目的: 探究灵芝酸A（ganoderic acid A,GAA）对大鼠脑缺血/再灌注（ischemia/reperfusion,I/R）损伤的治疗作用和机制。方法: 采用大脑中动脉栓塞法构建I/R大鼠模型,将I/R大鼠分为模型组、低剂量组（0.5 mg/kg GAA）、中剂量组（1 mg/kg GAA）、高剂量组（2.5 mg/kg GAA）和激活剂组（2.5 mg/kg GAA+0.1 mg/kg LPS）,健康SD大鼠设置为假手术组。对大鼠神经功能损伤进行评分,TTC染色检测脑梗死体积,HE染色检测脑组织损伤,尼氏染色检测神经元损伤,TUNEL染色检测神经元凋亡,试剂盒检测脑组织中丙二醛（malondialdehyde,MDA）、超氧化物歧化酶（superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）和活性氧（reactive oxygen species,ROS）水平。Western blot检测白细胞介素（interleukin,IL）-1β、IL-18、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、神经元核抗原（neuronal nuclear,NeuN）、脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）、Toll样受体4（Toll-like receptor 4,TLR4）、核因子κB（nuclear factor kappa-B,NF-κB）、p-NF-κB和NLR家族Pyrin域蛋白3（NLR family pyrin domain containing 3,NLRP3）蛋白表达水平。结果: 与模型组比较,低、中和高剂量组大鼠神经元和尼氏体数量、脑梗死体积分数、神经功能评分和神经细胞凋亡率均降低,IL-1β、IL-18、TNF-α、TLR4、NLRP3和p-NF-κB蛋白水平均降低,NeuN和BDNF蛋白水平均升高;SOD和GSH-Px水平升高,MDA和ROS水平降低。与高剂量组比较,激活剂处理抑制了高剂量GAA对I/R大鼠的治疗作用。结论: GAA可以减少大鼠脑I/R损伤后神经细胞凋亡,抑制炎症和氧化应激反应,缓解脑损伤,该作用可能与抑制TLR4/NF-κB/NLRP3信号通路有关。
Abstract
Objective To explore the therapeutic effect and mechanism of Ganoderic acid A (GAA) on cerebral ischemia/reperfusion (I/R) injury in rats. Methods The I/R rat model was constructed by middle cerebral artery embolization. The I/R rats were divided into the model group, low-dose group (0.5 mg/kg GAA), medium-dose group (1 mg/kg GAA), high-dose group (2.5 mg/kg GAA) and the activator group (2.5 mg/kg GAA+0.1 mg/kg LPS), and the healthy SD rats were set as the sham operation group, with 15 rats in each group. The neurological function impairment of rats was scored, the volume of cerebral infarction was detected by TTC staining, brain tissue injury was detected by HE staining, neuronal injury was detected by Nissl staining, and neuronal apoptosis was detected by TUNEL staining. The levels of MDA, SOD, GSH-Px and ROS in rat brain tissues were detected by reagent kits. The protein expression levels of IL-1β, IL-18, TNF-α, NeuN, BDNF, TLR4, NF-κB, p-NF-κB and NLRP3 were detected by Western blot. Results Compared with the model group, the number of neurons and Nissl bodies, cerebral infarction volume fraction, neurological function score and neural cell apoptosis rate of rats in the low-dose, medium-dose and high-dose groups were all decreased, the protein levels of IL-1β, IL-18, TNF-α, TLR4, NLRP3 and p-NF-κB were all decreased, and the levels of NeuN and BDNF were all increased. The levels of SOD and GSH-Px were all increased, while the levels of MDA and ROS were all decreased. Compared with the high-dose group, activator treatment inhibited the therapeutic effect of high-dose GAA on I/R rats. Conclusion GAA has a good therapeutic effect on cerebral I/R injury in rats. GAA can reduce the apoptosis of nerve cells after cerebral I/R injury in rats, inhibit inflammation and oxidative stress responses, and alleviate brain injury. This effect may be related to the inhibition of the TLR4/NF-κB/NLRP3 signaling pathway.
关键词
脑缺血/再灌注 /
灵芝酸A /
神经元损伤 /
炎症 /
氧化应激 /
TLR4/NF-κB/NLRP3信号通路
Key words
cerebral ischemia/reperfusion (I/R) /
ganoderic acid A (GAA) /
neuronal injury /
inflammation /
oxidative stress /
TLR4/NF-κB/NLRP3 signaling pathway
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基金
陕西省自然科学基础研究计划项目“KIF18B通过Notch信号通路调控胶质瘤进展的分子机制研究”（2023-JC-YB-829）; 西安医学院博士科研启动基金项目“CSRP2在胶质瘤中的功能及作用机制研究”（2023BS01）
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