The fruiting bodies of Gomphus floccosus (Gf), collected from Yunnan Province, China, were authenticated, and a voucher specimen (No. 251822) was deposited at the Fungarium (HMAS), Institute of Microbiology, Chinese Academy of Sciences. The fungal material used in this study, also sourced from Yunnan, was taxonomically identified by the Testing Department of the Shanghai Traditional Chinese Medicine Standardization Research Center (Testing No. JC21001).

The Gf extract (Batch No. 20220921) was prepared through sequential extraction of the dried fungal powder (40 mesh) using 95% and 70% ethanol. The combined extracts were concentrated under reduced pressure to obtain a primary active fraction, which was dissolved in water, filtered, and subjected to sequential liquid-liquid extraction with petroleum ether and ethyl acetate. The final aqueous phase was further purified using D101 macroporous resin, with water as the eluent, and concentrated to yield the final extract. Qualitative and quantitative analyses of the representative chemical constituents in the Gf extract were conducted using ultra-high-performance liquid chromatography coupled with Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS).

Eight-week-old male C57BL/6J mice (21 ± 1g) were obtained from Shanghai Jihui Experimental Animal Breeding Co., Ltd. (SCXK 2022-0009) and housed in the Specific Pathogen-Free (SPF) facility at Shanghai University of Traditional Chinese Medicine (SYXK 2020-0009). All animals were maintained under controlled environmental conditions. The experimental protocols were approved by the Institutional Animal Ethics Committee (Approval Nos. PZSHUTCM2312250011, PZSHUTCM250210001).

Biochemical studies of serum and liver homogenates were conducted in accordance with commercial kit methods. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured utilizing a microplate assay with 10 μL of serum. For hepatic lipid evaluation, liver tissue was homogenized in anhydrous ethanol, and the amounts of TC(A111-1-1) and TG(A110-1-1) in the resultant extracts were quantified. Moreover, tissue (100 mg) was homogenized in physiological saline to evaluate oxidative stress indicators (GSH, T-SOD, MDA)(A006-2, A001-3; S0131S, Beyotime, China) and glycolytic metabolites (lactate, pyruvate)(A019-2-1, A081-1-1) in the supernatant. All commercial kits were sourced from the Nanjing Jiancheng Bioengineering Institute (China), with the exception of the MDA kit.

Hepatic histopathology was evaluated on tissue sections stained with H&E and Oil Red O according to recognized techniques. Lipid accumulation was assessed by determining the area percentage of Oil Red O-positive lipid droplets from recorded pictures utilizing ImageJ software (v1.8.0) (25).

Tissue was deparaffinized and rehydrated. Sections underwent heat-induced epitope retrieval in citrate buffer (7–8 min at boiling, then 15 min at sub-boiling). Sections were cooled to room temperature, washed with TBST and incubated with 3% H₂O₂ in methanol to quench endogenous peroxidase. They were then blocked with 5% BSA for 30 min and incubated with primary antibody (30 μL/section) overnight at 4 °C. The next day, sections were washed and incubated with HRP-conjugated secondary antibody (1 h at RT), followed by 20-min SABC reagent incubation. DAB was used to develop color. Sections were counterstained with hematoxylin, dehydrated, cleared and mounted. The immunohistochemically stained area was quantified using ImageJ (v1.8.0).

TRIzol reagent (F919KB3054, Sangon Biotech, China) was used to extract total RNA from mouse liver tissues. After assessing purity, quantity, and integrity, transcriptome libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads. Raw sequencing data were processed with Fastp to remove low-quality sequences. Differential gene expression analysis was performed using the DESeq2 package in R (v4.3.2), with genes showing |log₂FC| > 1 and adjusted p-value<0.05 considered significantly upregulated or downregulated. Visualization of differential expression results was conducted using the Microbiostatistics platform.

GO and KEGG pathway enrichment analyses used a hypergeometric test algorithm. Significantly enriched terms and pathways were identified, and results were visualized using bubble plots (26).

Tissue sections were subjected to standard multiplex immunofluorescence staining using the tyramide signal amplification (TSA) method(10534100050, Panovue, China). Briefly, after deparaffinization, rehydration, and heat-induced antigen retrieval, sections were sequentially incubated with primary antibodies against F4/80(GB11027-100, Servicebio, China), iNOS, PKM2, and Arg-1(18985-1-AP, 60268-1-Ig, 66129-1-Ig, Proteintech, China), followed by corresponding secondary antibodies and TSA-conjugated fluorophores.

Between each staining cycle, antibody-TSA complexes were removed by repeating the antigen retrieval step to enable subsequent rounds of staining. Fluorophores were strategically assigned based on target abundance and emission intensity: 690 nm (F4/80), 520 nm (iNOS), 620 nm (PKM2), and 570 nm (Arg-1).

Following completion of all staining cycles, nuclei were counterstained with DAPI, then sections were mounted for later analysis.

Cytokine levels (IL-1β, IL-6, and IL-10)(88-7013-88, 88-7064-88, 88-7105-88, Invitrogen, USA) were quantified using ELISA kits.

Total protein was isolated using RIPA lysis buffer with inhibitors, then concentrations were measured via a BCA assay(WA322434, Thermofisher, USA). SDS-PAGE resolved protein samples, then were electrotransferred to PVDF membranes. The membranes were blocked and primed in a five-minute step before being probed with key target antibodies—GLUT1, GCK, HK2, LDHA (66290-1-Ig, 67216-1-Ig, 66974-1-Ig, 19987-1-AP, Proteintech, China), PKM2, iNOS, and Arg-1—as well as loading controls (GAPDH and Tubulin)(AF2823, AF2839, Beyotime, China). Subsequently, membranes were incubated with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized with an ECL(180-5001, Tanon, China) detection system and quantified by densitometric analysis using ImageJ software.

Total RNA was isolated and reverse-transcribed into cDNA using a Simply P kit (BSC52M1, BioFlux, China). qPCR then used SYBR Green chemistry, with GAPDH and ACTB serving as endogenous reference genes for normalization. The 2^(-ΔΔCt) method was used to determine relative mRNA expression levels, while primer sequences were supplied by Sangon Biotech and Guantai Biotech (Shanghai), as detailed in Table 1.

The RAW264.7 murine macrophage line and the THP-1 human monocyte line were obtained from the Cell Bank of the Chinese Academy of Sciences. RAW264.7 cells were routinely maintained in complete DMEM at 37 °C in a 5% CO₂ atmosphere. For experiments, cells were grown at appropriate densities, left to adhere for 24 h, and then switched to low-serum DMEM (1% FBS) containing 100 ng/mL lipopolysaccharide (LPS HY-D1056, MCE, USA) and 50 mM ethanol for 48 h. THP-1 cells were differentiated into macrophage-like cells by culture in complete RPMI-1640 medium containing 100 ng/mL phorbol 12-myristate 13-acetate (PMA ab120297, Abcam, UK) for 48 h. Following differentiation, THP-1 cells received the same stimulation and treatment regimen as RAW264.7 cells. Treatment groups were administered either 200 ng/mL Gf extract or 0.5 mM 2-deoxyglucose (2-DG, positive control)(103344-100, Agilent, USA) during the final 24h of stimulation. Post-treatment, cells and culture supernatants were collected for subsequent Western blot/RT-qPCR and ELISA analyses, respectively. (N: control group;M:100ng/ml LPS + 50mM ethanol; Gf: 100ng/ml LPS + 50mM ethanol+200ng/ml Gf;2-DG: 100ng/ml LPS + 50mM ethanol+0.5mM 2-DG).

The CCK8 (HY–K0301, MCE, USA) was used to evaluate cell viability release, following the manufacturer’s guidelines.

Following modeling and drug treatments, cells grown on coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 (4°C, 1 min) and immersed in 5% BSA for 60 min. Immunostaining was performed by probing the samples with primary antibodies against PKM2 (1:200) and iNOS (1:50), incubated either at 4 °C overnight. Post PBS washes, the cells were exposed to species-matched fluorescent secondary antibodies (FITC/Cy3, 1:2000)(ab6785, ab6939, Abcam, UK) for 60 min at RT in the dark. Following extensive washing, the nuclei underwent counterstaining with DAPI at a 1:2000 ratio for 15–20 min. The coverslips were eventually washed, affixed to glass slides, and viewed through a laser scanning confocal microscope.

Cellular metabolism was profiled with the help of an Agilent Seahorse XFe96 Analyzer. Cells were plated in XF96 microplates and equilibrated for 1 h at 37 °C in unbuffered XF assay medium (pH 7.4) (103575-100) containing 10 mM glucose(103577-100), 1 mM pyruvate(103578-100), and 2 mM glutamine(103579-100), under non-CO₂ conditions. Glycolytic function(103344-100) was assessed via sequential injection of 10 mM glucose, 1.5 µM oligomycin, and 50 mM 2-deoxyglucose. To evaluate mitochondrial respiration (103015-100), 2 µM oligomycin, 1.5 µM FCCP, and 0.5 µM antimycin A/rotenone were added one after the other. Metabolic parameters were automatically recorded and normalized using integrated Seahorse Software and a Cytation 7 imaging system. All reagents and kits were obtained from Agilent Technologies (USA).

Results are shown as mean ± standard deviation, and statistical analysis was conducted with SPSS 27.0. A one-way ANOVA with Tukey’s post hoc test was utilized for multiple group comparisons, provided the data adhered to normality and variance homogeneity assumptions. For data violating these assumptions, Dunn’s post hoc analysis was applied after the Kruskal-Wallis test. For two-group comparisons, the Mann-Whitney U test was utilized, contingent on parametric test assumptions. Statistical significance was assigned to p-values below 0.05. GraphPad Prism 10 was used for all graphical displays.

Comprehensive chemical analysis revealed that Gf extract contains diverse bioactive components, including alkaloids, flavonoids, carotenoids, terpenoids, sterols, saponins, polysaccharides, amino acids, and essential minerals (selenium, potassium, cobalt). Additionally, microbial components (A, B, C, D, E, and P) were detected. Seven representative bioactive compounds—ascorbic acid, gallic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, and cinnamic acid—were selected as reference standards based on literature.

The chemical characterization of these compounds was performed using UHPLC-Q-Exactive Orbitrap HRMS. Total ion chromatograms (TIC) of the extract and the mixed standards are shown in Figure 1, with detailed information on retention times, molecular weights, and fragment ions summarized in Table 2.

The ALD model and its treatment protocol are shown in Figure 2. In the 3-week model, mice were subjected to one week of adaptive feeding with an alcohol-containing diet, followed by one week of modeling during which drug treatment was administered concurrently (Figure 2A). Evaluation of this model revealed significantly elevated serum ALT and AST levels in the alcohol-fed group (Figures 2B, C), along with increased tissue TC and TG contents, heightened oxidative stress markers (MDA and NADH), and decreased T-SOD and GSH activities (Figures 2D–I), confirming successful induction of alcoholic liver disease. After one week of treatment with Gf extract, improvements were observed in liver function, lipid metabolism, and oxidative stress markers, with significant reductions in MDA and NADH levels and increased T-SOD and GSH activities. The high-dose Gf group demonstrated superior efficacy compared to the low-dose group, and it significantly outperformed the positive control drug Polyene Phosphatidylcholine in lowering NADH levels.

In the 4-week model, adaptive feeding was followed by two weeks of alcohol-induced liver injury, with drug treatment administered during the final week after modeling cessation (Figure 2J). Although the alcohol-fed group exhibited elevated serum and tissue indices similar to those in the 3-week model, the increases were less pronounced. Drug administration ameliorated all measured parameters, with the high-dose Gf group again showing stronger effects compared to the low-dose group (Figures 2K–O).

Histopathological analysis supported these findings. Liver tissues from alcohol-fed mice showed macrovesicular and microvesicular steatosis, ballooning degeneration, and neutrophil infiltration, while Oil Red O staining confirmed substantial lipid droplet accumulation in hepatocytes. Pathological injury was less severe in the 4-week model compared to the 3-week model. Treatment with Gf extract markedly alleviated hepatic steatosis, ballooning degeneration, and inflammatory cell infiltration, with the high-dose group exhibiting more substantial improvement than the low-dose group (Figures 2P–Q, S, T).

These results collectively demonstrate that Gf extract effectively ameliorates alcoholic liver disease, particularly at high doses, by improving liver function, reducing lipid accumulation, and mitigating oxidative stress and histopathological damage.

Transcriptome sequencing was performed on liver tissues from the alcohol-fed control (N), alcohol-fed model (M), and high-dose Gf extract-treated (H) groups. Venn diagram analysis identified 231 overlapping differentially expressed genes (DEGs) between the N vs. M and M vs. H comparisons (Figure 3A). Compared to the H group, the M group exhibited the upregulation of 252 genes and the downregulation of 258 genes (Figure 3B). The expression profiles of these DEGs across the N, M, and H groups are visualized in a heatmap (Figure 3C).

KEGG enrichment analysis of the 231 overlapping DEGs revealed that the top 20 enriched pathways included glycolysis/gluconeogenesis (Figure 3D). Key genes implicated in this pathway, such as Gck, Acss2, Pck1, Pck2, and Pgm2, were differentially expressed. Gene set enrichment analysis (GSEA) further confirmed the broad impact of Gf extract on hepatic metabolism. Although the glycolysis gene set did not reach statistical significance, significant enrichment was observed in the ‘glycogen metabolic process’ (Figure 3E). This finding suggests that Gf extract orchestrates a comprehensive reprogramming of carbohydrate metabolism in the liver. Specifically, it appears to attenuate glycolytic flux while modulating glycogen-related energy storage, thereby contributing to the restoration of metabolic homeostasis in alcoholic liver disease (ALD).

GO annotation further revealed that these DEGs were associated with biological processes (BP), cellular components (CC), and molecular functions (MF). Among the top 10 enriched BP terms were “negative regulation of macrophage chemotaxis,” “gluconeogenesis,” and “inflammatory response” (Figure 3F).

Immunohistochemical staining demonstrated prominent PKM2 expression localized to hepatic sinusoids (Figures 2R, U). Previous studies have established that macrophages undergo significant metabolic reprogramming during immune activation, with glycolysis serving as a core metabolic hallmark of their pro-inflammatory phenotype. Based on these findings, we hypothesize that Gf extract mitigates alcohol-induced liver injury by modulating glycolysis in macrophages, thereby attenuating the inflammatory response and restoring hepatic metabolic balance.

Multiplex immunohistochemical analysis demonstrated that Gf extract reversed the polarization imbalance of hepatic macrophages in a dose-dependent manner. Specifically, treatment with Gf extract significantly reduced the number of F4/80+PKM2+iNOS+ M1 macrophages while restoring the population of F4/80+PKM2+Arg-1+ M2 macrophages in the livers of the M group (Figures 4A, B). Consistent with these findings, Western blotting and qPCR analyses further confirmed that Gf extract downregulated the expression of the M1 polarization marker iNOS while upregulating the expression of the M2 polarization marker Arg-1 at both the protein and transcriptional levels (Figures 4F–J).

This phenotypic shift in macrophage polarization was accompanied by a reduction in the inflammatory response, as evidenced by decreased levels of pro-inflammatory cytokines and increased levels of anti-inflammatory cytokines (Figures 4C–E). These results collectively suggest that Gf extract exerts its anti-inflammatory effects by modulating macrophage polarization, specifically by suppressing the M1 pro-inflammatory phenotype and promoting the M2 anti-inflammatory phenotype, thereby contributing to the resolution of liver inflammation in alcoholic liver disease.

Metabolite analysis revealed that the levels of pyruvate, lactate, and lactate dehydrogenase (LDH) were significantly elevated in the M group compared to the N group. Treatment with Gf extract markedly reduced these levels, indicating a potential regulatory effect on glycolytic activity (Figures 5A–C).

To further investigate the molecular mechanisms underlying these metabolic changes, we analyzed the expression of key genes involved in the KEGG glycolysis/gluconeogenesis pathway, including Gck, Acss2, Pck1, Pck2, and Pgm2. Among these, only Gck and Pck2 exhibited significant intergroup differences (Figures 5D–H). Notably, Gck mRNA expression displayed the most pronounced upregulation in the model group, highlighting its role as a pivotal regulator of glycolysis initiation. This prompted further investigation into the expression of other key glycolytic enzymes, including Glut1, Pkm2, Hk2, and Ldha.

While no significant differences in Glut1 mRNA expression were observed among the groups (Figure 5I), Western blot analysis demonstrated a significant reduction in the protein levels of GLUT1, PKM2, HK2, and LDHA following Gf extract treatment (Figures 5M–S). Consistently, the mRNA levels of Pkm2, Hk2, and Ldha were also significantly downregulated in the treatment groups (Figures 5J–L).

These findings suggest that Gf extract exerts its therapeutic effects on alcoholic liver disease by modulating glycolytic enzyme expression and reducing the accumulation of glycolytic metabolites, thereby contributing to the restoration of metabolic homeostasis.

The CCK-8 assay identified 200 ng/mL as the optimal non-cytotoxic concentration of the Gf extract for in vitro experiments (Figure 6B). An in vitro M1 macrophage polarization model was established by treating RAW264.7 cells with 100 ng/mL LPS and 50 mM ethanol for 48 hours, followed by 24-hour co-incubation with the Gf extract.

Immunofluorescence co-staining showed increased PKM2 and iNOS expression in the model group, which was significantly suppressed by Gf extract treatment (Figure 6A). Similarly, ELISA results revealed elevated IL-1β and IL-6 levels in the model group, which were markedly reduced after treatment (Figures 6C, D). Western blotting and qPCR further confirmed that Gf extract downregulated iNOS expression at both mRNA and protein levels, while upregulating the M2 marker Arg-1 (Figures 6E–I).

These results suggest that Gf extract alleviates inflammation by suppressing M1 polarization through the downregulation of PKM2 and iNOS and promoting M2 polarization via Arg-1 upregulation.

To explore the metabolic reprogramming induced by Gf extract in RAW264.7 macrophages, we first assessed glycolytic function. The model group exhibited significantly elevated glycolytic rates, including basal and compensatory glycolysis, all of which were markedly suppressed by Gf extract, comparable to the effect of the glycolytic inhibitor 2-DG (Figures 7A–D). Despite these changes, the proportional contribution of glycolysis to basal energy production remained stable, suggesting that Gf extract reduces overall glycolytic capacity without altering energy source allocation.

Mitochondrial respiration analysis revealed severe dysfunction in the model group, with decreased basal respiration, ATP-linked respiration, and maximal respiratory capacity. Gf extract significantly restored these parameters, outperforming 2-DG, which had no notable effect on mitochondrial function (Figures 7E–H).

At the molecular level, the model group showed increased lactate and pyruvate accumulation, along with upregulation of glycolytic enzymes (Pkm2, Gck, Hk2, Glut1, and Ldha) at both mRNA and protein levels. These alterations were effectively reversed by Gf extract treatment (Figures 7I–V).

In summary, Gf extract reprograms macrophage metabolism by suppressing glycolysis and enhancing mitochondrial oxidative phosphorylation, restoring metabolic homeostasis in M1-polarized macrophages.

To evaluate the cross-species relevance of Gf extract, its effects were tested in PMA-differentiated human THP-1 macrophages. Similar to murine cells, Gf extract downregulated PKM2 and iNOS protein expression (Figure 8A) and significantly decreased extracellular lactate and pyruvate levels (Figures 8B, C). Additionally, the extract suppressed iNOS expression while enhancing Arg-1 at both mRNA and protein levels (Figures 8D–H), indicating effective inhibition of M1 polarization.

These findings confirm that the macrophage-polarizing effects of Gf extract are conserved between murine and human macrophages.

To determine whether Gf extract induces similar metabolic reprogramming in human macrophages, its effects were examined in PMA-differentiated THP-1 cells. Gf extract significantly suppressed glycolytic flux, including basal and compensatory glycolysis (Figures 9A–D).

Additionally, Gf extract restored mitochondrial respiratory function, improving basal respiration, ATP production, and maximal respiratory capacity, comparable to the effects of the glycolytic inhibitor 2-DG (Figures 9E–H).

At the molecular level, Gf extract reversed the accumulation of lactate and pyruvate in the supernatant and downregulated the expression of key glycolytic genes (Pkm2, Gck, Hk2, Glut1, and Ldha) and their protein products. Notably, the 50-fold upregulation of Gck observed in the model group was normalized by Gf extract treatment (Figures 9I–V).