Yan Min, Liu Ai-Ling, Zhou Shu-Jia, Tang Li-Peng, Ou Yan-Qiu, Yin Wei, Chen Xin-Ying, Su Xing-Wen, Qiu Peng-Xin, Huang Yi-Jun, Zhang Jing-Xia, Yan Guang-Mei, Leng Tian-Dong
Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, China.
School of pharmaceutical sciences, Sun Yat-Sen University, Guangzhou, China.
CNS Neurosci Ther. 2015 Jun;21(6):486-95. doi: 10.1111/cns.12378. Epub 2015 Feb 13.
Neuroactive steroids represent promising candidates for the treatment of neurological disorders. Our previous studies identified an endogenous steroid cholestane-3β, 5α, 6β-triol (Triol) as a novel neuroprotectant.
We aimed to identify a potent candidate for stroke treatment through a screening of Triol analogs.
Hypoxia- and glutamate-induced neuronal injury models in vitro, middle cerebral artery occlusion (MCAO)-induced cerebral ischemia model in vivo, fluorescein diacetate (FDA) for alive and propidium iodide (PI) for dead staining, LDH assay, and calcium imaging techniques were used.
24-keto-cholest-5-en-3β, 19-diol (Diol) showed the most potent neuroprotective effect among the screened structurally related compounds. FDA and PI staining showed that Diol concentration dependently increased the survival rate of cerebellar granule neurons (CGNs) challenged with glutamate or hypoxia, with an effective threshold concentration of 2.5 μM. Consistently, the quantitative LDH release assay showed the same concentration-dependent protection in both models. Diol, at 10 μM, potently decreased glutamate- and hypoxia-induced LDH release from 51.6 to 18.2% and 62.1 to 21.7%, respectively, which values are close to the normal LDH release (~16-18%). Moreover, we found Diol effectively decreased MCAO-induced infarction volume in mice from ~23% to 7%, at a dose of 6 mg/kg. We further explored the underlying mechanism and found that Diol attenuated NMDA-induced intracellular calcium ([Ca(2+) ]i ) increase in cortical neurons, suggesting a negative modulatory effect on NMDA receptor.
Taken together, we identified Diol as a potent neuroprotectant. It may represent a novel and promising neuroprotectant for stroke intervention.
神经活性甾体是治疗神经系统疾病的有潜力的候选药物。我们之前的研究确定内源性甾体胆甾烷-3β,5α,6β-三醇（三醇）为一种新型神经保护剂。
我们旨在通过筛选三醇类似物来确定一种有效的中风治疗候选药物。
采用体外缺氧和谷氨酸诱导的神经元损伤模型、体内大脑中动脉闭塞（MCAO）诱导的脑缺血模型、活细胞用二乙酸荧光素（FDA）染色和死细胞用碘化丙啶（PI）染色、乳酸脱氢酶（LDH）测定以及钙成像技术。
在筛选的结构相关化合物中，24-酮胆甾-5-烯-3β,19-二醇（二醇）显示出最有效的神经保护作用。FDA和PI染色表明，二醇浓度依赖性地提高了受到谷氨酸或缺氧刺激的小脑颗粒神经元（CGNs）的存活率，有效阈值浓度为2.5μM。同样，定量LDH释放测定在两种模型中均显示出相同的浓度依赖性保护作用。10μM的二醇能有效降低谷氨酸和缺氧诱导的LDH释放，分别从51.6%降至18.2%和从62.1%降至21.7%，这些值接近正常的LDH释放水平（约16 - 18%）。此外，我们发现剂量为6mg/kg时，二醇能有效将小鼠MCAO诱导的梗死体积从约23%降至7%。我们进一步探究其潜在机制，发现二醇可减弱NMDA诱导的皮质神经元细胞内钙（[Ca(2 +)]i）升高，提示其对NMDA受体有负性调节作用。
综上所述，我们确定二醇为一种有效的神经保护剂。它可能是一种用于中风干预的新型且有前景的神经保护剂。
