活血荣络方调控Bmal1改善脑梗死后坏死性凋亡的机制研究

Mechanistic Study on Huoxue Rongluo Formula Mediated Bmal1 Regulation of Necroptosis After Ischemic Stroke

关键词:  脑梗死  生物节律  坏死性凋亡  炎症级联反应  活血荣络方

Key words:  cerebral infarction  biological rhythm  necroptosis  inflammatory cascade  huoxue rongluo formula

摘要:

摘要：目的 探讨活血荣络方调控Bmal1改善脑梗死后坏死性凋亡的作用机制。方法 将雄性C57BL/6小鼠分别进行大脑中动脉阻塞模型(middle cerebral artery occlusion，MCAO)及生物节律紊乱模型制备（environmental circadian disruption model，ECD），并随机分至假手术组、脑梗死模型组、节律紊乱复合模型组、活血荣络方低、中、高剂量组及依达拉奉组；采用神经功能缺损(mNSS)评分、2,3,5-氯化三苯基四氮唑(TTC)染色评价生物节律紊乱及活血荣络方对各组小鼠神经功能缺损症状及脑梗死体积的影响；采用苏木素-伊红(HE)染色、尼氏(Nissl)染色评估各组小鼠海马区病理损伤情况；利用酶联免疫吸附试验(ELISA)检测TNF-α、IL-1β、IL-6的水平；通过免疫荧光(IF)检测各组海马区Bmal1与p-RIPK3及p-MLKL的表达水平变化；采用蛋白免疫印迹法(Western blot)检测各组Bmal1、RIPK1、p-RIPK1、RIPK3、p-RIPK3、MLKL及p-MLKL蛋白表达差异情况。结果 节律紊乱复合模型组较脑梗死模型组mNSS评分、梗死体积明显增加(P<0.01)；海马区神经元、胶质细胞结构损伤显著、排列紊乱、空泡样坏死明显增多，Nissl小体数量明显减少(P<0.05)；炎症因子水平明显增加(P<0.01或P<0.05)；脑组织中相关蛋白表达水平有明显差异(P<0.001,P<0.01或P<0.05)。活血荣络方各组较节律紊乱复合模型组mNSS评分、梗死体积、海马区神经元、胶质细胞排列、Nissl小体数量均有不同程度的改善，其中活血荣络方高剂量组改善程度最明显(P<0.01或P<0.001)，炎症因子水平及相关蛋白的表达均有显著差异(P<0.001)。结论 生物节律紊乱可加重脑梗死后坏死性凋亡进程，活血荣络方可通过调控Bmal1改善脑梗死后坏死性凋亡，减轻脑梗死后的炎症级联反应。

Abstract:

ABSTRACT: OBJECTIVE To investigate the mechanism on Huoxue Rongluo Formula mediated Bmal1 regulation of Necroptosis after Ischemic Stroke. METHODS Male C57BL/6 mice were respectively used to prepare the middle cerebral artery occlusion (MCAO) and the environmental circadian disruption model(ECD), and were randomly assigned to the sham operation group (Sham), the model group (MCAO), the environmental circadian disruption combined with cerebral infarction model group (ECD-MCAO), the low, medium, and high-dose Huoxue Rongluo Formula groups(HXRLF-L, HXRLF-M, HXRLF-H), and the edaravone group(EDA) Neurological deficit (mNSS) score and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the effects of environmental circadian disruption model and Huoxue Rongluo Formula on neurological deficit symptoms and cerebral infarct volume in each group of mice. HE staining and Nissl staining were used to evaluate the pathological damage in the hippocampus of mice in each group. ELISA was used to detect the levels of TNF-α, IL-1β and IL-6. Immunofluorescence (IF) was used to detect the expression levels of Bmal1, p-RIPK3 and p-MLKL in the hippocampus. Western blot was used to detect the differences in protein expression of Bmal1, RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL and p-MLKL among the groups. RESULTS The mNSS score and infarct volume in the environmental circadian disruption combined with cerebral infarction model group were significantly increased (P<0.01),the structural damage, disordered arrangement and vacuolar necrosis of neurons and glial cells in the hippocampus were significantly increased, the number of Nissl bodies was significantly decreased (P<0.05),the level of inflammatory factors was significantly increased (P<0.01 or P<0.05),and the expression of related proteins in brain tissues was significantly different from that in the middle cerebral artery occlusion group (P<0.001,P<0.01 or P<0.05).Compared with the environmental circadian disruption combined with cerebral infarction model group group, the mNSS score, infarct volume, hippocampal neurons and glial cell arrangement were improved to varying degrees in the Huoxue Rongluo Formula group, among which the Huoxue Rongluo Formula- High group had the most obvious improvement (P<0.01 or P<0.001).,and there were significant differences in the number of Nissl bodies, inflammatory factor levels and the expression of related proteins (P<0.001). CONCLUSION Environmental circadian disruption can intensify the process of necrotizing apoptosis after cerebral infarction, and activating Huoxue Rongluo Formula can regulate necrotizing apoptosis after cerebral infarction by targeting Bmal1 to reduce the inflammatory cascade after cerebral infarction.