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 years, the pharmacological activities of natural products have become a focal point in regenerative medicine research. Naphthoquinone compounds, in particular, have been widely studied because of their diverse biological effects⁴. Mollugin is a naphthoquinone compound derived from Rubiaceae plants and has shown anti-inflammatory, antioxidant, and cell cycle regulatory activities in previous studies^5,6. However, its mechanism of action in tissue regeneration remains unclear, and its potential role in modulating the inflammatory microenvironment and immune cell polarization warrants systematic investigation.

After injury, inflammation is rapidly activated and directly influences tissue repair. Moderate inflammatory signaling is essential for clearing necrotic tissue and initiating the regeneration program, whereas an excessive inflammatory cascade can exacerbate oxidative stress and lead to tissue fibrosis⁷. Cytokines, including IL-1β, IL-6, and TNF-α, increase markedly after injury and are accompanied by activation of TLR4/NF-κB signaling, leading to amplified inflammatory responses and tissue remodeling^8,9. Intercellular adhesion molecule-1 (ICAM-1) and CC chemokine ligand 2 (CCL-2) are also elevated after injury and are associated with increased recruitment of neutrophils and macrophages to the damaged tissues¹⁰. Notably, oxidative imbalance further aggravates tissue damage, and reduced superoxide dismutase (SOD) and catalase (CAT) activities indicate impaired antioxidant capacity^2,11.

Macrophages serve as key regulators of the inflammatory microenvironment, and their functional polarization significantly impacts the regenerative process¹². M1 macrophages are associated with inducible nitric oxide synthase (iNOS) expression and inflammatory activity, while M2 macrophages express arginase-1 (Arg-1) and are linked to tissue repair^13,14. Macrophage polarization is influenced by local cytokines, oxidative stress, and apoptosis-related signals. Within this context, the apoptotic regulatory network composed of B-cell lymphoma-2 (Bcl-2) family proteins and caspase-3 not only affects cell survival at the injury site but also indirectly modulates the chemotactic behavior of immune cells through the release of "find-me" signals¹⁵. Therefore, exploring intervention strategies that simultaneously modulate inflammatory responses, oxidative stress, and macrophage polarization may help improve tissue regeneration.

Natural compounds have demonstrated unique advantages in modulating these biological processes. Recent studies have confirmed that naphthoquinone substances, owing to their conjugated structures, possess the potential to scavenge free radicals; their benzoquinone core can affect redox-sensitive signaling pathways via electron transfer mechanisms^16,17. Mollugin has been reported to attenuate inflammatory responses in DSS-induced colitis, with reduced pro-inflammatory cytokine production and decreased TLR4 expression in colon tissues¹⁸. Mollugin has also been shown to inhibit TAK1-NF-κB/MAPK signaling and activate the Keap1-Nrf2 pathway in macrophages⁶; however, its impact on macrophage polarization and the apoptosis–anti-apoptosis network in an in vivo wound model has not yet been elucidated. Moreover, the dose-dependent biological effects of Mollugin and its regulatory role during the regeneration process remain to be systematically clarified.

The present study investigated the effects of Mollugin in a zebrafish tail fin regeneration model. Specifically, we examined tail regrowth, apoptosis-related gene expression, inflammatory signaling, antioxidant enzyme activity, and macrophage-associated responses after Mollugin treatment. Our findings suggest that Mollugin treatment is associated with improved tail regeneration and changes in multiple injury-response markers in this zebrafish model.

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 control of the macrophage-expressed mpeg1 promoter. All zebrafish lines were maintained at 28.5 °C under a 14-h/10-h light/dark cycle. Only morphologically normal embryos were used for experiments. For all experiments, embryos were incubated in Danieau’s buffer, which was prepared using reagent-grade chemicals including NaCl, KCl, Ca(NO₃)₂·4H₂O, MgSO₄·7H₂O, and HEPES adjusted to pH 7.2.

Drug preparation and safety determination
Mollugin (40 mg) was dissolved in 1 mL DMSO to generate a 40 mg/mL stock. The stock was first diluted 1:10 in Danieau’s buffer to 4 mg/mL, followed by further dilution to 22, 66, and 200 µg/mL for treatments. To determine the maximum safe concentration, 48-hour post-fertilization (hpf) AB strain embryos were dechorionated and disinfected with 0.003% sodium hypochlorite before being distributed into 6-well plates (n=20, triplicate wells per group). Embryos were incubated with Mollugin (22 µg/mL, 66 µg/mL, 200 µg/mL, 240 µg/mL, 280 µg/mL, 320 µg/mL, or 360 µg/mL) in E3 medium for 48 h at 28.5 °C. Survival was monitored under a stereomicroscope, and the highest concentration with ≥90% embryo viability was established as the safe upper limit for subsequent experiments.

Tail amputation and regeneration model construction
For regeneration studies, 48 hpf wild-type AB embryos were anesthetized with 0.03% tricaine prepared in embryo medium and positioned laterally under a stereomicroscope. Before tail amputation, embryos were maintained in Danieau’s buffer. A standardized transverse tail amputation was performed under stereomicroscopic guidance. The amputation line was placed immediately posterior to the notochord tip, with the posterior end of the notochord used as the anatomical landmark. The tail fin fold distal to this landmark was removed using a microscalpel to ensure a consistent injury level among specimens. Immediately following amputation (0 h), images were acquired with a stereo fluorescence microscope. After amputation, embryos were transferred to the corresponding E3-based treatment medium for the regeneration assay. Mollugin working solutions were prepared by diluting the Mollugin stock solution into E3 medium to final concentrations of 22, 66, and 200 µg/mL. Mollugin was administered by bath exposure during the regeneration period. To match vehicle exposure, the uninjured control group and injured model group were maintained in E3 medium containing 0.5% DMSO, corresponding to the final DMSO concentration in the 200 µg/mL Mollugin group. Embryos were maintained in 6-well plates at 28.5 °C for 2 days, with daily refreshment of the corresponding E3-based treatment medium. Regeneration was documented by imaging at 24 and 48 h post-amputation under both white light and fluorescence settings to assess tail regrowth and macrophage distribution. Image measurements were carried out in ImageJ.

For regeneration quantification, the recovered tail area was measured as an absolute area in µm² and was not normalized to embryo size or original tail size. To minimize variability, all embryos were amputated at the same anatomical landmark immediately posterior to the notochord tip, and the same landmark was used to define the measurement region across groups. Regions of interest were manually delineated in ImageJ based on the tail outline, and the analysis was performed by an investigator blinded to the treatment groups. For macrophage-positive area quantification, GFP fluorescence images of Tg(mpeg1:GFP) embryos were analyzed in ImageJ. The tail injury/regeneration region was manually defined as the region of interest using the same anatomical landmark applied for tail-area measurement. Images were converted to 8-bit grayscale, and background fluorescence was subtracted using the same background-correction settings for all images within the same experiment. A fixed fluorescence threshold was then applied uniformly to all images within each experiment to identify GFP-positive macrophage-associated signals. The macrophage-positive area was calculated as the threshold-positive GFP area within the defined ROI. Measurements were based on fluorescence-positive area rather than fluorescence intensity or manual cell counting. All image analyses were performed by an investigator blinded to the treatment groups.

RNA Extraction and quantitative real-time PCR (qRT-PCR)
Following completion of the regeneration assay, embryos from each experimental condition were collected and pooled (at least 20 embryos per group) for RNA analysis. For each condition, one biological replicate consisted of a pooled sample containing at least 20 embryos from the same experimental group. Three independent pooled biological replicates were prepared for each condition and used for statistical analysis. Total RNA was isolated after mechanical homogenization in a phenol/guanidine-based RNA extraction reagent. RNA quality and concentration were assessed spectrophotometrically by measuring A260/A280 ratios. cDNA was synthesized using a commercial reverse transcription kit. PCR amplification was performed with SYBR Green reagents. The qRT-PCR cycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. Expression levels of genes related to apoptosis (caspase-3 and Bcl-2), inflammation (TNF-α, IL-1β, IL-6, NF-κB1, TLR4, CCL2, and ICAM1), and macrophage phenotype (iNOS and arginase-1) were measured. β-Actin served as the reference gene. Fold changes in gene expression were determined using the 2−ΔΔCt approach. Primer information is listed in Table 1.

Activity assays for SOD and CAT
SOD and CAT levels were used to evaluate antioxidant status after tail injury. At 48 h post-amputation, zebrafish from each group were collected and homogenized in cold PBS. After centrifugation, the cleared extracts were used for SOD and CAT measurement with commercial activity assay kits. SOD activity was measured using a WST-1-based assay kit according to the manufacturer’s instructions. Samples were loaded onto 96-well assay plates together with standards, and absorbance was read on a microplate reader. Enzyme activities were obtained from standard curves. All assays were performed in triplicate.

Statistical analysis
Data are shown as mean ± SEM from at least three independent experiments. For the toxicity assay, each independent experiment consisted of one embryo batch, with 20 embryos per group and triplicate wells. For regeneration imaging and macrophage imaging in Tg(mpeg1:GFP) zebrafish, individual embryos were used as the measurement units, and 10 embryos were analyzed per group. For qRT-PCR analysis, one biological replicate consisted of a pooled sample containing at least 20 embryos from the same experimental group, and three independent pooled biological replicates were analyzed per condition. For SOD and CAT activity analysis, five independent pooled biological replicates were analyzed per condition. Group differences were analyzed by one-way ANOVA with Tukey’s post hoc test. Statistical analyses were performed in GraphPad Prism. Differences with p < 0.05 were regarded as significant.

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.