A total of forty-four male C57BL/6 SPF mice, aged 4 weeks, were acclimatized for 1 week under controlled conditions (12 h light/dark cycle, 22 ± 3°C) and fed normal chow (20% protein, 10% fat, 70% carbohydrates). Figure 1 outlines the workflow of the experimental protocol. Animals were then randomized into a control group (n = 13, normal chow) and a model group (n = 31, high-fat diet: 20% protein, 60% fat, 20% carbohydrates) for 12-week MASLD induction. Successful modeling was confirmed via serum biochemistry and histopathology in 4 mice/group at week 12. Remaining MASLD mice were reallocated into three cohorts (n = 9/group): HF (continued high-fat diet), HF-NC (switched to normal chow), and HF-MD (switched to MD) (MD: 15% protein, 35% fat, 50% carbohydrates). Control mice continued to be fed a normal chow named NC group (n = 9). Body weight and food intake were monitored weekly during the 18-week intervention, followed by terminal tissue collection. All procedures complied with ethical guidelines (Approval No. KY202405701, Qingdao Central Hospital).

Following a 12-h overnight fast with ad libitum water access, mice were anesthetized via intraperitoneal administration of 5% chloral hydrate (0.1 ml/10 g body weight) and blood samples were collected via retro-orbital puncture into EDTA-coated tubes, followed by euthanasia via cervical dislocation. Hepatic and epididymal adipose tissues were immediately excised for morphological imaging and gravimetric analysis (liver index = fresh liver weight/body weight). Liver specimens were processed into paraffin-embedded sections, cryosections, and snap-frozen aliquots (−80°C), while adipose tissues were preserved as cryosections and frozen samples. Fresh fecal samples (≥100 mg) were stabilized in 0.1% HCl-containing buffer prior to freezing. Whole blood in the EP tube was allowed to stand undisturbed for 2 h at room temperature. Subsequently, serum was separated by centrifugation (4,000 r/min, 15 min) and stored at −20°C for subsequent experiments.

Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GT), fasting glucose (Glu), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total protein (TP), total bile acid (TBA), total bilirubin (TBIL), unconjugated bilirubin (IBIL), and conjugated bilirubin (DBIL) were quantitatively analyzed using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China) with an XR220 Plus automatic biochemical analyzer (Xinrui, China). All biochemical determinations were performed in strict accordance with the manufacturer's standardized protocols, including calibration procedures and quality control measures as specified in the technical documentation.

Quantification of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in murine hepatic tissues was performed using commercially available ELISA kits (Medical Discovery Leader, China), in accordance with the manufacturer's standardized protocols.

The liver tissue samples underwent histopathological examination, with paraffin-embedded sections stained with hematoxylin and eosin (H&E) to evaluate cellular structure. Meanwhile, frozen liver sections were treated with Oil Red O stain to detect lipid accumulation. Additionally, morphological characterization of adipose tissue was conducted through H&E staining of frozen sections to evaluate structural alterations.

Genomic DNA was isolated from the samples using the CretMagTM Power Soil DNA Kit (Cretaceous, China). After extraction, DNA concentration was quantified with TBS-380, DNA purity was assessed with a NanoDrop200 (Thermo Fisher Scientific, USA), and DNA integrity was confirmed via 1% agarose gel electrophoresis. Fragment optimization was performed using magnetic bead-based size selection, targeting 200–400 bp fragments. Subsequent library preparation included end repair, A-tailing, and adapter ligation. PCR amplification is performed, the amplified product is recovered by product purification using magnetic beads, and the PCR product is cyclized to give a final library for pre-machine concentration testing. The final libraries were subjected to pre-sequencing (DNBSEQ, China).

Fastq software (13) processed raw Illumina sequencing data for quality assessment. Sequencing reads were assembled with MEGAHIT (14) (https://github.com/voutcn/megahit, version 1.1.2). Binning analysis was performed using vamb software and the results were screened against the following criteria: genome integrity >50% and contamination rate < 10%. Unigenes were aligned with sequences of bacteria, fungi, archaea and viruses extracted from NCBI's NR (Version: 2021.11) database using DIAMOND (15) software (blastp, evalue ≤ 1e−5) (16). Alpha diversity was measured to assess species diversity per sample. β-diversity was estimated using Bray-Curtis distance based on principal coordinate analysis (PCoA), non-metric multidimensional scale analysis (NMDS), and principal component analysis (PCA) as appropriate. Statistical comparisons of bacterial abundance and diversity were conducted using the Wilcoxon rank-sum test and Welch's t-test. Heatmaps visualizing phylum- and genus-level taxonomic profiles were generated based on non-parametric Wilcoxon test results (p < 0.05). Taxa with significant abundance differences were assessed using LEfSe (LDA effect size) analysis. Inter-group differential analysis of gut microbiota was performed using STAMP software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (KOBAS) with FDR correction (Benjamini-Hochberg, p < 0.05) identified significantly enriched pathways in differentially expressed genes.

Fecal samples were homogenized in 2 ml EP tubes with 1 ml of ultrapure water by vortexing (10 s) and mechanical disruption using steel beads in a tissue homogenizer (40 Hz, 4 min). Subsequent steps included ice-water bath ultrasonication (20 kHz, 5 min, repeated 3 × ) and centrifugation (5,000 rpm, 20 min, 4°C). A total of 0.8 ml of supernatant was transferred to a new 2 ml EP tube, mixed with 0.1 ml of 50% (v/v) sulfuric acid and 0.8 ml of extraction solution containing 2-methylvaleric acid (internal standard, 25 mg/L) in methyl tert-butyl ether. The mixture was vortexed (10 s), shaken (10 min), sonicated (10 min, ice-cooled), then spun (10,000 rpm, 15 min, 4°C). After static incubation (−20°C, 30 min), the supernatant was transferred to injection vials. SCFAs were quantified using a Shimadzu GC2030-QP2020 NX GC-MS system equipped with an Agilent HP-FFAP capillary column (30 m × 250 μm × 0.25 μm). Electron ionization (70 eV) and selected ion monitoring (SIM) were employed for detection. Quantitation was performed via internal standard calibration (linear range: 0.02–100 mg/L, R² > 0.99). Quality controls included blank samples, spiked recoveries (80%−120%), and technical replicates (RSD ≤ 20%).

Statistical analyses were done with GraphPad Prism 9 (GraphPad Software, San Diego, CA). Continuous data are shown as mean ± standard deviation (SD). Intergroup comparisons were conducted via unpaired Student's t-tests, while multigroup analyses utilized one-way ANOVA with post hoc Tukey's tests.

Following 12 weeks of a high-fat diet, the model group showed a notable increase in body mass in comparison to the control group (Figure 2A). Gross anatomical observations (Figure 2C) revealed that mice in the model group displayed enlarged body size, excessive intra-abdominal adiposity, and hepatomegaly characterized by pale discoloration, greasy appearance, soft consistency, mottled surface texture, and rounded hepatic margins. Conversely, mice in the control group maintained normal body proportions, minimal abdominal fat deposition, and livers with physiological dimensions, bright red coloration, firm texture, smooth glistening surfaces, and well-defined margins.

Serological analysis (Figure 2D) demonstrated that the model group exhibited significantly elevated levels of hepatic injury markers (ALT, AST, γ-GT), lipid metabolism parameters (LDL-C, TC, TG, TBA), and bilirubin profiles (TBIL, DBIL) compared to the control group, alongside a pronounced decrease in HDL-C (p < 0.05 for all comparisons). These biochemical alterations collectively indicated hepatic dysfunction and steatosis. Histopathological evaluation further corroborated these findings: HE staining revealed hepatic steatosis, hepatocyte ballooning, and inflammatory cell infiltration in portal areas, while Oil Red O staining highlighted extensive lipid droplet accumulation and vacuolar degeneration in the model group (Figure 2E). Thus, histopathology further confirms that the high-fat diet caused pathological changes in mouse livers, including lipid accumulation and hepatocyte injury. Integrated analysis confirmed the successful establishment of a MASLD mouse model in the model group following 12 weeks of high-fat diet intervention, recapitulating hallmark pathological features including dyslipidemia, ectopic lipid deposition, and progressive liver injury.

The high-fat diet (HF) is well-established for inducing MASLD in experimental animals (27). Consistent with previous findings (28), an HF-induced MASLD mouse model was successfully established in this study, recapitulating key pathological features including marked body weight gain, increased visceral adiposity, dysregulated glucose and lipid metabolism, hepatic steatosis, and inflammatory infiltration (Figure 2). These phenotypic alterations were accompanied by significant dysregulation of gut microbiota composition and microbial metabolites, characterized by reduced α-diversity, disrupted community structure, and decreased fecal SCFAs production. Notably, intervention with either normal chow or MD reversed hepatic steatosis, attenuated hepatic inflammation, and partially restored gut microbial homeostasis, with MD demonstrating superior efficacy in body weight reduction and metabolic improvement compared to normal chow.

Our findings align with meta-analytical evidence (29), which indicates that ≥5% body weight loss improves hepatic and metabolic parameters, while ≥7% loss significantly ameliorates liver histology and function. Pronounced weight reduction was observed in both dietary intervention groups, with MD achieving a more substantial decrease (>12%), underscoring the pivotal role of dietary patterns in modulating obesity-related metabolic dysregulation. Previous studies further highlight MD's potential to reduce central obesity and visceral adiposity (30, 31). In this study, dietary interventions reduced body weight and showed a downward trend in liver index, though intergroup differences in hepatic index improvement were not statistically significant. By contrast, eWAT mass was markedly reduced, particularly following MD intervention. This suggests adipose tissue may respond more rapidly to MD, whereas significant amelioration of visceral steatosis may require prolonged intervention.

Our results demonstrate that MD significantly reduced serum TG and TC levels compared to normal chow intervention (Figure 4), consistent with the meta-analytical evidence reported by Omid Asbaghi et al. (32). This metabolic advantage of MD further underscores its superior lipid-modulating properties, likely attributable to its unique nutritional profile enriched with monounsaturated/polyunsaturated fatty acids (MUFAs/PUFAs), polyphenols, and dietary fiber. Specifically, ω-3 PUFAs may mediate lipid homeostasis through PPARα activation, enhancing fatty acid oxidation, suppressing lipogenesis, promoting bile acid secretion and cholesterol excretion, and improving adipocyte function via adiponectin (33). Furthermore, polyphenolic compounds such as hydroxytyrosol (HT) and oleuropein (OLE) exhibit dual protective effects by preserving HDL from oxidative damage and enhancing cholesterol clearance through AMPK activation-mediated upregulation of LDL receptor expression, thereby optimizing lipid metabolism (34).

The bioactive components in MD demonstrate synergistic anti-inflammatory and antioxidant properties. Our analysis revealed markedly elevated serum IL-6 and TNF-α levels in MASLD mice, which were effectively suppressed by MD intervention, showing superior anti-inflammatory efficacy compared to normal chow (Figure 4). Recent investigations demonstrate that HT from olive mill wastewater (MD) suppresses IL-6 gene transcription (35), while OLE interrupts LPS-induced TLR4-MyD88-NF-κB/MAPK signaling (36), forming a dual anti-inflammatory barrier. Mitochondrial dysfunction in MASLD progression involves ROS-mediated membrane destabilization, electron transport chain impairment, and lipid peroxidation, forming a self-perpetuating pathogenic cycle (37). Mechanistically, HT enhances Nrf2/ARE-driven antioxidant enzymes (SOD, catalase), whereas resveratrol and quercetin cooperatively suppress NADPH oxidase activity (38, 39), collectively disrupting the “oxidative stress—mitochondrial damage—inflammatory amplification—secondary oxidative stress” axis to prevent steatosis-to-MASH transition. Although MD exhibited greater ALT/AST reduction than ND, clinical comparisons of dietary regimens remain inconclusive (40). Marin-Alejandre et al.'s RCT (41) reported equivalent ALT/γ-GT improvements with AHA diet and MD, yet divergent AST responses, suggesting intervention outcomes depend on metabolic heterogeneity and nutrient interactions.

The gut-liver axis (GLA), a bidirectional signaling network connecting intestinal and hepatic systems, critically regulates liver-gut homeostasis through microbial metabolism, immune signaling, and metabolite exchange. In MASLD pathogenesis, GLA dysfunction exhibits a dual-hit pathology: compromised intestinal barrier integrity synergizes with gut dysbiosis to drive disease progression (42). For instance, gut dysbiosis-induced barrier disruption facilitates translocation of microbial lipopolysaccharide (LPS) into circulation, triggering IL-6/TNF-α release and NADPH oxidase-mediated oxidative stress. This cascade not only directly induces hepatocyte injury but also activates hepatic stellate cells (HSCs) to accelerate fibrogenesis (10, 43, 44).

While current evidence remains fragmented regarding microbial dynamics in MASLD progression and dietary interventions, emerging studies consistently identify disease-associated dysbiotic patterns. Clinical observations reveal characteristic microbial shifts in MASLD patients: marked elevation of potential pathogens (Enterobacteriaceae, Escherichia) alongside depletion of metabolically beneficial commensals (Bacteroides, Bifidobacterium), with exacerbation during fibrotic progression (10). Systematic microbiota profiling revealed characteristic HF-induced restructuring in MASLD (Figures 7, 8). HF triggered phylum-level dysbiosis, marked by increased Proteobacteria/Actinobacteria and reduced Firmicutes abundance. Notably, genus-level analysis demonstrated contrasting Prevotella depletion and Alistipes enrichment, aligning with clinical observations of progressive Firmicutes reduction and early Prevotella decline in MASLD (45). While Firmicutes [often termed “obesity-associated bacteria” for their metabolic regulatory roles (46)] typically expand under HF, their suppression here may reflect chronic HF-induced intestinal inflammation that selectively inhibits oxidative stress-sensitive genera (e.g., Lactobacillus) while promoting inflammation-resistant Proteobacteria (e.g., Enterobacteriaceae). Intriguingly, Alistipes enrichment in this MASLD model contrasts with its reported depletion in cirrhosis studies (47, 48), despite proposed anti-inflammatory properties (49, 50). These findings suggest that MASLD mouse models partially recapitulate human gut microbial communities, although notable inter-species differences persist. Furthermore, microbial functionality exhibits significant context-dependence, with biological effects likely collectively modulated by host dietary components, local metabolites, and microbial interaction networks.

Both normal chow and Mediterranean diet (MD) interventions ameliorated gut dysbiosis in MASLD mice, characterized by increased abundance of Prevotella and Lachnospiraceae. While ND primarily reduced opportunistic pathogens, MD specifically enriched beneficial genera (Prevotella, Muribaculum, Duncaniella, Barnesiella), whose levels positively correlated with propionate production. The therapeutic efficacy of MD arises from multicomponent synergy: ω-3 PUFAs modulate the Firmicutes/Bacteroidetes ratio, enhancing Lachnospiraceae and Bifidobacteriaceae while suppressing LPS-producing Enterobacteriaceae (51); dietary fibers foster SCFA-producing bacteria through diversified fermentation substrates (52); and polyphenols selectively promote Lactobacillus growth, inhibit Enterococcus, and upregulate intestinal tight junction proteins (53). Notably, Prevotella may mediate MD's metabolic effects via carbon/BCAA-related fatty acid pathways (54), though conflicting clinical evidence links P. copri abundance to MASLD progression in Asian populations, suggesting strain-specific functionality (55). Importantly, not all studies support MD's microbial modulation—a 6-month trial (56) observed comparable gut microbiota alterations between MD and Western diets, highlighting challenges in achieving significant short-term restructuring of stable microbial ecosystems.

The downstream effects of gut-derived metabolites, particularly SCFAs, may constitute a pivotal mechanistic pathway. Beyond serving as primary energy substrates for colonocytes, SCFAs enhance intestinal barrier integrity by reducing systemic LPS levels (57). Their deficiency exacerbates hepatic inflammation and insulin resistance through impaired anti-inflammatory responses and attenuated HDAC3 activity (10, 44). Our targeted metabolomics revealed marked SCFA dysregulation in MASLD mice: decreased metabolically protective acetate, propionate, and butyrate alongside elevated detrimental isobutyrate/isovalerate (Figures 10, 11). MD intervention partially reversed this profile, promoting propionate accumulation while suppressing branched-chain fatty acids (BCFAs). MD's abundant dietary fibers reshape SCFAs synthesis by providing diverse fermentation substrates. Elevated propionate activates the APN-AMPK-PPARα axis, suppressing de novo lipogenesis while enhancing β-oxidation to alleviate hepatic lipid accumulation (58). Concurrent butyrate upregulation may mitigate mitochondrial dysfunction and ROS production through phage-bacterial LPS interactions, thereby reducing hepatocyte injury (59).

BCFAs, including isobutyrate and isovalerate, are microbial proteolytic metabolites derived from branched-chain amino acid (BCAA) fermentation, exhibiting detrimental effects on intestinal and metabolic homeostasis (60, 61). In the distal colon under high-fat diet conditions—characterized by limited fermentable carbohydrates—gut microbiota preferentially engage in proteolytic metabolism, driving BCFAs overproduction (62). This metabolic shift correlates with hepatic pathophysiology: murine models of macrovesicular steatosis demonstrate elevated cecal BCFAs levels concomitant with insulin resistance and hyperleptinemia (63), while microbial-derived phenylacetic acid exacerbates hepatic lipid accumulation via TCA cycle-potentiated BCAA catabolis (61). Collectively, these findings position proteolytic metabolites as critical mediators of steatosis progression. Dietary strategies favoring carbohydrate over proteolytic fermentation, such as increased fiber intake, may mitigate metabolic disorders including MASLD. The Mediterranean diet (MD) exemplifies this approach by supplying abundant fermentable fiber to suppress proteolytic pathway dominance and inhibit proteolytic taxa proliferation, as evidenced in our study. However, our study lacks comprehensive analysis of intestinal protein fermentation metabolites, including indole derivatives and branched-chain fatty acids. The mechanisms by which these metabolites influence MASLD pathogenesis remain unclear. Future research should investigate their roles in MASLD progression and evaluate dietary interventions targeting protein quality, intake levels, and fermentability to optimize clinical management.