山柰酚调控Wnt/β-catenin信号通路对急性缺血性脑卒中大鼠的保护作用研究

Protective effect of kaempferol on rats with acute ischemic stroke by regulating Wnt/β-catenin signaling pathway

关键词:  山柰酚  急性缺血性脑卒中  神经功能  炎症  无翅型MMTV整合位点家族  β-连环蛋白

Key words:  kaempferol  acute ischemic stroke  neurological function  inflammation  wingless MMTV integration site family  β-catenin

摘要:

[目的] 探究山柰酚对急性缺血性脑卒中（AIS）大鼠神经功能、炎症、脑梗死体积及脑组织病理损伤的影响，并分析对无翅型MMTV整合位点家族（Wnt）/β-连环蛋白（β-catenin）信号通路的调控作用是否是其作用机制。[方法] 通过大脑中动脉栓塞再灌注法建立AIS大鼠模型，将造模成功的72只AIS大鼠随机分为模型组、山柰酚组（50 mg/kg）、Wnt/β-catenin通路抑制剂组（ICG-001，5 mg/kg）、山柰酚+Wnt/β-catenin通路抑制剂组（50 mg/kg+5 mg/kg），每组18只。另外选取18只大鼠作为假手术组（进行相同的手术操作，不插入线栓）。各组进行相应干预14 d。用Zea-Longa评分对大鼠神经功能损伤进行评价；酶联免疫吸附法测定血清炎症因子[肿瘤坏死因子-α（TNF-α）、单核细胞趋化蛋白-1（MCP-1）、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、白细胞介素-1β（IL-1β）]水平；2，3，5-氯化三苯基四氮唑染色测定大鼠脑梗死体积百分比；苏木精-伊红染色观察大鼠缺血侧皮质区脑组织病理变化；荧光定量PCR和蛋白免疫印迹法分别测定大鼠缺血侧皮质区脑组织Wnt1、Wnt3a、β-catenin信使RNA（mRNA）和蛋白表达。[结果] 给药14 d后，与假手术组比较，模型组大鼠缺血侧皮质区脑组织神经元核变形且固缩、排列较松散，细胞呈多边形，可见坏死细胞，神经功能损伤评分、血清TNF-α、MCP-1、NLRP3、IL-1β水平、脑梗死体积百分比显著升高，缺血侧皮质区脑组织Wnt1、Wnt3a、β-catenin mRNA和蛋白表达水平显著降低（P<0.05）。与模型组比较，山柰酚组大鼠缺血侧皮质区脑组织神经元核变形及固缩、排列松散和细胞坏死现象明显改善，神经功能损伤评分、血清TNF-α、MCP-1、NLRP3、IL-1β水平、脑梗死体积百分比显著降低，缺血侧皮质区脑组织Wnt1、Wnt3a、β-catenin mRNA和蛋白表达水平显著升高（P<0.05），Wnt/β-catenin通路抑制剂组大鼠缺血侧皮质区脑组织神经元核变形及固缩、排列松散和细胞坏死现象加重，神经功能损伤评分、血清TNF-α、MCP-1、NLRP3、IL-1β水平、脑梗死体积百分比显著升高，缺血侧皮质区脑组织Wnt1、Wnt3a、β-catenin mRNA和蛋白表达水平显著降低（P<0.05）。山柰酚+Wnt/β-catenin通路抑制剂组大鼠缺血侧皮质区脑组织病理损伤和上述定量指标较山柰酚组恶化，较Wnt/β-catenin通路抑制剂组改善（P<0.05）。[结论] 对Wnt/β-catenin信号通路的激活效果可能是山柰酚改善AIS大鼠神经功能、炎症、脑梗死体积及脑组织病理损伤的作用机制。

Abstract:

[Objective] To explore the effects of kaempferol on neurological function，inflammation，cerebral infarction volume and pathological damage of brain tissue in rats with acute ischemic stroke（AIS），and analyze whether the regulatory effect on the wingless MMTV integration site family（Wnt）/β-catenin signaling pathway is its mechanism of action. [Methods] The rat model of AIS was established by middle cerebral artery embolization and reperfusion. The 72 successfully modeled AIS rats were randomly divided into model group，kaempferol group （50 mg/kg），Wnt/β-catenin pathway inhibitor group（ICG-001，5 mg/kg），kaempferol+Wnt/β-catenin pathway inhibitor group（50 mg/kg + 5 mg/kg），with 18 rats in each group. Another 18 rats were selected as the sham operation group（performing the same surgical operations without inserting the suture plugs）. Each group received corresponding intervention for 14 days. The neurological function impairment of rats was evaluated by the Zea-Longa score；the levels of serum inflammatory factors [tumor necrosis factor-α（TNF-α），monocyte chemoattractant protein-1（MCP-1），NOD-like receptor thermal protein domain-associated protein 3（NLRP3），interleukin-1β （IL-1β）] were determined by enzyme-linked immunosorbent assay；2，3，5-triphenyltetrazolium chloride staining was used to determine the percentage of cerebral infarction volume in rats；hematoxylin-eosin staining was used to observe the pathological changes of brain tissue in the ischemic cortical area of rats；the expressions of Wnt1， Wnt3a，β-catenin messenger RNA（mRNA）and protein in the brain tissue of the ischemic cortical area of rats were determined by fluorescence quantitative PCR and Western blotting，respectively. [Results] After 14 days administration，compared with sham operation group，the neuronal nuclei in the brain tissue of the ischemic cortical area of rats in model group were deformed and constricted，the arrangement was looser，the cells were polygonal，and necrotic cells could be seen，the neurological function impairment score，the levels of serum TNF-α，MCP-1， NLRP3，IL-1β，and the percentage of cerebral infarction volume were significantly increased，the Wnt1，Wnt3a，β-catenin mRNA and protein expression levels in the brain tissue of the ischemic cortical area were significantly decreased（P<0.05）. Compared with model group，the phenomena of neuronal nucleus deformation and condensation，loose arrangement and cell necrosis in the brain tissue of the ischemic cortical area of rats in kaempferol group were significantly improved，the neurological function impairment score，the levels of serum TNF-α，MCP-1，NLRP3，IL-1β，and the percentage of cerebral infarction volume were significantly decreased，the Wnt1， Wnt3a，β-catenin mRNA and protein expression levels in the brain tissue of the ischemic cortical area were significantly increased（P<0.05）；in the Wnt/β-catenin pathway inhibitor group，the phenomena of neuronal nucleus deformation and condensation，loose arrangement and cell necrosis in the brain tissue of the ischemic cortical area of rats were aggravated，the neurological function impairment score，the levels of serum TNF-α，MCP-1，NLRP3，IL-1β，and the percentage of cerebral infarction volume were significantly increased，the Wnt1，Wnt3a，β-catenin mRNA and protein expression levels in the brain tissue of the ischemic cortical area were significantly decreased（P <0.05）. The pathological damage of brain tissue in the ischemic cortical area and the above quantitative indicators of rats in the kaempferol+Wnt/β-catenin pathway inhibitor group were worse than those in the kaempferol group and better than those in the Wnt/β-catenin pathway inhibitor group（P<0.05）. [Conclusion] The activation effect on the Wnt/β-catenin signaling pathway may be the mechanism by which kaempferol improves neurological function， inflammation，cerebral infarction volume and pathological damage of brain tissue in AIS rats.