Fujian Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108, P.R. China.
College of Rehabilitation Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108, P.R. China.
Int J Mol Med. 2014 Mar;33(3):597-604. doi: 10.3892/ijmm.2013.1612. Epub 2013 Dec 30.
Gua Lou Gui Zhi decoction (GLGZD), a traditional Chinese medicine consisting of different herbal medicines, has been used for centuries in the treatment of muscular spasticity following stroke, epilepsy or spinal cord injury. However, the precise mechanisms involved remain poorly understood. In the present study, we investigated the neuroprotective effects of GLGZD on glutamate-induced apoptosis in cultured BV-2 cells, as well as the underlying mechanisms. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to assess the viability of the cells. An Annexin V/propidium iodide (PI) assay was utilized to analyze cellular apoptosis. Mitochondrial membrane potential (MMP) was evaluated by flow cytometry and laser scanning confocal microscopy. The gene and protein expression of the apoptosis-related genes, Bcl-2 and Bax, was analyzed by RT-PCR and western blot analysis, respectively. Furthermore, the expression of cleaved caspase-3 protein was detected by immunofluorescence. Glutamate treatment induced the loss of BV-2 cell viability, which was associated with an increase in the apoptotic rate, as well as an increase in the Bax/Bcl-2 ratio and the extracellular levels of cleaved caspase-3. Treatment with GLGZD significantly reversed these phenotypes, with its maximum protective effects observed at the concentration of 1,000 µg/ml. These results indicate that GLGZD protects BV-2 cells from glutamate-induced cytotoxicity. These protective effects may be ascribed to its anti-apoptotic activities, in part, associated with the decrease in the Bax/Bcl-2 ratio and caspase-3 expression, as well as with the stability of high mitochondrial membrane potential.
瓜蒌桂枝汤（GLGZD）是一种由多种草药组成的中药，几个世纪以来一直用于治疗中风、癫痫或脊髓损伤后的肌肉痉挛。然而，其确切的作用机制仍知之甚少。在本研究中，我们研究了 GLGZD 对谷氨酸诱导的培养 BV-2 细胞凋亡的神经保护作用及其潜在机制。应用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法评估细胞活力。用 Annexin V/碘化丙啶（PI）法分析细胞凋亡。通过流式细胞术和激光扫描共聚焦显微镜评估线粒体膜电位（MMP）。用 RT-PCR 和 Western blot 分析分别分析凋亡相关基因 Bcl-2 和 Bax 的基因和蛋白表达。此外，通过免疫荧光检测裂解的 caspase-3 蛋白的表达。谷氨酸处理诱导 BV-2 细胞活力丧失，这与凋亡率增加、Bax/Bcl-2 比值增加以及细胞外裂解的 caspase-3 水平增加有关。GLGZD 治疗显著逆转了这些表型，在 1000μg/ml 的浓度下观察到最大的保护作用。这些结果表明 GLGZD 可保护 BV-2 细胞免受谷氨酸诱导的细胞毒性。这些保护作用可能归因于其抗凋亡活性，部分与 Bax/Bcl-2 比值和 caspase-3 表达降低以及高线粒体膜电位的稳定性有关。
