in the LD group compared to the ND group, while GLP supplementation effectively reversed the upregulation (Fig. 2C).
Intestinal-derived FGF15 is secreted into the portal vein and circulates to the liver, where it binds to fibroblast growth factor receptor 4 (FGFR4) and acts to inhibit the synthesis of BAs from cholesterol [13].As shown in Fig. 2D, hepatic FGFR4 mRNA expression was increased in parallel with the increased ileal FGF15 mRNA and protein expression and serum FGF15 levels in LD-fed mice, while this upregulation was effectively reversed upon GLP supplementation.Additionally, both the mRNA and protein levels of SHP in the liver were significantly elevated in the LD group compared to the ND group, while GLP supplementation dramatically reduced its expression (Fig. 2E, F).
Taken together, these data suggested that GLP supplementation downregulated FXR-FGF15 signaling pathway in the intestine and FXR-SHP signaling pathway in the liver, both of which were activated by LD consumption.

GLP modulated synthesis and transport of BAs and cholesterol
Consistent with the activation of FXR induced by LD, we found that hepatic mRNA expression of CYP7A1, CYP8B1 and CYP7B1 were significantly decreased by LD consumption compared to the ND group.However, GLP supplementation significantly upregulated the mRNA expression of CYP7A1 and CYP7B1 but did not significantly affect the mRNA expression of CYP8B1.There was no significant difference in hepatic CYP27A1 mRNA expression among groups (Fig. 3A).Moreover, western blotting analysis confirmed that GLP supplementation effectively reversed the LD-induced reduction of hepatic CYP7A1 and CYP7B1 protein expression (Fig. 3B).Consistent with the increased BA biosynthesis from cholesterol, hepatic TC level was reduced by GLP supplementation (Fig. 1H).BSEP and MRP2 are hepatic BA efflux transporters.We observed that mice fed the LD showed higher BSEP mRNA levels compared to NDfed mice, but GLP did not significantly alter the mRNA expression of BSEP in the liver.Hepatic MRP2 mRNA  3C).Moreover, hepatic mRNA expression of ABCB4, a major phospholipid transporter, did not show significant differences among groups (Additional file 1: Fig. S5).
To further explore the role of cholesterol metabolism in liver, we detected the expression of genes involved in hepatic cholesterol synthesis and efflux.SREBP2 and SCAP are key regulators in cholesterol synthesis.GLP supplementation dramatically downregulated LD-induced mRNA expression of SREBP2 in the liver.However, hepatic SCAP mRNA expression was not significantly affected by GLP (Fig. 3D).Western blotting analysis further confirmed that GLP significantly inhibited LD-induced SREBP2 protein expression in liver (Fig. 3E).Moreover, both the mRNA and protein levels of ABCG5 and ABCG8, important cholesterol efflux transporters, were dramatically increased in the LD group compared to the ND group, while GLP significantly reversed the expression of these genes (Fig. 3F, G).These results were consistent with the reduction of cholesterol level in gallbladder bile by GLP supplementation (Fig. 1D and Additional file 1: Fig. S4B).
Taken together, these data indicated that GLP promoted BA synthesis while inhibiting cholesterol synthesis and efflux, which may contribute to the reduction of CSI in bile.

GLP reduced FXR-agonistic BAs and increased FXR-antagonistic BAs in ileum
BAs act as endogenous signaling molecules that bind to FXR and modulate BA and cholesterol metabolism [12].To determine the underlying mechanism by which GLP inhibits intestinal FXR signaling, we analyzed BA profiles in the distal ileum, where 95% of BAs are reabsorbed.The top 18 most abundant BA species and concentrations in the ileum were showed in Fig. 4A-C.The levels of total conjugated BAs and unconjugated BAs increased in the LD group, while GLP supplementation significantly reduced their levels (Fig. 4D).Notably, we found that the levels of CA, taurocholic acid (TCA), DCA and taurodeoxycholic acid (TDCA) were significantly elevated, but the β-MCA level was decreased in the ileum of LDfed mice.Interestingly, GLP supplementation reversed these alterations (Fig. 4A, B).These findings suggested In addition, the total BA levels in liver and serum were also measured, showing that hepatic BA level was increased and serum BA level was reduced by GLP supplementation (Additional file 1: Fig. S6A, B).

GLP reduced serum H 2 S level by improving microbiota dysbiosis
A previous study has demonstrated that Desulfovibrionales, along with its metabolic product H 2 S, can induce hepatic FXR and inhibit BA synthesis, thereby promoting CG formation [16].In order to further explore the potential mechanism by which GLP inhibits hepatic FXR signaling, we analyzed the intestinal microbiota using 16S rRNA gene sequencing.Alpha diversity indices, including Ace, Chao, Shannon and Sobs, were used to describe species richness, diversity and evenness.As shown in Fig. 5A, these indices exhibited significant decreases in LD-fed mice, while GLP supplementation significantly increased these indices.PCoA revealed that microbiota structure of LD-fed mice was distinct from that of NDfed mice, whereas GLP supplementation had a significant impact on the microbiota structure in LD-fed mice (Fig. 5B).
Next, we examined the gut microbiota structure at different levels.Figure 5C showed the top 7 most abundant phyla and relative abundances.LD-fed mice exhibited a lower abundance of Bacteroidota and higher abundances of Desulfobacterota and Proteobacteria compared to the ND group.However, GLP supplementation reversed these alterations (Fig. 5C, D).Previous studies have reported the beneficial impact of Bacteroidota on obesity [33], and the significant correlation between Proteobacteria and liver steatosis  [34].The top 20 most abundant families and relative abundances were showed in Fig. 5E.At the family level, the relative abundances of Muribaculaceae and Lactobacillaceae, which play important roles in ameliorating metabolic disorders [33], were significantly decreased in the LD group, however, GLP supplementation significantly increased the abundance of Muribaculaceae.Conversely, the LD group exhibited significant increases in the abundances of Desulfovibrionaceae and Erysipelotrichaceae, while GLP reduced their abundances (Fig. 5E, F).Desulfovibrionaceae and Erysipelotrichaceae have been reported to be associated with metabolic disorders and may be the potential causes of obesity [35].Notably, the relative abundances of the order Desulfovibrionales and the family Desulfovibrionaceae were increased in the LD group, while GLP reversed the upregulation (Fig. 5E, F and Additional file 1: Fig. S7A, B).In addition, GLP significantly decreased serum H 2 S levels, which were increased by LD consumption (Fig. 5G).
These results indicated that GLP may alleviate LDinduced activation of hepatic FXR signaling by improving gut microbiota dysbiosis, particularly by decreasing the relative abundances of the order Desulfovibrionales and the family Desulfovibrionaceae, and reducing serum H 2 S levels.

FXR activation attenuated the protective effects of GLP on CG formation
The results presented above suggested that GLP may improve CG formation by inhibiting intestinal and hepatic FXR signaling in LD-fed mice.To further confirm the beneficial effects of FXR inhibition by GLP, we conducted a follow-up recovery experiment using LD-fed mice treated with GLP (4% in LD), and GLP coupled with 100 mg kg −1 d −1 fexaramine, a intestine-specific FXR agonist [36], and GW4064, a non-selective FXR agonist [36], for 6 weeks.Fexaramine and GW4064 markedly reversed the protective effect of GLP against CG formation (Fig. 6A).The incidence and grade of gallstones were significantly increased by fexaramine and GW4064, but the increase was more remarkable with GW4064 (Fig. 6B,  C).There were no significant differences in body weight gain among groups (Additional file 1: Fig. S8A).Subsequently, we analyzed lipid composition in gallbladder bile and observed that both fexaramine and GW4064 significantly reversed the decrease in TC levels and the increase in total BA levels induced by GLP, resulting in an increase in the CSI of gallbladder bile (Fig. 6D, E and Additional file 1: Fig. S8B).Furthermore, GLP-induced reduction in serum TC level was significantly reversed by fexaramine and GW4064 (Fig. 6F).Importantly, the effects observed with GW4064 were more pronounced than those observed with fexaramine, suggesting that it was the global FXR inhibition that contributed to the improvement of CG formation by GLP, rather than solely intestinal FXR inhibition.Furthermore, we investigated the impact of fexaramine and GW4064 on FXR signaling in the ileum and liver.As shown in Fig. 6G-I, both fexaramine and GW4064 significantly increased the mRNA and protein expression of FGF15 in ileum as well as the serum concentration of FGF15, which were reduced by GLP supplementation.Hepatic FGFR4 mRNA expression was upregulated in parallel with the increased ileal FGF15 mRNA and protein expression and serum FGF15 levels with fexaramine and GW4064 intervention (Fig. 6J).In addition, both the mRNA and protein data indicated that hepatic SHP expression was markedly upregulated by GW4064, but not by the intestine-specific FXR agonist fexaramine (Fig. 6K, L).
In summary, these findings suggested that GLP attenuated CG formation through an intestinal and hepatic FXR-dependent mechanism.

Discussion
In the current study, we found that GLP supplementation effectively attenuated LD-induced CG formation in mice.GLP supplementation resulted in a reduction in the TC levels and an increase in the total BA levels, while also decreasing the CSI in gallbladder bile.Furthermore, GLP supplementation downregulated the FXR-FGF15 signaling pathway in the intestine and FXR-SHP signaling pathway in the liver, leading to enhanced hepatic BA synthesis and increased cholesterol depletion.Additionally, GLP supplementation reduced hepatic cholesterol synthesis and secretion.Mechanistically, GLP inhibited FXR signaling by (i) reducing the levels of FXR-agonistic BAs such as CA and DCA, while increasing the level of FXR-antagonistic BA (β-MCA) in the ileum; (ii) ameliorating LDinduced gut microbiota dysbiosis and reducing the serum level of H 2 S, a known hepatic FXR inducer [16].
The LD-induced mouse CG model has been widely utilized due to its simplicity, high success rate, good repeatability and easy observability.The LD is mainly characterized as high cholesterol levels, as excessive GLPH: high-dose Ganoderma lucidum polysaccharide; PCoA: principal coordinate analysis; H 2 S: hydrogen sulfide intake of cholesterol is a significant factor for CG formation.In 1964, Tepperman et al. [37] first discovered that incorporating a combination of cholesterol and CA in feed could effectively establish the mouse model of gallstones, suggesting the necessity of CA for gallstone formation.Since then, researchers have induced CG formation in mice by feeding them a high-fat, high-cholesterol diet with a certain proportion of CA [38,39].Based on previous studies, we employed a diet consisting of 15% fat, 1.25% cholesterol, 0.5% CA to induce a mouse CG model for investigating the impact of GLP on CG formation and exploring the potential mechanisms.
The occurrence of CGs is associated with metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease.There is a certain correlation between the development of CG diseases and metabolic disorders [40,41].For instance, the activation of hypoxia inducible factor 1α subunit (HIF1A) in steatotic liver contributed to CG formation [42].In patients with diabetes, hepatic insulin resistance (IR) could promote CG formation by upregulating the expression of hepatic ABCG5 and ABCG8 [43].Numerous studies have demonstrated that GLP can improve obesity, hyperlipidemia and hepatic steatosis [44,45].In addition, GLP has been shown to have hypoglycemic effects and can be used for the prevention and treatment of diabetes [46].Our study found that GLP not only reduced the CG formation but also alleviated hepatic steatosis and dyslipidemia in LD-fed mice.
The abnormal metabolism of BAs and cholesterol is the main cause of CG formation [40].FXR is well known for its role in regulating BA, carbohydrate and lipid metabolism [47].Intestinal FXR-FGF15 and hepatic FXR-SHP signaling are the main negative regulators of BA synthesis and are critical for the maintenance of BA homeostasis [12].When FXR is activated, it downregulates the expression of genes encoding BA synthetic enzymes in liver, thereby reducing BA synthesis.Our research found that GLP decreased the expression of FGF15 in ileum and SHP in liver, indicating that GLP could inhibit LDinduced activation of intestinal and hepatic FXR.Consequently, GLP increased the expression of CYP7A1 and CYP7B1 in liver, promoting BA synthesis from cholesterol and increasing cholesterol depletion.Hepatic hypersecretion of cholesterol is an important contributor to CG formation [40].Overexpression of sterol efflux transporters ABCG5/G8 increases the biliary cholesterol level, thus increasing the risk of cholesterol crystal precipitation and CG formation [16,41].Studies have showed that variants of ABCG5/G8 are associated with CGs in various populations [48].Our results showed that GLP decreased the expression of SREBP2, ABCG5 and ABCG8 in liver, suggesting that GLP can inhibit hepatic cholesterol synthesis and reduce the secretion of cholesterol into bile.Consistent with these findings, GLP also reduced the cholesterol levels in the liver and gallbladder bile.These results suggested that GLP can inhibit intestinal and hepatic FXR signaling, regulate BA and cholesterol metabolism, and attenuate LD-induced biliary cholesterol supersaturation.
As endogenous ligands of FXR, BAs play crucial roles in regulating various physiological processes in the host [49].Most BAs are reabsorbed in the terminal ileum.Dietary fibers have been shown to interact with BAs in the intestine.This interaction may partially prevent BAs from being reabsorbed into the enterohepatic circulation, resulting in an increase in BA excretion [50].In our study, we observed that the levels of CA and DCA were increased in the terminal ileum of LD-fed mice.This increase may be attributed to the high concentration of CA in the feed, which can be converted into DCA by intestinal bacteria.However, GLP supplementation resulted in decreased levels of CA and DCA in the ileum.This decrease may be due to the interactions between GLP and BAs in the intestine, leading to reduced reabsorption of BAs.The decrease of intestinal BA reabsorption is considered to be the main mechanism underlying the cholesterol-lowering effect of dietary fiber [51].In vivo studies showed that ingestion or administration of dietary fibers could increase the excretion of BAs in feces [52], leading to a reduction in cholesterol by diversion to BA synthesis [53][54][55].Our results showed that GLP upregulated the expression of CYP7A1 and CYP7B1 in the liver to promote BA synthesis, which may be due to increased BA excretion in feces.Consistent with the enhanced hepatic BA synthesis and reduced intestinal BA reabsorption, our data showed that GLP supplementation significantly increased hepatic levels and reduced serum levels of total BAs.In addition, we also found that GLP could increase the level of β-MCA in the ileum.As reported, β-MCA can prevent the formation of experimental CGs in mice [56].These results revealed that GLP reduced the levels of FXR-agonistic BAs and increased the levels of FXR-antagonistic BAs in the ileum, which may contribute to the inhibition of intestinal FXR signaling upon GLP supplementation.
There is a potential link between gut microbiota dysbiosis and the formation of CGs [57,58].Previous research has shown that alteration of indigenous gastrointestinal microbiota through bacteria transferring can induce CG formation in germ-free mice [57].Studies have revealed that GLP can improve the dysbiosis of gut microbiota [18,20].In our study, we observed that GLP supplementation alleviated the reduced-richness and diversity of intestinal microbiota and modulated the gut microbiota structure at different levels in LD-fed mice.Specifically, GLP decreased the abundances of bacteria that are detrimental to metabolism, such as Proteobacteria at the phylum level, Desulfovibrionales at the order level, and Desulfovibrionaceae and Erysipelotrichaceae at the family level.Conversely, GLP increased the abundances of bacteria that are beneficial to metabolism, such as the phylum Bacteroidota and the family Muribaculaceae.It is worth noting that the order Desulfovibrionales and the family Desulfovibrionaceae are types of bacteria that can reduce sulfate to H 2 S [59], and produce endotoxins [60].H 2 S can damage intestinal mucosa and disrupt intestinal barrier function, which may contribute to the development of intestinal inflammation and cancer [35].Endotoxin can trigger an immune response upon entering the host circulatory system, which may be the potential etiology of chronic diseases induced by high-fat diet (HFD) [35].A recent study [16] found that patients with CGs had an overabundance of Desulfovibrionales, and introducing Desulfovibrionales to gallstone-resistance mice could lead to gallstone formation.The increased abundance of Desulfovibrionales in the gut microbiota resulted in the production of H 2 S, which could activate hepatic FXR and suppress the expression of CYP7A1 and BA synthesis.This mechanism is believed to contribute to gallstone formation [16].Our study showed that LD-fed mice had higher levels of Desulfovibrionales and serum H 2 S, but these changes were reversed upon GLP supplementation.These results suggested that GLP can alleviate the dysbiosis caused by LD and drive a shift in gut microbiota composition towards a healthy condition.In addition, GLP also reduced serum H 2 S levels, which may be responsible for the inhibition of hepatic FXR signaling upon GLP supplementation.
The recovery study using FXR agonists further confirmed the important role of FXR signaling in the protective effect of GLP against CG formation.In our study, both the global FXR agonist GW4064 and the gut-restricted FXR agonist fexaramine markedly reversed the protective effect of GLP, leading to increased biliary CSI and promoting gallstone formation.In addition, we also found that both GW4064 and fexaramine counteracted the lowering effect of GLP on serum cholesterol.Notably, the reversal effect was more pronounced with GW4064 intervention compared to fexaramine.These findings demonstrated that both intestinal and hepatic FXR signaling are crucial for the protective effect of GLP against formation.
Considering that ABCG5/G8 expression can be regulated not only by FXR [61,62] but also by liver X receptor α (LXRα) [63,64], whether GLP ameliorates CG formation by regulating additional FXR-independent pathways is still unclear.In our study, the protective actions of GLP are primarily attributable to its regulation of the FXR signaling pathway.Although our findings suggest a new strategy for the prevention against CG formation, there are differences in BA composition between humans and mice [65] and additional research is needed before translation into clinical practice.

Conclusions
In conclusion, our study demonstrated that GLP effectively inhibited CG formation by suppressing intestinal and hepatic FXR signaling.The decrease in ileal CA, DCA, the increase in ileal β-MCA, the decrease in the abundance of Desulfovibrionales and concomitant decrease in serum H 2 S may be responsible for the inhibition of FXR by GLP, leading to increased hepatic BA synthesis.Additionally, GLP inhibited hepatic cholesterol synthesis and secretion.These alterations in BA and cholesterol metabolism resulted in a reduction in cholesterol supersaturation of gallbladder bile and a lower incidence of CGs by GLP.Based on these findings, GLP shows promise as a potential preventive strategy for CG disease.
ATA CGG GCT GAT TCG Reverse TCT ACA TCC TCC ACC ATC CTG AAC G FGFR4 Forward GGT GAC CGA GGA TGA TGT GAT GAA G Reverse AGA GAC AGC CAG CAG GAC CTTG SHP Forward CAG GTC GTC CGA CTA TTC TGT ATG C Reverse TAC CGC TGC TGG CTT CCT CTAG CYP7A1 Forward GTA TGC CTT CTG CTA CCG AGT GAT G Reverse GTG CGT CTT AGC CTT CTC CAT GTC CYP8B1 Forward TGG ACA GCG TGA TGG AGG AGAG Reverse GGT GGA TCT TCT TGC CCG ACTTG CYP27A1 Forward AAG GAC CAC CGA GAC CAC AAGG Reverse GGA GGG TTT CAG GCA GCC AATC CYP7B1 Forward TTG GCT TCC TTA TCT TGG CAT GGC Reverse TCG CTG ATA ATC GGC TGC TGAAC BSEP Forward GAG TTG GTG TGG CTG TCC TTA TCC Reverse ATC TGG TCG GCA ATG GCT TCATC MRP2 Forward CCT ATT CCT GCC TGT TCT TCG TCT C Reverse TCT GCT TCT TGG TCA ATC CGT GTG SCAP Forward TAC AGC AGC AGC AAC ACA GTGAC Reverse ACA GGA AGC GGT ACA GGT GAGG SREBP2 Forward CAA AGA AGG AGA GAG GCG GAC AAC Reverse GGC GGA GAC ATC AGA AGG ACA TTC ABCG5 Forward CAT CGC AGC CCA ACT CCT TCAG Reverse CCA CAG AAC ACC AAC TCT CCG TAA G ABCG8 Forward GCT CGT GTG GTT GGT GGT CTTC Reverse GTT GCC GAT TTG TGT GGT GTA AAG G GAPDH Forward AGG TCG GTG TGA ACG GAT TTG Reverse TGT AGA CCA TGT AGT TGA GGT CA assembled were discarded; and (iii) exact barcode matching, with 2 nucleotide mismatches in primer matching.

Fig. 1
Fig. 1 GLP ameliorated LD-induced CG formation and metabolic disorders.A Representative gallbladders and gallstones.B Incidence of gallstone.C Grades of experimental CGs.D Concentrations of TC, BA and PL in gallbladder bile.E CSI of gallbladder bile.F Representative images of H&E and Oil Red O staining of liver tissue sections.Scale bars, 50 μm.G Percentage of Oil Red O positive area.H Hepatic TC and TG levels.I Serum TC and TG levels.Data are shown as the mean ± SEM (n = 10).*p < 0.05, **p < 0.01.ND: normal diet; LD: lithogenic diet; GLPL: low-dose Ganoderma lucidum polysaccharide; GLPH: high-dose Ganoderma lucidum polysaccharide; CG: cholesterol gallstone; TC: total cholesterol; BA: bile acid; PL: phospholipid; CSI: cholesterol saturation index; H&E: hematoxylin and eosin; TG: triglyceride

Fig. 2
Fig. 2 GLP downregulated intestinal FXR-FGF15 and hepatic FXR-SHP signaling.A The mRNA expression of FXR and FGF15 in the ileum (n = 8).B The protein expression and quantification of FGF15 in the ileum (n = 6).C Serum FGF15 level (n = 10).D, E The mRNA expression of FGFR4, FXR and SHP in the liver (n = 8).F The protein expression and quantification of SHP in the liver (n = 6).The mRNA expression and protein expression were normalized to GAPDH and shown as fold changes relative to control group.Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01.ND: normal diet; LD: lithogenic diet; GLPL: low-dose Ganoderma lucidum polysaccharide; GLPH, high-dose Ganoderma lucidum polysaccharide

Fig. 3
Fig. 3 GLP promoted BA synthesis and inhibited cholesterol synthesis and efflux in liver.A The mRNA expression of CYP7A1, CYP8B1, CYP27A1 and CYP7B1 in the liver (n = 8).B The protein expression and quantification of CYP7A1 and CYP7B1 in the liver (n = 6).C, D The mRNA expression of BSEP, MRP2, SCAP and SREBP2 in the liver (n = 8).E The protein expression and quantification of SREBP2 in the liver (n = 6).F The mRNA expression of ABCG5 and ABCG8 in the liver (n = 8).G The protein expression and quantification of ABCG5 and ABCG8 in the liver (n = 6).The mRNA expression and protein expression were normalized to GAPDH and shown as fold changes relative to control group.Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01.ND: normal diet; LD: lithogenic diet; GLPL: low-dose Ganoderma lucidum polysaccharide; GLPH: high-dose Ganoderma lucidum polysaccharide; BA: bile acid

Fig. 4
Fig. 4 GLP changed the BA composition in the ileum.A-C The top 18 most abundant ileal BA species are shown.D Concentrations of total conjugated BAs and unconjugated BAs in the ileum.Data are shown as the mean ± SEM (n = 6).*p < 0.05, **p < 0.01.ND: normal diet; LD: lithogenic diet; GLPH: high-dose Ganoderma lucidum polysaccharide; BA: bile acid

Fig. 6
Fig. 6 FXR activation reversed the protective effects of GLP in LD-fed mice.A Representative gallbladders and gallstones.B Incidence of gallstone.C Grades of experimental CGs (n = 10).D Concentrations of TC, BA and PL in gallbladder bile (n = 10).E CSI of gallbladder bile (n = 10).F Serum TC level (n = 10).G The mRNA expression of FGF15 in the ileum (n = 8).H The protein expression and quantification of FGF15 in the ileum (n = 6).I Serum FGF15 level (n = 10).J, K The mRNA expression of FGFR4 and SHP in the liver (n = 8).L The protein expression and quantification of SHP in the liver (n = 6).The mRNA expression and protein expression were normalized to GAPDH and shown as fold changes relative to control group.Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01.LD: lithogenic diet; GLP: Ganoderma lucidum polysaccharide; CG: cholesterol gallstone; TC: total cholesterol; BA: bile acid; PL: phospholipid; CSI: cholesterol saturation index

Table 1
Primers for qPCR