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

The French Paradox, where high cholesterol consumption correlates with low coronary heart disease mortality, has been partially attributed to moderate alcohol intake. However, epidemiological studies on alcohol's effect on cardiovascular disease (CVD) risk have yielded conflicting results, with limitations in exposure measurement and confounding factors. Moderate alcohol consumption has shown associations with reduced CVD risk and all-cause mortality, but exceeding a certain threshold may negate these benefits. Furthermore, the effects may be confounded by other bioactive compounds in alcoholic beverages like resveratrol in wine. Baijiu, a Chinese liquor, offers a unique opportunity for study because of its complex composition of bioactive compounds and its widespread consumption. This research aims to elucidate the causal relationship between Baijiu consumption and CVD risk reduction by investigating the antithrombotic effects of Wuliangye Baijiu, a strong-aroma Baijiu, using a zebrafish model. The zebrafish model is chosen due to its genetic similarity to humans, and the use of arachidonic acid (AA) to induce thrombosis allows for quantitative evaluation of antithrombotic effects.

Numerous studies have explored the relationship between alcohol consumption and cardiovascular health. Some studies suggest a protective effect of moderate alcohol intake against CVD, while others highlight potential negative effects at higher levels. The French Paradox highlights a complex relationship, likely involving both the ethanol and other bioactive components in alcoholic drinks. Studies on resveratrol, a polyphenol in wine, demonstrate potential health benefits, but the amounts required are difficult to achieve without significant alcohol consumption. Therefore, a controlled study examining the impact of a complex alcoholic beverage like Baijiu is crucial to clarify the causal mechanisms involved.

A zebrafish thrombosis model was established using arachidonic acid (AA) to induce thrombus formation. The effects of Wuliangye Baijiu, ethanol (as a control), and aspirin (as a positive control) were compared. Several methods were employed to assess the impact of these substances: 1. **o-Dianisidine Staining:** To quantify red blood cell (RBC) levels in the heart and tail regions, indicating thrombus formation. 2. **Blood Flow Analysis:** Using DanioScope1 software to measure blood flow velocity in treated zebrafish. 3. **Oxidative Stress Assessment:** Using DCFH-DA, a fluorescent indicator of H2O2, to measure cellular oxidative stress. Catalase (CAT) activity was also measured to evaluate antioxidant capacity. 4. **RNA Sequencing (RNA-seq):** To identify differentially expressed genes (DEGs) between treatment groups, providing insights into the molecular mechanisms involved. Principal component analysis (PCA) and KEGG pathway analysis were performed on the RNA-seq data. 5. **Quantitative Real-Time PCR (qRT-PCR):** To validate the RNA-seq findings by measuring the expression levels of specific genes related to coagulation, platelet activation, and inflammation, including tissue factor (TF), coagulation factor II (f2), fibrinogen β chain (fgb), prostaglandin peroxide synthase 2A (ptgs2a), plasma plasminogen activator inhibitor PAI-1, tumor necrosis factor (TNF-α), interleukin-10 (IL-10), and interleukin-6 (IL-6). Zebrafish embryos were treated at 48 hours post-fertilization (hpf) with the test substances, and thrombosis was induced at 72 hpf with AA. Appropriate controls (DMSO, untreated zebrafish) were included. Statistical analysis was performed using one-way ANOVA and Student's t-test.

The key findings of this study are: 1. **Wuliangye Baijiu Reduces Thrombosis:** Wuliangye Baijiu significantly reduced thrombus formation in the AA-induced zebrafish model, as evidenced by increased RBC levels in the heart and decreased levels in the tail, compared to the AA-only group. This effect was not observed with ethanol at the same concentration. 2. **Improved Blood Flow:** Wuliangye Baijiu treatment significantly increased blood flow velocity in the AA-treated zebrafish, suggesting improved circulatory function. This improvement was superior to that observed with aspirin. 3. **Reduced Oxidative Stress:** Wuliangye Baijiu treatment significantly decreased cellular oxidative stress, as indicated by reduced DCFH-DA fluorescence intensity. Conversely, ethanol increased oxidative stress. CAT levels were significantly increased after treatment with Wuliangye Baijiu, confirming its antioxidant effects. 4. **Molecular Mechanisms:** RNA-seq analysis revealed a significant number of differentially expressed genes (DEGs) between the AA-treated group and the AA + Wuliangye group. KEGG pathway analysis indicated that these DEGs were enriched in pathways related to platelet aggregation, adhesion, inflammation, and oxidative stress. Wuliangye treatment downregulated genes involved in focal adhesion, ECM-receptor interaction, AGE-RAGE signaling, adherens junctions, and cell adhesion molecules (CAMs). 5. **qRT-PCR Validation:** qRT-PCR confirmed the RNA-seq findings by demonstrating that Wuliangye Baijiu significantly mitigated the changes in the expression levels of several key genes involved in coagulation, platelet activation, and inflammation compared to the AA-only group. Ethanol had a minimal effect on gene expression levels.

This study provides strong evidence that Wuliangye Baijiu possesses antithrombotic properties that are not solely attributable to its ethanol content. The observed effects appear to be mediated through a complex interplay of multiple bioactive compounds within the Baijiu. The reduction of oxidative stress and the modulation of genes involved in platelet aggregation, adhesion, and inflammation suggest multiple pathways are affected. The superior efficacy of Wuliangye Baijiu compared to aspirin in this model warrants further investigation to identify the specific bioactive compounds responsible for the beneficial effects. This study contributes to the understanding of the complex relationship between alcohol consumption, bioactive compounds, and cardiovascular health.

This research demonstrates that Wuliangye Baijiu effectively reduces thrombotic risks and oxidative stress in a zebrafish thrombosis model, independent of ethanol content. The observed effects are likely due to the combined action of multiple bioactive compounds, targeting several pathways involved in platelet activation and inflammation. Future research should focus on identifying the specific bioactive compounds responsible for these effects and exploring their potential therapeutic applications in CVD prevention and treatment.

The study used a zebrafish model, which, while advantageous for its genetic tractability and ease of observation, may not fully reflect the complexity of human physiology. The specific bioactive compounds in Wuliangye responsible for the observed effects were not identified. Further research is needed to translate these findings to human clinical settings.