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. 2026 Mar 2;14:e20863. doi: 10.7717/peerj.20863

Extract from crown-of-thorns starfish promotes zebrafish caudal fin regeneration by inhibition of neutrophil migration

Weibo Zhang 1,2,3,#, Zhehui Li 2,#, Yingkun Qiu 2, Zhewei Yu 2, Shunzhi Liu 2, Xin Liu 1, Wentao Niu 4, Jiaguang Xiao 4, Zhiqiang Wu 3,✉, Mingyu Li 1,2,✉
Editor: Mason Posner
PMCID: PMC12962133  PMID: 41800123

Abstract

Reparative regeneration is the process of repairing or replacing damaged or lost tissue. Starfish possess remarkable regenerative capacity and can regenerate nearly any part of their body, making them ideal models for studying regenerative medicine. In this study, we isolated four different extracts from crown-of-thorns starfish (COTS), Acanthaster planci, which recently experienced outbreaks in the South China Sea and pose a threat to the coral reef ecosystem. Our study found that one extract, the water-eluted fraction (TWE), which was proved to contain saponins by physical, chemical and spectroscopic identification, significantly promoted zebrafish larvae caudal fin regeneration. We further revealed that TWE inhibits neutrophil migration by reducing the expression of cxcl8-l1 in the tail tissue, thereby promoting caudal fin regeneration. Our results suggested that TWE may directly or indirectly enhance reparative regeneration in zebrafish by modulating neutrophil responses.

Keywords: Crown-of-thorns starfish, Regeneration, Zebrafish, Neutrophil migration, Extract

Introduction

Regenerative medicine is a rapidly advancing field that endeavors to replace or restore damaged or missing cells, tissues, and organs in organisms (Beetler et al., 2022). Tissue regeneration is a complex process requiring precise coordination among many different cell types and signaling pathways, in which the immune system plays an essential role (Altyar et al., 2022; Julier et al., 2017). Moreover, the immune response to tissue damage critically determines the speed and outcome of the healing process, including the extent of scarring and the recovery of organ function. Within the immune response, macrophages and neutrophils are crucial components of the innate immune response, engulfing invading microorganisms, transporting antibacterial compounds to the site of injury, participating in inflammation, and promoting tissue repair and regeneration (Ellett et al., 2011). Therefore, regulating the activity of macrophages and neutrophils is a powerful approach to modulating regeneration (Wynn & Ramalingam, 2012; Wynn & Vannella, 2016).

Regenerative capabilities vary among different species or even among different tissues and organs of the same species (Ben Khadra et al., 2017; Zheng, Zueva & Hinman, 2022). In mammals, including humans, wound healing typically results in scar repair, and most individuals are unable to regenerate tissues and organs after loss. In contrast, many invertebrates, such as starfish and sea urchins, can regenerate nearly any part of their body (Dai et al., 2016; Wolff & Hinman, 2021). The crown-of-thorns starfish (COTS), Acanthaster planci, belongs to the family Acanthasteridae, is a starfish named for the spines that cover its body and arms. COTS are considered harmful pests due to their predation on reef-building corals (Hall et al., 2017). In recent years, the rapid population increases of COTS have occurred in the Xisha Islands in the South China Sea, Okinawa Island in Japan, and the Great Barrier Reef in Australia, posing a serious threat to the local ecosystem (Deaker & Byrne, 2022; Xiao et al., 2022).

Several reports have revealed that substances isolated from COTS possess multiple bioactivities, including anti-bacterial, anti-oxidant, anti-cancer, anti-neurodegeneration, anti-atherosclerosis, and anti-inflammatory properties (Abd El Hafez et al., 2022; Ha et al., 2021; Lee et al., 2014; Sasayama et al., 2022; Vien et al., 2016). Although studies have been conducted on other types of starfish (Afshar et al., 2023; Dai et al., 2016), the regenerative activity of substances from COTS has never been evaluated.

In this study, we isolated several extracts from COTS and used zebrafish larvae as a model to survey their regenerative activities. Zebrafish have been demonstrated to be a powerful model organism for studying tissue regeneration, including the heart, spinal cord, musculoskeletal system, fin and pancreatic islet (Cigliola, Becker & Poss, 2020; Jia et al., 2022; Kaliya-Perumal & Ingham, 2022; Sehring & Weidinger, 2022; Tzahor & Poss, 2017). After the amputation of the caudal fin, larval zebrafish were incubated with culture medium or culture medium with extracts from COTS, and caudal fin regeneration was evaluated based on the increase in regenerative area. We also investigated the migration of neutrophils and macrophages associated with the caudal fin regeneration process, and examined the expression of several immune-related genes in zebrafish.

Materials and Methods

Animal collection and genetic species identification

Adult crown-of-thorns starfish (27–33 cm length) were collected by an aquanaut using tweezers from Xisha Islands of the South China Sea in 2021. Starfish were brought to the boat and cleaned with sterilized seawater, and then stored in −20 °C freezer immediately (The time between capture and storage was less than 20 min). After collection, the starfish were transported and stored at −80 °C in the laboratory of Xiamen University. For species identification, the DNA was extracted (Qiagen DNeasy blood and tissue kit) and PCR was performed to amplify the mitochondrial cytochrome oxidase subunit 1 (COI) gene using primer set (5′-GCCTGAGCAGGAATGGTTGGAAC-3′, 5′-GTGGGATATCATTCCAAATCCTGG-3′ (Vogler et al., 2008)). After sequencing, the cox1 sequence was submitted to the BOLD Systems and matched to crown-of-thorns starfish (Acanthaster planci) reference sequences with 100% similarity (Fig. S1). The nucleotide sequence has been deposited in GenBank (accession no. OR391930).

Extraction and separation of bioactive substances

According to methods described in previous studies (Ha et al., 2021; Kicha et al., 2014), nine fresh adult starfish (approximately 5 kg in total) were cut into small pieces and extracted three times with 70% ethanol (EtOH), each time for 1 h. The combined solution was concentrated to yield 160 g of the total alcohol extract (TAE). The TAE was suspended in 1 L of water and then extracted three times with petroleum ether (1 L each) to remove lipids. The aqueous layer was subsequently extracted three times with ethyl acetate (EtOAc, 1 L each) to obtain 33 g of the EtOAc-soluble extract (TEE). The remaining water layer was evaporated to remove residual EtOAc and then subjected to chromatography on a macroporous adsorption resin column (ADS-17; Cangzhou Bon Adsorber Technology, China; 45 cm × 15 mm i.d.). The column was first eluted with water, followed by 95% ethanol. The water eluate was evaporated and freeze-dried to afford the water-eluted fraction (TWE, 110 g). The ethanol eluate was concentrated to give the ethyl alcohol eluted extract (TEAE, 11.4 g), as summarized in Fig. 1.

Figure 1. Extraction and fractionation of starfish tissues (A. planci).

Figure 1

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Characterization of saponins in TWE extract

Saponins characterization of TWE by physical, chemical and spectroscopic identification using the following methods. (1) Foam test: 5 mg of TWE extract (crude saponin) was completely dissolved in 5 mL of triple-distilled water, shaken thoroughly for 1 min to produce a large amount of foam, left to stand, and foam persistence was observed within 15 min (Sun et al., 2022). (2) Liebermann-Burchard reaction: 5 mg of TWE extract (crude saponin) was placed in a test tube, 3 ml of acetic anhydride was added to fully dissolve it, then a drop of concentrated sulfuric acid was added, and the mixture was shaken well while observing the color change (Burke et al., 1974; Abel et al., 1952). (3) Molish reaction: 5 mg of TWE extract (crude saponin) was completely dissolved in 5 mL of triple-distilled water, and 2–3 drops of α-naphthol ethanol solution were added. After thorough shaking, 1 mL of concentrated sulfuric acid was added, and the appearance of purple rings at the junction of the two liquid phases was observed (Aziz, 2015). (4) Nuclear magnetic resonance (NMR) analysis: The 1H NMR and HSQC spectrum of TWE extracts were analyzed by using 600 MHz WB Solid-State Nuclear Magnetic Resonance Spectrometer (Bruker AV-600 MHz; Bruker, Zurich, Switzerland). (5) Saponin content analysis: The total content of steroidal saponins in TWE was determined using the sulfuric acid-methanol method (Le Bot et al., 2022). Dioscin (HPLC ≥ 98%; Yuanye Company, Shanghai, China) was used as the steroidal saponin standard, and the standard solution with concentration of 40, 80, 120, 160 and 200 μg/mL was prepared by methanol, take 1 mL into a 10 mL test tube and evaporated at 25 °C. Three groups were sampled in parallel in TWE, accurately weighed and prepared to 1 mg/mL sample solution, then take 1 mL and evaporated in the same way. A total of 5 mL of sulfuric acid-methanol (7:3) mixture was added to the dried standard and sample tube, the tube plug was covered, incubated in a 60 °C water bath for 60 min, and was then immediately placed in an ice bath. A Shimadzu UV-260 UV-Vis spectrophotometer (Shimadzu Corporation, Tokyo, Japan) was used to measure the absorption curve of the standard solution and the sample reaction solution at 200~800 nm, using sulfuric acid-methanol (7:3) solution as the blank group to deduct the background, it was found that the sample and standard reaction solution reached the maximum absorption value at 321 nm (Figs. S2A, S2B), which was consistent with measurements from previous literature (Le Bot et al., 2022) and complies with Lumber’s law in this concentration range. Taking the concentration as the abscissa and the absorption intensity of the reaction solution at 321 nm as the ordinate, the linear regression equation calculated by Graphpad Prism 8.0.2 is Y = 0.00690*X + 0.510 (Fig. S2C), showing a good linear relationship (R2 > 0.9999). Thus, the steroidal saponin concentration of the sample reaction solution was calculated to be 117.2 ± 17.7 μg/mL, indicating that the mass fraction of steroidal saponins from TWE was 11.72 ± 1.77% (w/w).

Zebrafish lines and maintenance

Zebrafish (Danio rerio) adult fish were raised in an aquaculture system (Haisheng, China) on a 14:10-h light-dark cycle at 28 °C. The embryos were obtained by mating fish pairwise in a plastic spawning trap and reared in 28.5 °C embryo culture medium according to Kimmel et al. (1995). In this study, AB strain, Tg(lyz: DsRed2) (Hall et al., 2007) and Tg(mpeg1: EGFP) (Ellett et al., 2011) transgenic lines were used. The fish in the experiment came from multiple pairs of parents. Embryos harvested from the same batch were merged into the same culture dish for subsequent experiments. Larvae were randomly selected for treatment in different groups. All procedures have been approved by the Animal Care and Utilization Committee of Xiamen University (Protocol No. XMULAC20170037 of February 28, 2017).

Tail fin amputation, drug treatment and regeneration area measurement

All isolated extracts (TAE, TEE, TWE, and TEAE) were made in 1,000× stock solution and stored in light-protected Eppendorf tubes at −20 °C, and dissolved in vehicle (DMSO) with 10, 50 and 100 μΜ solutions when used. For amputation, 3 dpf (days post fertilization) of AB strain larvae, which from multiple pairs of parents were used. In each experiment group, 12 larvae in the 24-well plate (extra holes are empty) were pre-treated for 2 h with extracts or vehicle (as negative controls), followed by anesthetization with 0.02% Tricaine and cut the caudal fin. The caudal fin of the larva was amputated approximately at the position of 5 mm (direction from head to tail. located proximal to the first point of the actinotrichia) using a surgical blade under a Leica M205 FCA stereomicroscope (Leica, Wetzlar, Germany) (Fig. 2A). After amputation, larvae were transferred back to 24-well plate and co-incubated with extracts or vehicle for another 4 h (He et al., 2020; Sun et al., 2019) (Fig. 2B).

Figure 2. Effects of extracts from COTS on zebrafish caudal fin regeneration.

Figure 2

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(A) Schematic diagram of 3 dpf zebrafish larva used for treatment, indicating the cut site, the measured regeneration area, and the imaging area. (B) Schema of the procedures for caudal fin amputation and drug treatment (pre-treatment for 2 h before amputation and treatment for 4 h after amputation), the regeneration area was measured at 72 hpa. (C) Representative images of the tail fins of zebrafish larvae at 0 and 72 hpa treated with vehicle or TWE, TEAE after amputation. The solid red lines indicated the cut site, and the yellow dotted line circles outline the regeneration area measured. (D) Quantification of the regeneration area in larvae treated with different concentration bioactive substances for 72 h. Data shown were means ± SD. n = 6, scale bar = 100 μm, ***p < 0.001.

After 4 h of treatment, the amputated zebrafish larvae were transferred to fresh egg water and placed in the incubator. For imaging, the treated larvae were anesthetized in 0.02% Tricaine and subsequently placed in a slide and imaged using Leica M205 FCA stereomicroscope at the appropriate time points. The regeneration area between the cut site and the newly grown region was quantified using ImageJ software (National Institutes of Health) (Schneider, Rasband & Eliceiri, 2012).

Imaging and quantification of neutrophils and macrophages

A total of 3 dpf Tg(lyz:DsRed2);Tg(mpeg1:EGFP) larvae (from multiple pairs of parents) were used for amputation and treatment. Then the larvae were fixed in 4% PFA and imaged utilizing Leica M205 FCA stereomicroscope at the time points of 0, 2, 4, 7, 24 hpa (hours post amputation) after anesthetization. The neutrophils were detected based on DsRed fluorescence, and macrophages were detected based on the GFP fluorescence. The total neutrophils/macrophages were counted manually based on the images in the defined area using Image J (Schneider, Rasband & Eliceiri, 2012), when the cell projections overlap, we count roughly by the amount of overlap or the intensity of the fluorescence. The recruited neutrophils/macrophages were calculated by the total neutrophils/macrophages of the next time point minus the previous time point.

CRISPR/Cas9 mutagenesis of zebrafish csf3r gene

CRISPR/Cas9 mutagenesis of csf3r was based on the method of Li et al. (2016). Briefly, the CRISPRscan (https://www.crisprscan.org/) was used to design a set of two sgRNAs targeting the zebrafish csf3r gene. Both two sgRNAs targeted exon 2. Each sgRNA contained a gene-specific spacer sequence followed by a Cas9 enzyme binding sequencing. These two sites were initially provided in different oligonucleotides called “site-specific” and “constant”, respectively. The sgRNA was synthesized by Genscript Biotech (Nanjing, China). The microinjection reaction system consisted of 0.5 μL sgRNA-1 and sgRNA-2 (400–500 ng/μL), 0.2 of 20 μM Cas9 Nuclease (New England Biolabs), 0.2 μL 0.5% Phenol red solution (Sigma) and 0.6 μL nuclease-free water. The mixture was mixed well and microinjected (500–1,000 pg) directly into 1-cell Tg(lyz:DsRed2) embryos.

Zebrafish tail tissue collection, RNA extraction and quantitative real-time RT-PCR

A total of 3 dpf larvae were used for amputation and treatment. At 7 hpa, the larvae were anesthetized and the posterior part of larvae (1/3 of body length) were cut with disposable sterile scalpel. Posteriors part from 30 larvae were collected for each group. Total RNA was extracted from collected samples using Trizol Reagents (Thermo Fisher Scientific, Waltham, MA, USA). To synthesize the first-strand cDNA, RNA was reverse transcribed using the M-MLV (Promega, Madison, WI, USA) with oligo(dT)16 primers. Relative gene expression was analyzed by Real-time polymerase chain reaction (PCR), using 2× SYBR Green PCR Master Mix (Applied Biosystems) with an ABI QuantStudio three instrument (Applied Biosystems). After a 30 s incubation at 95 °C, the amplification was performed as follows: 95 °C, 5 s; 60 °C, 30 s for 40 cycles. Results were calculated with 2−ΔΔCt, and the expression level was normalized to the housekeeping gene β-actin and expressed as relative fold change from the control group. For each gene, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in triplicate of each cDNA, from three biological samples. Primers for quantitative qRT-PCR are listed in Table S1.

Statistical analysis

The experiments were carried out in a randomized manner, and the data analyzed by a blinded reviewer. All statistical analyses were performed using GraphPad Prism v.9.0 (GraphPad Software, La Jolla, CA, USA). Results are presented as mean values ±SD. The statistics were performed using one-way ANOVA followed by Bonferroni post-hoc test or t-test. P < 0.05 was considered as significant. All statistical data are displayed in Table S2.

Results

Evaluation of the activities of extracts on zebrafish caudal fin regeneration

To evaluate the regeneration activities of COTS extracts, 3 dpf zebrafish larvae were pre-treated with different concentrations of isolated extracts (TAE, TEE, TWE, and TEAE) for 2 h. The caudal fin was then amputated and maintained with these extracts for another 4 h. The caudal fin regeneration areas were measured at 72 hpa (hours past amputation) (Figs. 2A and 2B). As shown in Figs. 2C and 2D, TWE significantly increased caudal fin regeneration at the concentrations of 10 and 50 μM, with 10 μM showing a greater effect than 50 μM. However, TAE, TEE, and TEAE did not promote the caudal fin regeneration after treatment at 10 μM (Fig. 2D). Furthermore, 50 and 100 μM of TAE, TEE, and TEAE were lethal to zebrafish larvae. These data suggested that TWE, but not the three other extracts, had activity to promote caudal fin regeneration in zebrafish larvae.

Since the TWE extract was isolated from the aqueous phase using a microporous column, a method previously reported to predominantly enrich saponins (Dahmoune et al., 2021; Ha et al., 2021; Malyarenko et al., 2014; Tangerina et al., 2018), we subsequently performed comprehensive physical, chemical, and spectroscopic characterization of TWE. Based on the physical and chemical tests such as foam test, positive Liebermann-Burchard reaction (showed green color) and positive Molish reaction (showed purple color), the TWE was proved to contain a large amount of saponins. We subsequently performed NMR analysis of the TWE, and found that TWE had proton signals of the saponin-like parent structure based on 1H NMR spectra (Fig. S2), as well as carbon-hydrogen correlation signals of the phases based on HSQC spectra (Fig. S3). Moreover, using dioscin as steroidal saponin standard, we measured that the saponins from TWE were 11.72 ± 1.77% (w/w) via sulfuric acid-methanol spectrophotometer method (Fig. S4). These data suggested that TWE contained a substantial amount of saponins.

TWE promotes caudal fin regeneration primarily on the 1st day after amputation

To further investigate the effects of 10 μM TWE on caudal fin regeneration, we then surveyed the gradient of regeneration over three consecutive days (Fig. 3A). As shown in Figs. 3B and 3C, the total fin regeneration areas were higher in the TWE treated group compared with the control group. To clarify at which time point TWE precisely increased caudal fin regeneration, we also measured the daily regeneration area, calculated by subtracting the total regeneration area of the previous day from that of the next day. Interestingly, TWE treated zebrafish larvae showed a significant increase regeneration area on the 1st day after amputation. However, the regeneration areas on the 2nd and 3rd day after amputation were comparable between TWE group and the control group (Fig. 3D). These data indicated that TWE increased the caudal fin regeneration primarily on the 1st day, since that the accumulated regeneration area keeps the TWE group higher than the control group.

Figure 3. TWE promotes caudal fin regeneration in zebrafish.

Figure 3

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(A) Schema of the procedures for caudal fin amputation, drug treatment, and regeneration observation at time course of 0, 24, 48 and 72 hpa. (B) Representative images of zebrafish larvae tail fins at 0, 24, 48 and 72 hpa after amputation, treated with vehicle or 10 μM TWE. (C) Quantification of total regeneration area at 24, 48 and 72 hpa after amputation. (D) Quantification of the daily regeneration area during the time windows of 0–24, 24–48 and 48–72 hpa, The daily regeneration area was calculated by the total regeneration area of the next day minus the previous day. Data shown were means ± SD. Scale bar = 100 μm, *p < 0.05, ***p < 0.001. ns, no significance.

TWE promotes caudal fin regeneration by inhibiting neutrophil migration

Previous studies have demonstrated that neutrophils and macrophages are the pivotal immune phagocytes that enter the wound after tissue injury to remove the cell debris and invaded microorganisms, thereby facilitating the regeneration of injured tissues (Pastar et al., 2021). To examine whether TWE affects the neutrophils and macrophages during the 1st day of fin regeneration, two transgenic lines Tg(lyz:DsRed2) and Tg(mpeg1:EGFP), which labeled neutrophils with DsRed2 and labeled macrophages with EGFP respectively, were employed. The Tg(lyz:DsRed2); Tg(mpeg1:EGFP) double-transgenic zebrafish larvae were treated with 10 μM TWE, and the number of neutrophils and macrophages was determined at 0, 2, 4, 7, and 24 hpa in the counting area surrounding the caudal fin (Figs. 4A and 4B).

Figure 4. TWE inhibits neutrophil migration during fin regeneration.

Figure 4

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(A) Schematic diagram of 3 dpf Tg(lyz: DsRed2); Tg(mpeg1: EGFP) zebrafish larva used for treatment, indicating the cut site, the immune cell counting area, and the imaging area. (B) Schema of the procedures for caudal fin amputation, drug treatment, and immune cell dynamics observation at 0, 2, 4, 7 and 24 hpa. (C) Representative fluorescence images of migration of neutrophils (red) and macrophages (green) after the treatment of TWE or vehicle at different time points. The white dot boxes indicated the area used for quantification of neutrophils and macrophages. (D and E) The total number of neutrophils (D) and macrophages (E) at different time points after treatment. (F and G) The number of recruited neutrophils (F) and macrophages (G) during different time windows from 0–24 hpa. The recruited neutrophils/macrophages were calculated by the total neutrophils/macrophages of the next time point minus the previous time point. Data shown were means ± SD. Scale bar = 100 μm. *p < 0.05.

As shown in Figs. 4C, 4D and 4E, both neutrophils and macrophages started to migrate to the wound region after 2 h of amputation, peaked at 7 hpa, and then decreased by 24 hpa. Intriguingly, TWE-treated larvae had fewer neutrophils in the tail region compared with the control (Fig. 4D). In addition, this trend persisted from 2–7 hpa (Fig. 4D). However, the number of macrophages was not changed during the 2–24 hpa (Fig. 4E). To further explore the details of phagocytes migration, we also measured the recruited (the next time point minus the previous time point) neutrophils and macrophages at different time windows from 0–24 hpa. Our results showed that the migration of neutrophils in TWE group was significantly inhibited during 0–2 hpa time window, but not for 2–4, 4–7 and 7–24 hpa (Fig. 4F). Moreover, the recruited macrophages were not different between TWE group and control group during 0–24 hpa (Fig. 4G).

To further verify whether the acceleration of caudal fin regeneration is caused by inhibition of neutrophil migration, we knockdown the csf3r gene of zebrafish embryos using sgRNA, resulting in reduced neutrophil development (Pazhakh et al., 2017) (Fig.5A, panel 1 and 2). Then the WT larva and csf3r crispants were incubated with DMSO or TWE. Our results indicate that the csf3r crispants significantly reduced neutrophil number, and csf3r crispants treated TWE had the lowest neutrophil (Fig. 5B). Across different time windows, neutrophils migration was remarkably inhibited in the crispants treated TWE (Fig. 5C). Moreover, the neutrophil numbers of csf3r crispants were similar to those in of WT larva treated with TWE at different time point hpa (Fig. 5B). Additionally, the fin regeneration areas and speed were also similar between csf3r crispants and TWE-treated WT larva (Figs. 5D and 5E). Interestingly, TWE-treated csf3r crispants exhibited the highest fin regeneration areas and speed (Figs. 5D and 5E). Taken together, these data suggested that TWE treatment suppressed the neutrophils migration to the wound after amputation, contributing to enhanced zebrafish caudal fin regeneration.

Figure 5. Knockdown of the csf3r gene further promotes caudal fin regeneration.

Figure 5

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(A) Representative fluorescence images of migration of neutrophils (red) after the treatment of TWE or DMSO at different time points of caudal fin regeneration. Four groups are wild-type larva with DMSO (control-WT), csf3r-knockdown larva with DMSO (control-csf3r-KD), wild-type larva incubated with TWE (TWE-WT), and csf3r knockdown larva incubated with TWE(TWE-csf3r-KD). (B) Quantitation of the total number of neutrophils at different time points after treatment in each group. (C) The number of recruited neutrophils at different time windows from 0–24 hpa. The recruited neutrophils were calculated by the total neutrophils of the next time point minus the previous time point. (D) The statistics of total regeneration area at the time point of 0, 2, 4, 7 and 24 hpa after amputation. (E) The statistics of regeneration area per day at the at the time windows of 0–2, 2–4, 4–7 and 7–24 hpa, regeneration area per day was calculated by the total regeneration area of the next day minus the previous day. Scale bar = 100 μm. Data shown were means ± SD. The different color of star-shaped means the relative group compared with control-WT. *p < 0.05, **p <0.01.

TWE regulates the mRNA level of immune-related genes in the regenerated caudal fin

To further investigate the molecular mechanism by which TWE promotes caudal fin regeneration, we examined the expression of several immune-related genes in the posterior part of zebrafish (1/3 of body length) at 7 hpa, which is the peak of neutrophils and macrophages infiltration. These genes include cxcl8-l1 (C-X-C motif chemokine ligand 8 like 1), ela2 (the gene encoding neutrophil elastase), mmp9 (matrix metallopeptidase 9), il-1β (interleukin-1 β) and tnf-α (tumour necrosis factor α). Cxcl8-l1 encoded CXCL8, one of the most potent chemoattractant molecules responsible for guiding neutrophils to reach the sites of injury (de Oliveira et al., 2013). The gene ela2 encodes the neutrophil elastase (NE), which degradation the extracellular matrix (ECM) and releases pro-inflammatory cytokines (Lebedeva et al., 2020) and MMP9 remodel matrix proteins to protect the healing organ or tissue and facilitate migrating cell replacement of lost tissue (Negron, Lun & May, 2014). Moreover, the inflammatory factors, IL-1β and TNF-α, also play important roles during caudal fin regeneration (Tsarouchas et al., 2018).

Our results showed that TWE treatment significantly inhibited the expression of cxcl8-l1 and ela2, and up-regulated mmp9 expression at 7 hpa (Figs. 6A, 6B and 6C). However, TWE did not alter the expression of il-1β and tnf-α (Figs. 6D, 6E). These data suggested the TWE treatment inhibited neutrophil marker gene ela2, and reduced the expression of cxcl8-l1 in the tail tissue, consistent with our observation that TWE suppressed neutrophil migration (Fig. 4D).

Figure 6. TWE modulates the expression of several immune-related genes.

Figure 6

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qRT-PCR analysis of immune relative genes expression at 7 hpa after treatment, including cxcl8-l1 (A), ela2 (B), mmp9 (C), il-1β (D) and tnf-α (E). RNA was isolated from the posterior part (1/3 of body length) of zebrafish larvae (n = 30). The expression of β-actin was used as an internal control for all real-time PCR experiments. Data in A-E are compared with control groups (vehicle = 1), and the values shown are means ± SD from three independent experiments. * p < 0.05, *** p < 0.001. ns, no significance.

Discussion

Reparative regeneration of injured or lost organs is a remarkable characteristic of starfish. Reports have revealed that starfishes are excellent models for regenerative medicine research, and many studies have been performed in order to understand their regenerative mechanisms (Ben Khadra et al., 2017). Moreover, extracts from other starfish (Archaster typicus, Brittle Star) have been shown to improve tissue regeneration in zebrafish (Dai et al., 2016) and wound healing in rat (Afshar et al., 2023). Dai et al. (2016) extracted and separated the active biomaterials from Archaster typicus starfish, and showed that compounds in normal hexane fractions (NHFs) and methanol-water fractions (MWFs) exhibit the ability to induce zebrafish wound tissue regeneration. Afshar et al. (2023) demonstrated that hydroalcoholic extracts of Ophiocoma cynthiae enhanced wound closure in rats, while polysaccharides from Ophiocoma erinaceus improved re-epithelialization, angiogenesis, and reduced inflammation in mammalian wound models. Despite differences in species, extraction methods, and outcome measures, these studies consistently support the regenerative potential of echinoderm extracts (Afshar et al., 2023). However, the regenerative effects of extracts from COTS on regeneration have never been evaluated. Therefore, the present study aimed to investigate the possibility and feasibility of applying COTS bioactive substances to induce reparative regeneration in zebrafish.

Firstly, we obtained the four different extracts (TAE, TEE, TWE, TEAE) from COTS through a series of extraction and isolation methods (Fig. 1). We then evaluated their regeneration activities using 3 dpf zebrafish caudal fin regeneration model. The data showed that TWE promotes caudal fin regeneration primarily on the 1st day after amputation (Fig. 3). We then characterized that the TWE contained a large amount of saponins by a series of physical and chemical experiments and nuclear magnetic resonance technology (1H NMR, HSQC) (Figs. S2–S4). Given the indispensable role of immune cells in zebrafish wound healing and tissue regeneration (Iribarne, 2021; Sipka et al., 2022), we analyzed the dynamic of neutrophils and macrophages during the TWE-induced caudal fin regeneration using double transgenic zebrafish Tg(lyz:DsRed2); Tg(mpeg1:EGFP). The results indicated that TWE treatment suppressed neutrophil migration into the amputated wound. Notably, knockdown of csf3r in zebrafish accelerated caudal fin regeneration regardless of TWE treatment, suggesting that reduced neutrophil accumulation induced by TWE is a contributing factor to enhanced regeneration. Actually, previous studies have also reported that neutrophil infiltration negatively affects on zebrafish fin regeneration (Li et al., 2012). Additionally, excessive neutrophil activation and release of neutrophil extracellular traps (NETs) impair wound healing in mice (Kruger et al., 2015).

A transient modulation of inflammation is known to be crucial for proper regeneration. For example, studies have demonstrated that a short and controlled inflammatory burst mediated by il-1β is required for fin fold regeneration in zebrafish, whereas persistent inflammation hampers regenerative processes (Hasegawa et al., 2017). Similarly, Tsarouchas et al. (2018) reported that macrophages dynamically regulate il-1β and tnf-α expression during zebrafish spinal cord regeneration, limiting inflammation once the initial wound response is established. Based on these studies, our findings suggest that TWE treatment down-regulated cxcl8-l1 and ela2, both involved in neutrophil activation and migration, while up-regulating mmp9, a positive regulator of extracellular matrix remodeling and regeneration. Despite the lack of significant changes in il-1β and tnf-α expression, TWE still enhanced regeneration. This indicates the promotion of caudal fin regeneration by TWE treatment may rely more on inhibiting neutrophil migration, rather than changing macrophage-derived cytokines.

Our study differs from previous works in several important aspects. First, earlier studies mainly focused on morphological outcomes such as blastema formation, angiogenesis, or wound closure (Afshar et al., 2023), whereas our work demonstrated that the COTS extract (TWE) promotes regeneration by suppressing neutrophil recruitment. Second, previous studies typically used prolonged exposure (24–72 h) or systemic administration in rodents, whereas our 6-h treatment in zebrafish was designed to capture the early inflammatory phase following injury. Finally, while other echinoderm extracts primarily acted through regulating cell proliferation (Afshar et al., 2023) and apoptosis (Dai et al., 2016), our findings suggest an immunomodulatory mode of action.

It should be noted that our research still has some limitations, including small sample sizes (only 6–12 subjects per group), used crude extract, and limited molecular mechanisms (how neutrophils are inhibited from migrating), which require further exploration.

Conclusions

Our study isolated four extracts from COTS and found that one extract, TWE which contained saponins, significantly promote zebrafish caudal fin regeneration by inhibition of neutrophil migration. Further work is required to identify which specific compound(s) in the TWE extract are responsible for promoting regeneration.

Supplemental Information

Supplemental Information 1. Supplemental materials.
peerj-14-20863-s001.docx (733.9KB, docx)
DOI: 10.7717/peerj.20863/supp-1
Supplemental Information 2. ARRIVE 2.0 Checklist.
peerj-14-20863-s002.pdf (164.1KB, pdf)
DOI: 10.7717/peerj.20863/supp-2
Supplemental Information 3. Quantitative Real-Time PCR checklist.
peerj-14-20863-s003.docx (825.1KB, docx)
DOI: 10.7717/peerj.20863/supp-3
Supplemental Information 4. Raw data.
peerj-14-20863-s004.xlsx (27KB, xlsx)
DOI: 10.7717/peerj.20863/supp-4

Acknowledgments

We thank members of the Li lab for constructive discussions. We also thank Zhen Li, Hong Yun, Yuhong Chen, Shan Jiang from School of Pharmaceutical Sciences, Xiamen University; Lei Huang from School of Life Sciences, Xiamen University, for technical support.

Funding Statement

This work was supported by grants from the Key Laboratory of Tropical Marine Ecosystem and Bioresource, Ministry of Natural Resources (2021ZD01 to Mingyu Li), Key Laboratory of Marine Biotechnology of Fujian Province (2022MB02 to Yingkun Qiu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Contributor Information

Zhiqiang Wu, Email: wuzhiqiang@ouc.edu.cn.

Mingyu Li, Email: limingyu@xmu.edu.cn.

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Weibo Zhang conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Zhehui Li performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Yingkun Qiu conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Zhewei Yu performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Shunzhi Liu performed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Xin Liu conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Wentao Niu performed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Jiaguang Xiao performed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Zhiqiang Wu conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Mingyu Li conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The Animal Care and Utilization Committee of Xiamen University approved the study (Protocol No. XMULAC20170037).

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

The nucleotide sequence is available at GenBank: OR391930.

Data Availability

The following information was supplied regarding data availability:

The raw data are available in the Supplemental File.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Information 1. Supplemental materials.
peerj-14-20863-s001.docx (733.9KB, docx)
DOI: 10.7717/peerj.20863/supp-1
Supplemental Information 2. ARRIVE 2.0 Checklist.
peerj-14-20863-s002.pdf (164.1KB, pdf)
DOI: 10.7717/peerj.20863/supp-2
Supplemental Information 3. Quantitative Real-Time PCR checklist.
peerj-14-20863-s003.docx (825.1KB, docx)
DOI: 10.7717/peerj.20863/supp-3
Supplemental Information 4. Raw data.
peerj-14-20863-s004.xlsx (27KB, xlsx)
DOI: 10.7717/peerj.20863/supp-4

Data Availability Statement

The following information was supplied regarding data availability:

The raw data are available in the Supplemental File.

Articles from PeerJ are provided here courtesy of PeerJ, Inc

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