基于TLR4/NF-κB通路探讨藏红花素对局灶性脑缺血再灌注损伤大鼠的神经保护作用

Neuroprotective Effect of Crocin on Rats with Focal Cerebral Ischemia-Reperfusion Injury Based on TLR4/NF-κB Pathway

目的:基于Toll样受体4(TLR4)/核因子-κB(NF-κB)通路研究藏红花素对局灶性脑缺血再灌注损伤(FCIR)大鼠的神经元保护作用,探讨藏红花素防治FCIR损伤可能的作用机制。方法:将128只SD大鼠按随机数字表法分为假手术组、模型组、藏红花素组、藏红花素+脂多糖组,每组32只。造模前7 d开始,藏红花素组通过腹腔注射藏红花素溶液;藏红花素+脂多糖组通过腹腔注射藏红花素溶液、脂多糖溶液;假手术组和模型组通过腹腔注射生理盐水。4组注射剂量均为5 mL/(kg·d),1次/d,连续干预7 d。采用线栓法复制FCIR大鼠模型。再灌注24 h后,采用改良神经功能缺损评分(mNSS)评价大鼠神经功能缺损状况;采用氯化三苯四氮唑(TTC)染色法、干湿重法分别计算脑梗死率和脑组织含水量;采用苏木精-伊红(HE)染色法、TUNEL染色法观察皮质神经元病理学改变和凋亡状况;采用ELISA法检测脑组织TNF-α、IL-1β、IL-6等炎症因子水平;采用Western blot法检测TLR4、核因子-κB p65(NF-κB p65)、p-NF-κB p65、IκBα、p-IκBα、Cleved Caspase-3、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)表达。结果:(1)神经功能、脑梗死率及脑含水量:与假手术组比较,模型组大鼠mNSS评分、脑梗死率、脑含水量明显升高(P<0.05);与模型组比较,藏红花素组和藏红花素+脂多糖组mNSS评分、脑梗死率、脑含水量均明显降低(P<0.05);与藏红花素组比较,藏红花素+脂多糖组mNSS评分、脑梗死率、脑含水量均明显升高(P<0.05)。(2)皮质神经元病理学改变及凋亡状况:假手术组皮质神经元形态正常、结构完整;模型组皮质神经元可见排列紊乱、数量减少、空泡变性、胞浆固缩深染、核膜边界不清、炎细胞浸润等病理学改变;藏红花素组皮质神经元病理学改变明显改善。与假手术组比较,模型组皮质神经元凋亡指数明显升高(P<0.05);与模型组比较,藏红花素组和藏红花素+脂多糖组凋亡指数明显降低(P<0.05);与藏红花素组比较,藏红花素+脂多糖组凋亡指数明显升高(P<0.05)。(3)炎症因子水平:与假手术组比较,模型组TNF-α、IL-1β、IL-6水平明显升高(P<0.05);与模型组比较,藏红花素组和藏红花素+脂多糖组TNF-α、IL-1β、IL-6水平明显降低(P<0.05);与藏红花素组比较,藏红花素+脂多糖组TNF-α、IL-1β、IL-6水平明显升高(P<0.05)。(4)蛋白表达:与假手术组比较,模型组TLR4、Cleved Caspase-3相对表达量及p-NF-κB p65/NF-κB p65、p-IκBα/IκBα、Bax/Bcl-2表达比值均明显升高(P<0.05);与模型组比较,藏红花素组和藏红花素+脂多糖组TLR4、Cleved Caspase-3相对表达量及p-NF-κB p65/NF-κB p65、p-IκBα/IκBα、Bax/Bcl-2表达比值均明显降低(P<0.05);与藏红花素组比较,藏红花素+脂多糖组TLR4、p-NF-κB、p-IκBα相对表达量及p-NF-κB p65/NF-κB p65、p-IκBα/IκBα表达比值明显升高(P<0.05)。结论:藏红花素能够抑制FCIR大鼠炎症反应和皮质神经元凋亡,减轻皮质神经元损伤,其作用机制可能与抑制TLR4/NF-κB通路活化有关。

Objective:To observe the protective effect of crocin on neurons of rats with focal cerebral ischemia-reperfusion(FCIR) injury based on the toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) pathway,and to explore the possible mechanism of crocin in the prevention and treatment on FCIR injury.Methods:A total of 128 SD rats were divided randomly into sham operation group,model group,crocin group,crocin + lipopolysaccharide group,with 32 rats in each group.From seven days before modeling,the crocin group was intraperitoneally injected with crocin solution;the crocin + lipopolysaccharide group was intraperitoneally injected with crocin solution and lipopolysaccharide solution;the sham operation group and the model group were intraperitoneally injected with normal saline.All groups were injected 5 mL/(kg·d),once a day,lasting for seven days.Suture occluded method was used to establish FCIR rats model.After reperfution for 24 h,modified neurological severity score(mNSS) was used to evaluate the neurological deficits;triphenyltetrazolium chloride(TTC) staining method was used to evaluate cerebral infarction rate,and drywet method was used to evaluate brain water content;hematoxylin-eosin staining(HE) staining and TUNEL staining were used to observe pathological changes and apoptosis of cortical neurons;ELISA method was used to detect the level of inflammatory factors in brain tissue(TNF-α,IL-1β,IL-6,etc.);western blot was used to detect the expression of toll-like receptor 4(TLR4),nuclear factor-κB p65(NF-κB p65),p-NF-κB p65,IκBα,p-IκBα,Cleved Caspase-3,B-lymphoma-2 gene(Bcl-2),Bcl-2-related X protein(Bax).Results:(1) Neurological function,cerebral infarction rate and cerebral water content:compared with the sham operation group,the mNSS score,cerebral infarction rate,cerebral water content in the model group increased significantly(P<0.05);compared with the model group,the mNSS score,cerebral infarction rate,cerebral water content in the crocin group and the crocin + lipopolysaccharide group decreased significantly(P<0.05);compared with the crocin group,the mNSS score,cerebral infarction rate,cerebral water content in the crocin + lipopolysaccharide group increased significantly(P<0.05).(2) Pathological changes and apoptosis of cortical neurons:the cortical neurons in the sham operation group showed normal morphology and complete structure;the pathological changes of cortical neurons such as disordered arrangement,decreased number,vacuolar degeneration,hyperchromatic cytoplasmic pyknosis,unclear nuclear membrane boundary,inflammatory cell infiltration were observed in the model group;the pathological changes of cortical neurons in the crocin group were significantly improved.Compared with the sham operation group,the apoptosis index of cortical neurons in the model group increased significantly(P<0.05);compared with the model group,the apoptosis index of cortical neurons in the crocin groupand the crocin + lipopolysaccharide group decreased significantly(P<0.05);compared with the crocin group,the apoptosis index of cortical neurons in the crocin + lipopolysaccharide group increased significantly(P<0.05).(3) Inflammatory factor level:compared with the sham operation group,the level of TNF-α,IL-1β,IL-6 in the model group increased significantly(P<0.05);compared with the model group,the level of TNF-α,IL-1β,IL-6 in the crocin group and the crocin + lipopolysaccharide group decreased significantly(P<0.05);compared with the crocin group,the level of TNF-α,IL-1β,IL-6 in the crocin + lipopolysaccharide group increased significantly(P<0.05).(4) Expression of proteins:compared with the sham operation group,the expression of TLR4,Cleved Caspase-3 and the ratio of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,Bax/Bcl-2 in the model group increased significantly(P<0.05);compared with the model group,the expression of TLR4,Cleved Caspase-3 and the ratios of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,Bax/Bcl-2 in the crocin group and the crocin + lipopolysaccharide group decreased significantly(P<0.05);compared with the crocin group,the expression of TLR4,Cleved Caspase-3 and the ratios of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,Bax/Bcl-2 in the crocin + lipopolysaccharide group increased significantly(P<0.05).Conclusion:Crocin can inhibit the inflammatory reaction and cortical neurons apoptosis in FCIR rats,and reduce the damage of cortical neurons.The mechanism may be related to the inhibition of TLR4/NF-κB pathway activation.