Application of (-)-epicatechin to inhibition of toxicity caused by MEZ

Abstract

The invention relates to the technical field of pharmaceutical biology, in particular to application of (-)-epicatechin to inhibition of toxicity caused by MEZ. The toxicity caused by the MEZ is developmental toxicity or hepatotoxicity. The developmental toxicity comprises the following symptoms: embryo malformation, embryo apoptosis and developmental morphology abnormality. The hepatotoxicity comprises liver development delaying, cholesterol and lipid metabolism disorder, liver injury and liver cell apoptosis. According to the research, the effect of the L-epicatechin on inhibiting MEZ-induced developmental toxicity and hepatotoxicity is found, embryo injury and liver injury caused by MEZ can be effectively relieved, and the pathogenesis of the MEZ-induced developmental toxicity and hepatotoxicity can be further understood.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical biology, and in particular to application of (-)-epicatechin in inhibiting toxicity caused by MEZ. Background Art

[0002] Metconazole (MEZ) is a triazole fungicide that acts as an effective C-14 demethylase inhibitor of ergosterol biosynthesis, thereby disrupting the growth and reproduction of pathogens. However, studies have shown that this triazole fungicide can induce hepatotoxicity in mice, and the presence of triazole fungicides in the aquatic environment poses a potential risk to aquatic organisms and humans. However, the mechanism of MEZ-induced toxicity remains largely unexplored.

[0003] Flavonoids are a class of natural products with low toxicity, antioxidant, anti-inflammatory and stability. Some flavonoids have the function of protecting liver function by scavenging free radicals, regulating enzymes and cytokines, and regulating cell apoptosis. Epicatechin (EC) is a flavonoid found in many Chinese herbal medicines. Studies have shown that it can effectively protect the liver from non-alcoholic fatty liver disease. However, the various symptoms of non-alcoholic fatty liver disease and the symptoms caused by MEZ toxicity are significantly different. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides the use of (-)-epicatechin in inhibiting the toxicity induced by MEZ.

[0005] The present invention discovered the function of (-)-epicatechin (hereinafter also referred to as L-epicatechin) in inhibiting the toxicity caused by MEZ, and then proposed related applications, which mainly alleviate the developmental toxicity and liver toxicity by inhibiting the cell apoptosis caused by MEZ. Other configurations of epicatechin have no related activity.

[0006] In a first aspect, the present invention provides use of (-)-epicatechin, or a composition comprising (-)-epicatechin, in inhibiting toxicity induced by MEZ.

[0007] The present invention further provides use of (-)-epicatechin, or a composition comprising (-)-epicatechin, in the preparation of a medicament for inhibiting toxicity induced by MEZ.

[0008] Furthermore, the toxicity induced by MEZ is developmental toxicity or hepatotoxicity.

[0009] Furthermore, the developmental toxicity includes the following symptoms: developmental abnormalities and embryonic apoptosis.

[0010] Furthermore, the developmental abnormalities include: developmental delay, shortened body length, reduced eye area, closure of the swim bladder, yolk sac edema, pericardial edema, spinal curvature, tail deformity, shortened mandible, delayed hatching, decreased embryo hatching rate, increased embryo mortality rate, embryo deformity or decreased motor level. One or more of the following.

[0011] Furthermore, the hepatotoxicity includes: Delaying liver development, cholesterol and lipid metabolism disorders and liver damage.

[0012] Furthermore, the delaying of liver development is to reduce the liver area; and / or, The cholesterol and lipid metabolism disorders include: abnormal levels of TCHO, TG and MDA, abnormal expression levels of hmgcra, hmgcrb, cyp2r1, cyp7a1, cyp51, less, dhcr7, pik3r3a, acox1, dgat2 and pparα, and accumulation of lipid droplets in hepatocytes; and / or, The liver damage includes: the gene expression level of fabp2, prox1, gata6, hadc1 or hadc3 is decreased. These genes are all genes related to liver development. The liver damage also includes: the level of AST and ALT is decreased.

[0013] The histone deacetylase encoding gene hadc1 mainly controls the embryonic development of the liver and exocrine pancreas, while hadc3 is mainly involved in the formation of the liver. Homeobox1 and GA-TA binding protein 6 have been reported to regulate the migration of hepatocytes during liver morphogenesis, highlighting their key roles in the development of this important organ. Ablation of prox1 leads to abnormal hepatocyte morphology and reduced expression of genes related to liver development, a process regulated by hadc1 and hadc3.

[0014] In a second aspect, the present invention provides a drug for inhibiting toxicity induced by MEZ, comprising: (-)-epicatechin.

[0015] Furthermore, the concentration of the (-)-epicatechin is 5-15 μM.

[0016] The present invention has the following beneficial effects: The present invention has found that L-epicatechin has an effect in inhibiting the developmental toxicity and hepatotoxicity induced by MEZ, and can effectively slow down the embryo damage and liver damage caused by MEZ, such as inhibiting the developmental delay, shortened body length, reduced eye area, swim bladder closure, yolk sac edema, pericardial edema, spinal curvature, tail deformity, mandibular shortening, hatching delay, decreased embryo hatching rate, increased embryo mortality rate and embryo deformity caused by MEZ. The application of L-epicatechin provided by the present invention in inhibiting the toxicity of MEZ has important application value for improving agricultural safety and promoting environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 1 is a graph showing the developmental toxicity of MEZ on zebrafish embryos provided in Example 1 of the present invention; wherein A is the main developmental changes in the 96 hpf MEZ exposure group at different MEZ concentrations; B is the tail deformity result; C is the embryo hatching rate result; D is the embryo mortality result; E is the body length result; and F is the eye size result.

[0019] Figure 2 It is the effect of MEZ on early liver development of zebrafish provided by Example 1 of the present invention; wherein A is the effect of different MEZ concentrations on zebrafish liver size and fluorescence intensity; B is the result of liver area; C is the fluorescence signal result of fabp-positive cells; D is the ALT result; E is the AST result; FJ is the result of fabp2, prox1, gata6, hadc1 and hadc3 gene expression levels.

[0020] Figure 3 is the effect of MEZ on zebrafish embryo apoptosis provided by Example 1 of the present invention; wherein A is the AO staining result of apoptotic cells; B is the relationship between the dosage of MEZ and the number of apoptotic cells; CE is the effect of different dosages of MEZ on the expression levels of bax, bcl2 and p53 apoptosis regulating genes.

[0021] Figure 4The figure is the effect of MEZ on cholesterol and lipid metabolism of zebrafish larvae provided by Example 1 of the present invention; wherein A and B are the changes in hepatocyte lipid levels under different MEZ concentrations; C is the result of TCHO level; D is the result of TG level; E is the result of MAD level; F is the result of cholesterol metabolism related genes hmgcra, hmgcrb, cyp2r1, cyp7a1, cyp51, less, dhcr7; G is the expression level result of acox1, dgat2 and pparα genes related to lipid synthesis.

[0022] Figure 5 The results of the effect of (-)-epicatechin on MEZ-induced zebrafish developmental damage and liver damage provided by Example 1 of the present invention; wherein A is the alleviation of EC on MEZ-induced deformity and liver area; B is the result of embryo body length after EC treatment; C and D are the results of liver fluorescence intensity after EC treatment; E is the result of liver area after EC treatment.

[0023] Figure 6 The results of the alleviation of MEZ toxicity by (-)-epicatechin provided in Example 1 of the present invention; wherein A is the inhibitory effect of (-)-epicatechin on MEZ-induced lipid droplet accumulation in hepatocytes; B and C are the results of AO staining of cell apoptosis; DF are the results of the expression levels of bax, bcl2 and p53.

[0024] Figure 7 The results of the alleviation of MEZ toxicity by (-)-epicatechin provided in Example 1 of the present invention; wherein A is the effect of (-)-epicatechin on the recovery of zebrafish locomotion; B and C are the effects of (-)-epicatechin on the total movement distance and speed recovery of zebrafish embryos. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0027] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0028] Example 1 Zebrafish embryos are used as an in vivo developmental model because they are transparent, easy to observe, have a short reproductive cycle, develop rapidly, and complete organogenesis at 96 hours after fertilization (hpf). In addition, approximately 87% of human homologous genes are present in the zebrafish genome. The zebrafish model can be used to evaluate the effects of drugs on the development of specific organs. Therefore, the present invention selects 96hpf zebrafish embryos as an animal model to study the toxicity of MEZ to zebrafish and the inhibitory effect of (-)-epicatechin (hereinafter referred to as EC) on this toxicity, including the following process: 1. MEZ exposure method The lethal effects of different concentrations of MEZ on zebrafish were evaluated at 96 hours post fertilization (96 hpf). To obtain the maximum effect of MEZ without causing lethality, MEZ was administered at concentrations of 0.15, 0.5, 2.5, and 4.5 mg·L -1 In the case of MEZ treatment, the experiment was performed with DMSO solvent control (n=60, 3 replicates). The final DMSO concentration in each group was 0.05%, which did not cause adverse reactions to zebrafish before the experiment and was used as a control in this study. During the exposure period, 50% of the original solution was replaced with fresh solution every 24 hours to ensure a constant concentration. After treatment, all embryos were incubated at 28°C. After 96 hours of MEZ treatment, samples were collected for subsequent experiments.

[0029] 2. Morphological evaluation Hatching rate and mortality were recorded every 24 h until 96 hpf. Zebrafish embryos were manually assessed for malformations such as spinal curvature (BS), tail malformation (TM), yolk sac edema (YSE), and pericardial edema (PE) at 96 hpf according to the basic guidelines of OECD.

[0030] To assess the developmental toxicity of MEZ at 96 hpf, zebrafish embryos were anesthetized with tricaine methanesulfonate and mounted on glass slides. Brightfield images of the whole body were captured using an OLYMPUS SZX2-1LLT stereomicroscope (n = 15, 3 replicates). Morphological abnormalities, including body and eye size, were observed and analyzed using Fiji software.

[0031] 3. Liver fluorescence imaging Tg(fabp10a:dsRed) transgenic zebrafish were treated with MEZ and observed and imaged using an OLYMPUS SZX2-1LLT stereomicroscope to visualize red fluorescent labeling within the liver region. Liver area was calculated using Fiji software and liver area and fluorescence intensity were evaluated (n = 12, 3 replicates).

[0032] 4. Staining detection method After treatment at all concentrations (96 hpf), embryos were collected and rinsed three times with circulating water to eliminate excess test solution. Acridine orange (AO) staining was used to selectively label nucleic acids with green fluorescence to evaluate apoptotic effects. The results show the level of apoptosis in each embryo after 20 µM AO staining for 30 min. Nile red staining was used to visualize triglycerides (tg) and fatty acids. Zebrafish embryos were stained with 2 µM Nile red for 30 min. The experiments were performed in a 28 °C incubator. After incubation, zebrafish embryos were rinsed with circulating water, anesthetized with Tricaine, and mounted on slides containing 3% methylcellulose. Dark field images of zebrafish embryos were captured using an OLYMPUS SZX2-1LLT fluorescence microscope. The fluorescence intensity of the images was determined using Fiji software, and the number of apoptotic cells was quantified using the “Analyze Particles” function. The experiments were repeated three times, with nine zebrafish embryos randomly selected for each group.

[0033] 5. Enzyme activity and biochemical measurements About 30 zebrafish embryos at 96 hpf were homogenized in 200 µL of phosphate-buffered saline and centrifuged at 8000 × g for 10 min at 4 °C. The supernatant was carefully transferred to a new tube for subsequent experiments. TG9 and total cholesterol (TCHO) levels as well as albumin aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using kits provided by Nanjing Jiancheng Biotechnology Institute according to the manufacturer's instructions.

[0034] The protein concentration was determined using the Bicinchoninic Acid Assay kit according to the manufacturer's instructions.

[0035] 6. EC treatment plan Based on the above exposure experiments, the MEZ concentration of 2.5 mg·L was selected to have the greatest effect and the highest hatching rate. -1 EC was added at 48 hpf. The experiment was divided into two groups: control group (0.05% DMSO); 2.5 mg·L -1 MEZ;15µmEC;2.5mg·L -1 MEZ+5µM EC;2.5mg·L -1 MEZ+15µM EC. The DMSO content of each group was adjusted to 0.05%. After 96h of treatment, body length, liver size, and fluorescence were measured as described above.

[0036] 7. Sports recording and analysis Zebrafish embryos (96 hpf) of each treatment group were transferred to 24-well plates, with 1 embryo per well and 2 mL of circulating water. Five embryos were analyzed for each concentration. After acclimation to the 24-well plate environment for 5 minutes, the swimming behavior of zebrafish embryos in a dark room at room temperature was recorded using the Noldus DanioVision system for 40 minutes. The recorded data included total distance (cm) and average speed (mm / s).

[0037] 8. Hematoxylin and eosin (H&E) staining Three zebrafish embryos at 96 hpf were collected from each treatment group for paraffin sectioning and hematoxylin and eosin staining. After agarose fixation, graded ethanol dehydration, paraffin embedding, and sectioning into 8-µm thick sections, the sections were stained using the H&E staining kit (TA&Clone, Shanghai). Finally, the sections were sealed and preserved in neutral gel. The samples were observed under an optical microscope (Cosmo Laboratory Equipment, India) and more than eight different fields of view were observed. The images were taken using a Nikon Eclipse E100 optical microscope (Tokyo, Japan) equipped with Scope Image 9.0 Windows software. The slides were examined to evaluate the pathological features of the tissues.

[0038] 9. RNA extraction and quantitative analysis of mRNA levels Real-time quantitative polymerase chain reaction (qRT-PCR) was used to evaluate the gene expression changes induced by MEZ and EC. -1 ) and EC (2.5mg·L -1 RNA was extracted from whole zebrafish embryos (n=50, 3 replicates) using TransZol Up solution after 96 h of exposure to MEZ+5µM EC and 2.5mg·L-1 MEZ+15µM EC. RNA purity was assessed by measuring the OD260 / OD280 ratio using an Unano-1000 microspectrophotometer. cDNA was synthesized using AccuPower RT premix. qRT-PCR analysis was performed on a CFX96TM Real-Time PCR System using the TransStart® TopTaq (TransGen-Biotech) method.

[0039] 10. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.5 (GraphPad Software, San Diego, CA, USA). Multiple comparisons were performed using one-way analysis of variance and Tukey's post hoc test.

[0040] 11. Experimental Results (1) Developmental toxicity of MEZ on zebrafish embryos The LC50 of MEZ was calculated using the Quest Graph LC50 calculator to be 12.96 mg·L -1 . Figure 1 Figure A summarizes the major developmental changes in the MEZ-exposed group at 96 hpf, including developmental delay, shortened body length, reduced eye area, swim bladder closure (SBC), YSE, PE, BS, shortened mandible, and delayed hatching. -1 Significant TM (p<0.01) was observed when ( Figure 1 B). When the MEZ concentration was >0.5 mg·L-1, the PE rate of each group was the highest (p<0.0001), and YSC and BS were dose-dependent (p<0.0001). -1 Hatching rate and mortality of embryos treated with MEZ ( Figure 1 C and D in Figure 1). As the concentration of MEZ increased, the hatching rate of embryos gradually decreased. At 48 hpf, the hatching rates were 77%, 58%, 52%, and 10%, respectively. -1 There was a significant inhibitory effect on the embryo hatching rate at 48 hpf (p<0.0001). -1 The hatching rate of 12 embryos at 96 hpf was 38% (p<0.0001) ( Figure 1 (C) Embryos were exposed to MEZ at 96 hpf at 2.5 and 4.5 mg·L -1 The mortality rates at the concentrations were 2% and 3%, respectively (p<0.001) ( Figure 1 D in ). Figure 1 As shown in A, the concentration is >2.5 mg·L -1 When zebrafish embryos are deformed, their body length is significantly shortened ( p <0.0001) ( Figure 1 E in the figure), the eyes became smaller (p<0.0001) ( Figure 1 F). These findings suggest that MEZ impedes normal embryonic development in zebrafish and gradually causes severe malformations as concentrations increase.

[0041] (2) Effects of MEZ on early liver development in zebrafish To evaluate the toxic effects of MEZ on early liver development, fluorescent images of the liver of transgenic zebrafish Tg(fabp10a:dsRed) were captured and liver size and fluorescence intensity ( Figure 2 Compared with the control group, the MEZ concentrations were 0.15, 0.5, 2.5 and 4.5 mg·L-1 When the liver area was 5% (p<0.05), 14% (p<0.05), 36% (p<0.0001) and 64% (p<0.0001), respectively ( Figure 2 Similarly, the fluorescence signal emitted by FABP-positive cells in the liver also decreased in a dose-dependent manner ( Figure 2 C). The levels of ALT and AST in the mez treatment group were significantly higher than those in the control group. -1 MEZ treatment had no significant effect on ALT and AST levels, but 0.5 and 2.5 mg·L -1 The MEZ group significantly increased the levels of ALT and AST. -1 ) have different responses and show a downward trend ( Figure 2 D and E in Figure 1). This may be due to compensatory effects, with inactivation of function beyond the threshold. In addition, the expression of five genes (fabp2, prox1, gata6, hadc1 and hadc3) was analyzed to determine the effects of different MEZ concentrations on liver development ( Figure 2 The results showed that after MEZ exposure at 96 hpf, the relative expression levels of prox1, gata6, hadc1, and hadc3 were significantly downregulated (p<0.0001), while the relative expression levels of 2.5 and 4.5 mg·L -1 The relative expression of fabp2 in the MEZ group was downregulated by 71% (p<0.001) and 53% (p<0.01), respectively. Taken together, these findings suggest that MEZ disrupts the complex liver development process during zebrafish embryogenesis and has a deleterious effect on liver tissue development.

[0042] ALT and AST are mainly present in hepatocytes. When the liver is damaged, transaminases enter the blood, resulting in elevated ALT and AST levels in the blood, which can be used as reliable biomarkers for assessing hepatocyte damage. Elevated ALT and AST levels have been shown to induce liver damage in zebrafish treated with perfluorooctane sulfonamide. In this study, a similar pattern was observed in zebrafish embryos treated with MEZ (more obvious at medium and low concentrations, while ALT and AST levels decreased at high concentrations due to compensatory effects), which provides strong evidence that MEZ induces severe liver damage in zebrafish embryos.

[0043] In addition, H&E staining also observed obvious vacuoles between zebrafish liver cells, indicating serious defects in the liver structure, unclear boundaries of liver cells, and damaged cell structure.

[0044] (4) MEZ induces apoptosis in zebrafish embryos The present invention further measured the level of apoptosis induced by MEZ exposure in zebrafish embryos. AO staining can be used to observe apoptotic cells in zebrafish embryos. The nuclear chromatin of early apoptotic cells is green and cylindrical ( Figure 3 A). After 96 h of MEZ exposure, the number of apoptotic cells increased in a dose-dependent manner ( Figure 3 The expression levels of bax, bcl2 and p53 apoptosis regulatory genes were analyzed and the results showed that the high concentration group (4.5 mg·L -1 ) is opposite to the expression trend of other groups ( Figure 3 Compared with the control group, 0.5 mg·L -1 and 2.5 mg·L -1 The expression of bax in the two groups increased by 3.52 times (p<0.0001) and 2.46 times (p<0.01), and the expression of p53 increased by 1.40 times and 1.73 times (p<0.01), respectively. -1 Compared with the control group, the expression of bax gene in the 0.5 and 2.5 mg·L -1 The expression of bcl2 mRNA in the 4.5 mg·L -1 The apoptosis signal was detected in the liver ( Figure 3 (A) MEZ affects zebrafish development by inducing apoptosis in embryos.

[0045] (5) MEZ disrupts cholesterol and lipid metabolism in zebrafish larvae Hepatotoxicity is closely related to lipid metabolism and cholesterol biosynthesis. Nile red is a lipophilic fluorescent probe used to quantify intracellular lipids. Nile red staining shows elevated lipid levels in hepatocytes ( Figure 4 A and B in the figure).

[0046] Therefore, the present invention studied lipid levels, related enzymes, cholesterol levels, and lipid metabolism-related gene expression in embryos to explore the potential mechanism of MEZ hepatotoxicity. TCHO levels were significantly reduced at all exposure concentrations ( Figure 4 Compared with the control group, 2.5 mg·L -1 The TG in the MEZ group was significantly increased by 1.27 times (p<0.01), and the TG in the 4.5 mg·L -1 The TG in the MEZ group was significantly downregulated by 35% (p<0.01) ( Figure 4 D in the figure). At all exposure concentrations, MDA content increased significantly in a dose-dependent manner ( Figure 4E in the figure). Cholesterol metabolism-related genes hmgcra, hmgcrb, cyp2r1, cyp7a1, cyp51, less, and dhcr7 were significantly increased in the high-concentration group (4.5 mg·L -1 MEZ) down-regulated, pik3r3a up-regulated ( Figure 4 Compared with the control group, 4.5 mg·L -1 The expression levels of acox1, dgat2, and pparα genes related to lipid synthesis in the group were upregulated by 1.25 times (p < 0.05), 1.74 times (p < 0.0001), and 1.49 times (p < 0.0001), respectively ( Figure 4 G in Figure 4). The mRNA expression levels of lipid metabolism-related genes were consistent with the results of Nile red staining. These results indicate that the disorder of cholesterol and lipid metabolism during zebrafish development is an important factor in MEZ-induced hepatotoxicity.

[0047] Cholesterol is essential for cells. It plays a vital role in the formation of cell membranes, is a component of bile acids, vitamin D and steroids, and is a component of lipids associated with lipid vesicles. Cholesterol biosynthesis is mainly completed in the liver, so dysregulation of hepatic cholesterol biosynthesis can lead to various liver diseases. Low cholesterol is the first step in catalyzing the biosynthesis of cholesterol, steroid hormones and vitamin D. In contrast, the dhcr7 gene acts as the final enzymatic catalyst in the cholesterol synthesis process by promoting the conversion of 7-dehydrocholesterol to cholesterol (Teng et al, 2019). HMGCR is the rate-limiting step in the catalytic process of cholesterol biosynthesis (Urlep et al, 2017). In the present invention, MEZ significantly downregulated the expression of cyp51, hmgcra, hmgcrb, lss and dhcr7, which is consistent with the observed reduction in TCHO content. Experimental evidence shows that specific knockdown of HMGCR can lead to severe hepatic steatosis accompanied by cell apoptosis (Sharpe and Brown, 2013).

[0048] In addition, Nile red staining performed after MEZ exposure showed increased TG accumulation in the intestinal and liver regions of zebrafish embryos. The acox1 gene encodes a crucial lipogenic enzyme that transports fatty acids from the outer membrane to the mitochondria, and pparα stimulates these responses (Wakil, 1989). Studies have shown that PIK3R3 regulates liver fatty acid responses through pparα (Yang et al., 2018). DGA-T, as the final catalyst for TG biosynthesis, promotes the conversion of fatty acids and glycerol to TG (Kawano and Cohen, 2013). The present invention observed that MEZ exposure led to increased TG content in zebrafish embryos, while the mRNA levels of lipid metabolism-related genes acox1, pparα, and dgat2 were upregulated. Previous studies have shown that propiconazole inhibits TCHO and increases TG levels, which may be related to insufficient energy supply (Teng et al., 2019). In addition, exposure to isoproconazole causes hepatotoxicity and disrupts lipid metabolism in zebrafish larvae (Jiang et al., 2020). The results of the present invention are consistent with those of previous studies.

[0049] It can therefore be concluded that MEZ exposure disrupts the expression of genes involved in lipogenesis and cholesterol biosynthesis.

[0050] (6) EC can alleviate early MEZ-induced developmental damage and liver injury in zebrafish After 48 h of MEZ exposure, EC was added to analyze the restorative effect of EC on MEZ-induced malformation and hepatotoxicity ( Figure 5 After adding EC, the alleviation of MEZ-induced deformity and liver area was observed ( Figure 5 The results showed that the embryo body length of the EC group was significantly increased compared with that of the MEZ group (p<0.001) ( Figure 5 In B, the fluorescence intensity of the liver increased (p<0.01) ( Figure 5 In D, the liver area increased (p<0.05) ( Figure 5 E in.

[0051] It is worth noting that there was no statistically significant difference between the EC group and the control group, indicating that the EC group did not harm zebrafish embryos. EC significantly inhibited MEZ-induced hepatocyte lipid droplet accumulation ( Figure 6 To further evaluate the inhibitory effect of EC on MEZ-induced cell apoptosis, AO staining was used to observe cell apoptosis ( Figure 6 In B). Compared with the MEZ group, the apoptotic cell counts in the adjacent liver areas of the MEZ+EC group were significantly reduced (p<0.001) ( Figure 6C in the figure). The present invention detected the effect of EC on the expression of apoptosis-related genes. qRT-PCR verification of key apoptosis-related genes showed that EC17 significantly restored the expression levels of bax, bcl2 and p53 inhibited by MEZ exposure ( Figure 6 DF in). Therefore, EC has an inhibitory effect on MEZ-induced hepatotoxicity by regulating cell apoptosis. MEZ-induced YSE disrupts the swimming balance of zebrafish embryos. Therefore, the present invention evaluates the effect of EC on zebrafish locomotion recovery by observing and analyzing swimming behavior ( Figure 7 Four groups were analyzed: control group; 2.5 mg·L -1 MEZ;15µm EC;2.5mg·L -1 MEZ+15µM EC group. The total movement distance and speed of embryos in the MEZ group were significantly lower than those in the control group (p<0.01). In addition, compared with the MEZ group, the MEZ+EC group showed recovery in terms of total movement distance and average speed ( Figure 7 B and C in the figure).

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of (-)-epicatechin or a composition comprising (-)-epicatechin for inhibiting toxicity induced by MEZ.

2. Use of (-)-epicatechin, or a composition comprising (-)-epicatechin, in the preparation of a medicament for inhibiting toxicity induced by MEZ.

3. The use according to claim 1 or 2, characterized in that: The toxicity induced by MEZ is developmental toxicity or hepatotoxicity.

4. The use according to claim 3, characterized in that: The developmental toxicity includes the following symptoms: developmental abnormalities and embryonic apoptosis.

5. The use according to claim 4, characterized in that: The developmental abnormalities include: developmental delay, shortened body length, reduced eye area, closure of the swim bladder, yolk sac edema, pericardial edema, spinal curvature, tail deformity, shortened mandible, delayed hatching, decreased embryo hatching rate, increased embryo mortality rate, embryo deformity or decreased motor level. One or more of the following.

6. The use according to claim 3, characterized in that: The hepatotoxicity includes: Delaying liver development, cholesterol and lipid metabolism disorders and liver damage.

7. The use according to claim 6, characterized in that: The delaying of liver development is to reduce the liver area; and / or, The cholesterol and lipid metabolism disorders include: abnormal levels of TCHO, TG and MDA, abnormal expression levels of hmgcra, hmgcrb, cyp2r1, cyp7a1, cyp51, less, dhcr7, pik3r3a, acox1, dgat2 and pparα, and accumulation of lipid droplets in hepatocytes; and / or, The liver damage includes: decreased gene expression levels of fabp2, prox1, gata6, hadc1 or hadc3, and decreased levels of AST and ALT.

8. A drug for inhibiting toxicity caused by MEZ, characterized in that include: (-)-Epicatechin.

9. The drug according to claim 8, characterized in that The concentration of the (-)-epicatechin is 5-15 μM.

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