目的 基于受体相互作用蛋白激酶1（receptor interacting protein kinase 1，RIP1）/RIP3/混合谱系激酶结构域样蛋白（mixed lineage kinase domain-like protein，MLKL）通路探讨桑色素对缺血性脑卒中（ischemic stroke，IS）大鼠神经元坏死性凋亡的影响。方法 采用改良Zea-Longa法建立IS大鼠模型，将造模成功的大鼠随机分为模型组及桑色素低、高剂量（15、30 mg/kg）组和依达拉奉（1.35 mL/kg）组、桑色素（30 mg/kg）＋重组蛋白RIP1（recombinant protein RIP1，rRIP1，8 µg/kg）组，每组10只，另取10只正常大鼠作为假手术组。给予药物干预1周后，对大鼠进行神经功能损伤评分，测定脑梗死面积比；检测血清中炎症因子水平；采用苏木素-伊红（hematoxylin eosin，HE）和Nissl染色检测脑组织病理变化及神经元数目；采用免疫组化法检测脑组织小胶质细胞数量；采用免疫荧光法检测脑组织神经元核抗原（neuronal nucleoprotein，NeuN）表达；采用TUNEL染色检测神经元细胞凋亡情况；采用qRT-PCR检测脑组织B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、活化的半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3，cleaved Caspase-3）mRNA表达；采用Western blotting检测脑组织RIP1/RIP3/MLKL通路相关蛋白表达。结果 与模型组比较，桑色素组神经功能损伤评分及血清中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）水平和脑梗死面积百分比、脑组织小胶质细胞数、神经元细胞凋亡率显著降低（P＜0.05），血清中IL-10水平、神经元数和NeuN表达显著升高（P＜0.05），脑组织Bax mRNA表达、磷酸化RIP1（phosphorylated RIP1，p-RIP1）/RIP1、RIP3、MLKL蛋白表达水平显著下调（P＜0.05），cleaved Caspase-3、Bcl-2 mRNA表达显著上调（P＜0.05）。rRIP1可以显著逆转桑色素对IS大鼠的保护作用（P＜0.05）。结论 桑色素通过抑制RIP1/RIP3/MLKL通路改善IS大鼠神经元坏死性凋亡。

Objective To explore the effect of morin on neuronal necrotic apoptosis in rats with ischemic stroke (IS) based on receptor interacting protein kinase 1 (RIP1)/RIP3/mixed lineage kinase domain like protein (MLKL) pathway. Methods An improved Zea-Longa method was used to establish an IS rat model. The successfully modeled rats were randomly divided into model group, morin low-, high-dose (15, 30 mg/kg) groups, edaravone (1.35 mL/kg) group and morin (30 mg/kg) + recombinant protein RIP1 (rRIP1, 8 µg/kg) group, with 10 rats in each group. Additionally, 10 normal rats were selected as sham group. After one week of drug intervention, rats were evaluated for neurological damage and the infarct area ratio was measured; The levels of inflammatory factors in serum was detected; Hematoxylin-eosin (HE) and Nissl staining were used to detect pathological changes and neuronal numbers in brain tissue; Immunohistochemistry was used to detect the number of microglia in brain tissue; Immunofluorescence method was used to detect the expression of neuronal nuclear protein (NeuN) in brain tissue; TUNEL staining was used to detect neuronal cell apoptosis; qRT-PCR was used to detect the mRNA expressions of B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and activated cysteine aspartate protease-3 (cleaved Caspase-3) in brain tissue; Western blotting was used to detect the expressions of RIP1/RIP3/MLKL pathway related proteins in brain tissue. Results Compared with model group, the neurological function damage score, levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) in serum, percentage of cerebral infarction area, number of microglia in brain tissue and neuronal apoptosis rate were significantly reduced of rats in morin group (P < 0.05), the level of IL-10 in serum, number of neurons and NeuN expression were significantly increased (P < 0.05), expression levels of Bax mRNA, phosphorylated RIP1(p-RIP1)/ RIP1, RIP3 and MLKL proteins in brain tissue were significantly down-regulated (P < 0.05), and expressions of cleaved Caspase-3 and Bcl-2 mRNA were significantly up-regulated (P < 0.05). rRIP1 could significantly reverse the protective effect of morin on IS rats (P < 0.05). Conclusion Morin improve neuronal necrotic apoptosis in IS rats by inhibiting RIP1/RIP3/MLKL pathway.

R285.5

河南省中医药传承与创新人才工程（仲景工程）中医药拔尖人才培养项目（豫卫中医函[2021]15号）；河南省国家中医药传承创新中心科研专项（2023ZXZX1042）；河南省中医药科学研究专项课题（2023ZY3001）