Mollugin Supports Zebrafish Tail Regeneration in Association with Altered Inflammatory and Oxidative Responses

Summary

In zebrafish, Mollugin at safe doses (≤200 µg/mL) was associated with improved tail regeneration, accompanied by changes in apoptosis-related gene expression, inflammatory markers, antioxidant enzyme activity, and macrophage-associated markers. These findings suggest that Mollugin may modulate the local injury response during zebrafish tail regeneration.

Abstract

Mollugin is a natural naphthoquinone compound with reported anti-inflammatory and antioxidant activities, yet its role in tissue regeneration remains incompletely understood. In this study, a zebrafish tail amputation model was used to investigate the effects of Mollugin on wound repair and associated immune and redox responses. Zebrafish embryos were treated with Mollugin at concentrations of 22 µg/mL, 66 µg/mL, and 200 µg/mL following tail injury. Mollugin treatment was associated with improved tail-area recovery in a dose-dependent manner, accompanied by reduced expression of apoptosis-related genes and suppression of pro-inflammatory mediators, including IL-1β, IL-6, TNF-α, TLR4, and NF-κB1. In parallel, Mollugin restored antioxidant enzyme activities of superoxide dismutase and catalase. Fluorescence imaging in Tg(mpeg1:GFP) zebrafish showed a time-dependent effect of Mollugin on macrophage accumulation, with no obvious reduction at 24 h, but decreased macrophage-positive area at 48 h in the 66 and 200 µg/mL groups compared with the injured model group. qRT-PCR analysis further showed reduced expression of the M1-associated marker iNOS and increased expression of the M2-associated marker arginase-1. Collectively, these findings suggest that Mollugin may support zebrafish tail regeneration in association with changes in inflammatory, oxidative stress, apoptosis-related, and macrophage-associated markers. Further studies are needed to define the underlying cellular and molecular mechanisms.

Introduction

Tissue regeneration is a critical physiological process by which organisms repair damage, involving complex regulation of cell proliferation, differentiation, and the maintenance of microenvironmental homeostasis¹. Among vertebrate models, zebrafish have emerged as a pivotal system for studying wound repair mechanisms due to their remarkable regenerative capacity. In particular, tail fin regeneration in zebrafish encompasses the spatiotemporal coordination of multiple biological events, including inflammation regulation, oxidative response balance, and apoptosis control^2,3. In recent ye....

Protocol

All animal procedures followed the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Fuzhou First General Hospital (approval no. 202408002).

Zebrafish breeding
All experiments were conducted using zebrafish (Danio rerio) maintained under standard laboratory conditions. Wild-type AB zebrafish were used for the toxicity assay and tail-amputation regeneration assay. Tg(mpeg1:GFP) zebrafish were used specifically for macrophage imaging. Tg(mpeg1:GFP) is a macrophage reporter line in which GFP expression labels macrophage-lineage cells under the....

Results

Toxicity and safe dose determination
To investigate the effects of Mollugin, we first performed toxicity tests. The results indicated that zebrafish embryo mortality remained low at concentrations up to 200 µg/mL, whereas increased mortality was observed at concentrations above 200 µg/mL (Table 2). Therefore, the highest safe dose was determined to be 200 µg/mL, while the low and medium doses were set at 22 and 66 µg/mL, respectively.

Tail amputati.......

Discussion

In this work, we examined the effects of Mollugin on zebrafish tail regeneration. Within the tested dose range, Mollugin treatment was associated with improved tail-area recovery after injury, along with changes in apoptosis-related gene expression, inflammatory markers, antioxidant enzyme activity, and macrophage-associated markers.

Zebrafish, as a prominent model for regeneration studies, exhibit tail regeneration that involves the coordinated action of multiple signaling pathways

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well tissue culture plates	Corning	3516
Catalase (CAT) assay kit	Nanjing Jiancheng Bioengineering Institute	A007-1-1
Chloroform	Sigma-Aldrich	288306
CMax Plus microplate reader	Molecular Devices	iD3s
Danieau’s buffer – Ca(NO3)2·4H2O	Guangzhou Pharmaceutical Holdings Ltd., China	N/A	Final concentration in Danieau’s buffer: 0.6 mM.
Danieau’s buffer – HEPES	Guangzhou Pharmaceutical Holdings Ltd., China	N/A	Final concentration in Danieau’s buffer: 5 mM; pH adjusted to 7.2.
Danieau’s buffer – KCl	Guangzhou Pharmaceutical Holdings Ltd., China	N/A	Final concentration in Danieau’s buffer: 0.7 mM.
Danieau’s buffer – MgSO4·7H2O	Guangzhou Pharmaceutical Holdings Ltd., China	N/A	Final concentration in Danieau’s buffer: 0.4 mM.
Danieau’s buffer – NaCl	Guangzhou Pharmaceutical Holdings Ltd., China	N/A	Final concentration in Danieau’s buffer: 58 mM.
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650
E3 embryo medium	Laboratory-prepared (standard recipe)	N/A	Standard recipe.
Gene-specific qPCR primers (tnfα, il1β, il6, nfkb1, tlr4, ccl2, icam1, inos, arginase-1, caspase-3, bcl2, β-actin)	Sangon Biotech	N/A
GraphPad Prism	GraphPad Software, San Diego, CA, USA	N/A	Version 8.0.
ImageJ software	National Institutes of Health (NIH)	N/A	Version 1.52n.
Mollugin (≥98%, HPLC)	Sigma-Aldrich	SMB00431	Active compound from Rubia cordifolia, dissolved in DMSO to 40 mg/mL and diluted to 22, 66 and 200 µg/mL in E3 medium.
Motorized tissue homogenizer	IKA, T10 basic ULTRA-TURRAX (or equivalent)	N/A
NovoScript 1st Strand cDNA Synthesis SuperMix	Novoprotein	abs60077
Phosphate-buffered saline (PBS)	Gibco, Thermo Fisher Scientific	10010023	pH 7.4
Real-time PCR system (Archimed X4)	Sansure Biotech Inc.	Archimed X4
Refrigerated microcentrifuge	SCILOGEX	CF1524R
RNA-free tips	Sangon Biotech	F603222-0010
RNAiso Plus	Takara Bio Inc.	9109	Phenol/guanidine-based RNA extraction reagent.
RNase-free microcentrifuge tubes, 1.5 mL	Axygen / Eppendorf	N/A
SMZ800N fluorescence stereomicroscope	Nikon	SMZ800N	Fluorescence stereo microscope used to image Tg(mpeg1:GFP) larvae and quantify macrophage aggregation.
Sodium hypochlorite solution	McLean, USA	N/A	0.003% working solution.
SYBR Green qPCR Mix (2× SYBR qPCR Mix)	Novoprotein	E096
SZX16 stereomicroscope	Olympus	SZX16	Used for morphological observations, toxicity assessment and tail fin amputation of larvae.
Total Superoxide Dismutase (T-SOD) assay kit	Nanjing Jiancheng Bioengineering Institute	A001-3
Transgenic zebrafish Tg (mpeg1:GFP)	Anburui, Fujian, China (or in-house colony)	N/A	Macrophage reporter line.
Tricaine methanesulfonate (MS-222)	Sigma-Aldrich	E10521	0.03% working solution.
UltraPure DNase/RNase-Free distilled water	Invitrogen	10977023
Wild-type zebrafish, CZ98 strain (Danio rerio)	Anburui, Fujian, China	N/A	Fertilized embryos and larvae were obtained from adult breeding stock.