Integrated analysis of NAFLD mitigation mechanism by Ganoderma lucidum insoluble dietary fiber based on transcriptomics, metabolomics and gut microbiota

Integrated analysis of NAFLD mitigation mechanism by Ganoderma lucidum insoluble dietary fiber based on transcriptomics, metabolomics and gut microbiota | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Integrated analysis of NAFLD mitigation mechanism by Ganoderma lucidum insoluble dietary fiber based on transcriptomics, metabolomics and gut microbiota Siqi Wang, Baitong Liu, Yunxia Ma, Guochuan Jiang, Xuejun Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7067699/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Nov, 2025 Read the published version in npj Science of Food → Version 1 posted 11 You are reading this latest preprint version Abstract The study established a mouse model of NAFLD induced by a 12-week high-fat diet (HFD). Utilizing hepatic transcriptomics, 16S rDNA sequencing, and metabolomics, the study examined the mechanisms by which GIDF exerts hepatic protection and modulates lipid metabolism in NAFLD through the gut-liver axis.The results demonstrated that GIDF supplementation significantly alleviated NAFLD characteristics in mice, including reducing biomarkers associated with hepatic lipid deposition and injury, modulating lipid disorders, and improving oxidative stress. Furthermore, GIDF likely regulated lipid metabolism through pathways such as Cytochrome P450, Retinol metabolism, and Peroxisome Proliferator-Activated Receptor signaling, while increasing the expression of ileal tight junction proteins (ZO-1 and Occludin), thereby repairing the intestinal barrier.GIDF altered gut microbiota composition, promoting the growth of Lactobacillus , Blautia , Clostridium , and Akkermansia .The results from the co-administration of antibiotics and GIDF further demonstrated that the ameliorative effects of GIDF on NAFLD are dependent on the presence of gut microbiota. Metabolomic analysis indicated that GIDF upregulated levels of L-cysteine, S-adenosylmethionine (SAMe), and 5'-methylthioadenosine (MTA), while downregulating 13(S)-HODE, thereby modulating amino acid and lipid metabolism. These findings validate that GIDF alleviates NAFLD via the gut-liver axis and may serve as a promising nutritional supplement. Biological sciences/Biochemistry Health sciences/Diseases Health sciences/Gastroenterology Biological sciences/Microbiology Insoluble dietary fiber NAFLD Transcriptomics Gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of fat in the liver, accounting for 5% or more of liver weight, in the absence of alcohol consumption or other specific conditions such as autoimmune diseases, medication effects, or viral hepatitis. 1 . The literature review established that NAFLD increases the risk of Type 2 diabetes and cardiovascular diseases (CVD) 2 . NAFLD is prevalent in approximately one-quarter of the world’s population, with an incidence rate of more than 30% in South America and the Middle East 3 . Presently, there are no pharmacological treatments recommended for NAFLD in clinical settings, despite its growing prevalence 4 . The term “multiple parallel hits” refers to the major pathogenesis of NAFLD, in which various factors, including insulin resistance, adipokines, bile acids, inflammation, and gut microbiota, simultaneously contribute to cause extensive fat accumulation in the liver, leading to significant metabolic derangement. These disturbances may extend to NAFLD, likely leading to cirrhosis 5,6 . The doctrine of “multiple parallel hits” emphasizes the role played by the gut-hepatic axis. In NAFLD patients, the integrity of the intestinal mucosal layer is compromised, allowing bacterial metabolites and lipopolysaccharides (LPS) to enter the liver via the portal vein. This invasion elicits a hepatocellular immune response and the subsequent production of pro-inflammatory cytokines including TNF-α and IL-1β, thereby worsening liver inflammation 7,8 . Given these dynamics, targeting gut microbes holds the potential for NAFLD therapy 9 . In disease research, especially in studies examining the influence of gut microbiota, antibiotic clearance models have gained popularity owing to their speed and ease of manipulation, enabling broad bacterial intervention in mice and facilitating gut microbiota management 10 . Dietary fiber, widely recognized as the seventh most essential nutrient alongside carbohydrates, proteins, fats, water, vitamins, and minerals, is classified into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) depending on its water solubility 11 . Traditional view typically posits that SDF ferments more readily.However, IDF typically exists as a complex network formed by diverse fiber polysaccharides, which is fermented by gut bacteria in the colon as a polymeric matrix, rather than being degraded as single purified fiber components.Research has demonstrated that insoluble substrates foster specific microbiota types; for example, IDF derived from highland barley has been shown to elevate Akkermansia populations in the fecal matter of obese model mice 12,13 . These results indicate that IDF may serve as an effective natural agent for modulating NAFLD. Ganoderma lucidum , a member of the genus Ganoderma within the Basidiomycetes order, has been esteemed in traditional Chinese edible fungi for over 4,000 years, earning its title as the king of mushrooms 14,15 . Moreover, Ganoderma lucidum and its extracts have been used in food processing 16 . This fungus reportedly possesses several physiological characteristics such as lipid-lowering, hypoglycemic, hepatoprotective, and anticancer properties 17,18 . Previous studies have shown that the polysaccharides from Ganoderma lucidum assist in controlling the gut microbiota and managing metabolic diseases in obesity patients 14 . In addition, the polysaccharide peptide from Ganoderma lucidum has been shown to significantly reduce NAFLD by inhibiting fatty acid synthesis via the FXR-SHP/FGF pathway 19 . Recent studies on Ganoderma lucidum extracts have mainly focused on polysaccharides, triterpenes and other bioactive compounds 15,20 . However, IDF, an understudied component, may hold the potential as a dietary supplement to ameliorate NAFLD. The study established a mouse model of NAFLD induced by a 12-week high-fat diet (HFD). The study utilizing hepatic transcriptomics, 16S rDNA sequencing, and metabolomics to examine the mechanisms by which Ganoderma lucidum insoluble dietary fiber (GIDF) exerts hepatic protection and modulates lipid metabolism in NAFLD through the gut-liver axis. This process not only opened new avenues for the treatment of NAFLD, but also expanded the potential for utilizing functional fibers from edible mushrooms to improve health. 2. Materials and methods 2.1. Materials and reagents The Ganoderma lucidum applied in this experiment was obtained from Kunming Xuanqing Biotechnology Co., Ltd. (Yunnan, China), while the biochemical enzymes such as α-amylase, neutral protease, and amyloglucosidase (AMG) were sourced from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). All the required experimental kits for determining the serum levels of lipids and liver enzymes, such as TC (Total Cholesterol), TG (Triglycerides), LDL-C (Low-Density Lipoprotein Cholesterol), HDL-C (High-Density Lipoprotein Cholesterol), AST (Aspartate Transaminase), ALT (Alanine Transaminase), MDA (Malondialdehyde), CAT (Catalase), and GSH (Glutathione), were purchased from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). Moreover, enzyme-linked immunosorbent assay kits were used for the identification of inflammatory markers such as IL-1β, IL-10, LPS and TNF-α from Yibo Biotechnology Co., Ltd. (Changchun, China). Primary antibodies against ZO-1, Occludin and GAPDH for protein detection were purchased from Servicebio Biotechnology Co., Ltd. (Wuhan, China) and all other chemical reagents used in this study were of analytical grade. 2.2. Preparation of Ganoderma lucidum insoluble dietary fiber To prepare GIDF, 10.0 g Ganoderma lucidum was combined with distilled water in a 1:10 w/v ratio and treated with 0.5% α-amylase in a 60°C water bath set at pH 5.5 for 90 minutes. Subsequently, the solution was cooled to 50°C, after which 0.5% protease was added. The solution then underwent an incubation period of 60 minutes at a pH of 7.0. Subsequently, 0.2% AMG was incorporated and the reaction continued in a 55°C water bath for another 60 minutes at pH 5.0. The enzymes were then inactivated by boiling the mixture for 10 minutes. After cooling, the solution was centrifuged at 11,100×g for 20 minutes using an XR1 centrifuge (Thermo Fisher Scientific, China). The precipitated solids were washed twice with distilled water, freeze-dried using a LGJ-25D freeze dryer, and collected as the final GIDF product. 2.3. Animal Experiments Six-week-old C57BL/6 mice (number 32) were maintained at 23 ± 2°C and 60% ± 5% humidity with a 12-hour light-dark cycle (Liaoning Changsheng Biotechnology Co., Ltd.). They underwent an adaptation phase to their new environment and diet for a week. During this period, the mice had continuous access to both food and water. Animal experiments have been approved by the Animal Ethics Committee of Jilin Agricultural University (approval number: 2021-10-12-001), in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The development of the NAFLD mouse model was initiated with a high-fat diet regimen. After a week-long adaptation phase, the mice were systematically assigned into four distinct groups, each comprising eight animals (Fig. 1 ): (1) NC group, sustained on a standard diet; (2) HFD group, receiving a diet comprising 60% fat; (3) GIDF group, high-fat diet with 60% fat and 500 mg/kg BW/d GIDF suspension by gavage; and (4) AN-GIDF group, high-fat diet with 60% fat and 500 mg/kg BW/d GIDF suspension by gavage, included antibiotics in their water, specifically ampicillin at 1.0 mg/mL and neomycin at 0.5 mg/mL. The NC and HFD groups were administered with equal amounts of physiological saline solution. After 12 weeks, following an eight-hour fasting period, the mice were sacrificed via cervical dislocation under ether anesthesia, and blood was drawn from the area surrounding the orbit. Furthermore, tissue samples, including those from the liver, ileum, and the contents of the cecum, were meticulously collected, analyzed, and subsequently stored at a temperature of -80℃ to ensure preservation. 2.4. Serum biochemical index detection The blood collected from the mice was allowed to clot at room temperature for two hours and serum was extracted by centrifuging the tubes at 3000 rpm for 10 minutes. The TC,TG, LDL-C, HDL-C, AST, ALT, MDA, CAT, and GSH analyses were performed according to the manufacturer’s protocol of the assay kits. Moreover, cytokines TNF-α, IL-10, LPS, and IL-1β were quantified in the culture medium by means of the specific ELISA kits and according to the manufacturer’s recommendations. 2.5. Histological analysis In the experimental phase, liver and colon specimens from the experimental mice were perfused with 4% paraformaldehyde and left in the same medium for 48 h. These tissues were then processed through a series of graded alcohol solutions to remove water, embedded in paraffin wax, and sectioned when the paraffin had set. Each section was subsequently stained with hematoxylin and eosin (H&E) for histological examination under an optical microscope. For liver tissues, Oil Red O staining was performed using an improved protocol obtained from previously described methods 21 . The sections were respectively treated by fixing in 10% formalin solution for 10 min, washing in distilled water for 3 min, and then in 60% isopropanol for 30 seconds. The sections were stained using 0.5% Oil Red O stain in 60% isopropanol for 15 min, followed by washing in 60% isopropanol and dried distilled water, and counterstained with Mayer’s hematoxylin for two minutes to improve the visualization of cellular details. 2.6.Transcriptome analysis RNA was extracted from the liver tissue, and mRNA was isolated using Oligo (dT) Beads. Following polymerase chain reaction (PCR) amplification, magnetic beads were used to purify and recover the target products ranging from 300–600 bp for sequencing. After library construction, the libraries were initially quantified using Qubit and diluted to 1 ng/µl. The insert size of the libraries was then assessed using a Qseq100 DNA Analyzer, and their effective concentrations were precisely quantified via Q-PCR. Libraries that passed the quality control were pooled according to their effective concentrations and required sequencing data output, followed by sequencing on an Illumina NovaSeq 6000 platform with a PE150 configuration. The RNA-seq standard analysis pipeline included quality control (QC), alignment, quantification, differential expression analysis, functional enrichment, as well as alternative splicing analysis, and Gene Set Enrichment Analysis (GSEA). 2.7. Western Blot (WB) Analysis Protein extraction for western blotting was performed by modifying a previously described method 22 . For protein extraction, 250 mg of tissue was homogenized in 1 ml of protein extraction buffer. This buffer consisted of 5 µL of protease inhibitor cocktail, 5 µL of PMSF, and 5 µL phosphatase inhibitor to eliminate the activity of any enzyme. This mixture was then homogenized for several periods of 30 seconds at low speed using a homogenizer until complete disruption of the tissue was achieved. The homogenate was further centrifuged at 14,000 xg for 15 min in a pre-chilled centrifuge. Subsequently, protein concentrations were estimated using a BCA assay kit. Before immunoblot analysis, protein samples were separated by electrophoresis. The proteins were gently blotted onto a PVDF membrane. This membrane was then moved to a hybridization bag, where the appropriate concentration of primary antibody was added, and incubated overnight at 4°C. The membrane was further processed by incubating with a diluted secondary antibody for an hour, followed by application of a freshly prepared ECL mixture on the protein-exposed side of the membrane for luminescence detection. 2.8. Intestinal Flora Analysis To analyze cecal microbiota, 16S rDNA sequencing was performed on the contents extracted from the mice cecum. Genomic DNA was isolated and subsequently amplified by PCR using the primers B341F (5'-CCTACGGGGNGGCWGCAG-3') and B785R (5'-GACTACHVGGGGTATCTAATCC-3'). Following amplification, the PCR products were electrophoresed using a 2% agarose gel to facilitate the isolation of DNA fragments. The targeted segments were carefully excised from the gel, and underwent a purification process, employing the QIQuick Gel Extraction Kit. This procedure was executed strictly in accordance with the detailed instructions provided by the manufacturer to ensure optimal recovery and purity of DNA. The purified libraries were then mixed, denatured, and loaded onto an Illumina Novaseq 6000 for high-throughput parallel sequencing. 2.9.Metabolomic analysis of mouse cecum contents During the sample preparation process, an exact amount of the sample was transferred into a 2 mL tube. To this tube, 600 µL of methanol, which was augmented with 2-chloro-L-phenylalanine at a concentration of 4 ppm, was carefully added to ensure proper mixing. This mixture was then vortexed, shaken for 30 seconds, ground, and subjected to sonication for 10 min using a KQ-800DE sonicator (Shumei, China). After the initial treatment, the samples were centrifuged at an acceleration of 5000 xg within a temperature-controlled environment maintained at 4°C. The centrifugation step was carefully timed to last 10 min to ensure the effective sedimentation of particulates. Subsequently, the mixture was centrifuged at 12000 rpm for 5 min, and the clear supernatant obtained was further filtered through a 0. 22 µm filter. The filtrate was then transferred to another analytical vial, which was to be used for LC-MS analysis. The sample was prepared in the correct manner for molecular analysis. Chromatographic analysis was performed using the state-of-the-art Vanquish UHPLC System bought from Thermo Fisher Scientific, USA. This system was specifically designed for LC-MS and was used in this study to achieve high sensitivity and selectivity. The samples were effectively separated using an ACQUITY UPLC® HSS T3 column (2. 1 x 100 mm, 1. 8 µm) obtained from Waters Corporation, Milford, MA, USA; the column was held at a constant temperature of 40°C. The injection volume was maintained constant at 2 µL and the flow rate of the system at an optimal level of 0. 3 mL/min in order to ensure good separation. To identify metabolites, an Orbitrap Exploris 120 mass spectrometer coupled with an ESI ion source was used, and both were obtained from Thermo Fisher Scientific. This instrument was set to acquire both MS1- and data-dependent MS/MS scans (Full MS-ddMS2 mode), and thus provided detailed information of the metabolic profiles of the samples. 2.10. Statistical analysis The results of the experiments were expressed in terms of mean ± SD, calculated from a series of three separate experiments. All statistical computations, including analysis of variance and other relevant tests, were performed using SPSS software. This criterion was applied to discern substantial differences across various experimental setups, highlighting the impact of the tested conditions. 3. Results 3.1. GIDF effectively ameliorated HFD-induced NAFLD Elevated body weight significantly exacerbates the risk of NAFLD. A study conducted over a 12-week period where mice were subjected to HFD demonstrated a pronounced increase in their body mass compared to their counterparts sustained on a normal diet. This significant divergence in weight gain between the two groups is illustrated in Fig. 2A, underscoring the impact of dietary fat on physiological health. By the end of the 12-week period, HFD, NC, GIDF, and AN-GIDF groups exhibited weight gains of 57.84%, 29.20%, 30.66%, and 37.61%, respectively. While GIDF therapy moderated the acceleration of weight gain among subjects, distinct differences in body weight were not evident after the 12-week regimen. Moreover, as illustrated in Fig. 2B, the liver mass recorded from the HFD group after this period significantly surpassed those of the NC cohort, highlighting the propensity for high-fat diets to promote substantial fat accumulation in the liver. Conversely, the liver mass in the GIDF group was discernibly reduced relative to that in the HFD group, demonstrating the potential of GIDF to mitigate fat deposition in the liver. No statistical difference was observed between the AN-GIDF and GIDF groups in terms of liver weight, indicating that antibiotic treatment did not influence liver size. Therefore, it can be concluded that GIDF can potentially improve the features related to NAFLD in mice. Liver morphology of the mice under GIDF intervention is depicted in Fig. 2C. The liver in the NC group was red-brown and elastic. In contrast, the liver of mice in the HFD group appeared pale and oily when touched. However, the liver in both GIDF and AN-GIDF groups exhibited a restored deep red color and a less oily surface. Oil Red O staining revealed extensive red lipid droplets in the livers of HFD-fed mice, along with hepatocellular steatosis, indicating significant fat accumulation in NAFLD-induced mice. Although the quantity of red lipid droplets was lower in both the GIDF and AN-GIDF groups compared to the HFD group, the AN-GIDF group continued to retain more lipid droplets than the GIDF group. H&E staining revealed that hepatocytes in the NC group were orderly with clearly discernible nuclei. In the HFD group, hepatocytes were disorganized, with numerous vacuoles and signs of inflammatory cell infiltration. In the GIDF group, there was a notable reduction in vacuoles and an absence of inflammatory cells, whereas only a few inflammatory cells persisted in the AN-GIDF group. These histological findings confirmed that GIDF intervention alleviated hepatic fat accumulation and tissue damage in NAFLD mice. Figure 2 Effects of GIDF and AN-GIDF on body weight, liver weight, liver injury,lipids and liver function in NAFLD mice.(A)Body weight.(B)Liver weight.(C)Liver epithelium,H&E-stained and oil red O-stained sections of liver.(H,I)Serum ALT, AST levels.(D,E,F,G)Serum TC,TG,LDL-C,and HDL-C levels.** p < 0.01,*** p < 0.01,compared to the HFD group.Different letters represent significant differences( p < 0.05). 3.2. Effect of GIDF on lipid metabolism in NAFLD mice Disruptions in lipid metabolism are closely linked to the onset of NAFLD. The study revealed that, relative to the NC group, both TG and TC levels were markedly elevated in the HFD group, indicating a significant disruption in lipid homeostasis owing to high-fat dietary intake, as depicted in Fig. 2D-G. Excessive accumulation of TG and TC in the liver may precipitate liver dysfunction, leading to cellular damage and eventually lead to fatty liver disease 23 . Administration of both GIDF and AN-GIDF resulted in a significant reduction in serum TC and TG levels in mice. Elevated LDL-C levels are linked to an increased risk of CVD, whereas HDL-C serves as a protective factor against such heart conditions 23 . GIDF intervention effectively lowered LDL-C levels, while increasing HDL-C levels. Although AN-GIDF treatment reduced serum LDL-C levels, it did not significantly impact HDL-C levels. 3.3 Effect of GIDF on liver injury ,oxidative stress and inflammatory status in NAFLD mice Liver damage was evaluated by measuring enzyme levels, specifically AST and ALT, as indicators of hepatic function and integrity. When liver cells are damaged, they release transaminases into the bloodstream, leading to a marked increase in aminotransferase activity 25 . Compared to the NC group, the HFD group exhibited markedly elevated levels of AST and ALT, as depicted in Fig. 2H-I. These findings underscore significant hepatic stress in the HFD group. GIDF administration helped restore liver function to some degree, effectively regulating AST and ALT levels. Additionally, the use of antibiotics in the AN-GIDF group resulted in higher AST and ALT levels than those in the GIDF group alone. The levels of MDA, GSH, and CAT activity in the liver tissue were measured to assess oxidative stress damage in NAFLD mice. As shown in Fig. 3 A-C, the HFD group demonstrated significantly increased MDA and GSH levels and markedly reduced CAT activity. After GIDF intervention, compared to the HFD group, MDA decreased by 36.27%, CAT activity increased by 22.88%, and GSH levels increased by 73.09%. The intervention using GIDF notably reduced the concentrations of the pro-inflammatory cytokines TNF-α and IL-1β. These inflammatory markers were previously observed to be substantially elevated in the HFD group, as illustrated in Fig. 3 D and F. The effective reduction in these cytokines underscores the therapeutic potential of GIDF in controlling inflammation induced by HFD intake. GIDF intervention notably reduced the levels of IL-10, an essential anti-inflammatory cytokine (Fig. 3 E). In contrast, the levels of TNF-α, IL-1β, and IL-10 showed no significant changes in the AN-GIDF group compared to the HFD group. These outcomes indicate that GIDF intervention can effectively alleviate liver injury and oxidative stress and mitigate systemic inflammation in these mice. 3.4. Effect of GIDF on gene expression in the liver To further investigate potential mechanisms by which GIDF alleviates liver injury in NAFLD, a transcriptomic analysis was performed on mouse liver tissues. Compared with the HFD group, GIDF intervention resulted in 1,126 differentially expressed genes (DEGs), including 654 upregulated and 472 downregulated (Fig. 4 A). Enrichment analyses of these DEGs were conducted using GO, KEGG, and GSEA, with functional categories illustrated in Fig. 4 B-G. Following GIDF intervention, enriched biological functions included drug metabolism, lipid metabolism, immune response, cellular processes, and signaling transduction mechanisms. Both KEGG and GSEA demonstrated enrichment results for signaling pathways; however, GSEA evaluated gene sets rather than individual gene expression changes, capturing subtle expression variations by ranking genes based on their differential expression between groups and testing whether predefined gene sets are enriched at the top or bottom of the ranked list. KEGG and GSEA jointly highlighted significant enrichment in pathways such as Cytochrome P450, Retinol metabolism, Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway, and fat digestion and absorption. These results indicate that GIDF intervention modulates hepatic gene expression, providing novel insights into its regulatory role in NAFLD. 3.5. Effect of GIDF on intestinal damage in NAFLD mice To assess intestinal damage in NAFLD mice resulting from HFD, histopathological analysis of ileal tissues from all groups was conducted. H&E staining revealed that, in the NC group, ileal villi were arranged in an orderly manner, whereas in the HFD and AN-GIDF groups, there was significant mucosal damage and inflammatory infiltration, accompanied by a noticeable reduction in villi height. However, this damage was ameliorated in the GIDF group (Fig. 5A). The prolonged high-fat dietary regime significantly reduced the expression of ZO-1 and Occludin proteins within the HFD group, as detailed in Fig. 53B-C. This reduction is indicative of compromised barrier function, including altered intestinal integrity and increased permeability. However, GIDF application markedly countered these adverse effects, effectively restoring the integrity of the intestinal mucosal barrier disrupted by the high-fat regimen. In contrast, when comparing the AN-GIDF and HFD groups, the levels of ZO-1 and Occludin were further reduced, suggesting that the addition of antibiotics may further exacerbate damage to the gut lining. Correspondingly, IL-1β, TNF-α, and LPS, previously elevated in the HFD group relative to the NC group, showed significant reductions post-GIDF intervention, as illustrated in Fig. 2E-G. These findings align with the hypothesis that intestinal barrier impairment facilitates LPS translocation, thereby elevating inflammatory factors in the gut and further exacerbating the organism’s inflammatory response, which is reversed by GIDF intervention. Figure 5 Effects of GIDF and AN-GIDF on intestinal damage in NAFLD mice.(A)H&E stained sections of ileum.(B,C,D)ZO-1 and Occludin protein expression in ileum.(E,F)IL-1βand Tnf- α level in ileum.(G) Serum LPS level.Different letters represent significant differences( p < 0.05). 3.6. GIDF alleviates gut microbial dysbiosis in NAFLD mice To assess the influence of GIDF on the intestinal microbiome of mice with NAFLD, researchers have implemented 16S rDNA sequencing techniques to analyze microbial populations in cecal contents. Microbial diversity analysis revealed that after 12 weeks of GIDF treatment, both the ace and chao1 indices showed significant increases compared to those in the HFD and AN-GIDF groups. In contrast, the Shannon and Simpson indices did not exhibit notable changes relative to the HFD group, but were nevertheless elevated compared to the AN-GIDF group, as displayed in Fig. 6 A-D. Principal component analysis (PCA) demonstrated that the microbial community structure in the GIDF group diverged from that in the HFD and AN-GIDF groups, as illustrated in Fig. 6 E. These findings indicate that GIDF supplementation enhances microbial richness and uniformity within the gut of NAFLD mice. To investigate the microbial communities associated with NAFLD progression, a detailed analysis of gut microbiota was conducted, examining both phylum and genus levels across various mouse cohorts. Over a 12-week period, there was a notable increase in the Firmicutes/Bacteroidetes(F/B) ratio among the HFD-fed mice, as indicated in the control group comparisons; this uptick was successfully counteracted by GIDF intervention, as illustrated in Fig. 7 B. Additionally, the intervention led to a decrease in Proteobacteria and an increase in Verrucomicrobia within the GIDF-treated group compared with those in the HFD group. At a more detailed genus level, a decrease in the relative abundance of Muribaculaceae_unclassified and Lachnospiraceae was observed in the HFD group, in contrast to the NC group, whereas genera such as Bacteroides and Desulfovibrionaceae_uncultured increased, as shown in Fig. 6 F. GIDF treatment adjusted these genera abundances back toward normal levels. Moreover, there was a notable enrichment of Lactobacillus , Blautia , Clostridia , Akkermansia , and Roseburia in the GIDF group (Fig. 6 G). Antibiotic treatment in the AN-GIDF group led to the depletion of most gut microbes and was marked by an enrichment of drug-resistant bacteria such as Muribaculaceae_ge , Enterobacteriaceae_unclassified , Enterobacter , Bacteroides , and Alloprevotella (Fig. 6 G). To identify biomarkers that differed significantly between groups, LDA was employed to evaluate the impact of GIDF intervention on specific species within the gut microbiota of NAFLD mice. The analysis identified 28 distinct species, with Lactobacillus , Blautia , Akkermansia , and Roseburia emerging as the significant differential species at the genus level (Fig. 7 A-I). These genera were distinctly associated with the GIDF group compared with the other groups, suggesting their potential as biomarkers for the therapeutic effects of GIDF. 3.7. Effect of GIDF on full-spectrum metabolism of cecum contents in NALFD mice The PCA method was used to visualize the overall distribution trends among the samples, reflecting the underlying biological differences. The PCA score plot revealed distinct separation of the NC group from the other two groups, with some overlap noted between the HFD and GIDF groups (Fig. 8 A). This indicates that HFD feeding influenced the metabolic profile in the the cecum of mice. As shown in Fig. 8 B, 231 differential metabolites were identified between the HFD and NC groups, of which 166 were upregulated and 65 downregulated. The analysis identified 90 metabolites differentially expressed between the GIDF and HFD groups, with 47 metabolites significantly upregulated and 43 downregulated, as depicted in Fig. 8 C. The volcano plot effectively illustrates the distribution and trends of these differential metabolites between the two groups. MetaboAnalyst software was used for KEGG pathway enrichment analysis of the differentially expressed metabolites to elucidate the molecular mechanisms by which GIDF mitigates NAFLD at the metabolite level. Overall, 20 pathways were identified as enriched among the metabolites differentiated between GIDF and HFD groups (Fig. 8 D). As shown in Fig. 8 I, in the D-Amino acid metabolism pathway, GIDF upregulated the levels of Diaminopimelic acid, D-Ornithine, L-Threonine, L-Cysteine, and Pyrrolidonecarboxylic acid, while downregulating Oxalacetic acid. In the citrate cycle (TCA) cycle pathway, GIDF increased the content of succinic acid, while reducing Oxalacetic acid and citric acid levels. These findings suggest that GIDF intervention can modulate amino acid and glucose metabolism. L-Cysteine participates in multiple metabolic pathways, including D-Amino acid metabolism; sulfur relay system; cysteine and methionine metabolism; glycine, serine, and threonine metabolism; and central carbon metabolism in cancer (Fig. 8 E). Notably, S-Adenosylmethionine (SAMe) and 5'-Methylthioadenosine (MTA), as metabolites in the cysteine and methionine metabolism pathway, were significantly upregulated following GIDF intervention (Fig. 8 F-G). Additionally, GIDF regulated the PPAR signaling pathway by modulating the levels of 13(S)-HODE (Fig. 8 H). This aligns with the hepatic transcriptomic results, indicating that GIDF’s amelioration of hepatic lipid accumulation in HFD-induced NAFLD mice may be mediated through the PPAR signaling pathway. Overall, these pathways underscore the potential pathogenesis of NAFLD and the therapeutic targets of GIDF, with the altered metabolites in these key pathways likely representing critical potential biomarkers. 3.8. The potential relationship between metabolites and gut microbiota To reflect the similarities and differences between the gut microbiota and metabolite expression patterns more intuitively, hierarchical clustering analysis of correlations was performed using Spearman correlation analysis on significantly differential gut microbiota and metabolites. As shown in the Fig. 9 , Akkermansia, Lactobacillus , and Turicibacter exhibited positive correlations with L-Cysteine and L-Threonine in amino acid metabolism, while Rikenella showed negative correlations with these metabolites. In the cysteine and methionine metabolism pathway, Anaerostipes, Roseburia, Akkermansia, Lactobacillus , and Turicibacter were positively correlated with S-Adenosylmethionine (SAMe) and 5'-Methylthioadenosine (MTA), whereas Desulfovibrionaceae_uncultured, Desulfovibrionaceae_unclassified , Helicobacter, Oscillibacter, Muribaculaceae_unclassified, Gemella , and Anaerofustis displayed negative correlations with these metabolites. Additionally, Akkermansia, Roseburia, Lachnospiraceae_unclassified, Lactobacillus , and Turicibacter were negatively correlated with 13(S)-HODE, while Rikenella, Desulfovibrionaceae_unclassified , and Betaproteobacteriales_unclassified showed positive correlations. These results indicate that GIDF modulates gut microbiota composition, thereby regulating amino acid and lipid metabolism, and alleviating NAFLD. 4. Discussion The growing prevalence of NAFLD in recent years has been closely linked to unhealthy high-fat dietary habits. Recent studies have underscored the role of diet, hepatic lipid accumulation, oxidative stress, inflammation, and gut microbiota dysbiosis in the progression of NAFLD 26 . Dietary fiber, particularly IDF, has received significant attention for its role in regulating lipid metabolism and reshaping the gut microbial community. Notably, edible mushrooms represent an underutilized, yet potent source of bioactive IDF, containing 3- to 10.5-fold higher IDF levels than SDF 27 . The unique properties of IDF allow it to bind to cholesterol and bile acids, thereby promoting the proliferation of beneficial intestinal bacteria 28 . Building on these findings, the study employed hepatic transcriptomics, 16S rDNA sequencing, and metabolomics to systematically examine how GIDF alleviates NAFLD in mice via the gut-liver axis. NAFLD is associated with factors such as obesity and lipid metabolism disorders. A murine model of NAFLD was established through long-term HFD feeding to simulate human dietary patterns characterized by excessive lipid intake. Hepatic lipid accumulation, considered a pivotal pathological hallmark of NAFLD progression, was significantly ameliorated by GIDF supplementation. This intervention effectively attenuated HFD-induced increases in body weight and liver index while normalizing serum parameters, including TC, TG, HDL-C, LDL-C, ALT, and AST. Histopathological analysis of liver sections demonstrated that GIDF administration substantially reduced lipid droplet deposition and restored normal hepatocyte architecture. Previous studies have demonstrated that IDF, which resists gastrointestinal digestion and absorption while providing negligible caloric value, enhances satiety through its hydration properties to reduce food intake and body weight 29 . Wang et al. revealed that Pleurotus ostreatus-derived IDF modulates lipid metabolism via PPAR signaling pathways and adipocytokine-regulated networks, thereby alleviating hepatic lipid deposition 30 . Based on these findings, this study confirms that GIDF effectively ameliorates dyslipidemia and attenuates hepatic lipid accumulation with concomitant injury in NAFLD mice. Sustained dysregulated lipid accumulation induces oxidative stress and inflammatory responses, where inflammation-promoted oxidative stress in turn exacerbates the inflammatory response, constituting a pivotal component of the "multiple-hit" pathogenesis in NAFLD progression 31 . The anti-inflammatory properties of IDF have been documented, demonstrating its capacity to scavenge free radicals in hepatocytes while upregulating superoxide dismutase and CAT activities, thereby mitigating oxidative damage and inflammatory reactions 32 . MDA, a terminal product of lipid peroxidation, showed decreased levels following GIDF intervention, concomitant with enhanced activities of CAT and GSH, critical endogenous antioxidant enzymes, and reductants. HFD-fed mice manifested elevated hepatic and serum concentrations of inflammatory cytokines including IL-1β, IL-10, and TNF-α. Mechanistically, TNF-α triggers hepatocyte apoptosis while exacerbating inflammatory responses through upregulation of pro-inflammatory cytokines, particularly IL-1β. However, these pathological elevations were substantially reversed by GIDF administration. Consequently, the study concludes that GIDF is capable of inhibiting lipid peroxidation and inflammatory responses and decelerate the development of NAFLD. The transcriptomic analysis of the differentially expressed hepatic genes elucidated the potential mechanisms underlying GIDF-mediated hepatoprotection. GIDF significantly modulated biological processes related to lipid and fatty acid metabolism, including steroid hormone biosynthesis, arachidonic acid metabolism, bile secretion, lipid biosynthesis proteins, linoleic acid metabolism, fatty acid degradation, fat digestion and absorption, biosynthesis of unsaturated fatty acids, fatty acid elongation, and primary bile acid biosynthesis. Moreover, GIDF was significantly enriched for Cytochrome P450 (CYP) and Retinol metabolism pathway. CYPs orchestrate both endogenous (steroidogenesis, cholesterol-to-bile acid conversion, arachidonate metabolism) and xenobiotic metabolic processes 33 .It undergoes hepatic transport via chylomicrons and participates in developmental regulation and metabolic homeostasis. Notably, supplementation with all-trans retinoic acid (atRA), a retinol metabolite, ameliorates HFD-induced hepatic steatosis and lipogenesis 34 .Particular emphasis should be placed on the PPAR signaling pathway. As nuclear receptors, PPARs exert multifunctional roles in lipid metabolism and immune regulation, with PPARα and PPARγ isoforms specifically enhancing fatty acid β-oxidation, adipocyte differentiation, and adiponectin secretion while improving insulin sensitivity.Furthermore, PPARα/γ isoforms demonstrate anti-inflammatory properties by suppressing the NF-κB signaling pathway, thereby attenuating inflammatory damage. Consistent with Zheng et al.'s 13 discovery that barley-derived dietary fiber modulates the PPAR signaling pathway to mitigate adipose tissue and hepatic injury in obese mice, this study substantiate that GIDF alleviates HFD-induced NAFLD through coordinated modulation of lipid metabolism via CYP, retinol metabolism, and PPAR signaling networks. The liver, which functions as a critical immune organ, possesses the capacity to recruit and activate immune cells in response to gut-derived metabolites or pathogen-associated signals. Receiving 70% of its blood supply through the portal vein from intestinal venous drainage, the liver maintains intimate bidirectional communication with the gut throughout life. Intestinal barrier leakage permits translocation of endotoxins and microbial metabolites into the liver, exacerbating inflammatory responses and consequently driving hepatic injury 35 . The mechanical barrier function of tight junctions between intestinal epithelial cells against microbiota and endotoxins is well-established 36 . The findings demonstrate that GIDF repairs ileal epithelial damage, upregulates the expression of intestinal tight junction proteins ZO-1 and Occludin, and enhances barrier integrity. GIDF has the expected effects on LPS, TNF-α, IL-1β. LPS, an endotoxin derived from gram-negative bacterial outer membranes, activates NADPH oxidase and the TLR-4/NF-κB pathway to generate excessive ROS and pro-inflammatory cytokines (TNF-α, IL-1β).During intestinal barrier dysfunction, LPS undergoes the enterohepatic circulation to accelerate the progression of liver injury. Post-GIDF intervention significantly reduced serum LPS concentrations and ileal TNF-α/IL-1β levels, consistent with this study. Conversely, antibiotic-induced depletion of the gut microbiota exacerbates intestinal mucosal damage and diminishes GIDF's therapeutic efficacy against NAFLD. Collectively, GIDF ameliorates hepatic pathology by rebalancing the gut-liver crosstalk, restoring intestinal barrier function, and mitigating systemic inflammation. The degree of hepatopathy in NAFLD is closely associated with alterations in the structure of the gut microbiota 37 . In this study, the HFD group had an increased F/B ratio, which is a typical change in gut microbial composition in relation to NAFLD 38 . GIDF treatment significantly attenuated the density of pathogenic bacteria, including Erysipelotrichaceae , Desulfovibrio, Helicobacter in NAFLD mice. Desulfovibrio and Erysipelotrichaceae have been identified as contributors to obesity and ulcerative colitis, positioning them as key targets in metabolic disease research 39–41 . Helicobacter , known for its pathogenicity, has been positively correlated with an increased risk of NAFLD 42 . In this context, GIDF notably enhanced the abundance of beneficial bacterial genera including Lactobacillus, Blautia, Clostridia, Akkermansia , and Roseburia in NAFLD mice. Lactobacillus , a Gram-positive bacterium, is crucial for synthesizing B vitamins and vitamin K, catabolizing bile salts, inhibiting inflammatory mediators, and exhibiting antimicrobial properties against various pathogens 43 . Blautia produces for producing short-chain fatty acids that help restore intestinal mucosa and are inversely related to visceral fat content 44 . Clostridia reduce host lipid absorption by affecting CD36 expression 45 . Both Akkermansia and Roseburia have been linked to a decreased risk of NAFLD, with an increases in their abundance improving dysregulated lipid metabolism in mice fed a high-fat diet; dietary fiber is known to support their growth 46,47 . Increasing and decreasing variations in Blautia and Clostridia levels have been observed in individuals with NAFLD across different ages and regions 48,49 . Surprisingly, Bifidobacterium levels were higher in the HFD group, suggesting that factors other than disease, such as environmental influences, also play a role in gut microbial dynamics. This underscores the need for more comprehensive investigations into the connections between gut microbiota and the evolution and exacerbation of NAFLD. Such research is crucial for fully elucidating the pathways through which these microorganisms influence disease onset and progression. Gut microbiota-derived metabolites exert direct regulatory effects on host physiological states and play indispensable roles in cellular signaling processes. Untargeted metabolomic profiling with KEGG pathway analysis of murine cecal contents demonstrated that GIDF intervention significantly modulated metabolic pathways, principally involving D-amino acid metabolism, TCA cycle, central carbon metabolism in cancer, and glucagon signaling pathway. GIDF significantly altered the levels of the metabolite L-cysteine. L-cysteine is involved in a variety of metabolic pathways regulated by GIDF. As a semi-essential amino acid, L-cysteine contributes to cellular redox homeostasis and hepatic phospholipid metabolism, conferring hepatoprotective effects against liver injury 48,49 . Furthermore, L-cysteine is a component of GSH, which, as an antioxidant, has been detected to be elevated after GIDF intervention. GIDF also upregulated hepatic concentrations of SAMe and MTA within the cysteine-methionine metabolic. SAMe, the principal methyl donor essential for numerous biological processes, has been demonstrated to play a critical role in hepatic pathophysiology, where its deficiency accelerates the development and progression of metabolic dysfunction-associated steatotic liver disease 51 . MTA induces apoptosis in hepatocellular carcinoma cells and inhibits proliferation, activation, and differentiation of human T cells by decreasing phosphorylation of protein kinase B (Akt). Furthermore, substantial evidence confirmed that MTA has anti-inflammatory properties mediated via suppression of pro-inflammatory cytokine release and inhibition of key regulatory factors such as NF-κB 52 . Additionally, HFD intervention elevated the concentration of 13 (S)-HODE, a linoleic acid derivative that not only potentiates oxidative stress oxidative stress (Barlic & Murphy, 2007), but also promoted atherosclerotic plaque formation (Shibata et al., 2009). Functioning as an endogenous PPARγ ligand, 13(S)-HODE mediates GIDF's regulatory effects on PPAR signaling pathway, thereby participating in systemic lipid and glucose homeostasis. Hepatic transcriptomic KEGG analysis confirmed GIDF's modulation of PPAR signaling, corroborating its therapeutic mechanism through gut-liver axis coordination to ameliorate NAFLD-associated metabolic dysregulation. Correlational analysis revealed positive associations between Lactobacillus , Akkermansia , and Turicibacter genera with L-cysteine concentrations. Given that L-cysteine can be converted from methionine in vivo and requires Vitamin B6 as a cofactor, coupled with GIDF's significant modulation of Vitamin B6 metabolic pathways by GIDF, it can be hypothesized that GIDF enhances L-cysteine levels by influencing Vitamin B6-producing microbial communities. Previous studies have shown that Vitamin B6, synthesized by gut flora, downregulates hepatic PPAR-γ protein expression in hyperlipidemic rats. Guo et al. found that GLH intervention upregulated Vitamin B6 levels, which enhanced the PPAR and TLR4/NF-κB signaling pathways, thereby attenuating NAFLD in mice 46,53 . 5. Conclusion In summary, the findings demonstrate that GIDF exerts significant protective effects against long-term HFD-induced NAFLD. GIDF ameliorated hepatic lipid accumulation and injury, suppressed biomarkers associated with oxidative stress and inflammatory responses, and restored both intestinal mechanical and biological barriers. Specifically, GIDF increased the abundance of beneficial gut microbiota, such as Lactobacillus, Akkermansia, and Turicibacter, while upregulating metabolites including L-cysteine, SAMe, and MTA, thereby attenuating NAFLD progression through the gut-liver axis. Results from the antibiotic-GIDF co-administration experiment further underscored the critical role of gut microbiota in mediating the hepatoprotective effects of GIDF, and integrated analysis of transcriptomic and metabolomic data revealed that lipid and amino acid metabolism are the central pathways through which GIDF alleviates NAFLD. Notably, the PPAR signaling pathway has been identified as a key mechanism by which GIDF inhibits hepatic lipid deposition and enhances energy metabolism. Future studies should explore the molecular mechanisms of PPAR in mitigating NAFLD across in vivo and in vitro models. The findings highlight that GIDF is a potent natural bioactive agent for NAFLD intervention, underscoring the need to enhance the bioavailability of edible fungal IDF. Declarations Ethical statement We acquired 32 male C57BL/6 mice from Liaoning Changsheng Biotechnology Co., Ltd. (License: SCXK (Liaoning) 2020-0001). Animal experiments have been approved by the Animal Ethics Committee of Jilin Agricultural University (approval number: 2021-10-12-001).The study is reported in accordance with ARRIVE guidelines. Funding This research was supported by Scientific Research Project of Jilin Provincial Department of Education (JJKH20250582KJ). Author Contribution Siqi Wang:Conceptualization, Data curation,Methodology, Writing – original draft, Writing – review & editing. Baitong Liu and Yunxia Ma:Data curation, Formal analysis, Software.Xuejun Liu:Supervision, Conceptualization, Writing – review & editing..Guochuan Jiang:Conceptualization，Funding acquisition. 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Supplementary Files WB.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Nov, 2025 Read the published version in npj Science of Food → Version 1 posted Editorial decision: Revision requested 22 Jul, 2025 Reviews received at journal 21 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers agreed at journal 10 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers invited by journal 09 Jul, 2025 Editor assigned by journal 09 Jul, 2025 Submission checks completed at journal 09 Jul, 2025 First submitted to journal 07 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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2","display":"","copyAsset":false,"role":"figure","size":103830,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of GIDF and AN-GIDF on body weight, liver weight , liver injury,lipids and liver function in NAFLD mice.(A)Body weight.(B)Liver weight.(C)Liver epithelium,H\u0026amp;E-stained and oil red O-stained sections of liver.(H,I)Serum ALT, AST levels.(D,E,F,G)Serum TC,TG,LDL-C,and HDL-C levels.**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01,***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01,compared to the HFD group.Different letters represent significant differences(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/3b34fa52af247070f35b2afb.jpeg"},{"id":86642605,"identity":"f00b49f0-ba75-4d71-a668-5c0e567aa4a2","added_by":"auto","created_at":"2025-07-14 08:28:48","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62659,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of GIDF and AN-GIDF on Liver oxidative stress and inflammatory factors in NAFLD mice.(A,B,C).Liver MDA, CAT and GSH levels(D,E,F).Serum TNF-α, IL-1β, and IL-10 levels.Different letters represent significant differences(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/3dd23a3c6c54f5725d68a862.jpeg"},{"id":86641537,"identity":"78b1fee4-0e38-46ee-b568-d2324076b3f6","added_by":"auto","created_at":"2025-07-14 08:20:48","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92310,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of GIDF on mouse liver gene expression. (A)The number of differentially expressed genes between the different groups. (B) GO analysis.(C) KEGG pathway.(D,E,F,G) GESA analysis.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/7ab01337868b36a3804f60a6.jpeg"},{"id":86641538,"identity":"955d84a3-65b0-4de2-9eaa-066280322ea3","added_by":"auto","created_at":"2025-07-14 08:20:48","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74015,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of GIDF and AN-GIDF on intestinal damage in NAFLD mice.(A)H\u0026amp;E stained sections of ileum.(B,C,D)ZO-1 and Occludin protein expression in ileum.(E,F)IL-1βand Tnf- α level in ileum.(G) Serum LPS level.Different letters represent significant differences(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/546256891da1172a50569bd6.jpeg"},{"id":86641547,"identity":"c3465bd7-3f40-4ad0-82c5-6338b97a14b1","added_by":"auto","created_at":"2025-07-14 08:20:48","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":76826,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of GIDF and AN-GIDF on the gut microbiota of NAFLD mice.(A-D)Ace index,Chao1 index,Shannon index.(E)Principal component analysis (PCA).(F)Relative abundance of gut microbiota at the phylum level for all groups.(G)Relative abundance of gut microbiota at the genus level for all groups.(*)\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.(**)\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.(***)\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/93fd2b97faf7166a8fb038bf.jpeg"},{"id":86642607,"identity":"c2f0f7d0-235d-41f8-9c55-979c071498bc","added_by":"auto","created_at":"2025-07-14 08:28:48","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70544,"visible":true,"origin":"","legend":"\u003cp\u003eKey differential microbial analysis of GIDF.(A)LDA distribution histogram.(B)Firmicutes/Bacteroidetes(F/B) ratio.(C,D,E,F,G,H,I)Effects of GIDF on several gut microbiota at genus level.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/97e439520f469524078498dc.jpeg"},{"id":86641543,"identity":"5ed65c24-66ba-4e38-b4ff-b726a4736a26","added_by":"auto","created_at":"2025-07-14 08:20:48","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":84011,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of GIDF on metabolites of intestinal contents..(A)PCA score of cecum metabolite in mice.(B,C)Volcano plot of differential metabolites in the HFD and NC groups,GIDF and HFD groups.(D)KEGG pathway analysis of the GIDF group.(E,F,G,H)Effects of GIDF on the levels of several metabolites.(I)Network diagrams between pathways and metabolites in GIDF group.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/61a1d1bc1bc14f2385bc839a.jpeg"},{"id":86643515,"identity":"27a9f393-f5a5-4282-9c2c-19a6bedb6d37","added_by":"auto","created_at":"2025-07-14 08:36:48","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":272464,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation plot of the relative abundance of gut microbiota and metabolites.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/6f721aa111e4d10488f17c33.jpeg"},{"id":96650072,"identity":"e65290af-124a-4b84-815c-ac4c41beac6d","added_by":"auto","created_at":"2025-11-24 16:06:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1957138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/9fa48580-eda7-40e9-ab1c-adb80c9732ae.pdf"},{"id":86641534,"identity":"4f4824d1-83ed-4b18-a12a-c65e8d63cbe5","added_by":"auto","created_at":"2025-07-14 08:20:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":606874,"visible":true,"origin":"","legend":"","description":"","filename":"WB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7067699/v1/f14deb1be8fbee8f68fadf80.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated analysis of NAFLD mitigation mechanism by Ganoderma lucidum insoluble dietary fiber based on transcriptomics, metabolomics and gut microbiota","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of fat in the liver, accounting for 5% or more of liver weight, in the absence of alcohol consumption or other specific conditions such as autoimmune diseases, medication effects, or viral hepatitis. \u003csup\u003e1\u003c/sup\u003e. The literature review established that NAFLD increases the risk of Type 2 diabetes and cardiovascular diseases (CVD)\u003csup\u003e2\u003c/sup\u003e. NAFLD is prevalent in approximately one-quarter of the world\u0026rsquo;s population, with an incidence rate of more than 30% in South America and the Middle East\u003csup\u003e3\u003c/sup\u003e. Presently, there are no pharmacological treatments recommended for NAFLD in clinical settings, despite its growing prevalence\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe term \u0026ldquo;multiple parallel hits\u0026rdquo; refers to the major pathogenesis of NAFLD, in which various factors, including insulin resistance, adipokines, bile acids, inflammation, and gut microbiota, simultaneously contribute to cause extensive fat accumulation in the liver, leading to significant metabolic derangement. These disturbances may extend to NAFLD, likely leading to cirrhosis \u003csup\u003e5,6\u003c/sup\u003e. The doctrine of \u0026ldquo;multiple parallel hits\u0026rdquo; emphasizes the role played by the gut-hepatic axis. In NAFLD patients, the integrity of the intestinal mucosal layer is compromised, allowing bacterial metabolites and lipopolysaccharides (LPS) to enter the liver via the portal vein. This invasion elicits a hepatocellular immune response and the subsequent production of pro-inflammatory cytokines including TNF-α and IL-1β, thereby worsening liver inflammation \u003csup\u003e7,8\u003c/sup\u003e. Given these dynamics, targeting gut microbes holds the potential for NAFLD therapy \u003csup\u003e9\u003c/sup\u003e. In disease research, especially in studies examining the influence of gut microbiota, antibiotic clearance models have gained popularity owing to their speed and ease of manipulation, enabling broad bacterial intervention in mice and facilitating gut microbiota management \u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDietary fiber, widely recognized as the seventh most essential nutrient alongside carbohydrates, proteins, fats, water, vitamins, and minerals, is classified into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) depending on its water solubility \u003csup\u003e11\u003c/sup\u003e. Traditional view typically posits that SDF ferments more readily.However, IDF typically exists as a complex network formed by diverse fiber polysaccharides, which is fermented by gut bacteria in the colon as a polymeric matrix, rather than being degraded as single purified fiber components.Research has demonstrated that insoluble substrates foster specific microbiota types; for example, IDF derived from highland barley has been shown to elevate \u003cem\u003eAkkermansia\u003c/em\u003e populations in the fecal matter of obese model mice \u003csup\u003e12,13\u003c/sup\u003e. These results indicate that IDF may serve as an effective natural agent for modulating NAFLD.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e, a member of the genus \u003cem\u003eGanoderma\u003c/em\u003e within the Basidiomycetes order, has been esteemed in traditional Chinese edible fungi for over 4,000 years, earning its title as the king of mushrooms \u003csup\u003e14,15\u003c/sup\u003e. Moreover, \u003cem\u003eGanoderma lucidum\u003c/em\u003e and its extracts have been used in food processing\u003csup\u003e16\u003c/sup\u003e. This fungus reportedly possesses several physiological characteristics such as lipid-lowering, hypoglycemic, hepatoprotective, and anticancer properties \u003csup\u003e17,18\u003c/sup\u003e. Previous studies have shown that the polysaccharides from \u003cem\u003eGanoderma lucidum\u003c/em\u003e assist in controlling the gut microbiota and managing metabolic diseases in obesity patients \u003csup\u003e14\u003c/sup\u003e. In addition, the polysaccharide peptide from Ganoderma lucidum has been shown to significantly reduce NAFLD by inhibiting fatty acid synthesis via the FXR-SHP/FGF pathway \u003csup\u003e19\u003c/sup\u003e. Recent studies on \u003cem\u003eGanoderma lucidum\u003c/em\u003e extracts have mainly focused on polysaccharides, triterpenes and other bioactive compounds\u003csup\u003e15,20\u003c/sup\u003e. However, IDF, an understudied component, may hold the potential as a dietary supplement to ameliorate NAFLD.\u003c/p\u003e\u003cp\u003eThe study established a mouse model of NAFLD induced by a 12-week high-fat diet (HFD). The study utilizing hepatic transcriptomics, 16S rDNA sequencing, and metabolomics to examine the mechanisms by which \u003cem\u003eGanoderma lucidum\u003c/em\u003e insoluble dietary fiber (GIDF) exerts hepatic protection and modulates lipid metabolism in NAFLD through the gut-liver axis. This process not only opened new avenues for the treatment of NAFLD, but also expanded the potential for utilizing functional fibers from edible mushrooms to improve health.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials and reagents\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eGanoderma lucidum\u003c/em\u003e applied in this experiment was obtained from Kunming Xuanqing Biotechnology Co., Ltd. (Yunnan, China), while the biochemical enzymes such as α-amylase, neutral protease, and amyloglucosidase (AMG) were sourced from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). All the required experimental kits for determining the serum levels of lipids and liver enzymes, such as TC (Total Cholesterol), TG (Triglycerides), LDL-C (Low-Density Lipoprotein Cholesterol), HDL-C (High-Density Lipoprotein Cholesterol), AST (Aspartate Transaminase), ALT (Alanine Transaminase), MDA (Malondialdehyde), CAT (Catalase), and GSH (Glutathione), were purchased from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). Moreover, enzyme-linked immunosorbent assay kits were used for the identification of inflammatory markers such as IL-1β, IL-10, LPS and TNF-α from Yibo Biotechnology Co., Ltd. (Changchun, China). Primary antibodies against ZO-1, Occludin and GAPDH for protein detection were purchased from Servicebio Biotechnology Co., Ltd. (Wuhan, China) and all other chemical reagents used in this study were of analytical grade.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of \u003cem\u003eGanoderma lucidum insoluble\u003c/em\u003e dietary fiber\u003c/h2\u003e\u003cp\u003eTo prepare GIDF, 10.0 g \u003cem\u003eGanoderma lucidum\u003c/em\u003e was combined with distilled water in a 1:10 w/v ratio and treated with 0.5% α-amylase in a 60\u0026deg;C water bath set at pH 5.5 for 90 minutes. Subsequently, the solution was cooled to 50\u0026deg;C, after which 0.5% protease was added. The solution then underwent an incubation period of 60 minutes at a pH of 7.0. Subsequently, 0.2% AMG was incorporated and the reaction continued in a 55\u0026deg;C water bath for another 60 minutes at pH 5.0. The enzymes were then inactivated by boiling the mixture for 10 minutes. After cooling, the solution was centrifuged at 11,100\u0026times;g for 20 minutes using an XR1 centrifuge (Thermo Fisher Scientific, China). The precipitated solids were washed twice with distilled water, freeze-dried using a LGJ-25D freeze dryer, and collected as the final GIDF product.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Animal Experiments\u003c/h2\u003e\u003cp\u003eSix-week-old C57BL/6 mice (number 32) were maintained at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60% \u0026plusmn; 5% humidity with a 12-hour light-dark cycle (Liaoning Changsheng Biotechnology Co., Ltd.). They underwent an adaptation phase to their new environment and diet for a week. During this period, the mice had continuous access to both food and water. Animal experiments have been approved by the Animal Ethics Committee of Jilin Agricultural University (approval number: 2021-10-12-001), in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The development of the NAFLD mouse model was initiated with a high-fat diet regimen. After a week-long adaptation phase, the mice were systematically assigned into four distinct groups, each comprising eight animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e): (1) NC group, sustained on a standard diet; (2) HFD group, receiving a diet comprising 60% fat; (3) GIDF group, high-fat diet with 60% fat and 500 mg/kg BW/d GIDF suspension by gavage; and (4) AN-GIDF group, high-fat diet with 60% fat and 500 mg/kg BW/d GIDF suspension by gavage, included antibiotics in their water, specifically ampicillin at 1.0 mg/mL and neomycin at 0.5 mg/mL. The NC and HFD groups were administered with equal amounts of physiological saline solution. After 12 weeks, following an eight-hour fasting period, the mice were sacrificed via cervical dislocation under ether anesthesia, and blood was drawn from the area surrounding the orbit. Furthermore, tissue samples, including those from the liver, ileum, and the contents of the cecum, were meticulously collected, analyzed, and subsequently stored at a temperature of -80℃ to ensure preservation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Serum biochemical index detection\u003c/h2\u003e\u003cp\u003eThe blood collected from the mice was allowed to clot at room temperature for two hours and serum was extracted by centrifuging the tubes at 3000 rpm for 10 minutes. The TC,TG, LDL-C, HDL-C, AST, ALT, MDA, CAT, and GSH analyses were performed according to the manufacturer\u0026rsquo;s protocol of the assay kits. Moreover, cytokines TNF-α, IL-10, LPS, and IL-1β were quantified in the culture medium by means of the specific ELISA kits and according to the manufacturer\u0026rsquo;s recommendations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Histological analysis\u003c/h2\u003e\u003cp\u003eIn the experimental phase, liver and colon specimens from the experimental mice were perfused with 4% paraformaldehyde and left in the same medium for 48 h. These tissues were then processed through a series of graded alcohol solutions to remove water, embedded in paraffin wax, and sectioned when the paraffin had set. Each section was subsequently stained with hematoxylin and eosin (H\u0026amp;E) for histological examination under an optical microscope. For liver tissues, Oil Red O staining was performed using an improved protocol obtained from previously described methods \u003csup\u003e21\u003c/sup\u003e. The sections were respectively treated by fixing in 10% formalin solution for 10 min, washing in distilled water for 3 min, and then in 60% isopropanol for 30 seconds. The sections were stained using 0.5% Oil Red O stain in 60% isopropanol for 15 min, followed by washing in 60% isopropanol and dried distilled water, and counterstained with Mayer\u0026rsquo;s hematoxylin for two minutes to improve the visualization of cellular details.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6.Transcriptome analysis\u003c/h2\u003e\u003cp\u003eRNA was extracted from the liver tissue, and mRNA was isolated using Oligo (dT) Beads. Following polymerase chain reaction (PCR) amplification, magnetic beads were used to purify and recover the target products ranging from 300\u0026ndash;600 bp for sequencing. After library construction, the libraries were initially quantified using Qubit and diluted to 1 ng/\u0026micro;l. The insert size of the libraries was then assessed using a Qseq100 DNA Analyzer, and their effective concentrations were precisely quantified via Q-PCR. Libraries that passed the quality control were pooled according to their effective concentrations and required sequencing data output, followed by sequencing on an Illumina NovaSeq 6000 platform with a PE150 configuration. The RNA-seq standard analysis pipeline included quality control (QC), alignment, quantification, differential expression analysis, functional enrichment, as well as alternative splicing analysis, and Gene Set Enrichment Analysis (GSEA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Western Blot (WB) Analysis\u003c/h2\u003e\u003cp\u003eProtein extraction for western blotting was performed by modifying a previously described method \u003csup\u003e22\u003c/sup\u003e. For protein extraction, 250 mg of tissue was homogenized in 1 ml of protein extraction buffer. This buffer consisted of 5 \u0026micro;L of protease inhibitor cocktail, 5 \u0026micro;L of PMSF, and 5 \u0026micro;L phosphatase inhibitor to eliminate the activity of any enzyme. This mixture was then homogenized for several periods of 30 seconds at low speed using a homogenizer until complete disruption of the tissue was achieved. The homogenate was further centrifuged at 14,000 xg for 15 min in a pre-chilled centrifuge. Subsequently, protein concentrations were estimated using a BCA assay kit. Before immunoblot analysis, protein samples were separated by electrophoresis. The proteins were gently blotted onto a PVDF membrane. This membrane was then moved to a hybridization bag, where the appropriate concentration of primary antibody was added, and incubated overnight at 4\u0026deg;C. The membrane was further processed by incubating with a diluted secondary antibody for an hour, followed by application of a freshly prepared ECL mixture on the protein-exposed side of the membrane for luminescence detection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Intestinal Flora Analysis\u003c/h2\u003e\u003cp\u003eTo analyze cecal microbiota, 16S rDNA sequencing was performed on the contents extracted from the mice cecum. Genomic DNA was isolated and subsequently amplified by PCR using the primers B341F (5'-CCTACGGGGNGGCWGCAG-3') and B785R (5'-GACTACHVGGGGTATCTAATCC-3'). Following amplification, the PCR products were electrophoresed using a 2% agarose gel to facilitate the isolation of DNA fragments. The targeted segments were carefully excised from the gel, and underwent a purification process, employing the QIQuick Gel Extraction Kit. This procedure was executed strictly in accordance with the detailed instructions provided by the manufacturer to ensure optimal recovery and purity of DNA. The purified libraries were then mixed, denatured, and loaded onto an Illumina Novaseq 6000 for high-throughput parallel sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9.Metabolomic analysis of mouse cecum contents\u003c/h2\u003e\u003cp\u003eDuring the sample preparation process, an exact amount of the sample was transferred into a 2 mL tube. To this tube, 600 \u0026micro;L of methanol, which was augmented with 2-chloro-L-phenylalanine at a concentration of 4 ppm, was carefully added to ensure proper mixing. This mixture was then vortexed, shaken for 30 seconds, ground, and subjected to sonication for 10 min using a KQ-800DE sonicator (Shumei, China). After the initial treatment, the samples were centrifuged at an acceleration of 5000 xg within a temperature-controlled environment maintained at 4\u0026deg;C. The centrifugation step was carefully timed to last 10 min to ensure the effective sedimentation of particulates. Subsequently, the mixture was centrifuged at 12000 rpm for 5 min, and the clear supernatant obtained was further filtered through a 0. 22 \u0026micro;m filter. The filtrate was then transferred to another analytical vial, which was to be used for LC-MS analysis. The sample was prepared in the correct manner for molecular analysis.\u003c/p\u003e\u003cp\u003eChromatographic analysis was performed using the state-of-the-art Vanquish UHPLC System bought from Thermo Fisher Scientific, USA. This system was specifically designed for LC-MS and was used in this study to achieve high sensitivity and selectivity. The samples were effectively separated using an ACQUITY UPLC\u0026reg; HSS T3 column (2. 1 x 100 mm, 1. 8 \u0026micro;m) obtained from Waters Corporation, Milford, MA, USA; the column was held at a constant temperature of 40\u0026deg;C. The injection volume was maintained constant at 2 \u0026micro;L and the flow rate of the system at an optimal level of 0. 3 mL/min in order to ensure good separation. To identify metabolites, an Orbitrap Exploris 120 mass spectrometer coupled with an ESI ion source was used, and both were obtained from Thermo Fisher Scientific. This instrument was set to acquire both MS1- and data-dependent MS/MS scans (Full MS-ddMS2 mode), and thus provided detailed information of the metabolic profiles of the samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe results of the experiments were expressed in terms of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, calculated from a series of three separate experiments. All statistical computations, including analysis of variance and other relevant tests, were performed using SPSS software. This criterion was applied to discern substantial differences across various experimental setups, highlighting the impact of the tested conditions.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1. GIDF effectively ameliorated HFD-induced NAFLD\u003c/h2\u003e\u003cp\u003eElevated body weight significantly exacerbates the risk of NAFLD. A study conducted over a 12-week period where mice were subjected to HFD demonstrated a pronounced increase in their body mass compared to their counterparts sustained on a normal diet. This significant divergence in weight gain between the two groups is illustrated in Fig.\u0026nbsp;2A, underscoring the impact of dietary fat on physiological health. By the end of the 12-week period, HFD, NC, GIDF, and AN-GIDF groups exhibited weight gains of 57.84%, 29.20%, 30.66%, and 37.61%, respectively. While GIDF therapy moderated the acceleration of weight gain among subjects, distinct differences in body weight were not evident after the 12-week regimen. Moreover, as illustrated in Fig.\u0026nbsp;2B, the liver mass recorded from the HFD group after this period significantly surpassed those of the NC cohort, highlighting the propensity for high-fat diets to promote substantial fat accumulation in the liver. Conversely, the liver mass in the GIDF group was discernibly reduced relative to that in the HFD group, demonstrating the potential of GIDF to mitigate fat deposition in the liver. No statistical difference was observed between the AN-GIDF and GIDF groups in terms of liver weight, indicating that antibiotic treatment did not influence liver size. Therefore, it can be concluded that GIDF can potentially improve the features related to NAFLD in mice.\u003c/p\u003e\u003cp\u003eLiver morphology of the mice under GIDF intervention is depicted in Fig.\u0026nbsp;2C. The liver in the NC group was red-brown and elastic. In contrast, the liver of mice in the HFD group appeared pale and oily when touched. However, the liver in both GIDF and AN-GIDF groups exhibited a restored deep red color and a less oily surface. Oil Red O staining revealed extensive red lipid droplets in the livers of HFD-fed mice, along with hepatocellular steatosis, indicating significant fat accumulation in NAFLD-induced mice. Although the quantity of red lipid droplets was lower in both the GIDF and AN-GIDF groups compared to the HFD group, the AN-GIDF group continued to retain more lipid droplets than the GIDF group. H\u0026amp;E staining revealed that hepatocytes in the NC group were orderly with clearly discernible nuclei. In the HFD group, hepatocytes were disorganized, with numerous vacuoles and signs of inflammatory cell infiltration. In the GIDF group, there was a notable reduction in vacuoles and an absence of inflammatory cells, whereas only a few inflammatory cells persisted in the AN-GIDF group. These histological findings confirmed that GIDF intervention alleviated hepatic fat accumulation and tissue damage in NAFLD mice.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;2 Effects of GIDF and AN-GIDF on body weight, liver weight, liver injury,lipids and liver function in NAFLD mice.(A)Body weight.(B)Liver weight.(C)Liver epithelium,H\u0026amp;E-stained and oil red O-stained sections of liver.(H,I)Serum ALT, AST levels.(D,E,F,G)Serum TC,TG,LDL-C,and HDL-C levels.**\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,compared to the HFD group.Different letters represent significant differences(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effect of GIDF on lipid metabolism in NAFLD mice\u003c/h2\u003e\u003cp\u003eDisruptions in lipid metabolism are closely linked to the onset of NAFLD. The study revealed that, relative to the NC group, both TG and TC levels were markedly elevated in the HFD group, indicating a significant disruption in lipid homeostasis owing to high-fat dietary intake, as depicted in Fig.\u0026nbsp;2D-G. Excessive accumulation of TG and TC in the liver may precipitate liver dysfunction, leading to cellular damage and eventually lead to fatty liver disease \u003csup\u003e23\u003c/sup\u003e. Administration of both GIDF and AN-GIDF resulted in a significant reduction in serum TC and TG levels in mice. Elevated LDL-C levels are linked to an increased risk of CVD, whereas HDL-C serves as a protective factor against such heart conditions \u003csup\u003e23\u003c/sup\u003e. GIDF intervention effectively lowered LDL-C levels, while increasing HDL-C levels. Although AN-GIDF treatment reduced serum LDL-C levels, it did not significantly impact HDL-C levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effect of GIDF on liver injury ,oxidative stress and inflammatory status in NAFLD mice\u003c/h2\u003e\u003cp\u003eLiver damage was evaluated by measuring enzyme levels, specifically AST and ALT, as indicators of hepatic function and integrity. When liver cells are damaged, they release transaminases into the bloodstream, leading to a marked increase in aminotransferase activity \u003csup\u003e25\u003c/sup\u003e. Compared to the NC group, the HFD group exhibited markedly elevated levels of AST and ALT, as depicted in Fig.\u0026nbsp;2H-I. These findings underscore significant hepatic stress in the HFD group. GIDF administration helped restore liver function to some degree, effectively regulating AST and ALT levels. Additionally, the use of antibiotics in the AN-GIDF group resulted in higher AST and ALT levels than those in the GIDF group alone.\u003c/p\u003e\u003cp\u003eThe levels of MDA, GSH, and CAT activity in the liver tissue were measured to assess oxidative stress damage in NAFLD mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C, the HFD group demonstrated significantly increased MDA and GSH levels and markedly reduced CAT activity. After GIDF intervention, compared to the HFD group, MDA decreased by 36.27%, CAT activity increased by 22.88%, and GSH levels increased by 73.09%.\u003c/p\u003e\u003cp\u003eThe intervention using GIDF notably reduced the concentrations of the pro-inflammatory cytokines TNF-α and IL-1β. These inflammatory markers were previously observed to be substantially elevated in the HFD group, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and F. The effective reduction in these cytokines underscores the therapeutic potential of GIDF in controlling inflammation induced by HFD intake. GIDF intervention notably reduced the levels of IL-10, an essential anti-inflammatory cytokine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In contrast, the levels of TNF-α, IL-1β, and IL-10 showed no significant changes in the AN-GIDF group compared to the HFD group. These outcomes indicate that GIDF intervention can effectively alleviate liver injury and oxidative stress and mitigate systemic inflammation in these mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect of GIDF on gene expression in the liver\u003c/h2\u003e\u003cp\u003eTo further investigate potential mechanisms by which GIDF alleviates liver injury in NAFLD, a transcriptomic analysis was performed on mouse liver tissues. Compared with the HFD group, GIDF intervention resulted in 1,126 differentially expressed genes (DEGs), including 654 upregulated and 472 downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Enrichment analyses of these DEGs were conducted using GO, KEGG, and GSEA, with functional categories illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-G. Following GIDF intervention, enriched biological functions included drug metabolism, lipid metabolism, immune response, cellular processes, and signaling transduction mechanisms. Both KEGG and GSEA demonstrated enrichment results for signaling pathways; however, GSEA evaluated gene sets rather than individual gene expression changes, capturing subtle expression variations by ranking genes based on their differential expression between groups and testing whether predefined gene sets are enriched at the top or bottom of the ranked list. KEGG and GSEA jointly highlighted significant enrichment in pathways such as Cytochrome P450, Retinol metabolism, Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway, and fat digestion and absorption. These results indicate that GIDF intervention modulates hepatic gene expression, providing novel insights into its regulatory role in NAFLD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Effect of GIDF on intestinal damage in NAFLD mice\u003c/h2\u003e\u003cp\u003eTo assess intestinal damage in NAFLD mice resulting from HFD, histopathological analysis of ileal tissues from all groups was conducted. H\u0026amp;E staining revealed that, in the NC group, ileal villi were arranged in an orderly manner, whereas in the HFD and AN-GIDF groups, there was significant mucosal damage and inflammatory infiltration, accompanied by a noticeable reduction in villi height. However, this damage was ameliorated in the GIDF group (Fig.\u0026nbsp;5A). The prolonged high-fat dietary regime significantly reduced the expression of ZO-1 and Occludin proteins within the HFD group, as detailed in Fig.\u0026nbsp;53B-C. This reduction is indicative of compromised barrier function, including altered intestinal integrity and increased permeability. However, GIDF application markedly countered these adverse effects, effectively restoring the integrity of the intestinal mucosal barrier disrupted by the high-fat regimen. In contrast, when comparing the AN-GIDF and HFD groups, the levels of ZO-1 and Occludin were further reduced, suggesting that the addition of antibiotics may further exacerbate damage to the gut lining.\u003c/p\u003e\u003cp\u003eCorrespondingly, IL-1β, TNF-α, and LPS, previously elevated in the HFD group relative to the NC group, showed significant reductions post-GIDF intervention, as illustrated in Fig.\u0026nbsp;2E-G. These findings align with the hypothesis that intestinal barrier impairment facilitates LPS translocation, thereby elevating inflammatory factors in the gut and further exacerbating the organism\u0026rsquo;s inflammatory response, which is reversed by GIDF intervention.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;5 Effects of GIDF and AN-GIDF on intestinal damage in NAFLD mice.(A)H\u0026amp;E stained sections of ileum.(B,C,D)ZO-1 and Occludin protein expression in ileum.(E,F)IL-1βand Tnf- α level in ileum.(G) Serum LPS level.Different letters represent significant differences(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6. GIDF alleviates gut microbial dysbiosis in NAFLD mice\u003c/h2\u003e\u003cp\u003eTo assess the influence of GIDF on the intestinal microbiome of mice with NAFLD, researchers have implemented 16S rDNA sequencing techniques to analyze microbial populations in cecal contents. Microbial diversity analysis revealed that after 12 weeks of GIDF treatment, both the ace and chao1 indices showed significant increases compared to those in the HFD and AN-GIDF groups. In contrast, the Shannon and Simpson indices did not exhibit notable changes relative to the HFD group, but were nevertheless elevated compared to the AN-GIDF group, as displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D. Principal component analysis (PCA) demonstrated that the microbial community structure in the GIDF group diverged from that in the HFD and AN-GIDF groups, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE. These findings indicate that GIDF supplementation enhances microbial richness and uniformity within the gut of NAFLD mice.\u003c/p\u003e\u003cp\u003eTo investigate the microbial communities associated with NAFLD progression, a detailed analysis of gut microbiota was conducted, examining both phylum and genus levels across various mouse cohorts. Over a 12-week period, there was a notable increase in the Firmicutes/Bacteroidetes(F/B) ratio among the HFD-fed mice, as indicated in the control group comparisons; this uptick was successfully counteracted by GIDF intervention, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. Additionally, the intervention led to a decrease in Proteobacteria and an increase in Verrucomicrobia within the GIDF-treated group compared with those in the HFD group. At a more detailed genus level, a decrease in the relative abundance of \u003cem\u003eMuribaculaceae_unclassified\u003c/em\u003e and \u003cem\u003eLachnospiraceae\u003c/em\u003e was observed in the HFD group, in contrast to the NC group, whereas genera such as \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eDesulfovibrionaceae_uncultured\u003c/em\u003e increased, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eF. GIDF treatment adjusted these genera abundances back toward normal levels. Moreover, there was a notable enrichment of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eClostridia\u003c/em\u003e, \u003cem\u003eAkkermansia\u003c/em\u003e, and \u003cem\u003eRoseburia\u003c/em\u003e in the GIDF group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Antibiotic treatment in the AN-GIDF group led to the depletion of most gut microbes and was marked by an enrichment of drug-resistant bacteria such as \u003cem\u003eMuribaculaceae_ge\u003c/em\u003e, \u003cem\u003eEnterobacteriaceae_unclassified\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, and \u003cem\u003eAlloprevotella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eTo identify biomarkers that differed significantly between groups, LDA was employed to evaluate the impact of GIDF intervention on specific species within the gut microbiota of NAFLD mice. The analysis identified 28 distinct species, with \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eAkkermansia\u003c/em\u003e, and \u003cem\u003eRoseburia\u003c/em\u003e emerging as the significant differential species at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-I). These genera were distinctly associated with the GIDF group compared with the other groups, suggesting their potential as biomarkers for the therapeutic effects of GIDF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Effect of GIDF on full-spectrum metabolism of cecum contents in NALFD mice\u003c/h2\u003e\u003cp\u003eThe PCA method was used to visualize the overall distribution trends among the samples, reflecting the underlying biological differences. The PCA score plot revealed distinct separation of the NC group from the other two groups, with some overlap noted between the HFD and GIDF groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). This indicates that HFD feeding influenced the metabolic profile in the the cecum of mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, 231 differential metabolites were identified between the HFD and NC groups, of which 166 were upregulated and 65 downregulated. The analysis identified 90 metabolites differentially expressed between the GIDF and HFD groups, with 47 metabolites significantly upregulated and 43 downregulated, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eC. The volcano plot effectively illustrates the distribution and trends of these differential metabolites between the two groups.\u003c/p\u003e\u003cp\u003eMetaboAnalyst software was used for KEGG pathway enrichment analysis of the differentially expressed metabolites to elucidate the molecular mechanisms by which GIDF mitigates NAFLD at the metabolite level. Overall, 20 pathways were identified as enriched among the metabolites differentiated between GIDF and HFD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, in the D-Amino acid metabolism pathway, GIDF upregulated the levels of Diaminopimelic acid, D-Ornithine, L-Threonine, L-Cysteine, and Pyrrolidonecarboxylic acid, while downregulating Oxalacetic acid. In the citrate cycle (TCA) cycle pathway, GIDF increased the content of succinic acid, while reducing Oxalacetic acid and citric acid levels. These findings suggest that GIDF intervention can modulate amino acid and glucose metabolism. L-Cysteine participates in multiple metabolic pathways, including D-Amino acid metabolism; sulfur relay system; cysteine and methionine metabolism; glycine, serine, and threonine metabolism; and central carbon metabolism in cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Notably, S-Adenosylmethionine (SAMe) and 5'-Methylthioadenosine (MTA), as metabolites in the cysteine and methionine metabolism pathway, were significantly upregulated following GIDF intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eF-G). Additionally, GIDF regulated the PPAR signaling pathway by modulating the levels of 13(S)-HODE (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). This aligns with the hepatic transcriptomic results, indicating that GIDF\u0026rsquo;s amelioration of hepatic lipid accumulation in HFD-induced NAFLD mice may be mediated through the PPAR signaling pathway. Overall, these pathways underscore the potential pathogenesis of NAFLD and the therapeutic targets of GIDF, with the altered metabolites in these key pathways likely representing critical potential biomarkers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.8. The potential relationship between metabolites and gut microbiota\u003c/h2\u003e\u003cp\u003eTo reflect the similarities and differences between the gut microbiota and metabolite expression patterns more intuitively, hierarchical clustering analysis of correlations was performed using Spearman correlation analysis on significantly differential gut microbiota and metabolites. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e, \u003cem\u003eAkkermansia, Lactobacillus\u003c/em\u003e, and \u003cem\u003eTuricibacter\u003c/em\u003e exhibited positive correlations with L-Cysteine and L-Threonine in amino acid metabolism, while \u003cem\u003eRikenella\u003c/em\u003e showed negative correlations with these metabolites. In the cysteine and methionine metabolism pathway, \u003cem\u003eAnaerostipes, Roseburia, Akkermansia, Lactobacillus\u003c/em\u003e, and \u003cem\u003eTuricibacter\u003c/em\u003e were positively correlated with S-Adenosylmethionine (SAMe) and 5'-Methylthioadenosine (MTA), whereas \u003cem\u003eDesulfovibrionaceae_uncultured, Desulfovibrionaceae_unclassified\u003c/em\u003e, \u003cem\u003eHelicobacter, Oscillibacter, Muribaculaceae_unclassified, Gemella\u003c/em\u003e, and \u003cem\u003eAnaerofustis\u003c/em\u003e displayed negative correlations with these metabolites. Additionally, \u003cem\u003eAkkermansia, Roseburia, Lachnospiraceae_unclassified, Lactobacillus\u003c/em\u003e, and \u003cem\u003eTuricibacter\u003c/em\u003e were negatively correlated with 13(S)-HODE, while \u003cem\u003eRikenella, Desulfovibrionaceae_unclassified\u003c/em\u003e, and \u003cem\u003eBetaproteobacteriales_unclassified\u003c/em\u003e showed positive correlations. These results indicate that GIDF modulates gut microbiota composition, thereby regulating amino acid and lipid metabolism, and alleviating NAFLD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe growing prevalence of NAFLD in recent years has been closely linked to unhealthy high-fat dietary habits. Recent studies have underscored the role of diet, hepatic lipid accumulation, oxidative stress, inflammation, and gut microbiota dysbiosis in the progression of NAFLD \u003csup\u003e26\u003c/sup\u003e. Dietary fiber, particularly IDF, has received significant attention for its role in regulating lipid metabolism and reshaping the gut microbial community. Notably, edible mushrooms represent an underutilized, yet potent source of bioactive IDF, containing 3- to 10.5-fold higher IDF levels than SDF \u003csup\u003e27\u003c/sup\u003e. The unique properties of IDF allow it to bind to cholesterol and bile acids, thereby promoting the proliferation of beneficial intestinal bacteria \u003csup\u003e28\u003c/sup\u003e. Building on these findings, the study employed hepatic transcriptomics, 16S rDNA sequencing, and metabolomics to systematically examine how GIDF alleviates NAFLD in mice via the gut-liver axis.\u003c/p\u003e\u003cp\u003eNAFLD is associated with factors such as obesity and lipid metabolism disorders. A murine model of NAFLD was established through long-term HFD feeding to simulate human dietary patterns characterized by excessive lipid intake. Hepatic lipid accumulation, considered a pivotal pathological hallmark of NAFLD progression, was significantly ameliorated by GIDF supplementation. This intervention effectively attenuated HFD-induced increases in body weight and liver index while normalizing serum parameters, including TC, TG, HDL-C, LDL-C, ALT, and AST. Histopathological analysis of liver sections demonstrated that GIDF administration substantially reduced lipid droplet deposition and restored normal hepatocyte architecture. Previous studies have demonstrated that IDF, which resists gastrointestinal digestion and absorption while providing negligible caloric value, enhances satiety through its hydration properties to reduce food intake and body weight \u003csup\u003e29\u003c/sup\u003e. Wang et al. revealed that Pleurotus ostreatus-derived IDF modulates lipid metabolism via PPAR signaling pathways and adipocytokine-regulated networks, thereby alleviating hepatic lipid deposition\u003csup\u003e30\u003c/sup\u003e. Based on these findings, this study confirms that GIDF effectively ameliorates dyslipidemia and attenuates hepatic lipid accumulation with concomitant injury in NAFLD mice.\u003c/p\u003e\u003cp\u003eSustained dysregulated lipid accumulation induces oxidative stress and inflammatory responses, where inflammation-promoted oxidative stress in turn exacerbates the inflammatory response, constituting a pivotal component of the \"multiple-hit\" pathogenesis in NAFLD progression\u003csup\u003e31\u003c/sup\u003e. The anti-inflammatory properties of IDF have been documented, demonstrating its capacity to scavenge free radicals in hepatocytes while upregulating superoxide dismutase and CAT activities, thereby mitigating oxidative damage and inflammatory reactions \u003csup\u003e32\u003c/sup\u003e. MDA, a terminal product of lipid peroxidation, showed decreased levels following GIDF intervention, concomitant with enhanced activities of CAT and GSH, critical endogenous antioxidant enzymes, and reductants. HFD-fed mice manifested elevated hepatic and serum concentrations of inflammatory cytokines including IL-1β, IL-10, and TNF-α. Mechanistically, TNF-α triggers hepatocyte apoptosis while exacerbating inflammatory responses through upregulation of pro-inflammatory cytokines, particularly IL-1β. However, these pathological elevations were substantially reversed by GIDF administration. Consequently, the study concludes that GIDF is capable of inhibiting lipid peroxidation and inflammatory responses and decelerate the development of NAFLD.\u003c/p\u003e\u003cp\u003eThe transcriptomic analysis of the differentially expressed hepatic genes elucidated the potential mechanisms underlying GIDF-mediated hepatoprotection. GIDF significantly modulated biological processes related to lipid and fatty acid metabolism, including steroid hormone biosynthesis, arachidonic acid metabolism, bile secretion, lipid biosynthesis proteins, linoleic acid metabolism, fatty acid degradation, fat digestion and absorption, biosynthesis of unsaturated fatty acids, fatty acid elongation, and primary bile acid biosynthesis. Moreover, GIDF was significantly enriched for Cytochrome P450 (CYP) and Retinol metabolism pathway. CYPs orchestrate both endogenous (steroidogenesis, cholesterol-to-bile acid conversion, arachidonate metabolism) and xenobiotic metabolic processes \u003csup\u003e33\u003c/sup\u003e.It undergoes hepatic transport via chylomicrons and participates in developmental regulation and metabolic homeostasis. Notably, supplementation with all-trans retinoic acid (atRA), a retinol metabolite, ameliorates HFD-induced hepatic steatosis and lipogenesis \u003csup\u003e34\u003c/sup\u003e.Particular emphasis should be placed on the PPAR signaling pathway. As nuclear receptors, PPARs exert multifunctional roles in lipid metabolism and immune regulation, with PPARα and PPARγ isoforms specifically enhancing fatty acid β-oxidation, adipocyte differentiation, and adiponectin secretion while improving insulin sensitivity.Furthermore, PPARα/γ isoforms demonstrate anti-inflammatory properties by suppressing the NF-κB signaling pathway, thereby attenuating inflammatory damage. Consistent with Zheng et al.'s \u003csup\u003e13\u003c/sup\u003e discovery that barley-derived dietary fiber modulates the PPAR signaling pathway to mitigate adipose tissue and hepatic injury in obese mice, this study substantiate that GIDF alleviates HFD-induced NAFLD through coordinated modulation of lipid metabolism via CYP, retinol metabolism, and PPAR signaling networks.\u003c/p\u003e\u003cp\u003eThe liver, which functions as a critical immune organ, possesses the capacity to recruit and activate immune cells in response to gut-derived metabolites or pathogen-associated signals. Receiving 70% of its blood supply through the portal vein from intestinal venous drainage, the liver maintains intimate bidirectional communication with the gut throughout life. Intestinal barrier leakage permits translocation of endotoxins and microbial metabolites into the liver, exacerbating inflammatory responses and consequently driving hepatic injury \u003csup\u003e35\u003c/sup\u003e. The mechanical barrier function of tight junctions between intestinal epithelial cells against microbiota and endotoxins is well-established \u003csup\u003e36\u003c/sup\u003e. The findings demonstrate that GIDF repairs ileal epithelial damage, upregulates the expression of intestinal tight junction proteins ZO-1 and Occludin, and enhances barrier integrity. GIDF has the expected effects on LPS, TNF-α, IL-1β. LPS, an endotoxin derived from gram-negative bacterial outer membranes, activates NADPH oxidase and the TLR-4/NF-κB pathway to generate excessive ROS and pro-inflammatory cytokines (TNF-α, IL-1β).During intestinal barrier dysfunction, LPS undergoes the enterohepatic circulation to accelerate the progression of liver injury. Post-GIDF intervention significantly reduced serum LPS concentrations and ileal TNF-α/IL-1β levels, consistent with this study. Conversely, antibiotic-induced depletion of the gut microbiota exacerbates intestinal mucosal damage and diminishes GIDF's therapeutic efficacy against NAFLD. Collectively, GIDF ameliorates hepatic pathology by rebalancing the gut-liver crosstalk, restoring intestinal barrier function, and mitigating systemic inflammation.\u003c/p\u003e\u003cp\u003eThe degree of hepatopathy in NAFLD is closely associated with alterations in the structure of the gut microbiota \u003csup\u003e37\u003c/sup\u003e. In this study, the HFD group had an increased F/B ratio, which is a typical change in gut microbial composition in relation to NAFLD \u003csup\u003e38\u003c/sup\u003e. GIDF treatment significantly attenuated the density of pathogenic bacteria, including \u003cem\u003eErysipelotrichaceae\u003c/em\u003e, \u003cem\u003eDesulfovibrio, Helicobacter\u003c/em\u003e in NAFLD mice.\u003cem\u003eDesulfovibrio\u003c/em\u003e and \u003cem\u003eErysipelotrichaceae\u003c/em\u003e have been identified as contributors to obesity and ulcerative colitis, positioning them as key targets in metabolic disease research \u003csup\u003e39\u0026ndash;41\u003c/sup\u003e. \u003cem\u003eHelicobacter\u003c/em\u003e, known for its pathogenicity, has been positively correlated with an increased risk of NAFLD \u003csup\u003e42\u003c/sup\u003e. In this context, GIDF notably enhanced the abundance of beneficial bacterial genera including \u003cem\u003eLactobacillus, Blautia, Clostridia, Akkermansia\u003c/em\u003e, and \u003cem\u003eRoseburia\u003c/em\u003e in NAFLD mice. \u003cem\u003eLactobacillus\u003c/em\u003e, a Gram-positive bacterium, is crucial for synthesizing B vitamins and vitamin K, catabolizing bile salts, inhibiting inflammatory mediators, and exhibiting antimicrobial properties against various pathogens \u003csup\u003e43\u003c/sup\u003e. \u003cem\u003eBlautia\u003c/em\u003e produces for producing short-chain fatty acids that help restore intestinal mucosa and are inversely related to visceral fat content \u003csup\u003e44\u003c/sup\u003e. \u003cem\u003eClostridia\u003c/em\u003e reduce host lipid absorption by affecting CD36 expression \u003csup\u003e45\u003c/sup\u003e. Both \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eRoseburia\u003c/em\u003e have been linked to a decreased risk of NAFLD, with an increases in their abundance improving dysregulated lipid metabolism in mice fed a high-fat diet; dietary fiber is known to support their growth \u003csup\u003e46,47\u003c/sup\u003e. Increasing and decreasing variations in \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eClostridia\u003c/em\u003e levels have been observed in individuals with NAFLD across different ages and regions \u003csup\u003e48,49\u003c/sup\u003e. Surprisingly, \u003cem\u003eBifidobacterium\u003c/em\u003e levels were higher in the HFD group, suggesting that factors other than disease, such as environmental influences, also play a role in gut microbial dynamics. This underscores the need for more comprehensive investigations into the connections between gut microbiota and the evolution and exacerbation of NAFLD. Such research is crucial for fully elucidating the pathways through which these microorganisms influence disease onset and progression.\u003c/p\u003e\u003cp\u003eGut microbiota-derived metabolites exert direct regulatory effects on host physiological states and play indispensable roles in cellular signaling processes. Untargeted metabolomic profiling with KEGG pathway analysis of murine cecal contents demonstrated that GIDF intervention significantly modulated metabolic pathways, principally involving D-amino acid metabolism, TCA cycle, central carbon metabolism in cancer, and glucagon signaling pathway. GIDF significantly altered the levels of the metabolite L-cysteine. L-cysteine is involved in a variety of metabolic pathways regulated by GIDF. As a semi-essential amino acid, L-cysteine contributes to cellular redox homeostasis and hepatic phospholipid metabolism, conferring hepatoprotective effects against liver injury \u003csup\u003e48,49\u003c/sup\u003e. Furthermore, L-cysteine is a component of GSH, which, as an antioxidant, has been detected to be elevated after GIDF intervention. GIDF also upregulated hepatic concentrations of SAMe and MTA within the cysteine-methionine metabolic. SAMe, the principal methyl donor essential for numerous biological processes, has been demonstrated to play a critical role in hepatic pathophysiology, where its deficiency accelerates the development and progression of metabolic dysfunction-associated steatotic liver disease\u003csup\u003e51\u003c/sup\u003e. MTA induces apoptosis in hepatocellular carcinoma cells and inhibits proliferation, activation, and differentiation of human T cells by decreasing phosphorylation of protein kinase B (Akt). Furthermore, substantial evidence confirmed that MTA has anti-inflammatory properties mediated via suppression of pro-inflammatory cytokine release and inhibition of key regulatory factors such as NF-κB \u003csup\u003e52\u003c/sup\u003e. Additionally, HFD intervention elevated the concentration of 13 (S)-HODE, a linoleic acid derivative that not only potentiates oxidative stress oxidative stress (Barlic \u0026amp; Murphy, 2007), but also promoted atherosclerotic plaque formation (Shibata et al., 2009). Functioning as an endogenous PPARγ ligand, 13(S)-HODE mediates GIDF's regulatory effects on PPAR signaling pathway, thereby participating in systemic lipid and glucose homeostasis. Hepatic transcriptomic KEGG analysis confirmed GIDF's modulation of PPAR signaling, corroborating its therapeutic mechanism through gut-liver axis coordination to ameliorate NAFLD-associated metabolic dysregulation.\u003c/p\u003e\u003cp\u003eCorrelational analysis revealed positive associations between \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eAkkermansia\u003c/em\u003e, and \u003cem\u003eTuricibacter\u003c/em\u003e genera with L-cysteine concentrations. Given that L-cysteine can be converted from methionine in vivo and requires Vitamin B6 as a cofactor, coupled with GIDF's significant modulation of Vitamin B6 metabolic pathways by GIDF, it can be hypothesized that GIDF enhances L-cysteine levels by influencing Vitamin B6-producing microbial communities. Previous studies have shown that Vitamin B6, synthesized by gut flora, downregulates hepatic PPAR-γ protein expression in hyperlipidemic rats. Guo et al. found that GLH intervention upregulated Vitamin B6 levels, which enhanced the PPAR and TLR4/NF-κB signaling pathways, thereby attenuating NAFLD in mice \u003csup\u003e46,53\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, the findings demonstrate that GIDF exerts significant protective effects against long-term HFD-induced NAFLD. GIDF ameliorated hepatic lipid accumulation and injury, suppressed biomarkers associated with oxidative stress and inflammatory responses, and restored both intestinal mechanical and biological barriers. Specifically, GIDF increased the abundance of beneficial gut microbiota, such as Lactobacillus, Akkermansia, and Turicibacter, while upregulating metabolites including L-cysteine, SAMe, and MTA, thereby attenuating NAFLD progression through the gut-liver axis. Results from the antibiotic-GIDF co-administration experiment further underscored the critical role of gut microbiota in mediating the hepatoprotective effects of GIDF, and integrated analysis of transcriptomic and metabolomic data revealed that lipid and amino acid metabolism are the central pathways through which GIDF alleviates NAFLD. Notably, the PPAR signaling pathway has been identified as a key mechanism by which GIDF inhibits hepatic lipid deposition and enhances energy metabolism. Future studies should explore the molecular mechanisms of PPAR in mitigating NAFLD across in vivo and in vitro models. The findings highlight that GIDF is a potent natural bioactive agent for NAFLD intervention, underscoring the need to enhance the bioavailability of edible fungal IDF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical statement\u003c/h2\u003e\u003cp\u003eWe acquired 32 male C57BL/6 mice from Liaoning Changsheng Biotechnology Co., Ltd. (License: SCXK (Liaoning) 2020-0001). Animal experiments have been approved by the Animal Ethics Committee of Jilin Agricultural University (approval number: 2021-10-12-001).The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by Scientific Research Project of Jilin Provincial Department of Education (JJKH20250582KJ).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSiqi Wang:Conceptualization, Data curation,Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Baitong Liu and Yunxia Ma:Data curation, Formal analysis, Software.Xuejun Liu:Supervision, Conceptualization, Writing \u0026ndash; review \u0026amp; editing..Guochuan Jiang:Conceptualization，Funding acquisition.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDong, Y., Guo, Y., Li, Q., Zhao, Y. \u0026amp; Cao, J., Soluble dietary fiber from Dendrocalamus brandisii (Munro) Kurz shoot improves liver injury by regulating gut microbial disorder in mice. \u003cem\u003eFOOD CHEM X\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e 101472 (2024).\u003c/li\u003e\n\u003cli\u003eFernando, D. H., Forbes, J. M., Angus, P. W. \u0026amp; Herath, C. 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C.\u003cem\u003e et al.\u003c/em\u003e, Suppressive effects of tumor cell-derived 5\u0026prime;-deoxy-5\u0026prime;-methylthioadenosine on human T cells. \u003cem\u003eONCOIMMUNOLOGY\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e 15 (2016).\u003c/li\u003e\n\u003cli\u003eZhang, Q.\u003cem\u003e et al.\u003c/em\u003e, Antihyperlipidemic and Hepatoprotective Properties of Vitamin B6 Supplementation in Rats with High-Fat Diet-Induced Hyperlipidemia. \u003cem\u003eENDOCR METAB IMMUNE\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e 2260 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Insoluble dietary fiber, NAFLD, Transcriptomics, Gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-7067699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7067699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study established a mouse model of NAFLD induced by a 12-week high-fat diet (HFD). Utilizing hepatic transcriptomics, 16S rDNA sequencing, and metabolomics, the study examined the mechanisms by which GIDF exerts hepatic protection and modulates lipid metabolism in NAFLD through the gut-liver axis.The results demonstrated that GIDF supplementation significantly alleviated NAFLD characteristics in mice, including reducing biomarkers associated with hepatic lipid deposition and injury, modulating lipid disorders, and improving oxidative stress. Furthermore, GIDF likely regulated lipid metabolism through pathways such as Cytochrome P450, Retinol metabolism, and Peroxisome Proliferator-Activated Receptor signaling, while increasing the expression of ileal tight junction proteins (ZO-1 and Occludin), thereby repairing the intestinal barrier.GIDF altered gut microbiota composition, promoting the growth of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, and \u003cem\u003eAkkermansia\u003c/em\u003e.The results from the co-administration of antibiotics and GIDF further demonstrated that the ameliorative effects of GIDF on NAFLD are dependent on the presence of gut microbiota. Metabolomic analysis indicated that GIDF upregulated levels of L-cysteine, S-adenosylmethionine (SAMe), and 5'-methylthioadenosine (MTA), while downregulating 13(S)-HODE, thereby modulating amino acid and lipid metabolism. These findings validate that GIDF alleviates NAFLD via the gut-liver axis and may serve as a promising nutritional supplement.\u003c/p\u003e","manuscriptTitle":"Integrated analysis of NAFLD mitigation mechanism by Ganoderma lucidum insoluble dietary fiber based on transcriptomics, metabolomics and gut microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 08:20:44","doi":"10.21203/rs.3.rs-7067699/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-22T08:38:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T00:23:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T08:04:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297487998135763054828806543686183930603","date":"2025-07-15T07:04:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190572962466862336927998222064656570027","date":"2025-07-14T22:40:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271787039369518891343260165031230654119","date":"2025-07-10T08:35:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99157496153799964042045955114462729789","date":"2025-07-10T01:26:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-09T22:15:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T22:10:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-09T16:03:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Science of Food","date":"2025-07-07T16:58:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa73376f-f717-4dba-9fcd-ad614477c50b","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51325841,"name":"Biological sciences/Biochemistry"},{"id":51325842,"name":"Health sciences/Diseases"},{"id":51325843,"name":"Health sciences/Gastroenterology"},{"id":51325844,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-11-24T16:00:29+00:00","versionOfRecord":{"articleIdentity":"rs-7067699","link":"https://doi.org/10.1038/s41538-025-00586-6","journal":{"identity":"npj-science-of-food","isVorOnly":false,"title":"npj Science of Food"},"publishedOn":"2025-11-17 15:57:21","publishedOnDateReadable":"November 17th, 2025"},"versionCreatedAt":"2025-07-14 08:20:44","video":"","vorDoi":"10.1038/s41538-025-00586-6","vorDoiUrl":"https://doi.org/10.1038/s41538-025-00586-6","workflowStages":[]},"version":"v1","identity":"rs-7067699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7067699","identity":"rs-7067699","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}