Wuliangye Baijiu but not ethanol reduces cardiovascular disease risks in a zebrafish thrombosis model

Epidemiologic studies suggest moderate alcohol consumption may correlate with reduced cardiovascular disease (CVD) risk (the “French Paradox”), potentially via hemostatic effects on platelets. However, confounding, exposure misclassification, and mixed causal evidence limit conclusions, and alcohol is also associated with higher risks of hypertension, stroke, cancer, and liver disease. Wine polyphenols like resveratrol can be beneficial, but required intake levels conflict with alcohol toxicity, impeding causal inference. Mechanistic model-based studies are needed to test causality. Baijiu, a traditional Chinese distilled spirit produced by complex solid-state fermentation, contains numerous bioactive compounds (phenols, acids, pyrazines, sulfur compounds, terpenes, esters, etc.) with reported antioxidant and cardioprotective activities. Thrombosis is a major pathological driver of CVD, initiated by platelet aggregation with ensuing oxidative stress. This study uses an AA-induced zebrafish thrombosis model to test whether Wuliangye Baijiu (a strong-aroma Baijiu) exerts antithrombotic effects beyond ethanol alone, and to explore underlying mechanisms via imaging and transcriptomic/profiling approaches.

Prior large-scale observational studies report lower CVD and all-cause mortality with low-to-moderate alcohol intake, but genetic and prospective evidence indicates limited or non-causal protection for stroke and potential harms (hypertension, cancer, cirrhosis). Measurement bias and dietary confounding limit epidemiologic inference. Wine bioactives (e.g., resveratrol) have mechanistic benefits but are present at levels that may require harmful alcohol doses. Baijiu’s complex fermentation yields hundreds of volatile and non-volatile compounds; several constituents (e.g., α-linolenic acid, ethyl linoleate, ferulic acid, pyrazines, sulfur compounds, terpenes) show antioxidant, anti-inflammatory, vasodilatory, and antithrombotic activities in prior studies. Arachidonic acid (AA) is a known inducer of platelet activation/aggregation and is used to model thrombosis in zebrafish, which share substantial hemostatic homology with humans. These data motivate testing whether Baijiu’s bioactives confer antithrombotic effects independent of ethanol.

Design: An AA-induced zebrafish thrombosis model was used to assess antithrombotic effects of Wuliangye Baijiu compared with aspirin (positive control) and ethanol at equivalent alcohol concentration. Outcomes included red blood cell (RBC) distribution via o-dianisidine staining, blood flow velocity, RBC aggregation in Tg(gata1:DsRed) embryos, oxidative stress (ROS via DCFH-DA fluorescence; catalase [CAT] activity), and molecular profiling (RNA-seq, qRT-PCR). Ethics: Experiments followed Animal Ethics Committee guidelines (Gannan Normal University). Animals and husbandry: Transgenic Tg(gata1:DsRed) zebrafish were maintained at 28 ± 0.5 °C, 14/10 h light/dark cycle; embryos collected and reared at 28.5 °C with PTU to inhibit pigmentation. Thrombosis induction and treatments: AA concentrations (30–60 μM) and exposure durations (30–90 min) were optimized; 30 μM AA for 45 min at 72 hpf was selected. Toxicity screening identified ≥0.5% ethanol (v/v) causing increased malformations and impaired hatchability; 0.3% ethanol (v/v) showed no obvious developmental effects and was used for both Wuliangye and ethanol groups. From 48 hpf, embryos were pre-incubated in 22.5 mg/L aspirin, Wuliangye (0.3% ethanol, v/v), or ethanol (0.3% v/v) until sampling. AA was dissolved in 0.075% DMSO; 0.075% DMSO served as vehicle control. Groups: control, AA, AA-aspirin, AA-Wuliangye, AA-ethanol. Imaging and hemodynamics: o-dianisidine staining quantified RBCs in heart and tail (n=10 per group). Blood flow videos were recorded (embryos in 1% low-melt agarose), velocity quantified with DanioScope; RBC aggregation assessed in anesthetized Tg(gata1:DsRed) embryos under Leica M205 FA microscope. Oxidative stress assays: ROS measured by DCFH-DA fluorescence (1,000×, 20 min, 28.5 °C, dark; imaged on Leica M205 FA; ImageJ quantification). CAT activity quantified using commercial kits, normalized to total protein; 50 embryos per group, three biological replicates, each measured in triplicate. RNA-seq: Total RNA from 30 embryos/group (3 dpf) extracted with Trizol; libraries sequenced on Illumina HiSeq X Ten (150 bp paired-end). Differential expression performed with DESeq2; GO/KEGG enrichment via clusterProfiler (adjusted P<0.05 considered significant). qRT-PCR: RNA from 50 embryos/group reverse-transcribed; SYBR Green qPCR performed (β-actin control) on Step-One-Plus/qTower 3G; primers in Supplementary Table 1; triplicate reactions. Target panel included TF, f2, fgb, ptgs2a, PAI-1, TNF-α, IL-10, IL-6. Statistics: One-way ANOVA and Student’s t test (GraphPad Prism 5.0); data as mean ± SD; significance annotated as *P<0.05, **P<0.01, ***P<0.001. Data availability: RNA-seq data in GSA (CRA006632).

• AA-induced thrombosis: AA (30 μM, 45 min) reduced heart RBCs and increased tail accumulation; blood flow velocity significantly decreased vs control (P<0.001). - Antithrombotic effects: Pretreatment with Wuliangye (0.3% ethanol, v/v) or aspirin restored heart RBC levels and reduced tail RBC accumulation toward control (P<0.001 vs AA), while ethanol (0.3% v/v) showed no significant effect (P>0.05). Wuliangye improved blood flow velocity significantly vs AA (P<0.001) and showed better efficacy than aspirin. - Oxidative stress: AA increased ROS (DCFH-DA fluorescence) and decreased CAT activity (P<0.001 vs control). Wuliangye and aspirin significantly reduced ROS and restored CAT (P<0.001 vs AA), whereas ethanol increased ROS and did not improve CAT. - Transcriptomics: PCA showed distinct clustering by group. Compared with AA, AA-Wuliangye had 14,193 DEGs (6,762 up; 7,431 down); AA-ethanol had 2,127 DEGs (1,098 up; 1,029 down). KEGG enrichment indicated Wuliangye modulated pathways related to platelet activation/adhesion and thrombus biology, including focal adhesion, ECM-receptor interaction, AGE-RAGE signaling, adherens junctions, CAMs, PPAR signaling, inflammation, and oxidative stress pathways; many key genes were downregulated with Wuliangye. - qRT-PCR: AA dysregulated coagulation and inflammatory genes (elevated TF, f2, fgb, ptgs2a, PAI-1, TNF-α, IL-6; reduced IL-10). Wuliangye significantly mitigated changes in TF, f2, ptgs2a, PAI-1, TNF-α, IL-10, and IL-6; aspirin significantly mitigated f2, ptgs2a, IL-10, and IL-6; ethanol only affected IL-6. - Safety window: Ethanol concentrations ≥0.5% (v/v) in Wuliangye or pure ethanol increased malformation rates and affected hatchability; 0.3% showed no obvious developmental effects and was used for efficacy comparisons.

Using a mechanistic zebrafish model, the study demonstrates that Wuliangye Baijiu exerts antithrombotic and antioxidant effects independent of ethanol. Wuliangye restored hemodynamics and RBC distribution, reduced ROS, and normalized antioxidant enzyme activity. Molecular profiling showed broad transcriptomic remodeling with downregulation of pathways mediating platelet adhesion/activation (focal adhesion, ECM interactions, CAMs), oxidative stress, and inflammatory signaling, consistent with decreased thrombus formation. qRT-PCR confirmed inhibition of coagulation cascade components (TF, f2, fibrinogen) and pro-inflammatory mediators (TNF-α, IL-6) with restoration of anti-inflammatory IL-10, indicating modulation of both coagulation and inflammation. The superior performance of Wuliangye over ethanol (and in some measures over aspirin) supports a role for non-ethanol bioactive constituents acting synergistically across multiple pathways to reduce thrombosis risk. These findings address the research question by providing causal, model-based evidence that Baijiu’s effects are attributable to its bioactive compounds rather than ethanol per se.

Wuliangye Baijiu, at an ethanol-equivalent concentration that is developmentally tolerated (0.3% v/v), prevents and reduces AA-induced thrombosis in zebrafish by improving blood flow, normalizing RBC distribution, decreasing oxidative stress, and modulating coagulation, platelet activation/adhesion, and inflammatory pathways. Ethanol alone at the same concentration does not confer these benefits. The study provides mechanistic support for CVD risk reduction associated with Baijiu’s bioactives. Future work should isolate and quantify specific active compounds, define dose-response relationships, validate in mammalian thrombosis models, and assess translational relevance to human cardiovascular outcomes.

• Model specificity: Findings are from an AA-induced zebrafish thrombosis model, which, while informative and genetically conserved, may not capture the full complexity of human thrombosis and CVD. - Compound attribution: The specific bioactive constituents responsible for effects were not identified; Wuliangye’s complex composition precludes attribution to individual compounds. - Single dose/timepoint: Efficacy was primarily evaluated at one ethanol-equivalent concentration (0.3% v/v) and a single AA regimen; broader dose–response and timing analyses are needed. - Outcome scope: Functional cardiovascular endpoints beyond thrombosis (e.g., bleeding risk, long-term vascular remodeling) were not assessed. - Ethanol control: Only one ethanol concentration was tested for comparison; higher or lower non-toxic doses might yield different molecular responses. - Generalizability: Results may not generalize across Baijiu brands or batches with different compositions; batch-to-batch variation was not evaluated.