Ursolic acid derivative UA312 ameliorates ionizing radiation-induced cardiotoxicity and neurodevelopmental toxicity in zebrafish via targeting chrna3 and grik5

Abstract

The biological damage caused by ionizing radiation (IR) depends not only on the time and doses of exposure to tissue components but also on the developmental state of the cells. Currently, amifostine is the only radiation-protective agent used for clinical indications related to radiation therapy, but this compound has multiple drawbacks including high toxicity, short half-life and no protective effect on the nervous system. Ursolic acid (UA), a natural pentacyclic triterpenoid that exhibits multiple protective effects including anti-inflammatory, anticarcinogenic, and antioxidant effects. Due to its poor solubility and bioavailability, UA is mostly administered with liposomes. In this study we investigated the impact of UA312, an optimized derivative of UA, on radiation-induced developmental toxicity in zebrafish embryos and larvae. Embryo and larvae survival were observed at 4, 24, 48, and 72 hpf. UA312 was administered at 3 hpf, while embryos were irradiated with 6 Gy of γ-irradiation (dose rate: 0.88 Gy/min) at 4 hpf, then the embryos were moved to a fresh buffer. We determined that 40 µM of UA312 was a safe concentration for zebrafish embryos and larvae. We found that treatment with UA312 (40 µM) restored IR-induced early developmental dysplasia of the zebrafish embryos and larvae. Transcriptomic analysis revealed that exposure to IR inhibited multiple pathways related to neurodevelopment and cardiomyocyte function in zebrafish, which were validated by assessing abnormal cardiac morphology, variations in neurotransmitter levels and alterations in locomotor behavior; and that UA312 treatment ameliorated these alterations. We demonstrated that UA312 treatment significantly reversed the related signaling pathways by targeting chrna3 and grik5. In conclusion, this study identified a promising radioprotective drug, UA312, which alleviates IR-induced cardiotoxicity and neurodevelopmental toxicity in zebrafish by targeting chrna3 and grik5. UA312 may be developed as a novel radioprotective agent against acute IR damage in humans.

Introduction

In recent years, the wide use of nuclear technology in modern science and technology has increased the risk of exposure of living organisms to ionizing radiation (IR) [1]. In particular, with the rapid increase of nuclear power units and the occurrence of the Fukushima accident, the amount of nuclear-contaminated water discharged into the ocean has increased, raising concerns regarding the effect of radiation on marine life. Exposure to different doses of IR leads to the generation of free radicals and reactive oxygen species that cause disorganization, multiple organ dysfunction, and permanent cellular damage, including necrosis, infection, DNA damage, and metabolic dysfunction [2]. The degree of biological damage depends not only on the time and doses of exposure to tissue components but also on the developmental state of the cells. Immature, undifferentiated, and actively dividing cells are more susceptible to IR-induced effects than mature and differentiated cells. In terms of radiation protection drugs, only amifostine, a radioprotector for clinical indications related to radiotherapy, is available. However, there are still several drawbacks associated with the use of amifostine. For example, this compound has been shown to cause various adverse reactions, including nausea, vomiting, hypotension, sneezing, hypocalcemia, and skin reactions, which indirectly reflect its cytotoxicity. Moreover, the half-life of amifostine is very short, with an elimination half-life of only approximately 8 min, and it cannot cross the blood-brain barrier and protect the central nervous system from radiation. The most important limitation exists in terms of the administration method, i.e., intravenous injection, which has the disadvantages of causing adverse reactions and acting over a short period of time, greatly limiting the application of amifostine as a protective agent against acute radiation syndrome [3–5]. Therefore, safe and effective chemical and biological radioprotectors that can reduce the risk or damage caused by IR are urgently needed.

Ursolic acid (UA), which is a pentacyclic triterpene acid, is widely distributed in several fruits, vegetables, and whole grains. Due to its poor solubility and bioavailability, UA is mostly administered with liposomes. UA exerts several protective effects, including anti-inflammatory, anticarcinogenic, and antioxidant effects, by regulating nuclear factor κB, mitogen-activated protein kinase, cyclooxygenase 2, rapamycin, and p53. In a previous study, UA ameliorated exercise tolerance and decreased sarcopenia [6]. In another study, UA prevented skeletal muscle atrophy [7]. Furthermore, UA has been reported to exert positive effects on cardiovascular health, suggesting that UA is a beneficial exercise mimetic [8]. UA has also been reported to mediate cardioprotective effects through its membrane-stabilizing action [9]. However, whether UA can regulate and control IR-induced acute damage and its significance in neurodevelopment and cardiomyocyte function remain unknown.

Zebrafish have been widely used in preclinical drug evaluation in vivo due to their high fecundity, high genetic homology with humans (approximately 70%), and well-characterized developmental stages, thereby reducing discrepancies between in vitro and in vivo studies [10, 11]. The physiological parameters and organ systems of zebrafish are similar to those of mammals. This aids in understanding the functions and biological mechanisms of drugs. Potential drugs for treating spinal cord injury in mammals have been identified using a zebrafish model [12]. Zebrafish have also been used to elucidate how sleep pressure precisely modulates the number of neuronal synapses [13]. It has been shown that exposure to isavuconazole may induce neurodevelopmental defects and behavioral disorders in zebrafish larvae [14]. In addition, the effects of Per- and polyfluoroalkyl substances (PFAS) on heart development and function have been demonstrated using a zebrafish model [15]. Furthermore, zebrafish embryos are ideal for radiation research because embryonic cells divide rapidly and are the most sensitive to radiation. Studies have indicated that lower doses of IR have a minimal impact on the overall phenotype of zebrafish embryos [16]. It has been revealed that when the dose exceeded 0.01 and 0.05 Gy, the survival and hatching rates of embryos were significantly reduced. With increasing radiation doses, zebrafish exhibited a more pronounced sensitivity to IR, which manifested as reduced hatchability, shorter body length, higher mortality, and malformations [17]. At radiation doses up to 20 Gy, none of the embryos at 4 h post fertilization (hpf) survived beyond 120 hpf [18]. Thus, zebrafish embryos provide a rapid and simple screening system for radioprotectors or sensitizers. Overall, zebrafish can be used to evaluate drug toxicity, systemic circulation, and therapeutic efficacy and can serve as an effective alternative vertebrate screening model to reduce the number of experiments in higher vertebrates.

In the present study, we investigated the impact of an optimized derivative of UA, namely, UA312, on radiation-induced developmental toxicity in zebrafish embryos and larvae. UA312 demonstrated low toxicity to zebrafish embryos and significantly reversed radiation-induced developmental abnormalities, such as embryonic death, malformation, and shortened body length. In addition, UA312 significantly improved radiation-induced damage to heart development and the nervous system. UA312 mainly reversed the myocardial contraction and nerve signaling pathways inhibited by IR by targeting the upregulation of chrna3 and grik5. Our findings indicate that UA312 may be a promising radioprotector against radiation for human beings and aquatic organisms, providing a new direction for the protection of nuclear pollution and radiological medicine.

Materials and methods

Synthesis, optimization, and physicochemical properties of UA311 and UA312

The UA derivatives, namely UA311 and UA312 compounds, were synthesized using UA (77–52–1, Shanghai Yaxing Biotech Company, Shanghai, China) as the precursor. The synthesis process is presented in brief in Supplementary Materials. The zebrafish model was used to evaluate the efficacy of the UA derivatives. In brief, zebrafish were fertilized to obtain embryos. These embryos were treated with different concentrations of UA, UA311, and UA312 compounds to observe survival and malformation rates. The lead compounds obtained by screening were further studied for their ability to be used as drugs, including structural optimization and preparation process optimization. Based on these data, UA311 and UA312 were finally selected as the most suitable candidates.

Zebrafish feeding and embryo collection

Adult wild AB zebrafish (3–6 months old) and Tg (cmlc2: EGFP, 3–4 months old) were purchased from the National Zebrafish Resource Center (Wuhan, China). Embryos were obtained by mating adult male and female zebrafish. Zebrafish were cultured in a freshwater recirculation system at 28 ± 1 °C (Shanghai Haisheng Biotech Company, Shanghai, China) with a 14-h light/10-h dark cycle. Zebrafish were fed artemia twice a day to ensure spawning. The night before mating, sexually mature male and female zebrafish were separated and housed overnight in a dedicated breeding tank separated by a transparent baffle. The next morning, the baffle was removed, and the light was turned on to induce spawning. The embryos were collected within 15–30 min, washed, selected with standard zebrafish Holt buffer (NaCl 3.5 g/L, KCl 0.05 g/L, CaCl₂ 0.1 g/L, NaHCO₃ 0.025 g/L), and grown or irradiated and treated with drugs. A stereomicroscope (SMZ1270, Nikon, Tokyo, Japan) was used to observe embryonic development and perform embryo counting. The experiments on zebrafish were conducted in compliance with the guidelines of the Animal Care and Use Committee and approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College (Approval ID: IRM2-IACUC-2409-081).

Drug exposure and embryo irradiation

Fertilized embryos were transferred to a 24-well plate. Each well contained 20 embryos and 2 mL of Holt buffer. At 3 hpf, working doses of 0, 10, 20, 50, and 100 μM were administered. At 4 hpf, embryos were irradiated with 6 Gy of γ-irradiation (dose rate: 0.88 Gy/min). The buffer was replaced with fresh buffer and maintained at 28 ± 1 °C with a 14-h light/10-h dark cycle. Holt buffer and drugs were replaced once a day. The experiment was repeated at least three times.

Embryonic development

Embryo and larvae survival were observed at 4, 24, 48, and 72 hpf using a stereomicroscope connected to a camera. Morphological indicators, such as larval mortality, deformity rate, hatching rate, hatching process, body length, and eye periphery, were observed. The membranes of surviving embryos and larvae were transparent and developed a morphology, whereas the membranes of dead embryos and larvae were black and had no morphology. Dead embryos and larvae were removed, and their number was recorded to calculate the survival rate as follows: (total number of embryos and larvae—number of dead embryos and larvae) / total number of embryos and larvae × 100%. Deformity rate was calculated as follows: (total number of embryos and larvae—number of deformed embryos and larvae) / total number of embryos and larvae × 100%. Deformity types were divided into curved tails, pericardial edema, and microphthalmia.

At 72 hpf, 8 embryos and larvae were randomly selected from each group to photograph embryos and larvae head fields of view. The embryonic eye area was measured using DanioScope software. At 72 hpf, 8 embryos and larvae were randomly selected from each group to photograph embryonic whole-body fields. Embryonic body length was measured using DanioScope software. At 72 hpf, 3 embryos and larvae were randomly selected from each group, and the embryonic heartbeat video was recorded for 1 min under a stereomicroscope. Heartbeats were analyzed and counted using DanioScope Heartbeat Detector software (Noldus Information Technology, Wageningen, Netherlands). All measurements were repeated at least three times.

Behavioral analysis

Zebrafish larvae at 120 hpf were collected and pipetted into a 24-well plate with one larva per well, and 10 replicates per group were set up. We added 500 μL of 1× Holt buffer to each well and placed the wells in a collection box. System water was introduced through a water pipe and heated to 28.5 °C. After testing the lighting conditions (light on and off), we waited for collection. The square well where each larva was located was defined as the collection area and analysis area. The collection procedure was as follows: 5 min for adaptation, collection initiated, free swimming for 20 min, and collection stopped. The collection was performed using the behavioral analysis system and EthoVision XT software (DanioVision, Noldus, Netherlands). The swimming trajectory and heat map were exported, and movement parameters, such as total swimming distance, average speed, cumulative movement time, activity frequency, cumulative activity time, and cumulative stop time, were statistically analyzed.

Acridine orange (AO) staining

The drug- and/or IR-treated embryos to be stained were washed with PTU-Holt buffer before 24 hpf to remove the pigment. At 72 hpf, the AO fluorescent probe stock solution (17502, AAT Bioquest, Pleasanton, California, America) was diluted to 1× Holt buffer at a ratio of 1:5000, and 6 larvae per group were exposed to the diluted AO dye solution. The larvae were then stained in an incubator at 37 °C in the dark for 30 min. After staining, they were washed in 1× Holt buffer to remove excess unbound fluorescent probes. The washed larvae were placed in methylcellulose gel and photographed under the green fluorescence channel of a stereo fluorescence microscope. Green, fluorescent bright spots were considered positive signals. Adiphenine hydrochloride (HY-B0379A), UBP 302 (HY-107604), and Kainic acid (HY-N2309) were purchased from Medchem Express (Shanghai, China). Nicotine (612057) was purchased from Sigma-Aldrich (Shanghai, China).

O-Dianisidine staining

Drug- and/or IR-treated larvae (6 embryos per well, 18 embryos per group) were transferred to new tubes and stained with o-dianisidine (D9143-5G, Sigma-Aldrich, Shanghai, China). After staining for 30 min in the dark, the larvae were washed three times with PBST. Then, the stained larvae were fixed overnight in 4% PFA (P6148, Sigma-Aldrich, Shanghai, China) and stored in 80% glycerol for imaging under a confocal microscope (7CSSP8, Leica, Mannheim, Baden-Württemberg, Germany).

Transcriptomic analysis

Total RNA was extracted from the collected larvae (200 larvae per group, n = 3) with TRIzol reagent and quantified using NanoDrop (ND-1000, Wilmington, Delaware, USA). RNA quality was evaluated using Bioanalyzer 2100, and RNA samples with RIN values of >7.0 were selected. RNA samples were used to construct sequencing libraries for transcriptome sequencing. The sequencing was performed on Illumina NovaSeq 6000 (Hangzhou Lianchuang Biotechnology Co., Ltd., Hangzhou, China). DESeq2 (https://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) was used to analyze significantly differentially expressed genes with the following parameters: P < 0.05 and absolute fold-change ≥2. Enrichment analysis was performed using GO (https://geneontology.org/), KEGG (https://www.kegg.jp/), and GSEA (https://www.gsea-msigdb.org/gsea/index.jsp) pathway enrichment analyses.

Enzyme-linked immunosorbent assay (ELISA)

At 72 hpf, 4 replicate samples of 30 zebrafish larvae were randomly selected to measure the levels of the neurotransmitters 5-hydroxytryptamine (5-HT, ml874512), dopamine (DA, ml993244), and gamma-aminobutyric acid (GABA, ml325412). The larvae were mixed with phosphate-buffered saline (PBS, pH 7.4) in a homogenizer, crushed, and centrifuged to obtain the supernatant. The expression levels of 5-HT, DA, and GABA were measured according to the instructions of the ELISA kit (Enzyme-linked Biotechnology, Shanghai, China).

Western blotting

The total proteins obtained from zebrafish larvae at 72 hpf were ground and tracked using RIPA lysis buffer. The lysates were centrifuged at 12,000 rpm at 4 °C for 15 min to collect the supernatant. The bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to quantify the protein concentration. Then, the samples were loaded and separated on 12% SDS-PAGE gels and transferred onto PVDF membranes. These membranes were blocked with 5% skim milk for 60 min at room temperature and then incubated with primary antibodies (grik5 Cat# 28550-1-AP-50, chrna3 Cat# 10333-1-AP-50, and β-actin Cat# 66009-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight and with the HRP-coupled secondary antibody for over 2 h at room temperature. The ECL Western Blotting Substrate kit (Beyotime Biotechnology, Shanghai, China) was used to detect the signals.

Molecular docking

Molecular docking was conducted using Schrödinger software. First, we minimized the energy of the ligand, set the charge, added polar and nonpolar hydrogens, and assigned rotatable chemical bonds using the LigPrep module. Second, the crystal structure of the protein (PDB code: 3OM1, 4ZK4, 6DLS, 8OP9, 8PVB, 7QN5) was downloaded from the Protein Data Bank (https://www.rcsb.org/). Subsequently, the protein was hydrogenated, solvent was removed, and bond level was specified using Protein Preparation Wizard. The structure was used to evaluate possible binding sites using the SiteMap module. A grid file was designed with the optimal site as the center, with a box size of 15 Å. The generated receptor grid file and the ligand structure were docked using the docking module. The results were analyzed using Pymol.

Molecular dynamics (MD) simulation

Following molecular docking, the stability of the selected docking model was validated through MD simulations using the Amber22 and AmberTools22 software packages. The ff14SB force field and TIP3P water model were employed for protein systems, whereas the GAFF force field was utilized for small molecules. Ligands were prepared using the antechamber module, and proteins were preprocessed using the Tleap module. Following energy minimization, the system was heated from 0 K to 300 K over a period of 30 ps. The system was equilibrated sequentially under NVT and NPT ensembles at 300 K. MD simulations were conducted for 50 ns under constant temperature and pressure conditions, with periodic boundary conditions applied. The root mean square deviation (RMSD) was computed to analyze the molecular dynamics trajectories, and the results were visualized using QtGrace.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

The total RNA of zebrafish larvae was extracted using TRIzol reagent (Invitrogen, CA, USA) and reversed into cDNA using a reverse transcription kit (Invitrogen, CA, USA) according to the manufacturer’s instructions. Subsequently, qRT-PCR was performed using SYBR^® Green Master Mix (TIANGEN Biotechnology, Beijing, China), with three replicates for each sample (50 larvae). The expression level of the target gene was determined by normalizing its reaction threshold cycle (Ct) value to that of the housekeeping gene GAPDH. mRNA fold changes in the target gene relative to GAPDH were assessed using the 2^−△△Ct method. Experiments were conducted in triplicate. The primer sequences are listed in the Supplementary Table S2.

Mouse experiments

All experimental procedures were approved by the Animal Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College (approval number: IRM2–IACUC–2502–010). SPF-grade male C57BL/6 mice (6–8 weeks old) were obtained from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and housed in the SPF Laboratory Animal Center of the Institute of Radiation Medicine (IRM), Chinese Academy of Medical Sciences. All animal experimental protocols were performed in compliance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, mice were maintained in a facility with a 12–h light cycle (06:00–18:00), temperature controlled at 20–22 °C, and humidity maintained at 50%. Mice were randomly assigned to four groups (n = 5 per group): saline control; IR (9 Gy); UA312 (80 mg/kg); and IR (9 Gy) + UA312 (80 mg/kg). Mice received UA312 or saline via oral gavage. Seven days later, the irradiation group was subjected to a dose (9 Gy) of ¹³⁷Cs γ-irradiation, and drug administration continued for an additional 3 days post–irradiation. All irradiation procedures were conducted using a ¹³⁷Cs γ–ray irradiator (Gammacell–40, Atomic Energy of Canada, Ontario, Canada) at a dose rate of 0.88 Gy/min.

Locomotor activity

The locomotor ability of mice was assessed in an open–field apparatus. Specifically, each mouse was placed in a plastic chamber (50 cm × 50 cm × 50 cm) divided into central and peripheral fields and allowed to explore freely for 5 min. Tests were conducted in a soundproof room, and mouse movements were tracked using a video tracking system (Tracking Master V4.10 software, Zhongshi Co., Ltd., Beijing, China) positioned above the chamber.

Statistical analysis

Shapiro-Wilk and Levene’s tests were used to analyze the normal distribution and homogeneity of the data, respectively. The significance for multiple groups was calculated using one-way analysis of variance and Tukey’s test. The values were analyzed in SPSS 22.0 (SPAA, Chicago, USA) and GraphPad Prism 5 for Windows (GraphPad Software, CA, America). The 4-parameter logistic curve fitting was used to analyze ELISA data in ELISAcalc. All experiments were separately performed at least three times, and data were presented as mean ± standard deviation (SD). A P value of <0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and not significant [NS]).

Results

UA312 is safe and less toxic for zebrafish embryos and larvae

We screened UA311 and UA312 which were prepared through the structural modification of the lead compound UA (Fig. 1a, Supplementary Fig. S1a). To test the toxic effects of UA, UA311, and UA312 on the early development of zebrafish embryos and larvae, we treated embryos with different concentrations of UA, UA311, and UA312 at 3, 24, 48, and 72 hpf (Fig. 1b). UA (10 µM) caused obvious toxic effects and malformation in zebrafish embryos and larvae (Fig. 1c, d). We further tested the effect of various concentrations of UA311 on zebrafish. The findings revealed that UA311 inhibited developmental processes and induced different degrees of deformities in zebrafish embryos and larvae (Fig. 1e, f). By contrast, 0–50 µM UA312 had few toxic effects (Fig. 1g, h). To further determine the safest concentration of UA312 in zebrafish, the embryos and larvae were subsequently treated with 0, 30, 40, and 50 µM of UA312. The results demonstrated that the hatchability of zebrafish embryos and larvae was inhibited by approximately 44% (P = 0.0006) and 50% (P = 0.0002) at concentrations of 30 µM and 50 µM, respectively, whereas at a concentration of 40 µM, this hatchability was reduced by 22% (P = 0.0760, Fig. 1i, j). Concurrently, at a concentration of 50 µM, the survival rate of zebrafish was significantly decreased, and the malformation rate was markedly elevated. In contrast, the concentrations of 30 µM and 40 µM had no significant impact on either the survival or malformation rates (Fig. 1k, Supplementary Fig. S1b). Therefore, after a comprehensive evaluation, we selected the concentration of 40 µM as the optimal dose for subsequent studies, given its minimal impact on the survival rate, malformation rate, and hatching rate. Overall, these data provide compelling evidence that UA312 (40 µM) is the safest concentration for zebrafish embryos and larvae.

Fig. 1. UA312 is safe and less toxic for zebrafish embryos and larvae.

a Chemical structure of ursolic acid (UA) and its derivatives UA311 and UA312. b Flow diagram of drug screening for zebrafish developmental toxicity. Zebrafish were mated to generate embryos, then 20 embryos were collected and arrayed into 24-well plates each well. The compounds of UA, UA311, and UA312 dissolved to 0, 10, 20, 50, and 100 μM in DMSO were added to each well, respectively. Screening was performed by monitoring the survival and malformation rate at 3, 24, 48, and 72 h post-fertilization (hpf). The survival (c) and malformation (d) rate of zebrafish treated with UA (0, 10, 20, 50, and 100 μM) at 3, 24, 48, and 72 hpf (n = 3/group, each replicate comprising 20 larvae). The survival (e) and malformation (f) rate of zebrafish treated with UA311 (0, 10, 20, 50, and 100 μM) at 3, 24, 48, and 72 hpf (n = 3/group, each replicate comprising 20 larvae). The survival (g) and malformation (h) rate of zebrafish treated with UA312 (0, 10, 20, 50, and 100 μM) at 3, 24, 48, and 72 hpf (n = 3/group, each replicate comprising 20 larvae). i Representative images of zebrafish exposed to UA312 (0, 30, 40, and 50 μM) from 3 to 72 hpf. j The hatchability rate of zebrafish treated with UA312 (0, 30, 40, and 50 μM) at 72 hpf (n = 4/group, each replicate comprising 20 larvae). k The survival rate of zebrafish treated with UA312 (0, 30, 40, and 50 μM) at 3, 24, 48, and 72 hpf (n = 4/group, each replicate comprising 20 larvae). Data are displayed as the mean ± SD. *P < 0.05, ***P < 0.001.

UA312 significantly improved IR-induced developmental toxicity in zebrafish embryos and larvae

Zebrafish, as a model organism with a highly similar genome to that of humans, is an effective model for drug evaluation [19, 20]. In zebrafish, IR aggravated radiation-induced early dysplasia, especially under the 6 Gy γ-irradiation explosion [21]. Therefore, we evaluated the effect of UA312 on IR-induced developmental toxicity under 6 Gy γ-irradiation at 3, 24, 48, and 72 hpf (Fig. 2a). Embryos and larvae exposed to IR exhibited severe injury in a time-dependent manner, and UA312 rescued the injury (Fig. 2b, c). UA312 significantly increased the survival rate of embryos and larvae from 3 hpf to 72 hpf (Fig. 2d). The survival rate inhibited by IR at 72 hpf was recovered by UA312, as observed under a stereomicroscope (Fig. 2e). After 6 Gy radiation exposure, the body length of larvae was shortened by approximately 79.8%. UA312 rescued the inhibition by approximately 89.6% (Fig. 2f–h). Notably, the above phenomenon was consistently by the atrophied eye size at 72 hpf, in detail, the relative inhibition rates were 76.5% (IR) and 87.6% (IR + UA312) for eye size (Fig. 2i–k). Consistently, UA312 recovered the pericardial area induced by IR exposure (Fig. 2l). Upon exposure to IR, a variety of ectopic morphological patterns were induced, with the incidence rates being 30% for curved tails, 26.25% for pericardial edema, 23.75% for microphthalmia, and 8.75% for other deformities. Treatment with UA312 significantly reduced the prevalence of most malformations, with the rates decreasing to 17.5% for curved tails, 10% for pericardial edema, 8.75% for microphthalmia, and 12.5% for other deformities (Fig. 2m). These results support the hypothesis that UA312 significantly ameliorates the effects of IR in zebrafish embryos and larvae.

Fig. 2. UA312 significantly improved IR-induced developmental toxicity in zebrafish embryos and larvae.

a Flow diagram of the experimental design in the zebrafish for drug evaluation. Zebrafish were mated to generate embryos, then embryos were collected and arrayed into 24-well plates each well. The UA312 compound dissolved to 40 μM in DMSO was added to each well and exposed to 6 Gy γ-irradiation. Evaluation was performed by monitoring the survival rate, malformation rate, body length, eye perimeter, pericardial area, heart rate, and ethology at 3, 24, 48, and 72 hpf. b Representative images of zebrafish exposed to UA312 (40 μM) and/or IR (6 Gy) from 3 to 72 hpf. The survival (c) and malformation (d) rate of zebrafish treated with UA312 (40 μM) and/or IR (6 Gy) at 3, 24, 48, and 72 hpf (n = 4/group, each replicate comprising 20 larvae). e The survival rate of zebrafish at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 80). f The representative images of the body length of zebrafish at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 8). g Quantification of the body length of zebrafish at 72 hpf (n = 8). h Inhibition rate of body length for zebrafish at 72 hpf (n = 8). i The representative images of the eye perimeter and pericardial area of zebrafish at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 8). j Quantification of the eye perimeter of zebrafish (n = 8). k Inhibition rate of eye perimeter for zebrafish at 72 hpf (n = 8). l Quantification of the pericardial area of zebrafish at 72 hpf (n = 8). m The rate of different malformation patterns, including normal embryo, curved tail, pericardial edema, microphthalmia, and other kinds of deformities (n = 4/group, each replicate comprising 20 larvae). Data are displayed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Transcriptome analysis predicted that UA312 mediates cardiac protection and neuroprotection after IR damage

UA312 significantly restored IR-induced developmental toxicity in zebrafish larvae. We used RNA sequencing to explore the underlying mechanisms. The genes were distinguishable between the IR and control groups as well as between the IR + UA312 and IR groups (Fig. 3a, b). Compared with those in the ctrl group, 4229 mRNAs, including 2734 upregulated and 1495 downregulated, were significantly differentially expressed in the IR group. Compared with those in the IR group, 5162 aberrantly expressed genes, including 2296 downregulated and 2866 upregulated, were identified in the IR + UA312 group (P < 0.05 and fold-change ≥5 or ≤0.2) (Fig. 3c). Surprisingly, the downregulated genes in the IR or ctrl group were significantly enriched in neuroactive ligand-receptor interactions, adrenergic signaling in cardiomyocytes, and cardiac muscle contraction pathways (Fig. 3d). Consistently, neuroactive ligand-receptor interactions, adrenergic signaling in cardiomyocytes, phototransduction, and cardiac muscle contraction pathways were reversed by UA312 (Fig. 3e). We further investigated the core genes between the IR and ctrl groups using GSEA. The results showed that neurotransmitter receptor activity, neurotransmitter transport, and heart contraction pathways were inhibited in the IR group (Fig. 3f). UA312 treatment enriched these pathways (Fig. 3g). These data indicate that UA312 protects against IR-induced damage by increasing the activation of cardiac and neuro-related signaling pathways. Next, to analyze IR-regulated genes that are inhibited by UA312, a Venn diagram was constructed. In total, 2670 differentially expressed genes were shared among the three groups, from which we selected ccka, grik5, and grik2 to confirm the results (Fig. 3h, i). Overall, we investigated the effect of UA312 on IR-induced signaling pathways and found that in zebrafish embryos and larvae, UA312 significantly reversed the effects of IR-induced damage to the cardiac and neurological systems.

Fig. 3. Transcriptome analysis predicted that UA312 mediates cardiac protection and neuroprotection after IR damage.

a Volcano plot of DEGs after 6 Gy γ-irradiation exposure. The red color represents the up-regulated genes in the IR group relative to the Ctrl. The blue color represents the down-regulated genes in the IR group relative to Ctrl. b Volcano plot of DEGs after 6 Gy γ-irradiation exposure and/or UA312 (40 μM) treatment. The red color represents the up-regulated genes in the IR + UA312 group relative to the IR. The blue color represents the down-regulated genes in the IR + UA312 group relative to the IR. c The statistics of DEGs between the IR and Ctrl group, as well as the IR + UA312 group relative to the IR. d The top 12 of KEGG enrichment analysis from the down-DEGs in the IR group relative to the Ctrl by RNA-sequencing analysis. e The top 12 of KEGG enrichment analysis from the up-DEGs in the IR + UA312 group relative to the IR group by RNA-sequencing analysis. f GSEA analysis for KEGG neurotransmitter receptor activity, neurotransmitter transport, and heart contraction signaling pathway in the IR group compared with Ctrl. g GSEA analysis for KEGG neurotransmitter receptor activity, neurotransmitter transport, and heart contraction signaling pathway in the IR + UA312 group relative to the IR group. h Venn diagram of DEGs among Ctrl, IR, and IR + UA312 group. i Relative mRNA expression of ccka, grik5, and grik2 in Ctrl, UA312, IR, and IR + UA312 groups detected by qRT-PCR assay (200 larvae per group, n = 3). Data are displayed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

UA312 alleviated IR-induced cardiac damage in zebrafish

The results of the transcriptome analysis revealed that UA312 has potential cardiac-protective roles after IR-induced damage. The pericardial area of zebrafish affected by IR at 72 hpf was recovered by UA312. Furthermore, UA312 treatment restored the heart rate of zebrafish larvae that was significantly reduced after IR treatment (Fig. 4a). Heart rate, preload, afterload, and contractility are the major factors that regulate pump performance, which represents cardiac function [22]. Cardiac function is usually examined by assessing heart rate and contractility [23]. Therefore, these results indicated that UA312 affects the radiation-inhibited zebrafish embryonic heart. Similarly, o-dianisidine staining of larvae showed that red blood cells accumulated in the blood vessel in the tail region (marked by black arrow) after exposure to IR, indicating a significant increase in the formation of tail venous thrombus. After UA312 treatment, the thrombus almost disappeared (Fig. 4b, c). Subsequently, we employed the heart-specific expression of green-fluorescent transgenic Tg (cmlc2: EGFP) zebrafish to assess heart-related indicators across different groups. The cmlc2 (myl7) gene, highly expressed in cardiac myocytes encircling both the atria and ventricles, serves as a marker for observing morphological heart development [24, 25]. Upon side dissection of Tg (cmlc2: EGFP) zebrafish, we observed that, compared to the control group, IR-exposed zebrafish showed reduced overlap between the atria and ventricles, as well as an increased sinus-venosus and bulbus-arteriosus (SV-BA) distance. Furthermore, from ventral anatomical observations, IR induced abnormalities and asymmetries in the morphology and structure of the atrium (A) and ventricle (V), which were significantly ameliorated following UA312 treatment (Fig. 4d). Consequently, the results indicated that IR possesses significant cardiotoxic effects on zebrafish. The administration of UA312 notably reversed the enlarged cardiac morphology and abnormal atrioventricular structure. Additionally, we observed the effect of IR on apoptosis in embryos exposed to 72 hpf through AO staining. The results revealed pronounced apoptosis, characterized by green fluorescence, within the cardiac region of the IR group. In contrast, UA312 treatment significantly inhibited the formation of apoptotic cells induced by IR, implying that UA312 may ameliorate radiation-induced cardiomyocyte apoptosis (Fig. 4e, Supplementary Fig. S2). Observing abnormal cardiac development in IR-treated zebrafish embryos prompted us to investigate the expression levels of key genes implicated in heart development. The expression of nkx2.5, nppa, vmhc, myh6, and tbx5α was significantly upregulated after IR treatment, whereas the expression of tbx2 was significantly downregulated. Compared with those in the IR group, the expression levels of nkx2.5, nppa, vmhc, myh6, and tbx5α were markedly reduced in the UA312 + IR group, and there was a trend of reduced tbx2 expression after UA312 + IR exposure (Fig. 4f). Overall, these results suggest that in zebrafish, IR may cause dysregulation of the expression of genes related to cardiac development, and UA312 can reverse IR-induced cardiac damage.

Fig. 4. UA312 alleviated IR-induced cardiac damage in zebrafish.

a Heart rate of zebrafish at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 3). b Respective images of o-dianisidine staining of zebrafish at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 3/group, each replicate comprising 6 larvae). c Erythrocyte staining intensity of zebrafish stained by o-dianisidine in the Ctrl, UA312, IR, and IR + UA312 groups (n = 3/group, each replicate comprising 6 larvae). d The cardiac morphology of zebrafish larvae at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups. Pericardial edema changes (bright field), sinus-venosus and bulbus-arteriosus (SV-BA) distance (fluorescent, lateral view), cardiac circuit integrity and symmetry (fluorescent, ventral view). The red line represents the SV-BA distance. The green arrows represent the atrium (A) and ventricle (V). Scale bar = 100 μm, n = 8. e Acridine orange (AO) staining of zebrafish larvae at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups. The green arrow represents the apoptosis cells. Scale bar = 100 μm, n = 6. f Relative mRNA expression of nkx2.5, nppa, vmhc, myh6, tbx5α, and tbx2 at 72 hpf in Ctrl, UA312, IR, and IR + UA312 groups detected by qRT-PCR assay (n = 3/group, each replicate comprising 20 larvae). Data are displayed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

UA312 altered IR-reduced motor behavior in zebrafish larvae

Transcriptome analysis indicated that UA312 has effects on the neuroactive ligand-receptor interaction in IR-damaged zebrafish, which refers to the binding of ligands (such as neurotransmitters and hormones) to receptors, triggering intracellular signaling processes. Therefore, we first explored the effects of IR and UA312 on neurotransmitters in zebrafish and found that IR significantly inhibited 5-hydroxytryptamine (5-HT) levels (Fig. 5a), whereas the UA312 treatment significantly improved 5-HT levels. Similarly, dopamine (DA), and gamma-aminobutyric acid (GABA) levels were significantly reduced after IR exposure and increased under the UA312 treatment (Fig. 5b, c). Considering that behavior is governed by the nervous system, we then explored the influence of UA312 and IR on the motor behavior of zebrafish larvae. A photoperiod stimulation experiment was performed to monitor the behavioral traces of zebrafish larvae at 120 hpf. IR attenuated locomotor behavior. The larvae resumed active behavior in the UA312 + IR group compared with the IR group (Fig. 5d, Supplementary Fig. S3a). Total distance, total velocity, active frequency, and total movement times decreased by >50% after IR exposure, and UA312 + IR rescued these values (Fig. 5e–h). IR stimulation decreased total swimming distance compared with the ctrl group during light-dark transition stimulation, which was restored by UA312 (Fig. 5i). We hypothesize that UA312 promotes locomotory behavior role by regulating the brain. To further support this idea, we used AO staining to assess the apoptosis level in the brain. The level of apoptosis was upregulated in the IR group, and the number of AO-positive cells in the UA312 + IR group significantly decreased (Fig. 5j, Supplementary Fig. S3b). We then detected genes associated with developmental neurotoxicity, including shha, gap43, elav13, gfap, and sox2. We found that the levels of these genes decreased after IR exposure, whereas UA312 increased their expression (Fig. 5k). These data strongly corroborate the notion that UA312 ameliorates the IR-induced decrease in locomotor activity in zebrafish larvae through neuroprotection.

Fig. 5. UA312 altered IR-reduced motor behavior in zebrafish larvae.

The neurotransmitter levels of 5-HT (a), DA (b), and GABA (c) of zebrafish larvae at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 3/group, each replicate comprising 40 larvae). d The larval motion trials of larvae at 120 hpf in Ctrl, UA312, IR, and IR + UA312 groups within ~1 min (n = 10). The total movement distance (e), total movement velocity (f), active frequency (g), and total movement time (h) in Ctrl, UA312, IR, and IR + UA312 groups were computed (n = 10). i light-dark photoperiod stimulation test (70 min) for zebrafish larvae at 120 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 10). j AO staining of zebrafish larvae at 72 hpf in the Ctrl, UA312, IR, and IR + UA312 groups (n = 6). k Relative mRNA expression of shha, gap43, elav13, gfap, and sox2 in Ctrl, UA312, IR, and IR + UA312 groups detected by qRT-PCR assay (n = 3/group, each replicate comprising 20 larvae). Data are displayed as the mean ± SD. *P < 0.05, ***P < 0.001.

UA312 improved the cardiac protection and neuroprotection function of IR damage by targeting chrna3 and grik5

To elucidate the targets through which U312 provides radioprotection to zebrafish, we further analyzed the top 30 regulated genes involved in neuroactive ligand-receptor interactions, adrenergic signaling in cardiomyocytes, and cardiac muscle contraction pathways. PPI network analysis was performed using the STRING database (https://cn.string-db.org/) to construct a PPI network and perform module analysis on differentially expressed genes. Interaction between these differentially expressed genes and several central genes was important for the network (chrna2, gabrd, chrna5, chrna6, gabra1, gabrr1, chrna3, grik5, and gabrd3) (Fig. 6a). To further screen the core genes, we performed qRT-PCR. The results indicated that chrna3 and grik5 were the most significantly upregulated genes by UA312 after IR-induced decrease (Fig. 6b). Subsequently, we obtained the crystal structures of gabrd (PDB ID: 7QN5), gabra1 (PDB ID: 6D1S), gabrr1 (PDB ID: 8OP9), gabrb3 (PDB ID: 8PVB), chrna3 (PDB ID: 4ZK4), and grik5 (PDB ID: 3OM1) from the RCSB database. However, we could not find crystal structures for chrna2, chrna5 and chrna6. The predominant binding conformations of UA312 with gabra1, gabrr1, gabrd3, chrna3, and grik5 were determined using the induced-fit docking approach. This method also validated that chrna3 and grik5 exhibited the highest docking scores with UA312 (Supplementary Table S1). In detail, chrna3 consists of five subunits, and UA312 (pink) was located in one deep groove at the interface of adjacent subunits. Although no additional interaction was observed, the complex of UA312/chrna3 had a relatively high docking score of -4.562. One possible reason was that the hydrophobic rigid backbone of UA312 perfectly fit the groove, which consisted of hydrophobic amino acid residues, such as tryptophan, isoleucine, and tyrosine (Fig. 6c, Supplementary Fig. S4a). To investigate the stability and dynamic interactions of the protein-ligand complex, molecular dynamics (MD) simulations were employed to validate the docking results and elucidate the molecular motion of the chrna3 upon binding to UA312. The docking complex was subjected to MD simulations for 50 ns, during which the temperature, kinetic energy, and potential energy were maintained at stable levels, while the root mean square deviation (RMSD) exhibited minor fluctuations, thereby confirming the reliability of the docking conformation (Fig. 6d–f). Meanwhile, the docking score of the docking complex of UA312/grik5 was -4.488. UA312 (pink) was located in the hydrophobic cleft of grik5 (light blue), and the carboxyl group of UA312 was embedded in the basic hydrophobic bottom formed by Pro81, Lys148, Arg154, Ala199, and Asn200. One hydrogen bond and salt bridge were observed between the carboxyl group of UA312, the ε-amino of Lys148, and the guanidyl group of Arg154, stabilizing the bonding complex. Although the A ring of UA312 was exposed to the solvent, the additional arene-H stacking interaction between 4-fluorobenzene and Asp176 stabilized the complex. In addition, no covalent bond was found in the docking complexes, even with the reactive michael acceptor of 2-benzylidene-1-one in the structure of UA312 (Fig. 6g, Supplementary Fig. S4b). Consistent with the aforementioned findings, MD simulations further corroborated the stability of UA312 binding to the grik5 target (Fig. 6h-j). Subsequently, we aimed to validate the functions of chrna3 and grik5. Initially, we assessed the efficacy of Adiphenine hydrochloride (AH) [26] and UBP 302 [27]in inhibiting chrna3 and grik5, confirming their significant inhibitory effects (Supplementary Fig. S4c). Then, we investigated the cardioprotective effects of these two targets on UA312 in transgenic Tg (cmlc2: EGFP) zebrafish. Following treatment with AH or UBP 302 post-UA312 and IR exposure, the zebrafish hearts consistently showed signs such as an increased SV-BA distance, and abnormalities and asymmetries in the morphology and structure of the A and V, as observed from lateral and ventral views, respectively (Fig. 6k). Meanwhile, the inhibition of chrna3 and grik5 resulted in a reduction of cardiac cell apoptosis induced by IR + UA312, as evidenced by AO staining (Fig. 6l, Supplementary Fig. S4d). Additionally, the levels of neurotransmitters including 5-HT, DA, and GABA were suppressed by AH and UBP 302 following IR + UA312 treatment, as determined by ELISA, confirming the important role of chrna3 and grik5 in the neuroprotection function of UA312 (Fig. 6m).

Fig. 6. UA312 improved the cardiac protection and neuroprotection function of IR damage by targeting chrna3 and grik5.

a STRING analysis of different expressed genes. The nodes represent proteins, and each edge represents the interaction between proteins. The thicker the line, the greater the correlation. b Relative mRNA expression of chrna2, gabrd, chrna5, chrna6, gabra1, gabrr1, chrna3, grik5, and gabrd3 genes in Ctrl, UA312, IR, and IR + UA312 groups detected by qRT-PCR assay (n = 3/group, each replicate comprising 20 larvae). c Molecular docking of UA312 and chrna3 protein. d Total, kinetic, and potential energy variations in the MDs. e Temperature changes in the MDs. f RMSD plot of UA312 complexed with chrna3 during 50 ns. g Molecular docking of UA312 and grik5 protein. h Total, kinetic, and potential energy variations in the MDs. i Temperature changes in the MDs. j RMSD plot of UA312 complexed with grik5 during 50 ns. k The cardiac morphology of zebrafish larvae at 72 hpf in the IR, IR + UA312, IR + UA312 + AH (0.5 μg/L), and IR + UA312 + UBP 302 (10 μM) groups. Adiphenine hydrochloride (AH), the inhibitor of chrna3. UBP 302, the inhibitor of grik5. Pericardial edema changes (bright field), SV-BA distance (fluorescent, lateral view), cardiac circuit integrity, and symmetry (fluorescent, ventral view). The red line represents the SV-BA distance. The green arrows represent A and V. Scale bar = 100 μm, n = 8. l AO staining of zebrafish larvae at 72 hpf in the IR, IR + UA312, IR + UA312 + AH (0.5 μg/L), and IR + UA312 + UBP 302 (10 μM) groups. The green arrow represents the apoptosis cells. Scale bar = 100 μm, n = 6. m The neurotransmitter levels of 5-HT, DA, and GABA of zebrafish larvae at 72 hpf in the IR, IR + UA312, IR + UA312 + AH (0.5 μg/L), and IR + UA312 + UBP 302 (10 μM) groups (n = 4/group, each replicate comprising 30 larvae). Data are displayed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

To further elucidate the role of the targets, we intended to stimulate zebrafish treated with IR + UA312 by using activators of chrna3 and grik5. However, there are currently no well-established and widely recognized grik5 and chrna3 activators. Kainic acid (KA), employed in some studies, may exert an activating effect on grik5 expression [28]. Subsequently, we utilized qRT-PCR to assess the mRNA levels of the grik5 following KA treatment, revealing that KA treatment significantly upregulated grik5 expression following irradiation (Supplementary Fig. S4e). Chrna3 is a member of the nicotinic acetylcholine receptor (nAChR) family. Cytisine, a plant alkaloid derived from the seeds of laburnum anagyroides and other fabaceae plants, exhibits a high binding affinity for nAChRs [29, 30]. It acts as a partial agonist and can activate these receptors to alleviate withdrawal symptoms. However, there is currently no evidence regarding the regulatory effects of cytisine on chrna3. Therefore, we examined the effect of cytisine on chrna3 expression in zebrafish and found that cytisine can also upregulate the IR-induced chrna3 mRNA level (Supplementary Fig. S4e). As anticipated, AO staining revealed that the activation of grik5 and chrna3 significantly enhanced the radioprotective effect of UA312, as evidenced by a reduction in the number of apoptotic cells in the heart and brain (Supplementary Fig. S4f, g). Collectively, these results indicate that chrna3 and grik5 are important targets of UA312 for its radioprotective effect in zebrafish.

Motivated by our findings in zebrafish, we sought to explore the effects of UA312 on irradiated mammals. To this end, we employed the open field test to evaluate spontaneous activity in mice (Fig. 7a). Notably, irradiated mice exhibited diminished activity and attenuated excitability, as evidenced by reduced running distance, average speed, and frequency of center entries. However, administration of UA312 to irradiated mice restored this abnormal activity, mirroring the effects observed in zebrafish (Fig. 7b-d). Collectively, all these data suggest that UA312 has a potential radioprotective effect not only in lower vertebrates but also in mammals.

Fig. 7. UA312 improved the cardiac protection and neuroprotection function of IR damage in mice.

a Diagram and movement trajectory diagram of mouse movement monitored in an open field. b Total movement distance in Ctrl, UA312, IR, and IR + UA312 groups was computed (n = 5). c Average speed in Ctrl, UA312, IR, and IR + UA312 groups was computed (n = 5). d Number of entries into the central area in Ctrl, UA312, IR, and IR + UA312 groups was computed (n = 5). Data are displayed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

Nuclear energy is clean, affordable, and efficient, thereby having an economic value. However, its production results in a large amount of liquid effluent being discharged into the ocean, negatively affecting the environment and humans. In this regard, it should be noted that the release of nuclear wastewater from the Fukushima power plant into the sea in 2011 has increased the impact of radiation on the marine environment. Therefore, the Chernobyl disaster, environmental radiation pollution, nuclear power plant leaks, and the use of radioactive drugs have gradually increased concerns about IR and intensified the demand for radioprotective drugs [31–33]. Developing effective radioprotectors and applying them to radiotherapy and accidental nuclear exposure are essential. At present, amifostine is the most effective radioprotector. However, it can cause serious side effects, such as vomiting, diarrhea, and neurotoxicity [34, 35]. Therefore, developing new, effective, and nontoxic radiation protection drugs is important for clinical radiotherapy or prevention and treatment of IR damage caused by accidents. Here, we report that a UA derivative, UA312, improved IR-induced developmental toxicity by regulating cardiac function and neurodevelopment with low toxicity in zebrafish and mice. Most importantly, UA312 can be administered orally, thereby addressing the issues with amifostine in multiple ways. Therefore, UA312 can serve as a potential radiation protection drug for marine organisms. However, further research is needed to determine whether UA312 can also exert radioprotective effects in rats and humans, as well as elucidate its specific mechanism of action.

Recently, natural products have received widespread attention for the research and application of antiradiation therapeutics. These products have many advantages, such as low toxicity and wide availability [36, 37]. UA is an organic compound widely found in fruits and plants, such as bearberries, apples, and pears. Moreover, it has various biological activities, including anti-inflammatory, antioxidant, and antitumor effects. Several research and clinical studies have pointed out that UA has a potential medicinal value [38–40]. However, in this study, we found that UA was mildly toxic to zebrafish. Using UA as the substrate, two new derivatives, UA311 and UA312, were synthesized through a series of reactions and the introduction of multiple pharmacophores. UA312 was identified as a less toxic compound.

As an aquatic model organism, zebrafish exhibit high homology with the human genome. The majority of DNA repair genes are conserved in zebrafish [18], and their high sensitivity to environmental changes renders them excellent indicators for environmental monitoring. Consequently, employing zebrafish to investigate the effects of radiation on organisms is gaining popularity. Currently, research on zebrafish radioactivity primarily concentrates on the uptake and excretion of radioactive nuclides, the impact of gamma rays, tritium, and X-rays on the growth and development of zebrafish embryos, as well as the study of zebrafish in radiation protection and radiosensitization. During the early stages of embryonic development (approximately 24 hpf), radiation toxicity is positively correlated with the radiation dose at levels ≥ 1 Gy [41–43]. Following exposure to gamma radiation (0, 1, 2, 5, 10 Gy) from a Co⁶⁰ source at 26 hpf, zebrafish embryos exhibited a decrease in mortality and hatching rates with increasing doses [44]. Nonetheless, at a 10 Gy dose, there was a significant reduction in the body length, head length, and eye diameter of the juvenile fish [45]. Therefore, zebrafish embryos are highly sensitive to infrared radiation and have been used to evaluate various types of radiation that have adverse effects on many tissue and organ features.

The heart is the first organ to form during vertebrate development and is also the first organ to mature in structure and function during zebrafish embryonic development, which is an important stage in zebrafish early embryonic development [46, 47]. The hemostasis system in zebrafish is similar to that in humans. Zebrafish have coagulation factors and platelet receptors and respond well to clinically used anticoagulant and antithrombotic drugs, making them suitable for the evaluation of thrombosis mechanisms and therapeutic drugs [48, 49]. Therefore, zebrafish are recognized as a suitable model for studying human hemostasis and thrombosis. In hematology research, zebrafish are also widely used in thrombosis research and antithrombotic drug screening. Advances in transcriptome sequencing have provided a new platform for exploring the molecular mechanism, using which we concluded that UA312 may play a radioprotective role by regulating neuroactive ligand-receptor interactions, adrenergic signaling in cardiomyocytes, and cardiac contraction pathways in zebrafish. Further experiments proved that in zebrafish, UA312 has a good recovery effect on the IR-induced reduction in heart rate and increase in thrombus formation, suggesting that UA312 can be used to protect against radiation by regulating myocardial cells.

Zebrafish is a new model animal for studying neurobehavior. The results of the present study showed that the nervous system of zebrafish was damaged following exposure to IR. This damage manifested as a significant attenuation of neurotransmitter expression, total movement distance, cumulative movement duration of the larvae, a decrease in activity frequency, and a shortening of the cumulative active time. The results of AO staining of the brain showed an increase in the number of apoptotic cells in the brain after irradiation. However, UA312 treatment significantly reversed the IR-induced damage to the nervous system. In addition, our previous research results showed that under IR, Xenopus laevis showed behavioral abnormalities and slower activity, indicating that irradiation has a strong neural damage effect on the embryo, which is consistent with our results [50]. Importantly, in terms of radiation protection, amifostine cannot penetrate the blood-brain barrier, which prevents it from protecting the nervous system from radiation-induced damage [34, 35, 51, 52]. In our study, for the first time, we showed that UA312 has a good protective effect against radiation-induced nerve damage, which also compensates for the shortcomings of amifostine.

Further exploration of the targets revealed that chrna3 and grik5 may be the targets of UA312. The chrna3 plays a key role in autonomic ganglionic transmission. This subunit forms a “ganglionic” nAChR with other subunits (most commonly α3β4). Mutations or deletions in chrna3 can lead to autonomic dysfunction in humans and mice, including bladder enlargement and dribbling urination [53]. This further confirms our conclusion that UA312 plays a radioprotective role by targeting chrna3. By contrast, glutamate is an important excitatory neurotransmitter in the mammalian central nervous system, where it is involved in synaptic transmission, memory, and neuronal development [54]. Glutamate activates neural signals after binding to metabotropic glutamate receptors and ionotropic glutamate receptors. Studies have shown that ionotropic glutamate receptors play a common role in the etiology of mental illness [55]. The kainate receptor (grik) is a member of the ionotropic glutamate receptors subfamily [56]. Grik5 is a member of the grik family and is involved in neurotransmission [57]. This result also further proved our conclusion. However, the chrna3 and grik5 targets and radioprotective function of UA312 need to be further verified in zebrafish, mice, and clinical settings.

In conclusion, our results showed that exposure to IR can cause developmental toxicity, cardiac damage, and behavioral retardation in zebrafish embryos/larvae as well as alteration of multiple signaling pathways. The UA312 (40 µM) developed in this study significantly mitigates this damage and causes low toxicity to zebrafish. Therefore, UA312 is a novel and effective radioprotective drug that may have a key role in addressing the issues of medical radiation exposure.

Conclusion

This study showed that UA312 can protect zebrafish from IR by targeting chran3 and grik5 to improve cardiac toxicity and neurological damage (Fig. 8). These findings suggest that environmentally relevant radiation can have serious effects on the heart and nerves of zebrafish or humans. UA312 may serve as a new potential low-toxic and effective radiation protection drug, playing a protective role in medicine.

Fig. 8. UA312 protects zebrafish from IR by targeting chran3 and grik5 to improve cardiac toxicity and neurological damage.

Exposure to IR can cause developmental toxicity, cardiac damage, and behavioral retardation in zebrafish embryos/larvae as well as alteration of multiple signaling pathways. The UA312 (40 µM) developed in this study significantly mitigates this damage and causes low toxicity to zebrafish. Therefore, UA312 is a novel and effective radioprotective drug that may have a key role in addressing the issues of medical radiation exposure.

Author contributions

FFX, YS, HQW, and WYZ designed, conducted, and validated the experiments. FFX and YS drafted the manuscript. HQW and WYZ collected and analyzed the data. LXW and TH performed zebrafish feeding and embryo collection. SQZ, YLL, HHS, and WBH involved in regular discussions and manuscript revisions. WFG, SJF, and YLL designed, reviewed and revised the paper. All the authors revised and approved the manuscript. FFX, HQW, WFG, SJF, and YLL provided funding support.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-025-01564-0.