ABSTRACT

Betaine is a bioactive peptide whose beneficial effects on diabetes complications have been considered, previously. The present study aimed to investigate the possible protective effects of betaine against hyperglycemia-induced steroidogenesis impairment and apoptosis in mice granulosa cells. Ovarian granulosa cells were isolated from C57/BL6 mice and cultured in steroidogenesis medium (SM) containing 30 ng/ml FSH and 0.5 µM testosterone. The cells were cultured in SM containing low (5 mM) or high (30 mM) glucose concentrations for 24 h in the presence or absence of betaine (5 mM). At the end of the experiment, estradiol and progesterone were measured by ELISA in the culture medium. Expression of apoptosis and steroidogenesis associated genes and caspase-3 activity were determined by qRT-PCR and colorimetric assays, respectively. Exposure of mice granulosa cells to high glucose concentration inhibited the steroidogenesis by decreasing estradiol and progesterone secretion and downregulation of steroidogenesis-related genes including 3βHSD, Cyp11a1, Cyp19a1, and StAR. Betaine treatment could ameliorate the steroidogenesis impairment at molecular and biochemical levels. High glucose concentration also enhanced apoptosis in mice granulosa cells that were characterized by elevation of caspase-3 activity, upregulation of bax gene and downregulation of bcl2 gene. Betaine treatment could attenuate the apoptotic-related changes induced by high glucose concentration in granulosa cells. According to the results of the present study, betaine could ameliorate the adverse effects of hyperglycemia on the physiological function of ovarian granulosa cells. The results highlight the potential role of betaine for the intervention of ovarian dysfunction in diabetic patients.

Abbreviations: AABA: Betaine-α-aminobutyric acid; AGEs: Advanced glycation end products; bax: bcl2 Associated X; bcl2: B-cell lymphoma 2; AMPK: AMP-activated protein kinase; BHMT: Betaine homocysteine methyltransferase; C/EBP: CCAAT-enhancer-binding proteins; Cyp11a1: Cholesterol side-chain cleavage cytochrome P450; Cyp19a1: Cytochrome P450 aromatase; DM: Diabetes mellitus; E2: Estradiol; ERS: Endoplasmic reticulum stress; GCs: Granulosa cells; GLUT: Glucose transporter; FSH: Follicle-stimulating hormone; 3βHSD: 3β-hydroxysteroid dehydrogenase; IL-1β: interleukin-1ß; LH: Luteinizing hormone; MDCK: Madin-Darby Canine Kidney cell; MT: Methionine synthase, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NLRP3: NLR Family Pyrin Domain Containing 3; NF-κB: Nuclear factor κB; P4: Progesterone; ROS: Reactive oxygen species; SGLT: Sodium dependent glucose transporter; SLC7A6: Solute Carrier Family 7 Member 6; StAR: Steroidogenic acute regulatory protein; STZ: Streptozotocin; Tumor necrosis factor α: TNF-α; TXNIP: Thioredoxin interacting protein.

摘要

甜菜碱是一种生物活性肽，以前曾考虑过其对糖尿病并发症的有益作用。本研究旨在研究甜菜碱对小鼠颗粒细胞中高血糖诱导的类固醇生成障碍和细胞凋亡的可能保护作用。从 C57/BL6 小鼠中分离出卵巢颗粒细胞，并在含有 30 ng/ml FSH 和 0.5 µM 睾酮的类固醇生成培养基 (SM) 中培养。在甜菜碱 (5 mM) 存在或不存在的情况下，将细胞在含有低 (5 mM) 或高 (30 mM) 葡萄糖浓度的 SM 中培养 24 小时。在实验结束时，通过ELISA测量培养基中的雌二醇和孕酮。细胞凋亡和类固醇生成相关基因的表达和 caspase-3 活性分别通过 qRT-PCR 和比色测定确定。3βHSD、Cyp11a1、Cyp19a1和StAR。甜菜碱治疗可以在分子和生化水平上改善类固醇生成障碍。高葡萄糖浓度还增强了小鼠颗粒细胞的凋亡，其特征是 caspase-3 活性升高、bax基因上调和bcl2基因下调。甜菜碱处理可以减弱颗粒细胞中高葡萄糖浓度引起的凋亡相关变化。根据本研究结果，甜菜碱可改善高血糖对卵巢颗粒细胞生理功能的不良影响。结果突出了甜菜碱在干预糖尿病患者卵巢功能障碍方面的潜在作用。

缩写：AABA：甜菜碱-α-氨基丁酸；AGEs：晚期糖基化终产物；bax：bcl2关联 X；bcl2：B 细胞淋巴瘤 2；AMPK：AMP活化蛋白激酶；BHMT：甜菜碱同型半胱氨酸甲基转移酶；C/EBP：CCAAT 增强子结合蛋白；Cyp11a1：胆固醇侧链裂解细胞色素 P450；Cyp19a1：细胞色素 P450 芳香化酶；DM：糖尿病；E2：雌二醇；ERS：内质网应激；GCs：颗粒细胞；GLUT：葡萄糖转运蛋白；FSH：促卵泡激素；3βHSD：3β-羟基类固醇脱氢酶；IL-1β：白细胞介素-1β；LH：黄体生成素；MDCK：Madin-Darby 犬肾细胞；MT：甲硫氨酸合酶，MTT：3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑；NLRP3：NLR 家族 Pyrin 域包含 3；NF-κB：核因子κB；P4：孕酮；ROS：活性氧；SGLT：钠依赖性葡萄糖转运蛋白；SLC7A6：溶质载体家族 7 成员 6；StAR：类固醇生成急性调节蛋白；STZ：链脲佐菌素；肿瘤坏死因子α：TNF-α；TXNIP：硫氧还蛋白相互作用蛋白。