Characterization of a Synthetic Steroid 24‐keto‐cholest‐5‐en‐3β, 19‐diol as a Neuroprotectant

• Published in: CNS neuroscience & therapeutics 2015-06, Vol.21 (6), p.486-495
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Calcium - metabolism; Cell Hypoxia - drug effects; Cells, Cultured; Cerebellum - cytology; Cholestanols - chemistry; Cholestanols - therapeutic use; Disease Models, Animal; Excitatory Amino Acid Agonists - toxicity; Glutamic Acid - toxicity; Infarction, Middle Cerebral Artery - drug therapy; L-Lactate Dehydrogenase - metabolism; N-Methylaspartate - toxicity; Neurons - drug effects; Neuroprotection; Neuroprotective Agents - pharmacology; NMDA; Original; Oxygen - administration & dosage; Rats; Rats, Sprague-Dawley; Steroid; Stroke
• ISSN: 1755-5930; 1755-5949
• DOI: 10.1111/cns.12378

Summary Background 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. Aim We aimed to identify a potent candidate for stroke treatment through a screening of Triol analogs. Methods 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. Results 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 ([Ca2+]i) increase in cortical neurons, suggesting a negative modulatory effect on NMDA receptor. Conclusion Taken together, we identified Diol as a potent neuroprotectant. It may represent a novel and promising neuroprotectant for stroke intervention.