Tongyangxiao Lotion promotes postoperative wound healing in a rat model of anal fistula by downregulating inflammatory factors and suppressing inflammation

doi:10.12122/j.issn.1673-4254.2024.07.05

Abstract

Abstract:

Objective To explore the mechanism of Tongyangxiao Lotion (TYX) for promoting wound healing following surgery for anal fistula. Methods The active ingredients and drug targets of TYX were explored using TCMSP and BATMAN databases, and the targets associated with wound healing were screened using GeneCards and OMIM databases; the intersecting drug and wound-related targets were analyzed with protein-protein interaction (PPI) analysis and GO and KEGG enrichment analyses. In 25 SD rat models with simulated anal fistula surgery, the effect of wound dressing with TYX at low, medium and high doses (once daily for 14 days) on wound healing were assessed in comparison with potassium permanganate (PP) solution. The granulation tissues collected from the wounds were examined for pathological changes with HE staining and for TNF-α expression using immunohistochemistry. The expressions of 1β, TNF-α, IL-6 mRNA and proteins in the granulation tissue were detected using RT-qPCR, Western blotting or ELISA. Results Network pharmacology analysis yielded 156 common targets between TYX and wound healing, and among them IL-1β, TNF-α, and IL-6 were identified as potential targets of TYX for promoting wound healing. Six core components of TYX were capable of binding to IL-1β, TNF-α, and IL-6 with binding energies all below -6.0 Kcal/mol. In the rat models, the wounds with TYX and PP solution dressing showed significantly reduced inflammatory cell infiltration and increased fibroblasts and collagen deposition. TYX at the 3 doses and PP solution all significantly reduced the expressions of IL-6, IL-1β, TNF-α mRNA and IL-6 protein in the granulation tissues, but TYX at the medium and high doses produced significantly stronger effects than PP solution for lowering TNF-α protein expression and mRNA expressions of TNF-α and IL-6. Conclusion TYX accelerates wound healing by down-regulating the inflammatory factors and reducing inflammation in the wounds.

Key words: wound healing, anal fistula, network pharmacology, interleukin, tumor necrosis factor-α

Linyue WANG, Wenyue QI, Jihua GAO, Maosheng TIAN, Jiancheng XU. Tongyangxiao Lotion promotes postoperative wound healing in a rat model of anal fistula by downregulating inflammatory factors and suppressing inflammation[J]. Journal of Southern Medical University, 2024, 44(7): 1256-1265.

Figures/Tables 13

Fig.1 Establishment of rat models following simulated anal fistula surgery. A: Preparation of a depilation area on the back. B: Creating a 2 cm-diameter circular wound and excision of the whole skin. C: Spraying E. coli suspension and application of waterproof stickers. D: Purulent secretion visible on the wound surface.

Tab.1 Primer sequences for RT-qPCR

Gene	bp	Sequence of primer
GAPDH	74	F: AGGAAATGATGACCTCCTGAACT
GAPDH	74	R: TGTTTTTGTAAGTATCTTGGTGCCT
IL-6	85	F: AGAGACTTCCAGCCAGTTGC
IL-6	85	R: AGTCTCCTCTCCGGACTTGT
TNF-α	164	F: GGCTTTCGGAACTCACTGGA
TNF-α	164	R: GGGAACAGTCTGGGAAGCTC
IL-1β	121	F: TGGCAACTGTCCCTGAACTC
	121	R: CCCAAGTCAAGGGCTTGGAA

Fig.2 Drug-compound-target mapping for TYX Lotion.

Fig.3 PPI map of the core targets of TYX Lotion for promoting wound healing.

Fig.4 GO enrichment analysis of intersecting TYX Lotion and wound targets.

Fig.5 KEGG enrichment of intersecting TYX Lotion and wound targets.

Fig.6 Heat map of molecular docking of the core components to the core targets.

Fig.7 Molecular docking between the active ingredients and the core targets. A: β-sitosterol+IL-1β. B: Apigenin+IL-6. C: Wogonin+TNF-α. Hydrogen bonding and hydrophobic interactions are shown in yellow and blue, respectively.

Fig. 8 HE staining of granulation tissue in each group (Original magnification: ×40). A: Model. B: PP. C: TYX-L. D: TYX-M. E: TYX-H.

Fig.9 Expression of TNF-α in the granulation tissue of rats in each group detected by immunohistochemical staining. A: Model. B: PP. C: TYX-L. D: TYX-M. E: TYX-H. F: AOD of TNF‑α. *P<0.05.

Fig. 10 Relative expression of TNF-α (A), IL-1β (B) and IL-6 (C) mRNA in granulation tissue in each group. *P<0.05 vs Model group. #P<0.05 vs PP group.

Tab.2 Expression level of IL-6 in the granulation tissue in each group detected by ELISA

Group	IL-6 (pg/mL)
Model	18.77±1.63
TYX-L	12.70±3.06*
TYX-M	14.45±1.38*
TYX-H	14.05±2.25*
F	4.361
P	0.04

Fig. 11 TNF-α and IL-1β protein expression in the granulation tissue in each group. A: Western blotting for detecting protein expression of TNF-α and IL-1β. B, C: Quantitative analysis of TNF-α and IL-1β protein levels. *P<0.05 vs Model group.

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