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PeerJ
2026 Mar 02;14:e20863. doi: 10.7717/peerj.20863.
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Extract from crown-of-thorns starfish promotes zebrafish caudal fin regeneration by inhibition of neutrophil migration.
Zhang W, Li Z, Qiu Y, Yu Z, Liu S, Liu X, Niu W, Xiao J, Wu Z, Li M.
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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.
Figure 1. Extraction and fractionation of starfish tissues (A. planci).
Figure 2. Effects of extracts from COTS on zebrafish caudal fin regeneration.(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.
Figure 3. TWE promotes caudal fin regeneration in zebrafish.(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.
Figure 4. TWE inhibits neutrophil migration during fin regeneration.(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.
Figure 5. Knockdown of the csf3r gene further promotes caudal fin regeneration.(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.
Figure 6. TWE modulates the expression of several immune-related genes.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.
