Exploring the mechanism of Panax Notoginseng in the treatment of skin wound based on network pharmacology and experimental verification

This study integrates network pharmacology with experimental verification to demonstrate that Panax notoginseng accelerates skin wound healing in rats by modulating key inflammatory cytokines (TNF-α, IL-6, IL-10) through multi-component, multi-target regulation of NF-κB, MAPK, and JAK-STAT signaling pathways.

The Gist

Imagine your skin is like a bustling construction site. When you get a cut or a wound, the body sends in a "cleanup crew" (immune cells) to clear away debris and fight off germs. This is the inflammation phase. Once the site is clean, a "construction crew" (fibroblasts) arrives to build new tissue and patch the hole.

The problem, as the authors of this paper explain, is that sometimes the cleanup crew gets too excited. They stay too long, shouting and fighting so much that they accidentally damage the new construction, or they refuse to leave, preventing the builders from starting their work. This leads to slow healing, infection, or ugly scars.

This study investigates Panax Notoginseng (PN), a traditional Chinese herb, to see how it helps fix this traffic jam at the construction site.

Here is the breakdown of their findings, translated into everyday language:

1. The Digital Detective Work (Network Pharmacology)

Before they even touched a rat, the researchers acted like digital detectives. They used computer databases to map out how the herb works.

• The Ingredients: They found 8 main "active ingredients" in the herb (like different tools in a toolbox).
• The Targets: They discovered these ingredients talk to 156 different parts of the body's immune system.
• The Big Three: They narrowed it down to three key "foremen" that the herb controls: TNF-α, IL-6, and IL-10.
  • Think of TNF-α and IL-6 as the "Alarm Sirens." They scream, "We have a problem! Send help!" (This starts inflammation).
  • Think of IL-10 as the "Peacekeeper." It says, "Okay, the problem is handled, everyone calm down and go home." (This stops inflammation).

The computer model suggested that PN doesn't just turn the sirens off; it teaches them when to ring and when to stop, ensuring the Peacekeeper arrives at the right time.

2. The Real-World Test (The Rat Experiment)

To prove the computer was right, they tested this on 48 rats with large skin wounds.

• Group A (The Control): Got a saltwater injection (a placebo).
• Group B (The PN Group): Got the Panax Notoginseng injection.

The Results:

• Day 1: Both groups looked the same. The "Alarm Sirens" were ringing loudly in both groups. This is normal; you need the alarm to start the cleanup.
• Day 4 & 7: This is where the magic happened.
  • The Control Group: The alarm sirens (TNF-α and IL-6) kept screaming for too long. The site was still chaotic, filled with angry cleanup crews (inflammatory cells), and the builders hadn't started working yet. The wounds were still big.
  • The PN Group: The herb acted like a traffic controller. It let the alarm ring for a bit to clear the debris, but then it quickly told the sirens to "Quiet down!" The cleanup crew left the site, and the construction crew (fibroblasts) rushed in to build new skin. The wounds healed much faster and were significantly smaller.

3. The "Why" (The Mechanism)

The researchers looked at the cells under a microscope and found the secret sauce:

• Timing is Everything: In the PN group, the "Alarm Sirens" (TNF-α and IL-6) peaked early and then dropped off quickly. In the control group, they stayed high, keeping the wound in a state of panic.
• The Peacekeeper's Role: The herb helped the "Peacekeeper" (IL-10) do its job effectively, turning the angry cleanup crew into calm builders.
• The Result: The PN group had less swelling, fewer angry immune cells, and a lot more new skin cells building a strong, neat patch.

The Bottom Line

Think of Panax Notoginseng not as a "cure-all" that stops the wound from hurting, but as a smart project manager.

• Without it: The construction site gets stuck in a loop of chaos. The cleanup crew never leaves, and the builders can't start.
• With it: The project manager ensures the cleanup happens efficiently, then immediately signals the builders to start. The result is a wound that heals faster, with less risk of infection, and a much lower chance of leaving a messy scar.

In short, this herb helps your body's natural healing process run on a perfect schedule: Clean fast, build fast, heal well.

Technical Summary

1. Problem Statement

Large skin wound defects often exceed the body's capacity for spontaneous healing, leading to prolonged recovery times and a high risk of infection. The healing process involves complex phases (hemostasis, inflammation, proliferation, and remodeling), where an imbalance in cytokine secretion (specifically pro-inflammatory factors like TNF-α and IL-6 versus anti-inflammatory factors like IL-10) can cause uncontrolled inflammation, delaying repair and increasing infection susceptibility. While Panax notoginseng (PN) is clinically known to promote wound healing, its specific molecular mechanisms and target pathways remain unclear. This study aims to elucidate these mechanisms using a combination of network pharmacology and in vivo experimental verification.

2. Methodology

The study employed a two-pronged approach: computational prediction via network pharmacology and biological validation via animal experiments.

A. Network Pharmacology Analysis:

• Data Mining: Active components of PN were retrieved from the TCMSP database (filtering for Oral Bioavailability ≥ 30% and Drug-likeness ≥ 0.18). Skin wound-related targets were obtained from GeneCards (Score > median).
• Target Intersection: Common targets between PN and skin wounds were identified using Venny 2.1.0.
• Network Construction: A Protein-Protein Interaction (PPI) network was built using the STRING database and visualized in Cytoscape 3.8.2.
• Core Analysis: The top 10 key genes were identified using the CytoHubba plugin (Maximal Clique Centrality), and functional modules were analyzed using MCODE.
• Enrichment Analysis: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the DAVID database to identify biological processes and signaling pathways.

B. Experimental Verification (In Vivo):

• Subjects: 48 Sprague-Dawley (SD) male rats were randomly divided into a Control Group (saline) and a PN Group (Panax notoginseng injection, 1.5 mL/animal, intraperitoneal).
• Model: A full-thickness skin excision (1 cm diameter) was created on the dorsal spine.
• Intervention: Treatment began 2 hours post-surgery and continued daily for 7 days.
• Assessments:
  • Wound Area: Measured on days 1, 4, and 7 using Image-Pro Plus software.
  • Histopathology: Hematoxylin and Eosin (HE) staining to observe cellular infiltration, fibroblast proliferation, and collagen secretion.
  • Immunohistochemistry (IHC): Localization of TNF-α, IL-6, and IL-10 in tissue sections.
  • Molecular Detection: Quantitative Real-Time PCR (qRT-PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) to quantify mRNA and protein expression levels of TNF-α, IL-6, and IL-10.

3. Key Contributions

• Identification of Active Components: Identified 8 major active components of PN (including quercetin, ginsenoside Rg2, and beta-sitosterol) and mapped them to 156 potential targets.
• Mechanistic Elucidation: Defined the core regulatory axis of PN as the TNF-α / IL-6 / IL-10 network, primarily operating through the NF-κB, MAPK, and JAK-STAT signaling pathways.
• Temporal Regulation Insight: Demonstrated that PN does not merely suppress inflammation but optimizes the temporal sequence of the healing process, facilitating a smooth transition from the inflammatory phase to the proliferative/remodeling phase.
• Experimental Validation: Provided robust in vivo evidence confirming that PN accelerates wound closure, reduces inflammatory cell infiltration, and promotes fibroblast proliferation compared to controls.

4. Key Results

Network Pharmacology Findings:

• Core Targets: The top 10 key genes included TNF, IL-6, IL-10, CCL2, IL1B, CXCL8, JUN, and RELA.
• Pathways: Enrichment analysis highlighted the TNF signaling pathway, NF-κB signaling pathway, MAPK signaling pathway, and JAK-STAT signaling pathway as central to PN's therapeutic effect.
• Mechanism Hypothesis: PN acts via a "multi-component, multi-target, multi-pathway" synergy to reshape the dynamic balance between pro-inflammatory and anti-inflammatory cytokines.

Experimental Findings:

• Wound Healing Rate: At days 4 and 7, the PN group showed significantly smaller wound areas compared to the control group ($P < 0.05$).
• Histopathology (HE Staining):
  • Day 7: The PN group exhibited significantly reduced neutrophil and inflammatory cell infiltration compared to the control group.
  • Tissue Regeneration: The PN group showed pronounced fibroblast proliferation and collagen secretion, indicating an advanced stage of tissue remodeling.
• Cytokine Expression (qRT-PCR & ELISA):
  • TNF-α: Expression was significantly lower in the PN group at days 4 and 7 ($P < 0.01$), indicating effective suppression of prolonged inflammation.
  • IL-6: Expression peaked at day 4 in both groups but was significantly lower in the PN group at all time points ($P < 0.01$), suggesting a dampening of the inflammatory amplification loop.
  • IL-10: Expression was significantly lower in the PN group at days 1 and 7 ($P < 0.01$). This suggests PN prevents the compensatory, excessive upregulation of IL-10 seen in the control group, maintaining a more efficient equilibrium.
• Localization: IHC confirmed that TNF-α, IL-6, and IL-10 are primarily distributed in the cytoplasm and matrix of epidermal cells, inflammatory cells, and fibroblasts.

5. Significance

This study provides a comprehensive molecular explanation for the clinical efficacy of Panax notoginseng in wound care.

• Therapeutic Strategy: It establishes that PN promotes healing not by simply inhibiting inflammation, but by optimizing the temporal progression of the healing cascade: "Efficient debridement → Orderly repair → Low scar risk."
• Mechanism of Action: PN achieves this by regulating the "Pro-inflammatory/Anti-inflammatory" control axis (TNF-α/IL-6 vs. IL-10). It ensures the initiation of necessary inflammation for debridement while preventing the "vicious cycle" of chronic inflammation that delays healing and causes scarring.
• Clinical Application: The findings validate PN as an ideal natural drug for accelerating skin wound healing and reducing infection risks, offering a theoretical basis for its use in clinical formulations and future drug development.