Introduction

Glycyrrhiza, a traditional Chinese herb widely recognized in Asian pharmacopoeias1, is a natural chalcone flavonoid. Its active component, ISL, is derived from the root of Glycyrrhiza uralensis2,3. Previous studies have elucidated the diverse pharmacological properties of ISL, including its anti-inflammatory4, antimicrobial5, and anti-diabetic6effects. Notably, ISL demonstrates distinct and pronounced anti-tumor activities across a wide range of tumor cell types7,8,9,10. The US Food and Drug Administration (FDA) has deemed glycyrrhiza and its derivatives safe for use in food and animal feed, as they have long been approved as additives for human consumption11. ISL has found extensive application as a sweetener in various products, including chewing gum, chocolate, tobacco products, toothpaste, cough mixtures, cosmetics, herbal teas, and beverages3. However, the potential harmful effects of ISL on humans and aquatic organisms remain insufficiently studied.

It is estimated that the average daily exposure to ISL for human ranges from 1 to 2 mg/kg12. Notably, dietary supplements containing ISL are particularly popular among women seeking relief from symptoms associated with premenopausal syndrome and menopause, resulting in frequent exposure to this compound13. Excessive use of products containing ISL and related compounds can lead to the accumulation of ISL within the human body14. Studies have demonstrated that ISL exposure results in ovarian disorders in mice, specifically affecting neutral steroid hormones15. Additionally, ISL induces developmental toxicity and oxidative stress-induced apoptosis in zebrafish embryos and larvae through the nRF2-HO1/JNK-ERK/mitochondrial pathway16. Our previous research has also indicated that ISL disrupts immune system function by promoting the expression of proinflammatory factors17, suggesting potential health risks associated with ISL exposure. However, it remains unclear whether ISL induces endoplasmic reticulum stress. Therefore, this study aims to elucidate the toxic mechanisms of ISL.

Zebrafish larvae are increasingly utilized as an experimental model for assessing drug compound toxicity and are particularly favored for studying developmental toxicology due to their advantages, such as embryonic transparency (enabling non-invasive studies on heart and liver function), small size (facilitating behavioral assessments), and high genetic, physiological, and anatomical similarity to mammals18,19. Consequently, zebrafish were selected as the experimental model for this study20,21,22. Therefore, we hypothesized that ISL may induce malformations in zebrafish embryos by triggering endoplasmic reticulum stress, leading to adverse aquatic ecological environments. To assess endoplasmic reticulum stress-related changes induced by ISL treatment, we employed TM as a positive control group known to induce ER stress responses23, and examined alterations in ER-related genes and analyzed enrichment pathways associated with endoplasmic reticulum stress using RNA transcriptome sequencing. The results demonstrated that ISL induced malformations in zebrafish embryos by triggering endoplasmic reticulum stress and the UPR signaling pathway. We should pay attention to the impact of ISL on the environment to reduce ecological risks.

Materials and methods

Chemicals and materials

Isoliquitigenin (98% purity) was procured from Aladdin Company, Shanghai, China, while tunicamycin (TM) (CAS No. 11089-65-9) was sourced from Dalian Melonepharma Co., Ltd., Dalian, C hina. Both compounds were dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 0.001%. All chemicals used in this study were of analytical grade.

Zebrafish husbandry and ISL exposure

Wild-type AB zebrafish embryos were procured from A and provided by Wanyuan Laboratory, Jiangxia District, Wuhan. And the zebrafish embryos provided by the company can be used for live research experiments, which meet the requirements of toxicology, pharmacology, etc. Quality assessment of the embryos was conducted under a stereomicroscope at 20 h post-fertilization (hpf).

For each well in a 6-well plate, 50 embryos at 22 hpf were randomly distributed in a volume of 3 ml. The control group consisted of embryos treated with 0.001% DMSO, while the positive control group for endoplasmic reticulum stress was treated with TM. The exposure concentrations of ISL were set as follows: 1.88µM, 3.75µM, 7.50µM, and 15.00µM, respectively. The temperature for embryo growth was maintained between 27.5 and 28.5 °C, and fresh solution (50%) was added every twelve hours to monitor hatching rate, mortality rate, and malformation rate. Three independent replicates were performed for each experimental group.

Cell cultures and treatments

HL-7702 originates from the Department of Digestive Diseases, Tongji Affiliated Hospital of Huazhong University of Science and Technology. The cell line was cultured in high-glucose Dulbecco’s Modified Eagle Medium (HYCLONE) supplemented with 10% fetal bovine serum (HYCLONE) at 37 °C in a 5% CO2 incubator. HL-7702 cells were treated with ISL (75.00µM and 150.00µM) and TM for 48 h. Each cell culture experiment was repeated at least three times.

RNA extraction and reverse transcription

After isoglycyrrhizin exposure, total RNA was extracted from zebrafish embryos using the MonPureTM Universal RNA Kit from Monad Biotech Co., Ltd. Subsequently, the extracted RNA underwent reverse transcription utilizing the MonScriptTM RTIII All-in-One Mix with dsDNase kit, also from Monad Biotech Co., Ltd.

Real-time fluorescence quantitative PCR

High-purity cDNA obtained through reverse transcription reaction was subjected to real-time fluorescence quantitative PCR analysis employing TB Green chimeric fluorescence method (TB Green® Premix Ex TaqTM II (Tli RNaseH Plus), Takara Bio). The primer sequences used in this study are listed in Table 1.Amplification conditions included initial denaturation at 95°C for 30s, followed by denaturation at 95°C for 5s, annealing at 60°C for 30s, and extension at 72°C for 30s, repeated over 36 cycles. All reactions were performed on an AB Applied Biosystems PCR machine. Values were calculated using the formula 2ΔCT (not exposed)-ΔCT (exposed). Each experimental group underwent three replicates.

Table 1 List of primers for each gene.
Full size table

Biochemical analysis

120 zebrafish larvae exposed to ISL (1:10, w/v) were homogenized and the supernatant was collected by centrifugation at 3000 rpm at 4° to detect the biochemical indexes of T-AOC and MDA (Solarbio), with 3 repetitions in each group.

Histological evaluation

In order to check the histopathological changes of liver, the zebrafish larvae of control group and ISL exposure group were fixed in 4% PFA at 96 hpf and embedded, and then the tissue was cut into thin slices for histological analysis by hematoxylin and eosin (H&E) staining.

Western blot analysis

After the treatment of HL-7702 cells, the total protein of the cells was cracked on ice with the protein lysis solution (Solarbio, China) according to the instructions. The working dilution and distributor of each antibody were as follows, GAPDH (1:200000) (Cat No 60004-1-Ig), ATF4 Monoclonal antibody (1: 2000) (Cat No 60035-1-Ig), ATF6 Polyclonal antibody (1:300) (Cat No 24169-1-AP) and Pdia4 Polyclonal antibody (1:5000) (Cat No 14712-1-AP) was obtained from proteintech (China). Anti-pEIF2a antibody (1:1000) (ser51) was obtained from CST (Boston, Massachusetts, USA).

Transcriptome sequencing

Transcriptome sequencing was conducted by Shanghai Meiji Biotechnology Co., Ltd. (China). Each sample comprised 150 embryos at 96 h post-fertilization (hpf), and RNA sequencing was performed for the experimental group (7.50 µM ISL) and control group (0.001% DMSO), each with three replicates. Total RNA was extracted and assessed for RNA integrity using agarose gel electrophoresis. mRNA was isolated from total RNA using magnetic beads with Oligo (dT) to perform A-T base pairing with poly (A). The complete RNA sequences were enriched and sequenced on the Illumina Novaseq 6000 platform after sample processing. Under the action of reverse transcriptase, random hexamers were added to reversely synthesize single-strand cDNA using mRNA as a template, followed by second-strand synthesis to form a stable double-stranded structure. End Repair Mix was added to create blunt ends, followed by the addition of an“A”base at the 3’end for ligation of a Y-shaped linker. Finally, sequencing was performed on the Illumina platform, and the ER stress gene set was specifically curated for separate data and image analysis.

Statistical analysis

Data were processed using Graph-Pad Prism 9 software, and statistical analysis was performed using Dunnett’s post-test two-way analysis of variance. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001.

Results

Developmental malformations observed in zebrafish embryos exposure to ISL

To assess the survival state of zebrafish embryos under different concentrations of ISL, a single batch of zebrafish embryos was exposed to varying concentrations (1.88, 3.75, 7.50, and 15.00 µM ISL). Hatching rate, embryo malformation rate, and mortality were observed to directly evaluate the toxicity of ISL on zebrafish embryos. After exposure until reaching a developmental stage of 96 hpf, the results indicated no significant difference in the effect of ISL on the hatching rate of zebrafish embryos (Fig. 1A). However, there was a significant increase in the malformation rate of zebrafish at ISL concentrations of 7.50 µM and above; additionally, mortality substantially increased at a concentration of 15.00 µM ISL (Fig. 1B and C). Although no differences were observed in hatching rates across different concentrations of ISL, distinct variations were noted in zebrafish malformation rates and mortality under high concentrations of ISL. These findings suggest that prolonged exposure to high concentrations of ISL during embryo cultivation can lead to an elevated incidence of malformations and mortality in zebrafish embryos.

Fig. 1
figure 1

Acute exposure analysis of zebrafish embryos to ISL. (A) Hatching rate of embryos at 72 hpf. (B) Malformation rate at 96 hpf. (C) Mortality rate at 96 hpf. Each group was repeated 3 times, Dunnett’s post-test two-way analysis of variance, data represent mean ± SME (n = 3). ***p < 0.001, ****p < 0.0001.

Full size image

Histological changes in liver of zebrafish after exposure to ISL

In order to study whether ISL has liver toxicity, we performed HE staining on zebrafish larvae treated with different concentrations of ISL at 96 hpf for histological analysis to observe the histological changes of liver cells. As shown in Fig. 2, compared with the control group, the liver of zebrafish treated with ISL showed obvious histopathological changes. The liver cells of the control group had normal appearance, tightly connected cells, uniform distribution of cytoplasm, and clear cell boundaries. Compared with the control group, 1.88µM ISL-exposed group showed obvious enlarged vacuolated structures in hepatocytes, loose hepatic architecture, and could be evaluated as grade 1 according to the pathological damage of hepatocytes. In the 3.75µM group, hepatocytes showed uneven cytoplasm, irregular staining distribution, vacuolization of cytoplasm, eccentric nucleus, and slight fibrosis, with a pathological damage score of 2. In the 7.50µM group, the liver underwent severe changes, with obvious disorder in tissue and cell structure, loose cell-cell contact, loss of cell boundaries, and patchy necrosis, accompanied by increased connective tissue, with a pathological score of 3. However, in the 15.00µM ISL-exposed group, the normal physiological structure of hepatocytes had completely disappeared, and the liver parenchyma showed obvious fibrosis, with a pathological score of 4. In addition, we tested the levels of T-AOC and MDA in zebrafish embryos, compared with the control group, the levels of MDA and T-AOC in zebrafish larvae exposed to 15.00 µM ISL were markedly increased (Fig. 2B and C). Therefore, we speculated that ISL exposure would lead to severe changes in the pathological phenotype of hepatocytes and induce liver toxicity.

Fig. 2
figure 2

Liver toxicity induced by ISL exposure. (A) The liver cells in the control group had normal appearance, cells were tightly connected, and cytoplasm was evenly distributed. The liver cells were round, with a central nucleus and clear cell boundaries, the liver of zebrafish in the 15.00µM ISL group showed serious changes, with obvious tissue and cell disorder, loose intercellular contacts and large vacuoles, and loss of cell boundaries. Each group was repeated 3 times. (B) and (C) Detection of MDA and T-AOC.

Full size image

ISL activates the eif2ak3-eif2s1 pathway in zebrafish embryos

Firstly, we selected the eif2ak3-eif2s1 related gene in the unfolded protein response (UPR) pathway and directly assessed its transcription to reflect the quantity of misfolded proteins in the endoplasmic reticulum (ER). To evaluate the expression of the ER stress pathway eif2ak3-eif2s1 at different concentrations, we examined the levels of genes involved in this pathway within each group. The TM group was set as a positive control for ER stress. Following exposure from 24 to 96 hpf, there was a significant increase in eif2ak3 gene expression observed in both the 15.00µM ISL and 0.5 mg/L Tm positive groups (Fig. 3A). Additionally, key gene eif2s1 (the zebrafish homolog of eIF2α) showed a significant increase in expression within both the 7.50µM ISL and 15.00µM ISL groups (Fig. 3B). Furthermore, gene atf4 exhibited increased expression within both the 1.88µM and 15.00µM groups (Fig. 3C). Furthermore, western blot analysis revealed that the levels of protein EIF2A and ATF4 were considerably higher in the HL-7702 cell line after ISL treatment compared to the control group (Fig. 3D). It demonstrated that UPR pathway transcripts such as eif2ak3-eif21 were dramatically altered in embryos treated with high concentrations of ISL and TM.

Fig. 3
figure 3

ISL activates the eif2ak3-eif2s1 pathways in zebrafish embryos. (A) (B) and (C) Zebrafish embryos were exposed to 1.88, 3.75, 7.50, 15.00µM ISL and 0.5 mg/L TM (positive control) from 22hpf to 96hpf, mRNA was subjected for qRT-PCR. all data normalized with β-actin, and relative mRNA expressions were calculated. Each bar represents the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001vs. control). (D) The level of protein EIF2A was detected by western blot.

Full size image

ISL activation of ire1/ern1-xbp1 pathway in zebrafish embryos

Subsequently, we selected additional genes related to ire1/ern1-xbp1 and atf6 on the unfolded protein response (UPR) pathway to observe their transcript levels. It was found that gene ern1 displayed strikingly increased expression within both the 15.00µM ISL and 0.5 mg/L Tm positive groups (Fig. 4A). Gene xbp-1s exhibited increased expression specifically within the 7.50µM group (Fig. 4B), while gene aft6 showed increased expression only within both the 15.00µM ISL and TM positive groups (Fig. 4C). And the levels of protein ATF6 was markedly higher in the HL-7702 cell line after ISL treatment compared to the control group (Fig. 4D). These results strongly indicate significant differences between embryos treated with ISL or TM compared to untreated embryos regarding UPR pathway gene transcripts such as eif2ak3-eif2s1 and ire1/ern1-xbp1. These findings suggest that ISL may induce accumulation and aggregation of unfolded proteins within the endoplasmic reticulum which further activates UPR pathways.

Fig. 4
figure 4

ISL activates the ire1/ern1-xbp1 pathways in zebrafish embryos. (A) (B) and (C) mRNA was subjected for qRT-PCR and relative mRNA expressions were calculated. Each bar represents the mean ± SEM of three independent experiments (*P < 0.05; **P < 0.01vs. control). (D) The level of protein ATF6 was detected by western blot.

Full size image

ISL increases the expression of ER stress chaperone in zebrafish embryos

Based on the aforementioned results, it is evident that exposure to ISL activates the eif2ak3-eif2s1 and ire1/ern1-xbp1 pathways in zebrafish embryos. Therefore, we further investigated whether ISL exposure could induce expression of ER stress chaperones. Following a certain period of exposure, compared to the control group, we observed a significant increase in the expression level of ER stress chaperone hspa5 in the 7.50µM ISL and 0.5 mg/L TM groups (Fig. 5A). Additionally, there was also a remarkably increase in the expression level of ER stress chaperone hsp90b1 in the 7.50µM ISL, 15.00µM ISL, and 0.5 mg/L TM groups (Fig. 5B). Furthermore, varying degrees of increased expression were observed for ER stress-related apoptosis factor chop in the 7.50µM ISL and 0.5 mg/L TM groups (Fig. 5C). Our data analysis revealed significant differences in the expression levels of ER stress chaperones such as hsp90b1. These findings demonstrate that ISL can activate UPR signaling pathway, enhance expression levels of ER stress chaperones like hsp90b1, and stimulate expression of ER stress-related apoptosis factors. The results indicate that treatment with ISL leads to alterations in the expression profile of ER stress chaperones, thus suggesting that endoplasmic reticulum overload-induced apoptosis may be responsible for death among ISL-exposed zebrafish embryos.

Fig. 5
figure 5

ISL enhances the expression of ER stress chaperones in zebrafish embryos. mRNA was extracted and subjected to qRT-PCR analysis for chop, hspa5 and hsp90b1 genes with β-actin used for normalization. The asterisk represents a statistically significant difference when compared with the corresponding controls (*P < 0.05). (D) The level of protein PDIA4 was detected by western blot.

Full size image

ER differentially expressed genes

After the Read Counts of genes were obtained by gene expression analysis, DESeq2 was used to analyze the gene differential expression of multiple copies (n = 3) between groups, and the differentially expressed genes were identified (q-value, fdr, padj ≤ 0.05). Comparison with DMSO control group, ISL (7.50 µM) exposure group had 107 downregulated and 129 upregulated differentially expressed genes (Fig. 6A). We established a set of endoplasmic reticulum stress genes and analyzed their expression patterns by clustering. The stratified clustering heat map of significant differences in the expression of endoplasmic reticulum stress-related genes in zebrafish larvae after ISL exposure is shown in Fig. 6B. What’s more, we validated the level of protein PDIA4 through western blot analysis, as shown in the Fig. 5D, where the expression of PDIA4 protein in the HL-7702 cell line after ISL exposure was apparently increased.

Fig. 6
figure 6

ER differential expression genes. (A) Scatter plot of expression difference. (B) ER differential gene clustering diagram: clustering by log10 (FPKM + 1) value, red indicates up-regulated genes, and blue indicates down-regulated genes.

Full size image

GO enrichment analysis and KEGG analysis of endoplasmic reticulum stress

The GO enrichment bubble chart visually represents the functional enrichment of differentially expressed genes in the context of Gene Ontology (GO). The top 20 significantly enriched GO terms have been selected to underscore their significance. In organisms, genes coordinate to fulfill biological functions. Through rigorous pathway enrichment analysis, we identified several key pathways associated with numerous genes/transcripts within the Gene Ontology (GO) framework, including cellular response to stress, response to endoplasmic reticulum stress, organelle function, and biological regulation. Notably enriched pathways include the positive regulation of transcription from RNA polymerase II promoter in response to endoplasmic reticulum stress, regulation of the ERAD pathway (endoplasmic reticulum-associated degradation), intrinsic apoptotic signaling pathway in response to external stimuli, and regulation of the response to endoplasmic reticulum stress. Furthermore, our results demonstrate that the unfolded protein response (UPR) plays a crucial role in maintaining endoplasmic reticulum function by managing the unfolded protein load during stress conditions, as evidenced by the GO enrichment results obtained from RNA transcriptome analysis (Fig. 7A).

Fig. 7
figure 7

GO enrichment analysis and KEGG metabolic pathway analysis. (A) GO enrichment analysis displayed on a graph where the vertical axis represents GO Term while the horizontal axis represents Rich factor indicating the level of enrichment (larger value indicates greater enrichment). The size and color of each dot corresponded respectively to the number of genes/transcripts annotated within each GO Term category and different Padjust ranges. (B) KEGG metabolic pathway analysis represented by a graph where each pathway name is shown on the vertical axis while the number of annotated genes or transcripts is displayed on the horizontal axis.

Full size image

The histogram generated from KEGG analysis highlights seven major categories encompassing metabolic pathways, Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, Human Diseases, and Drug Development. ISL exposure significantly impacts categories including cell growth and death processes, infectious diseases caused by viruses, signal transduction mechanisms, as well as folding, sorting, and degradation processes. These findings indicate that ISL exposure primarily affects cellular processes related to human diseases, as well as genetic and environmental factors (Fig. 7B).

Discussion

The cumulative usage of ISL in daily necessities is gradually increasing, underscoring the need to confirm the ecological harm caused by ISL. Previous studies have reported that high concentrations of ISL can result in the death and abnormal development of zebrafish embryos24, which aligns with our findings. The embryonic mortality and malformation rates significantly increased in the ISL treatment groups (7.50µM, 15.00µM), and these rates furth er escalated with prolonged exposure, indicating evident developmental toxicity induced by ISL in zebrafish embryos. Building upon this, our study explored the involvement of the endoplasmic reticulum stress pathway in ISL-induced developmental toxicity in zebrafish.

In our investigation, our histological results demonstrated that zebrafish hepatocytes in the 1.88 µM ISL exposure group had uneven eosinophilic cytoplasm, irregular staining distribution, and cytoplasmic vacuolation, liver tissue and cells in the 15.00 µM ISL treatment group were obviously disordered, with loose intercellular contacts and large vacuoles, as well as loss of cell boundaries. This suggests that ISL exposure affects liver toxicity, and the effect on the liver is greater with the increase of exposure concentration. And ISL disrupted the homeostasis of the zebrafish endoplasmic reticulum, leading to ER stress and the accumulation of misfolded proteins, thereby initiating a complex signaling network known as the unfolded protein response (UPR). The unfolded protein response enables cells to adapt to ER stress and plays a vital role in maintaining ER function25. There are three distinct UPR pathways, the protein kinase RNA-like ER kinase (PERK) pathway (also referred to as the eif2ak3-eif2 pathway), the inositol-requiring enzyme 1 (ire1)/ern1-xbox binding protein 1(xbp1) pathway, and the activating transcription factor 6 (atf6) pathway26. Studies have demonstrated that short or low exposures to various external stressors can induce the UPR, effectively addressing the unfolded protein load within the ER while preserving secretion pathway function during mild or acute stress27. Importantly, these different UPR responses yield diverse outcomes that may either prevent or promote diseases28. Our results indicated that at ISL concentrations of 7.50 µM and 15.00 µM, the upr-related gene eif2s1 was substantially augmented, particularly at 7.50 µM. At a concentration of 7.50 µM, ISL dramatically upregulated the expression levels of the ire1/ern1 and xbp-1s genes. Notably, at a concentration of 15.00 µM, the transcriptome levels of ire1/ern1 and xbp-1s were even higher. This suggests that with the increase in ISL exposure concentration, more endoplasmic reticulum stress genes, namely ire1/ern1 and xbp-1s, were expressed in zebrafish, indirectly indicating that the toxicity of ISL is concentration-dependent and can activate endoplasmic reticulum stress.

Studies have demonstrated that ISL induces the generation of reactive oxygen species (ROS) and triggers apoptosis in liver cancer cells by impairing mitochondrial redox processes29,30. Disruption of the redox state is crucial for maintaining proper protein folding within the endoplasmic reticulum, while excessive ROS can damage this redox state, leading to misfolding or accumulation of misfolded proteins31. Our previous research has shown that ISL promotes ROS production as an indicator of oxidative stress17. Our research data also clearly showed that the MDA and T-AOC indexes in zebrafish larvae treated with ISL were substantially increased. Therefore, we hypothesize that endoplasmic reticulum stress may be an important mechanism underlying ISL-induced embryonic developmental disorders in zebrafish.

Endoplasmic reticulum pathways can upregulate the expression of ER chaperone proteins such as hspa5 and hsp90b126. Notably, these pathways can interconnect under different cellular stresses28. The primary purpose of the unfolded protein response is to facilitate cellular adaptation to environmental changes and restore normal ER function. However, if the unfolded protein response fails to alleviate endoplasmic reticulum overload, it activates apoptotic pathways. Chop, also known as the growth arrest and DNA damage-inducible gene, plays a pivotal role in apoptosis induced by endoplasmic reticulum stress. When endoplasmic reticulum stress becomes severe and prolonged, the unfolded protein response also leads to cell death. The induction of apoptosis by endoplasmic reticulum stress is believed to contribute to the development of various diseases32,33,34,35. To confirm that endoplasmic reticulum stress is a mechanism through which ISL negatively impacts zebrafish embryonic development, this study further examined the expression of ER chaperone proteins, hspa5, hsp90b1, and the ER-related apoptosis factor chop. Following exposure for a specific duration, we observed a significant increase in the expression of the endoplasmic reticulum stress molecular chaperone hspa5 in the 7.50 µM ISL and 0.5 mg/L Tm groups. The expression of another endoplasmic reticulum stress molecular chaperone, hsp90b1, was significantly increased in the 7.50 µM ISL, 15.00 µM ISL, and 0.5 mg/L TM groups compared to the control group. Additionally, there was an increase in the expression of the endoplasmic reticulum stress-related apoptosis factor chop to varying degrees within the 7.50 µM ISL and 0.5 mg/L TM groups. Data analysis revealed significant differences in the expression levels of endoplasmic reticulum stress molecular chaperones such as hsp90b1 between embryos treated with ISL or TM and untreated embryos. These results demonstrate that ISL can activate the UPR signaling pathway, leading to increased expression levels of endoplasmic reticulum stress molecular chaperones like hsp90b1 and related apoptotic factors associated with ER stress.

Furthermore, our RNA transcriptome sequencing results provide additional evidence supporting a relationship between ISL exposure and endoplasmic reticular stress response. GO Term analysis revealed that pathways related to the response to ER stress and stimulus are the most numerous and enriched after ISL exposure. Notably, the unfolded protein response within the ER was significantly increased following such exposure according to KEGG metabolic pathways analysis. Affected pathways include cell growth and death, infectious diseases caused by viruses, signal transduction, and folding-sorting-degradation processes, indicating that ISL impacts human diseases and the ecological environment.

Considering the variations in toxic concentrations between the non-mammalian lower vertebrate zebrafish model and mammalian models such as mice or human cells, our results strongly demonstrate that ISL exposure is closely associated with the activation of the endoplasmic reticulum pathway. However, further research is needed to elucidate the molecular mechanisms of action, which has so far been limited to relatively simple validation methods such as RNA transcriptome sequencing, RT-PCR, and cell line WB. We need to further explore the specific molecular interactions that contribute to ISL toxicity, not just in zebrafish, but also in other animal models. In addition, while the results of the acute exposure experiment showed that as the duration and dose of ISL exposure increased, the embryo malformation rate increased significantly, unfortunately, we have not yet been able to specifically demonstrate the dose- and time-dependent response pattern of ISL. In summary, this study systematically examined whether exposure to ISL could induce ER stress and UPR in a zebrafish model system. Our findings indicate that exposure to ISL triggers an ER stress response by activating the eif2ak3-eif2s1 and ire1/ern1-xbp1 pathways of the UPR, resulting in abnormal development and hepatotoxicity of zebrafish embryos.