Department of Microbiology and Immunology, Medical School of Southeast University, Nanjing, China.
Department of Pharmacology, Poonch Medical College, Rawalakot Azad Jammu & Kashmir, Pakistan.
Biomed Res Int. 2022 May 29;2022:1557010. doi: 10.1155/2022/1557010. eCollection 2022.
Ischemic stroke is a devastating CNS insult with few clinical cures. Poor understanding of underlying mechanistic network is the primary limitation to develop novel curative therapies. Extracellular accumulation of granzyme B subsequent ischemia promotes neurodegeneration. Inhibition of granzyme B can be one of the potent strategies to mitigate neuronal damage. In present study, we investigated the effect of murine Serpina3n and human (homolog) SERPINA3 against cerebral ischemia through granzyme B inactivation. Recombinant Serpina3n/SERPINA3 were expressed by transfected 293 T cells, and eluted proteins were examined for postischemic influence both and . During test, Serpina3n was found effective enough to inhibit granzyme B, while SERPINA3 was ineffectual to counter cytotoxic protease. Treatment of hypoxic culture with recombinant Serpina3n/SERPINA3 significantly increased cell viability in dosage-dependent manner, recorded maximum at the highest concentration (4 mM). Infarct volume analysis confirmed that 50 mg/kg dosage of exogenous Serpina3n was adequate to reduce disease severity, while SERPINA3 lacked behind in analeptic effect. Immunohistochemical test, western blot analysis, and protease activity assay's results illustrated successful diffusion of applied protein to the ischemic lesion and reactivity with the target protease. Taken together, our findings demonstrate therapeutic potential of Serpina3n by interfering granzyme B-mediated neuronal death subsequent cerebral ischemia.
缺血性中风是一种具有很少临床治愈方法的中枢神经系统破坏性损伤。对潜在机制网络的理解不足是开发新的治疗方法的主要限制。缺血后细胞外 granzyme B 的积累会促进神经退行性变。抑制 granzyme B 可以是减轻神经元损伤的有效策略之一。在本研究中，我们通过 granzyme B 失活研究了鼠源 Serpina3n 和人源（同源）SERPINA3 对脑缺血的影响。通过转染的 293T 细胞表达重组 Serpina3n/SERPINA3，并用离体和在体实验检测缺血后对蛋白的影响。实验中发现，Serpina3n 能够有效抑制 granzyme B，而 SERPINA3 则不能抑制细胞毒性蛋白酶。用重组 Serpina3n/SERPINA3 处理缺氧培养物，以剂量依赖性方式显著增加细胞活力，在最高浓度（4 mM）时达到最大值。梗塞体积分析证实，50 mg/kg 剂量的外源性 Serpina3n 足以减轻疾病严重程度，而 SERPINA3 在复苏作用方面落后。免疫组织化学试验、western blot 分析和蛋白酶活性测定的结果表明，所应用的蛋白能够成功扩散到缺血性损伤部位并与靶蛋白酶发生反应。总之，我们的研究结果表明，Serpina3n 通过干扰缺血后 granzyme B 介导的神经元死亡具有治疗潜力。
