Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 20032, China.
J Ethnopharmacol. 2021 Apr 6;269:113669. doi: 10.1016/j.jep.2020.113669. Epub 2020 Dec 15.
Tiao Geng (TG) decoction is a Chinese herbal medicine extract that has been utilized for the treatment of menopausal symptoms for a history of over 30 years. In our previous study, we suggest that TG decoction possibly exerts an anti-apoptotic effect on hypothalamic neurons of ovariectomized rats via the ASK1/MKK7/JNK pathway. Tributyltin chloride (TBTC) causes oxidative damage and induces apoptosis of primary hypothalamic neurons in rats.
The present work aimed to explore the inhibition of TG decoction on TBTC-induced GT1-7 cell apoptosis and its possible molecular mechanism.
The GT1-7 cell line was exposed to TG decoction at diverse doses (31.25, 62.5, 125 μg/mL) for 24 h and later with TBTC (1 mg/L) for 1 h, with 17β-E (100 nM) treatment being the positive control. Then, CCK8 assay was conducted to evaluate cell viability, while flow cytometric analysis was conducted to examine the apoptosis level. Related pathways and differentially expressed proteins were identified by tandem mass tag (TMT)-based quantitative phosphoproteomics. qRT-PCR was carried out to examine mRNA levels of Bax and B-cell lymphoma-2 (Bcl-2). Western blotting was performed to detect the levels of Bax, Bcl-2, c-Jun, c-Jun N-terminal kinase (JNK), Caspase-3 (Casp3), Mitogen-activated protein kinase kinase 7 (MKK7), and apoptosis signal-regulating kinase 1 (ASK1) . Finally, cells were pretreated with SP600125, an inhibitor of JNK, later the expression of JNK and Casp3 was measured.
Application of TG decoction mitigated the GT1-7 cell apoptosis and injury caused by TBTC; besides, it inhibited the activation of the ASK1/MKK7/JNK pathway. Moreover, Bcl-2/Bax ratio became higher, and the MKK7, ASK1, Casp3 and c-Jun levels were inhibited. Besides, TG decoction combined with SP600125 (the JNK inhibitor) more significantly inhibited GT1-7 cell apoptosis caused by TBTC.
As discovered from the experiment in this study, TG decoction has a neuroprotective effect, which is achieved through inhibiting the ASK1/MKK7/JNK signal transduction pathway to reduce GT1-7 cell apoptosis.
调更（TG）汤是一种中药提取物，已被用于治疗更年期症状超过 30 年。在我们之前的研究中，我们认为 TG 汤可能通过 ASK1/MKK7/JNK 途径对去卵巢大鼠下丘脑神经元发挥抗凋亡作用。三丁基锡氯化物（TBTC）可引起氧化损伤，并诱导大鼠原代下丘脑神经元凋亡。
本研究旨在探讨 TG 汤对 TBTC 诱导的 GT1-7 细胞凋亡的抑制作用及其可能的分子机制。
将 GT1-7 细胞系用不同剂量（31.25、62.5、125μg/ml）的 TG 汤处理 24 小时，然后用 1mg/L 的 TBTC 处理 1 小时，以 100nM 17β-E 处理作为阳性对照。然后，通过 CCK8 测定评估细胞活力，通过流式细胞术分析检测细胞凋亡水平。通过串联质量标签（TMT）定量磷酸蛋白质组学鉴定相关途径和差异表达蛋白。通过 qRT-PCR 检测 Bax 和 B 细胞淋巴瘤-2（Bcl-2）的 mRNA 水平。通过 Western blot 检测 Bax、Bcl-2、c-Jun、c-Jun N 端激酶（JNK）、半胱天冬酶-3（Casp3）、丝裂原活化蛋白激酶激酶 7（MKK7）和凋亡信号调节激酶 1（ASK1）的水平。最后，用 JNK 抑制剂 SP600125 预处理细胞，然后测量 JNK 和 Casp3 的表达。
TG 汤减轻了 TBTC 引起的 GT1-7 细胞凋亡和损伤；此外，它抑制了 ASK1/MKK7/JNK 通路的激活。此外，Bcl-2/Bax 比值升高，MKK7、ASK1、Casp3 和 c-Jun 水平受到抑制。此外，TG 汤与 SP600125（JNK 抑制剂）联合应用可更显著抑制 TBTC 引起的 GT1-7 细胞凋亡。
本研究实验发现，TG 汤具有神经保护作用，通过抑制 ASK1/MKK7/JNK 信号转导通路减少 GT1-7 细胞凋亡来实现。
