目的 探究西红花提取物调控自噬和焦亡发挥缺血性脑卒中大鼠神经损伤的保护机制。方法 采用大鼠中脑动脉闭塞(middle cerebral artery acclusion,MCAO)模型,第1组:MCAO大鼠随机分为假手术组、模型组、西红花提取物低、中、高剂量组(3、6、12 mg·kg-1)、依达拉奉组(10 mg·kg-1)。造模7 d后进行神经功能缺陷评分,并采用2,3,5-三苯基氯化四氮唑(TTC)染色检测脑梗死体积。Western blot检测脑组织自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、p62以及焦亡相关蛋白NLRP3、cleaved-caspase-1、IL-1β、IL-18表达。第2组MCAO大鼠随机分为:假手术组、模型组、西红花提取物高剂量(12 mg·kg-1)、西红花提取物高剂量+自噬抑制剂三甲基腺嘌呤组(3-MA,200 mmol·L-1,2 μL)、自噬抑制剂三甲基腺嘌呤组(3-MA)。MCAO大鼠腹腔注射西红花提取物(12 mg·kg-1),每日1次,持续7 d,并采用大鼠侧脑室注射自噬抑制剂3-MA进行干预。Zea-Longa法评估神经功能损伤,并采用TTC染色检测脑梗死体积;Western blot和免疫荧光检测自噬水平以及焦亡相关蛋白NLRP3、cleaved-caspase-1、IL-1β、IL-18表达。第3组MCAO大鼠随机分为:假手术组、模型组、西红花提取物高剂量(12 mg·kg-1)、caspase-1抑制剂Z-YVAD组(8 μg·kg-1,2 μL)、西红花提取物高剂量+Z-YVAD组。Western blot和免疫荧光检测脑组织cleaved-caspase-1及神经元LC3阳性表达率。结果 与假手术组相比,模型组大鼠神经功能评分增加,脑梗死体积增大,LC3-Ⅱ/LC3-Ⅰ比值、NLRP3、cleaved-aspase-1、IL-1β、IL-18表达升高(P<0.01),p62表达降低(P<0.01)。与模型组相比,各剂量西红花提取物组大鼠神经功能评分和脑梗死体积均明显降低(P<0.05,P<0.01),中、高剂量西红花提取物组LC3-Ⅱ/LC3-Ⅰ比值升高,p62和焦亡相关蛋白表达降低(P<0.05,P<0.01)。与模型组和西红提取物组相比,大鼠侧脑室注射3-MA能够抑制自噬水平,诱导细胞焦亡相关蛋白表达,增大脑梗死体积,加重脑神经功能损伤。MCAO大鼠脑组织高表达cleaved-caspase-1能够被caspase-1抑制剂Z-YVAD阻断,但对神经元LC3阳性表达率无显著性影响(P>0.05)。结论 西红花提取物能够改善MCAO大鼠神经损伤,机制与促进细胞自噬、抑制NLRP3炎症小体诱发的细胞焦亡相关。
Abstract
OBJECTIVE To explore the protective mechanism of saffron extract on nerve injury in ischemic stroke rats through regulating autophagy and pyroptosis. METHODS Rat middle cerebral artery occlusion (MCAO) model was used. Group 1: MCAO rats were randomly divided into sham operation group, model group, low, medium and high dose groups of saffron extract (3, 6, 12 mg·kg-1), edaravone group (10 mg·kg-1). Seven days after modeling, neurological deficits were scored, and TTC staining was used to detect the volume of cerebral infarction. Western blot was used to detect the expressions of autophagy-related proteins LC3-Ⅱ/LC3-Ⅰ, p62 and pyroptosis-related proteins NLRP3, cleaved-caspase-1, IL-1β and IL-18 in brain tissue. Group 2: MCAO rats were randomly divided into sham operation group, model group, high dose of saffron extract (12 mg·kg-1), high dose of saffron extract+autophagy inhibitor trimethyladenine group (3-MA, 200 mmol·L-1, 2 μL), autophagy inhibitor trimethyladenine group (3-MA). MCAO rats were intraperitoneally injected with saffron extract (12 mg·kg-1), once a day, for 7 days, and the autophagy inhibitor 3-MA was injected into the lateral ventricle of rats for intervention. Neurological damage was assessed by Zea-Longa method, and cerebral infarct volume was detected by TTC staining. Western blot and immunofluorescence were used to detect the level of autophagy and the expressions of pyroptosis-related proteins NLRP3, cleaved-caspase-1, IL-1β, and IL-18. Group 3: MCAO rats were randomly divided into sham operation group, model group, high dose of saffron extract (12 mg·kg-1), caspase-1 inhibitor Z-YVAD group (8 μg·kg-1, 2 μL), high dose crocus extract+Z-YVAD group. Western blot and immunofluorescence were used to detect the expression of cleaved caspase-1 in brain tissue and the positive rate of neuron LC3. RESULTS Compared with the sham-operated group, the rats in the model group had increased neurological function scores, cerebral infarction volume LC3-Ⅱ/LC3-Ⅰ ratio, and increased expressions of NLRP3, cleaved-caspase-1, IL-1β, and IL-18 (P<0.01), while p62 expression decreased (P<0.01). Compared with the model group, the neurological function score and cerebral infarction volume of the rats were significantly decreased in each dose of saffron extract group (P<0.05, P<0.01), the ratio of LC3-Ⅱ/LC3-Ⅰ increased, and the expressions of p62 and pyroptosis-related proteins decreased in the middle and high doses of saffron extract groups (P<0.05, P<0.01). Compared with the model group and saffron extract group, injection of 3-MA into the lateral ventricle of rats could inhibit the level of autophagy, induce the expression of pyroptosis-related proteins, increase the volume of cerebral infarction, and aggravate the damage of cerebral nerve function. The high expression of cleaved caspase-1 in brain tissue of MCAO rats could be blocked by caspase-1 inhibitor Z-YVAD but had no significant effect on the positive expression rate of LC3 in neurons (P>0.05). CONCLUSION Saffron extract can improve nerve injury in MCAO rats, and its mechanism is related to promoting autophagy and inhibiting NLRP3 inflammation-induced cell pyroptosis.
关键词
西红花提取物 /
缺血性脑卒中 /
自噬 /
焦亡 /
神经损伤
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Key words
saffron extract /
ischemic stroke /
autophagy /
pyroptosis /
nerve injury
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脚注
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基金
河南省中医药科学研究专项课题资助(2019zY1020);河南省医学科技攻关计划项目资助(2018020776)
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