穗花牡荆总黄酮通过线粒体依赖性途径保护星形胶质细胞免受 HO 诱导的凋亡。
Total flavones of Dracocephalum moldavica L. protect astrocytes against HO-induced apoptosis through a mitochondria-dependent pathway.
机构信息
Xinjiang Key Laboratory of Uighur Medicines, Xinjiang Institute of Materia Medica, 140 Xinhua South Road, Urumchi, 830004, Xinjiang, China.
Xinjiang Medical University, Urumchi, 830000, Xinjiang, China.
出版信息
BMC Complement Med Ther. 2020 Mar 12;20(1):78. doi: 10.1186/s12906-020-2846-4.
BACKGROUND
The active components of Dracocephalum moldavica L. (TFDM) can inhibit myocardial ischemia by inhibiting oxidative stress. However, the effects of TFDM on astrocytes have not been investigated in vitro. The current study aimed to explore whether TFDM protects astrocytes against HO-induced apoptosis through a mitochondria-dependent pathway.
METHODS
The human glioma cell line U87 was used to investigate the ability of TFDM to protect astrocytes against HO-induced apoptosis. The cell counting kit-8 assay and flow cytometry were used to detect cell viability, apoptosis, MMP, Ca influx and reactive oxygen species (ROS). Lactate dehydrogenase (LDH) and malonic dialdehyde (MDA) levels were measured by ELISA. In addition, protein and mRNA expression changes were detected by Western blotting and qRT-PCR.
RESULTS
TFDM (0.78~200 μg/ml) had limited cytotoxic effects on the viability of U87 cells. Compared with the model group (treated with H2O2 only), cells treated with medium- and high-dose TFDM exhibited reduced MDA concentrations (P < 0.05) and ROS production (P < 0.05) and decreased MMP (P < 0.05) and reduced apoptosis (P < 0.05). The percentage of annexin V-FITC-stained cells was markedly suppressed by TFDM, confirming its anti-apoptotic properties. WB results showed that protein expression of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, Caspase-12, and B-cell leukemia/lymphoma 2 (Bcl2) was reduced in the TFDM group compared with that in the model group (P < 0.05) and that expression of these proteins was normalized by TFDM treatment in a dose-dependent manner. According to RT-qPCR results, TFDM pretreatment resulted in reduced mRNA expression of BAX, Caspase-9, Caspase-12, p38MAPK, and CaMKII and increased mRNA expression of mTOR compared with the model group.
CONCLUSIONS
The current study revealed the protective effects of TFDM on U87 cells under oxidative stress conditions through the inhibition of a mitochondria-dependent pathway that is associated with the CaMKII/P38MAPK/ERK1/2 and PI3K/AKT/mTOR pathways.
背景
穗花牡荆（TFDM）的活性成分可通过抑制氧化应激抑制心肌缺血。然而，TFDM 对体外星形胶质细胞的影响尚未得到研究。本研究旨在探讨 TFDM 是否通过线粒体依赖性途径保护星形胶质细胞免受 HO 诱导的凋亡。
方法
用人神经胶质瘤细胞系 U87 研究 TFDM 保护星形胶质细胞免受 HO 诱导凋亡的能力。细胞计数试剂盒-8 检测法和流式细胞术检测细胞活力、凋亡、MMP、Ca 内流和活性氧（ROS）。通过 ELISA 测定乳酸脱氢酶（LDH）和丙二醛（MDA）水平。此外，通过 Western 印迹和 qRT-PCR 检测蛋白和 mRNA 表达变化。
结果
TFDM（0.78~200μg/ml）对 U87 细胞活力的细胞毒性作用有限。与模型组（仅用 H2O2 处理）相比，中、高剂量 TFDM 处理组 MDA 浓度降低（P<0.05），ROS 生成减少（P<0.05），MMP 降低（P<0.05），凋亡减少（P<0.05）。TFDM 显著抑制 Annexin V-FITC 染色细胞的百分比，证实其具有抗凋亡作用。WB 结果显示，与模型组相比，TFDM 组 Bcl-2 相关 X 蛋白（BAX）、Caspase-3、Caspase-9、Caspase-12 和 B 细胞白血病/淋巴瘤 2（Bcl2）的蛋白表达减少，并且 TFDM 处理以剂量依赖性方式使这些蛋白的表达正常化。根据 RT-qPCR 结果，TFDM 预处理可降低模型组 Bax、Caspase-9、Caspase-12、p38MAPK 和 CaMKII 的 mRNA 表达，并增加 mTOR 的 mRNA 表达。
结论
本研究揭示了 TFDM 通过抑制与 CaMKII/P38MAPK/ERK1/2 和 PI3K/AKT/mTOR 通路相关的线粒体依赖性途径，对 U87 细胞在氧化应激条件下的保护作用。
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