Modified Danggui Sini formula (加味当归四逆汤) promotes wound healing via regulating natural killer cell function: a network pharmacology-based study targeting killer cell lectin like receptor K1

doi:10.19852/j.cnki.jtcm.2026.05.013

Abstract

Abstract:

OBJECTIVE: To explore the potential mechanism of the modified Danggui Sini formula (加味当归四逆汤, M-DGSNF) in the treatment of freezing cold injury (FCI).

METHODS: FCI model rats were used to evaluate the effects of M-DGSNF on wound appearance viahistopathological analysis. Furthermore, the potential targets of M-DGSNF were identified viaultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF/MS) analysis, network pharmacology and RNA sequencing. Frozen mouse skin sections were analysed by Western blotting and immunohistochemistry. Interleukin (IL)-6, interferon-gamma (IFN-γ), IL-4, IL-10 and vascular endothelial growth factor (VEGF) were assessed by enzyme-linked immunosorbent assay. The effects of M-DGSNF on macrophage polarization were monitored in vitroby flow cytometry. Depletion of natural killer (NK) cells in vivo was used to confirm the role of NK cells in the effects of M-DGSNF.

RESULTS: M-DGSNF promoted wound healing and ameliorated the inflammatory response to frostbite wounds in rats with FCI. Additionally, the expression of CD31, alpha-smooth muscle actin (α-SMA), and type I collagen, which promote wound healing, increased after M-DGSNF treatment. Furthermore, killer cell lectin-like receptor k1 (KLRK1) and indoleamine 2,3-dioxygenase 1 (IDO1), which are associated with the function of NK cells, were identified by UPLC-high-resolution mass spectro-metry analysis, network pharmacology and RNA-seq. Accordingly, an increase in the release of inflammatory factors such as VEGF, IFN-γ, IL-6, IL-4, and IL-10 was observed in the early stage of FCI, after which the expression of IFN-γ and IL-6 decreased in the late stage of frostbite. Furthermore, M-DGSNF treatment contributed to M2 macrophage polarization both in vivo and in vitro. Depletion of NK cells abolished the effects of M-DGSNF on wound healing, suggesting that NK cells play a key role in the mechanism by which M-DGSNF affects mice with FCI.

CONCLUSION: M-DGSNF may promote wound healing in mouse models of FCI by regulating the function of NK cells through targeting KLRK1.

Key words: freezing cold injury, killer cells, natural, wound healing, network pharmacology, modified Danggui Sini formula

XU Yannan, TIAN Yuan, WANG Xionghui, XIE Wei, ZHENG Tianxiao, PEI Zhi, LING Changquan, DU Juan. Modified Danggui Sini formula (加味当归四逆汤) promotes wound healing via regulating natural killer cell function: a network pharmacology-based study targeting killer cell lectin like receptor K1[J]. Journal of Traditional Chinese Medicine, 2026, 46(5): 1194-1205.

Figures/Tables 4

Figure 1 M-DGSNF promotes healing of frostbite wounds in dry ice-induced FCI rats A: HE staining (n = 6). A1: Model group; A2: M-DGSNF group; A3: DGSNF group; B: skin appearance images (n = 6). B1: Model group; B2: alcohol extract group; B3: 1:3 of alcohol to water ratio group; B4: 3:1 of alcohol to water ratio group; B5: water extract group; C: wound healing speed (n = 6). C1-C4: M-DGSNF group; C1: 4 d after administration; C2: 8 d after administration; C3: 12 d after administration; C4: 16 d after administration; C5-C8: Model group; C5: 4 d after administration; C6: 8 d after administration; C7: 12 d after administration; C8: 16 d after administration; D: images of immunofluorescence and proteins expression, the nuclei were stained with DAPI (blue). D1: Model group; D2: M-DGSNF group; E: images of Western blot analysis of protein expression. Model: treated with deionized water for 3 d; DGSNF: treated with DGSNF of 4.5 g/kg for 3 d; M-DGSNF: treated with M-DGSNF of 6.4 g/kg for 3 d. M-DGSNF: modified Danggui Sini formula; FCI: freezing cold injury; HE: hematoxylin-eosin; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 2 UPLC-Q-TOF/MS and network pharmacology analysis predicts potential targets of M-DGSNF A: the quantity of intersecting M-DGSNF and FCI target genes; B: KEGG enrichment analysis of co-targeted genes of M-DGSNF and FCI, including top 20 most significant pathways; C: GO enrichment analysis of co-targeted genes of M-DGSNF and FCI, including the top 10 significant enrichment terms of BP, CC, and MF. UPLC-Q-TOF/MS: ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry; M-DGSNF: modified Danggui Sini formula; FCI: freezing cold injury; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes; BP: biological process; CC: cellular component; MF: molecular function.

Figure 3 RNA-sequence screens therapeutic targets of M-DGSNF in frostbite model rats A: bar chart of GO analysis of DEGs; B: bubble chart of KEGG pathways of DEGs; C: venn diagram between potential targets from UPLC-Q-TOF/MS and network pharmacology and DEGs from RNA-sequencing; D: images of Western blot analysis of protein expression. Model: treated with deionized water for 3 d; M-DGSNF: treated with M-DGSNF of 6.4 g/kg for 3 d. M-DGSNF: modified Danggui Sini formula; DEGs: differentially expressed genes; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes; UPLC-Q-TOF/MS: ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Figure 4 NK cells play an important role in the effect of M-DGSNF on FCI A: HE staining (n = 3). A1: Model group; A2: M-DGSNF group; A3: M-DGSNF group (NK cell deficient). B: Western blot images of α-SMA and NKG2D (n = 3). Model: treated with deionized water for 3 d; M-DGSNF: treated with M-DGSNF of 6.4 g/kg for 3 d; M-DGSNF group (NK cell deficient): treated with M-DGSNF of 6.4 g/kg for 3 d. M-DGSNF: modified Danggui Sini formula; FCI: freezing cold injury; HE: hematoxylin-eosin; α-SMA: alpha-smooth muscle actin; NKG2D: natural killer group 2, member d.

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