表没食子儿茶素没食子酸酯对高糖诱导的斑马鱼胰岛 β 细胞发育的影响及机制研究

Effect and mechanism of epigallocatechin gallate on the development of pancreatic β-cells induced by high glucose in zebrafish

摘要

探讨表没食子儿茶素没食子酸酯（epigallocatechin gallate，EGCG）对高糖诱导斑马鱼胰岛 β 细胞发育异常的干预作用及潜在机制。以 2% 葡萄糖建立高糖致斑马鱼胰岛 β 细胞发育损伤模型，分别用 10，50 μmol/L EGCG 预处理。观察斑马鱼死亡率、体长变化；RT-PCR 检测胰岛 β 细胞发育和功能相关基因的表达；荧光显微镜评估 Tg(ins:mCherry) 斑马鱼胰岛 β 细胞形态；试剂盒测定幼鱼体内葡萄糖、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPx）、过氧化氢酶（CAT）活性及丙二醛（MDA）和活性氧（ROS）水平。结果表明：高糖处理导致斑马鱼幼鱼死亡率升高、体长增加、调控 β 细胞发育的转录因子的表达持续下调，胰岛素 a 基因（insa）表达先升后降；并诱导胰腺碎片化和中空等畸形增加，胰岛面积变大并伴随体内葡萄糖含量升高；高糖处理还导致斑马鱼幼鱼体内 SOD、GPx 和 CAT 酶活异常，MDA 和 ROS 含量显著升高。而 50 μmol/L EGCG 干预显著地逆转上述异常，恢复 β 细胞正常形态与功能，并显著改善氧化应激状态。综上可知 EGCG 显著改善高糖诱导的斑马鱼胰岛 β 细胞发育异常，其机制可能与其改善斑马鱼机体的氧化应激和葡萄糖水平有关。

Abstract

This study aimed to elucidate the protective effect and underlying mechanism of epigallocatechin gallate (EGCG) against high glucose-induced pancreatic β-cell developmental anomalies in zebrafish. Zebrafish embryos were exposed to 2 % glucose to induce abnormal pancreatic β-cell development and were pretreated with 10 μmol/L or 50 μmol/L EGCG. Mortality and body length were recorded; and the expression of key genes related to pancreatic β-cell development and function of pancreatic β-cells was detected by RT-PCR. The morphology of pancreatic β-cells in Tg(ins:mCherry) zebrafish was examined under a fluorescent microscope; and glucose levels, as well as the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), were measured using commercial kits. The results showed that high-glucose treatment led to an increased mortality rate and body length in zebrafish larvae, along with sustained downregulation in the expression of transcription factors regulating β-cell development. The expression of the insulin a gene (insa) initially increased and then decreased. High-glucose treatment also induced an increase in pancreatic fragmentation and hollow malformations, enlarged islet area, and elevated glucose levels in the body. Furthermore, it caused abnormal activities of SOD, GPx, and CAT enzymes, along with a significant rise in MDA and ROS content in zebrafish larvae. However, intervention with 50 μmol/L EGCG significantly reversed these abnormalities, restored normal β-cell morphology and function, and notably improved the oxidative stress status. In conclusion, EGCG significantly ameliorates high glucose-induced abnormal development of pancreatic β-cells in zebrafish, and its mechanism may be related to the improvement of oxidative stress and glucose levels in zebrafish.

关键词

表没食子儿茶素没食子酸酯 / β 细胞 / 发育 / 氧化应激 / 妊娠期糖尿病

Key words

epigallocatechin gallate / β-cell / development / oxidative stress / gestational diabetes mellitus

参考文献

[1] O’Donnell KJ, Meaney MJ. Fetal origins of mental health: the developmental origins of health and disease hypothesis[J]. Am J Psychiatry, 2017, 174(4): 319-328.

[2] Firmin S, Bahi—Jaber N, Abdennebi—Najar L. Food contaminants and programming of type 2 diabetes: recent findings from animal studies[J]. J Dev Orig Health Dis, 2016, 7(5): 505-512.

[3] Agarwal P, Brar N, Morriseau TS, et al. Gestational diabetes adversely affects pancreatic islet architecture and function in the male rat offspring[J]. Endocrinology, 2019, 160(8): 1907-1925.

[4] Saucedo R, Ortega—Camarillo C, Ferreira—Hermosillo A, et al. Role of oxidative stress and inflammation in gestational diabetes mellitus[J]. Antioxidants (Basel), 2023, 12(10): 1812.

[5] Gerber PA, Rutter GA. The role of oxidative stress and hypoxia in pancreatic beta—cell dysfunction in diabetes mellitus[J]. Antioxid Redox Signal, 2017, 26(10): 501-518.

[6] Zhao J, Gao JR, Li H. Ferulic acid confers protection on islet β cells and placental tissues of rats with gestational diabetes mellitus[J]. Cell Mol Biol (Noisy—le—grand), 2020, 66(1): 37-41.

[7] Dong JY, Kimura T, Ikehara S, et al. Soy consumption and incidence of gestational diabetes mellitus: the Japan environment and children’s study[J]. Eur J Nutr, 2021, 60(2): 897-904.

[8] Kim HS, Quon MJ, Kim JA. New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3—gallate[J]. Redox Biol, 2014, 2: 187-195.

[9] Eng QY, Thanikachalam PV, Ramamurthy S. Molecular understanding of epigallocatechin gallate (EGCG) in cardiovascular and metabolic diseases[J]. J Ethnopharmacol, 2018, 210: 296-310.

[10] Shao YX, Zhang YH, Zou SY, et al. (−)—Epigallocatechin 3—gallate protects pancreatic β—cell against excessive autophagy—induced injury through promoting FTO degradation[J]. Autophagy, 2024, 20(11): 2460-2477.

[11] Mi JR, Ren LP, Andersson O. Leveraging zebrafish to investigate pancreatic development, regeneration, and diabetes[J]. Trends Mol Med, 2024, 30(10): 932-949.

[12] Huang J, Chen YL. Zebrafish as a preclinical model for diabetes mellitus and its complications: From monogenic to gestational diabetes and beyond[J]. World J Diabetes, 2025, 16(5): 100574.

[13] Wang C, Juan J, et al. A summary of Chinese guidelines on diagnosis and management of hyperglycemia in pregnancy (2022)[J]. Maternal Fetal Med, 2023, 5(1): 4-8.

[14] Pan FC, Brissova M. Pancreas development in humans[J]. Curr Opin Endocrinol Diabetes Obes, 2014, 21(2): 77-82.

[15] Singh A, Castillo HA, Brown J, et al. High glucose levels affect retinal patterning during zebrafish embryogenesis[J]. Sci Rep, 2019, 9(1): 4121.

[16] Titialii K, Morris A. Embryonic hyperglycemia causes a reduction in photoreceptor cells and increased oxidative stress in the developing retina[J]. FASEB J, 2020, 34(S1): 1.

[17] Li YQ, Chen QQ, Liu YN, et al. High glucose—induced ROS—accumulation in embryo—larval stages of zebrafish leads to mitochondria—mediated apoptosis[J]. Apoptosis, 2022, 27(7/8): 509-520.

[18] Lombó M, González—Rojo S, Fernández—Díez C, et al. Cardiogenesis impairment promoted by bisphenol A exposure is successfully counteracted by epigallocatechin gallate[J]. Environ Pollut, 2019, 246: 1008-1019.

[19] Tian YN. Potential mechanisms of butylparaben—induced cardiotoxicity in zebrafish at early developmental stages and the protective role of EGCG (EGCG 缓解对羟基苯甲酸丁酯斑马鱼胚胎心脏发育毒性及其分子机制研究)[D]. JinZhou Medical University(锦州医科大学), 2023.

[20] Hu BX, Dong X, Fan HW, et al. Evaluation of antiosteoporotic activity for micro—amount asperosaponins V and VI by on the osteoporosis model using zebrafish[J]. J China Pharm Univ(中国药科大学学报), 2019, 50(4): 452—458.

[21] Sant KE, Jacobs HM, Xu JL , et al. Assessment of toxicological perturbations and variants of pancreatic islet development in the zebrafish model[J]. Toxics, 2016, 4(3): 20.

[22] Jacobs HM, Sant KE, Basnet A, et al. Embryonic exposure to Mono(2—ethylhexyl) phthalate (MEHP) disrupts pancreatic organogenesis in zebrafish (Danio rerio) [J]. Chemosphere, 2018, 195: 498-507.

[23] Kahraman S, Dirice E, De Jesus DF, et al. Maternal insulin resistance and transient hyperglycemia impact the metabolic and endocrine phenotypes of offspring[J]. Am J Physiol Endocrinol Metab, 2014, 307(10): E906-E918.

[24] Hu F, Qiu XH, Bu SZ. Pancreatic islet dysfunction in type 2 diabetes mellitus[J]. Arch Physiol Biochem, 2020, 126(3): 235-241.

[25] Lushchak VI. Free radicals, reactive oxygen species, oxidative stress and its classification[J]. Chem Biol Interact, 2014, 224: 164-175.

[26] Wong CN, Lim YM, Liew KB, et al. A review on mechanistic actions of epigallocatechin—3—gallate in targeting the ominous octet of type 2 diabetes mellitus[J]. J Integr Med, 2025, 23(4): 344-356.

[27] Chu KO, Wang CC, Chu CY, et al. Uptake and distribution of catechins in fetal organs following in utero exposure in rats[J]. Hum Reprod, 2007, 22(1): 280-287.

基金资助

江苏省卫生健康委医学科研项目(H2023128)