Core targets and immune regulatory mechanisms of Huoluo Xiaoling Pellet for promoting zebrafish fin regeneration

doi:10.12122/j.issn.1673-4254.2025.03.07

Abstract

Abstract:

Objective To investigate the core targets and immunomodulatory mechanisms of Huoluo Xiaoling Pellet (HLXLP) for promoting tissue repair. Methods Network pharmacology and protein-protein interaction network were used to screen active components in HLXLP, the disease-related targets and the core targets, followed by GO and KEGG enrichment analyses and molecular docking to predict the pharmacological mechanisms. The toxicity of HLXLP was evaluated in zebrafish, and in a tissue regeneration model established in 3 dpf zebrafish larvae by amputating 95% of the tail fin, the effects of a formulated zebrafish embryo culture medium and 10, 20, and 40 μg/mL of aqueous extract of HLXLP on tissue regeneration was evaluated; RT-qPCR was performed to detect mRNA expressions of tissue regeneration marker genes and the core target genes. Transgenic zebrafish with fluorescently labeled macrophages and neutrophils were used to observe immune cell migration during tissue regeneration, and macrophage polarization at different stages was assessed with RT-qPCR. Results We identified a total of 149 intersected targets between HLXLP active components and tissue repair and 5 core targets (AKT1, IL-6, TNF-α, EGFR and STAT3). GO and KEGG analyses suggested that the effects of HLXLP were mediated primarily through the JAK-STAT pathway, adhesion junctions and positive regulation of cell migration. HLXLP was minimally toxic below 40 μg/mL and lethal at 320 μg/mL in zebrafish, and caused renal and pericardial edema and vascular defects above 80 μg/mL. In zebrafish with tail fin amputation, HLXLP significantly promoted tissue regeneration, reduced IL-6 and TNF-α and enhanced AKT1, EGFR and STAT3 mRNA expressions, modulated neutrophil and macrophage recruitment to the injury sites, and regulated M1/M2 macrophage polarization during tissue regeneration. Conclusion HLXLP promotes zebrafish tail fin regeneration through multiple active components, targets and pathways for immunomodulation of immune cell migration and macrophage polarization to suppress inflammation and accelerate healing.

Key words: Huoluo Xiaoling Pellet, tissue repair, immunologic mechanism, core target, zebrafish

Yan HUANG, Xi CHEN, Mengchen QIN, Lei GAO. Core targets and immune regulatory mechanisms of Huoluo Xiaoling Pellet for promoting zebrafish fin regeneration[J]. Journal of Southern Medical University, 2025, 45(3): 494-505.

Figures/Tables 12

Tab.1 Primers used to quantify mRNA expression

Gene	Sequence of FP (5'-3')	Sequence of RP (5'-3')
Zf β-actin	ATGGATGAGGAAATCGCTGCC	CTCCTGATGTCTGGGTCGTC
Zf PCNA	TATGGACTCCTCTCATGTGTCTCTG	GTGATGATGTCTTCATTTCCAGCAC
Zf AKT1	GATTACGGTCGTGCGGTGGA	TCCACCTAACCGCTGCATGG
Zf EGFR	GTGACCCGTCCCGCTATCTG	GTGGGGTCTGGGATGTAGCG
Zf STAT3	AACGTGCAGCCCATTTTCGC	CCACGGAAACAAGCGCAGTC
Zf TLR4	GACCGTGTTTGTTCGCTGCC	TTCAACTACAGCCCCACGGC
Zf NOS2	CGGATCAGGAGGAGGATCGC	CGTGCTGCAAGTGCCGAAAT
Zf IL10	CGCTTCTTCTTTGCGACTGTGCT	TCACCATATCCCGCTTGAGTTCC
Zf TNF-α	GCTGGATCTTCAAAGTCGGGTGTA	TGTGAGTCTCAGCACACTTCCATC
Zf TGF-β	GAACTCGCTTTGTCTCCA	TACAGTCGCAGTATAACCTCA
Zf IL-6	CGGCATTTGAAGGGGTCAGGA	TGGCCTCCAGCAGTCGTTTG
Zf IL-1β	TGGACTTCGCAGCACAAAATG	CACTTCACGCTCTTGGATGA
Zf fgf20a	AAAAGCTGTCAGCCGAGTGT	TGGACGTCCCATCTTTGTTG
Zf msxb	ACACTTTGTCGAGCGTTTCGG	TCTTGTGCTTGCGTAAGGTGC
Zf IL4	GGGATCCTGAATGGGAAAGGGG	AAGCCATTCCTGCTTGGCAGA
Zf IL13	CTGGCCTGTCCGGTGTCAAAT	CTTCATCAGCAGCGTGACTCCT
Zf ARG1	GGCCAGCCGATGTCTTACCT	GGGTCCAGCGCATCAATGTC
Zf ifng1	GATGGGCTTTGCCTGGGGAG	TGCCAGCCTCTCCTTTGTAGC

Fig.1 Screening process for the intersection targets of Huoluo Xiaoling Pellet and tissue repair.

Fig.2 Core target screening strategy and PPI network visualization.

Fig.3 GO function (A) and KEGG pathway (B) enrichment analysis diagram of Huoluo Xiaoling Pellet-tissue repair intersection targets.

Fig.4 "Active ingredient-target-signaling pathway" network of Huoluo Xiaoling Pellet for promoting tissue repair. Elliptical markers represent active ingredients, rectangular markers represent the targets, and inverted triangle markers represent the signaling pathways. DG, DS, RX, and MY denote Danggui, Danshen, Ruxiang, and Moyao, respectively.

Tab.2 Top 10 active ingredients in Huoluo Xiaoling Pellet

Number	Mol ID	Active ingredient	Degree	Betweenness centrality	Closeness centrality
MY39	MOL000490	petunidin	37	0.02587001	0.4317817
MY15	MOL001033	diayangambin	36	0.02263804	0.42630745
DS9	MOL000006	luteolin	35	0.01654172	0.42902711
MY37	MOL001164	[(5S, 6R, 8R, 9Z)-8-methoxy-3, 6, 10-trimethyl-4-oxo-6, 7, 8, 11-tetrahydro-5H-cyclodeca[b]furan-5-yl] acetate	35	0.01612	0.42630745
DS15	MOL007049	4-methylenemiltirone	34	0.01770958	0.42362205
DS20	MOL007061	methylenetanshinquinone	29	0.01130007	0.41965679
MY28	MOL001095	isofouquierone	29	0.0106332	0.40573152
DS22	MOL007064	przewalskin b	28	0.00999823	0.41194487
MY17	MOL001045	(13E, 17E, 21E)-8-hydroxypolypodo-13, 17, 21-trien-3-one	27	0.01052943	0.40943683
MY35	MOL001148	3β-hydroxydammar-24-ene	27	0.00947679	0.41194487

Tab.3 Molecular docking binding energy prediction

Active ingredient	AKT1	IL6	TNF	EGFR	STAT3
Petunidin	-8.945	-5.695	-8.657	-6.666	-5.256
Diayangambin	-9.256	-4.711	-6.801	-6.224	-4.331
Luteolin	-9.539	-6.244	-8.509	-7.556	-5.601
[(5S, 6R, 8R, 9Z)-8-methoxy-3, 6, 10-trimethyl-4-oxo-6, 7, 8, 11- tetrahydro-5H-cyclodeca[b]furan-5-yl] acetate	-8.202	-5.165	-7.881	-6.628	-4.732
4-methylenemiltirone	-10.652	-6.558	-8.393	-8.194	-5.372

Fig.5 Toxic effects of Huoluo Xiaoling Pellet extract on zebrafish larvae (Mean±SD, n=30). A: Effect of Huoluo Xiaoling Pellet extract on zebrafish larvae growth and development (VD: Vasodilation; RE: Renal edema; PE: Pericardial edema). B: Survival rate. C: Hatching rate. D: Heart rate. ***P<0.001 vs 0 μg/mL group.

Fig.6 Effect of different concentrations of Huoluo Xiaoling Pellet extract on tail fin tissue defect repair in the zebrafish. A: Changes in tail fin defect of the zebrafish after treatment with formulated medium (model group) and low-, medium-, and high-dose Huoluo Xiaoling Pellet extract. B: Relative area of tail fin tissue repair measured 72 h post-surgery in different groups (Mean±SD, n=4). C-E: Expression levels of PCNA, msxb and fgf20a mRNA in zebrafish 72 h after tail fin amputation (Mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001 vs Model group.

Fig.7 Effect of different concentrations of Huoluo Xiaoling Pellet extract on expressions of the core targets related to tail fin repair in zebrafish (Mean±SD, n=3). A-E: The mRNA expression levels of AKT1, EGFR, STAT3, IL-6, and TNF-α in zebrafish 72 h after tail fin amputation. *P<0.05, **P<0.01, ***P<0.001 vs Model group.

Fig.8 Huoluo Xiaoling Pellet regulates migration of macrophages and neutrophils in the wound of zebrafish tail fin (Mean±SD, n=3). A, B: Distribution of macrophages and neutrophils at the injury site of the tail fin of zebrafish at 24 h and 48 h. C, D: Statistical analysis of macrophage and neutrophil migration to the tissue repair site at 24 h and 48 h. E: The mRNA expression levels of ifng1 in zebrafish detected by qPCR. *P<0.05, **P<0.01, ***P<0.001 vs Model group; #P<0.05 vs 48 h Model group.

Fig.9 Huoluo Xiaoling Pellet regulates expressions of macrophage subset markers at different stages of tissue regeneration (Mean±SD, n=3). A-I: The mRNA expression levels of TLR4, TNF-α, IL-1β, NOS2, IL-4, TGF-β, IL-13, IL-10 and ARG1 in zebrafish detected by qPCR. *P<0.05, **P<0.01, ***P<0.001 vs Model group; #P<0.05, ##P<0.01, ###P<0.001 vs 48 h Model group.

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