基于斑马鱼模型评估黄连根提取物在治疗炎症性肠病中的安全性和有效性

中药鬼臼（Sophorae Flavescentis Radix）通过调节FoxO4/NOD-like/Apoptosis和MAPK信号通路改善肠道免疫，低剂量（≤500 μg/mL）安全有效，高剂量（>800 μg/mL）诱导肝细胞凋亡。

　　
Sophorae Flavescentis Radix (Kushen) is a traditional Chinese medicinal herb with a 2,000-year history of use in treating intestinal inflammation, heat clearance, and dampness elimination. Recent studies have expanded its therapeutic scope to include potential benefits in inflammatory bowel disease (IBD), a chronic gastrointestinal disorder characterized by persistent inflammation and barrier dysfunction. This research employs a zebrafish model to systematically evaluate the safety and efficacy of Kushen extract in IBD, while integrating network pharmacology and transcriptomics to elucidate its molecular mechanisms.

### Safety Evaluation
The study first established the safety profile of Kushen extract using zebrafish larvae. Dose-dependent hepatotoxicity was observed at concentrations exceeding 500 μg/mL, marked by hepatic morphological abnormalities and increased apoptosis. Histopathological analysis revealed disordered hepatocyte arrangement and reduced cytoplasmic density in high-dose groups, while low-dose groups (≤500 μg/mL) showed no significant toxicity. This identified a critical safety threshold (LC?: 500 μg/mL) and toxicity window (LC??: 1,000 μg/mL), emphasizing the importance of precise clinical dosing to avoid adverse effects.

### Therapeutic Efficacy in IBD Model
Following induction of IBD using trinitrobenzenesulfonic acid (TNBS), Kushen extract demonstrated marked therapeutic effects at safe doses. Key outcomes included:
1. **Neutrophil Infiltration Reduction**: Low-dose (300 μg/mL) and high-dose (500 μg/mL) treatments significantly decreased intestinal neutrophil counts compared to the model group, with the high dose showing more pronounced efficacy.
2. **Cytokine Suppression**: Pro-inflammatory cytokines (IL-1β, TNF-α, PGE?) were剂量依赖性地降低，验证了Kushen的的抗炎作用。
3. **Intestinal Barrier Protection**: Neutral red and alcian blue staining revealed improved endocytic function and goblet cell mucin secretion in treated groups, indicating restored intestinal integrity.
4. **Apoptosis Inhibition**: Histological and molecular analyses confirmed reduced epithelial cell apoptosis in treated zebrafish.

### Mechanistic Insights
The study combined network pharmacology and transcriptomics to uncover the molecular basis:
- **Network Pharmacology**: Identified 207 shared targets between Kushen components and IBD pathways. Key hubs included AKT1 (Protein kinase B1), TNF, EGFR, and BCL-2. Pathway analysis highlighted involvement in FoxO4/NOD-like/Apoptosis and MAPK signaling.
- **Transcriptomics**: Identified 2631 upregulated and 2505 downregulated genes in low-dose groups, with 3592 upregulated and 3499 downregulated in high-dose. Enrichment analysis revealed significant modulation in calcium signaling, tight junction formation, and cell adhesion pathways.
- **RT-qPCR Validation**: Confirmed downregulation of FoxO4, NLRP6, and apoptosis-related genes (CASP9, CASP2) in treated groups, while upregulation of BCL-2 and EGF suggested anti-apoptotic and pro-repair effects.
- **Molecular Docking**: demonstrated strong interactions between oxymatrine and EGFR (-10.4 kcal/mol), and matrine with BCL-2 (-8.1 kcal/mol), supporting their roles in pathway modulation.

### Pathway-Specific Effects
1. **FoxO Signaling**: Suppressed NF-κB activation, reducing pro-inflammatory cytokine production. FoxO4 downregulation in treated groups aligned with its role in inhibiting TNF-α and IL-6.
2. **NOD-Like Receptor Pathway**: Reduced NLRP3 inflammasome activation, lowering IL-1β secretion. NLRP6 upregulation in treated groups supported epithelial barrier repair.
3. **Apoptosis Regulation**: Decreased BAD, BID, and caspase activity, indicating inhibition of mitochondrial apoptosis pathways. BCL-2 upregulation blocked cytochrome c release.
4. **MAPK Pathway**: Suppressed BRAF-MEK-ERK signaling, reducing TNF-mediated activation of downstream kinases (MAPK3, MAPK8).

### Clinical Implications and Limitations
The study established a clear safety-efficacy window (≤500 μg/mL) for Kushen extract in IBD treatment, with no hepatotoxicity observed below this threshold. Mechanistically, it identified a multi-target approach involving immune modulation, epithelial repair, and anti-apoptotic effects. However, limitations include:
- **Batch Variability**: Natural product inconsistency may affect reproducibility.
- **Post-Transcriptional Regulation**: Lack of protein-level validation hinders causal link establishment.
- **Model Limitations**: Zebrafish models may not fully capture human IBD complexity, particularly regarding microbiota interactions.

### Future Directions
1. **Murine Validation**: Test in rodent models to assess translational potential.
2. **Protein-Level Confirmation**: Perform immunohistochemistry or Western blotting to validate transcriptomic findings.
3. **Component-Specific Analysis**: Isolate major alkaloids (matrine, oxymatrine) to determine individual contributions to hepatotoxicity and efficacy.
4. **Clinical Trial Design**: Use established biomarkers (e.g., calprotectin, fecal lactoferrin) to standardize dosing and monitor adverse effects.

### Conclusion
This integrative approach provides a robust framework for understanding Kushen extract's therapeutic potential in IBD. By defining a safe dosage range (≤500 μg/mL) and identifying key pathways (FoxO4/NOD-like/Apoptosis, MAPK), the study bridges traditional medicine use with modern pharmacological mechanisms. While zebrafish models offer advantages in throughput and cost, future work must validate findings in mammalian systems and clinical settings to fully realize the herb's potential.