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1. Introduction

Leeches have been employed in medical practice for thousands of years, with historical records dating back to ancient Egypt, Greece, and China . Among the various medicinal leech species, Hirudo nipponia (Japanese medicinal leech or Asian medicinal leech) holds a prominent position in traditional Chinese medicine, where it has been officially documented in pharmacopoeias for the treatment of cardiovascular and cerebrovascular diseases . Unlike its European counterpart Hirudo medicinalis, H. nipponia is native to East Asia and has been subject to increasing scientific scrutiny in recent years.

The therapeutic efficacy of medicinal leeches derives from the complex cocktail of bioactive molecules secreted in their saliva. These compounds serve to facilitate blood-feeding by inhibiting host hemostatic mechanisms, including coagulation, platelet aggregation, and vasoconstriction. For medical applications, these same molecules offer potent pharmacological activities with relatively low toxicity. Modern research has expanded our understanding of leech-derived compounds beyond traditional anticoagulation to include antimicrobial, anti-inflammatory, and even potential antitumor activities .

This article reviews the contemporary medical applications of Hirudo nipponia, highlighting recent discoveries enabled by genomic and transcriptomic technologies, as well as established clinical practices supported by hemodynamic evidence.

2. Anticoagulant and Antiplatelet Agents

2.1 Hirudin-HN: A Potent Thrombin Inhibitor

The most well-known leech-derived anticoagulant is hirudin, a direct thrombin inhibitor originally isolated from Hirudo medicinalis. In H. nipponia, researchers have identified and characterized a homologous protein designated hirudin-HN . Transcriptome sequencing of salivary glands from H. nipponia generated 6.5 Gb of data, from which a transcript annotated to hirudin HV3 was identified and validated.

Hirudin-HN is encoded by a 270-bp cDNA that translates to an 89-amino acid protein containing a 20-amino acid signal peptide. The mature protein possesses the canonical structural features of the hirudin family, including three conserved disulfide bonds and characteristic PKP and DFxxIP motifs . Functional assays using chromogenic substrates (S2238) and surface plasmon resonance (SPR) analysis confirmed that hirudin-HN exhibits potent anticoagulant activity through direct binding to thrombin.

A critical finding from this research was that the N-terminal structure of the mature protein is essential for its anticoagulant activity. Comparison between the full-length recombinant protein (Hir) and a truncated version (M-Hir) revealed that the N-terminal domain is indispensable for thrombin affinity . This structure-function relationship provides a foundation for rational drug design aimed at optimizing antithrombotic efficacy.

2.2 HnSaratin: A Collagen-Binding Antiplatelet Agent

Beyond thrombin inhibition, H. nipponia produces proteins that interfere with platelet aggregation through alternative mechanisms. One such molecule is HnSaratin, a 12.37 kDa protein encoded by a 387-bp open reading frame that yields a 128-amino acid precursor .

HnSaratin exhibits a distinctive structural architecture: the N-terminal region folds into a globular domain stabilized by three disulfide bonds with a ββαβββ topology, while the C-terminal region remains flexible. Two glutamate residues within the globular domain are responsible for binding to collagenous lysine residues, thereby competitively inhibiting the interaction between von Willebrand factor (vWF) and collagen . Since vWF-mediated platelet adhesion is a critical step in thrombus formation, this inhibition effectively prevents platelet aggregation at sites of vascular injury.

Functional validation in rat models demonstrated that HnSaratin prevents blood clotting and exhibits anti-platelet aggregation activity . Notably, expression of HnSaratin mRNA in salivary glands is significantly upregulated following bloodmeal ingestion, consistent with its biological role in facilitating feeding . The therapeutic potential of saratin has been further supported by studies in animal models of carotid endarterectomy and intimal hyperplasia, where it reduced platelet adhesion and decreased pathological vascular remodeling .

2.3 Comprehensive Genomic Characterization of Antithrombotic Genes

Recent advances in comparative genomics have provided a comprehensive inventory of antithrombotic genes in H. nipponia. A high-quality genome assembly coupled with RNA-seq-based expression analysis identified 22 antithrombotic gene families, including 14 coagulation inhibitors, 4 platelet aggregation inhibitors, 3 fibrinolysis enhancers, and 1 tissue penetration enhancer . The total number of antithrombotic genes in H. nipponia was determined to be 86, with comparable numbers observed in the closely related species Hirudo tianjinensis.

Molecular evolution analysis revealed that antithrombotic genes in H. nipponia are under purifying selection, suggesting their functional importance is evolutionarily conserved. Expression patterns of these gene families were not significantly different between the two species, indicating functional similarity . This genomic resource represents the most comprehensive collection of antithrombotic biomacromolecules from Asian medicinal leeches to date and will facilitate future drug development efforts.

3. Antimicrobial Peptides: Combating Antibiotic Resistance

The global crisis of antibiotic resistance has prompted intensive search for novel antimicrobial agents with new mechanisms of action. Natural products, particularly those from organisms that feed on blood or inhabit microbe-rich environments, represent promising sources of such compounds. H. nipponia has recently yielded a novel antimicrobial peptide with remarkable activity against multidrug-resistant bacteria.

3.1 Discovery of Hirunipin-2

Using an innovative approach combining AI-based bioinformatic analysis and three-dimensional holotomography high-throughput screening (3D HT-HTS), a Korean research group discovered a novel antimicrobial peptide named Hirunipin-2 from the salivary glands of Hirudo nipponia . The discovery pipeline involved:

1. Transcriptome analysis: AI-based bioinformatic tools evaluated the structural stability, antibacterial, and anti-inflammatory functions of salivary gland transcripts, yielding 19 candidate peptides.

2. High-throughput screening: 3D HT-HTS technology enabled simultaneous evaluation of multiple candidates with real-time imaging of antibacterial mechanisms.

3. Functional validation: Hirunipin-2 demonstrated potent activity against multidrug-resistant bacteria (MDR-bacteria, also known as superbacteria).

3.2 Antibacterial and Antibiofilm Activity

The efficacy of Hirunipin-2 was assessed using label-free 3D holotomography imaging, which allows real-time observation of bacterial responses without the need for staining or other preprocessing that might interfere with natural biological processes . This technology revealed that Hirunipin-2 effectively inhibits bacterial growth and, importantly, disrupts pre-formed biofilms produced by multidrug-resistant bacteria.

Biofilms represent a significant clinical challenge because they protect bacteria from both host immune responses and antibiotic treatment. The ability of Hirunipin-2 to destroy existing biofilms is therefore particularly noteworthy. The research demonstrated that treatment with Hirunipin-2 led to progressive biofilm disruption over 12 hours, as visualized through changes in the refractive index tomograms of bacterial communities .

3.3 Synergistic Effects with Conventional Antibiotics

A particularly promising finding was that Hirunipin-2 exhibits synergistic effects when combined with existing antibiotics, including tetracycline, and rifampicin . This synergy suggests that Hirunipin-2 could serve as an antibiotic adjuvant, enhancing the efficacy of conventional drugs while potentially allowing for reduced dosing and decreased toxicity.

The discovery of Hirunipin-2 is significant for several reasons. First, antimicrobial peptides derived from natural products generally have a low propensity for inducing resistance and exhibit low toxicity to human cells. Second, the multitarget mechanism of action of such peptides makes it difficult for bacteria to develop resistance through single mutations. Third, the combination of natural product databases with advanced imaging technologies represents a paradigm shift in antimicrobial drug discovery .

4. Clinical Applications in Reconstructive Surgery

Beyond the development of pharmaceutical agents, living leeches themselves continue to play a role in modern medicine, particularly in reconstructive surgery. Leech therapy, or hirudotherapy, is employed for the salvage of congested flaps and replanted digits where venous outflow is compromised .

4.1 Mechanism of Action in Flap Congestion

When a surgical flap or replanted tissue has adequate arterial inflow but insufficient venous drainage, blood accumulates in the microcirculation, leading to venous congestion, tissue edema, and ultimately necrosis if left untreated. Leeches applied to congested tissue serve to:

• Remove accumulated blood mechanically

• Inject anticoagulant and vasodilatory compounds that promote continued bleeding after the leech detaches

• Reduce tissue pressure and restore microcirculatory flow

4.2 Hemodynamic Evidence

A quantitative study using rabbit island flap models provided important insights into the hemodynamic effects of leech therapy . In this study, congested flaps were treated with either one leech, three leeches, or no leeches (control). Flap survival area was significantly larger in the three-leech group compared to both the one-leech and control groups.

Monitoring of transcutaneous oxygen and carbon dioxide tensions (TcPO₂ and TcPCO₂) revealed that effective leech therapy leads to:

• Significant reduction in TcPCO₂, indicating clearance of accumulated metabolic waste

• Decreased diameters of arterioles and venules, suggesting resolution of congestion

• Increased flow velocity following clamp release

The study concluded that leech therapy salvages compromised flaps by replacing congested blood with fresh arterial blood, thereby maintaining tissue viability. Importantly, TcPO₂ and TcPCO₂ monitoring was identified as a useful tool for evaluating therapeutic efficacy and determining the appropriate number of leeches and duration of treatment .

4.3 Clinical Considerations

While leech therapy is effective, it is not without risks. The primary complications include:

• Infection: Leeches harbor symbiotic bacteria, particularly Aeromonas species, in their gut. Prophylactic antibiotics are therefore recommended during leech therapy.

• Bleeding: The anticoagulant effects can persist for hours after leech detachment, necessitating careful monitoring of blood loss.

• Allergic reactions: Some patients may develop hypersensitivity to leech saliva components.

Despite these considerations, leech therapy remains an established procedure in microsurgical units worldwide, particularly for salvage of congested flaps and replanted digits where other interventions have failed.

5. Future Perspectives and Conclusion

The convergence of genomics, transcriptomics, and advanced imaging technologies has dramatically accelerated the discovery and characterization of bioactive compounds from Hirudo nipponia. Several promising directions for future research and development can be identified:

5.1 Next-Generation Antithrombotics

Recombinant hirudin derivatives are already in clinical use as anticoagulants, but the discovery of novel inhibitors such as HnSaratin offers opportunities for developing agents with distinct mechanisms of action. Combined inhibition of thrombin and platelet adhesion may provide superior antithrombotic effects with fewer bleeding complications. The successful expression of HnSaratin in prokaryotic systems  provides a pathway for large-scale production and pharmaceutical development.

5.2 Antimicrobial Peptides as Antibiotic Adjuvants

The emergence of multidrug-resistant bacteria represents one of the most urgent threats to global health. Hirunipin-2 and similar peptides offer a dual mechanism of direct antibacterial activity and synergistic enhancement of existing antibiotics . Future research should focus on elucidating the precise molecular mechanism of Hirunipin-2, optimizing its structure for enhanced activity and stability, and evaluating its safety and efficacy in animal models of infection.

5.3 Personalized Leech Therapy

The identification of biomarkers such as TcPO₂ and TcPCO₂ that predict response to leech therapy  suggests the possibility of personalized treatment protocols. Real-time monitoring could enable clinicians to tailor the number of leeches and treatment duration to individual patient needs, optimizing outcomes while minimizing risks.

5.4 Integrative Approaches

The traditional use of H. nipponia in Chinese medicine for cardiovascular diseases  is now being validated by molecular studies that identify specific bioactive compounds responsible for therapeutic effects. Continued integration of traditional knowledge with modern scientific methods promises to yield additional discoveries.

In conclusion, Hirudo nipponia exemplifies the value of biodiverse natural resources for drug discovery and medical therapy. From anticoagulant and antiplatelet agents to novel antimicrobial peptides and living leech therapy in reconstructive surgery, the medical applications of this species continue to expand. As genomic and analytical technologies advance, further therapeutic molecules from H. nipponia and related species are likely to emerge, offering new solutions to pressing medical challenges including antibiotic resistance and thrombotic disease.

References

[1] Lee, S., et al. (2025). Real-time 3D visualization of leech peptide reveals potent antibacterial and antibiofilm activity. Advanced Science. 

[2] Cheng, B., et al. (2023). Molecular cloning and functional analysis of HnSaratin from Hirudo nipponia. Gene, 869, 147401. 

[3] Kashiwagi, K., et al. (2013). Quantitative analysis of hemodynamics of congested island flaps under leech therapy. Journal of Medical Investigation, 60(3-4), 213-220. 

[4] Nature Research Intelligence. (n.d.). Medicinal Leech Therapy. Nature Portfolio. 

[5] Yang, C., et al. (2024). Herbal Textual Research on Hirudo. Chinese Journal of Modern Applied Pharmacy, 41(19), 2648-2657. 

[6] Kashiwagi, K., Hashimoto, I., Abe, Y., Kotsu, K., Yamano, M., & Nakanishi, H. (2013). Quantitative analysis of hemodynamics of congested island flaps under leech therapy. Journal of Medical Investigation, 60(3-4), 213-220. 

[7] Cheng, B., et al. (2019). Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia. PeerJ, 7, e7716. 

[8] Zhao, F., et al. (2024). Comparative genomics of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis: With emphasis on antithrombotic genes and their corresponding proteins. International Journal of Biological Macromolecules, 269, 132278. 

[9] Cheng, B., et al. (2023). Molecular cloning and functional analysis of HnSaratin from Hirudo nipponia. Gene, 869, 147401.