姜黄素通过抑制炎症反应和GPX4介导的铁死亡减轻大鼠脑缺血-再灌注损伤

Curcumin attenuates cerebral ischemia reperfusion injury in rats by inhibiting the inflammatory response and GPX4-mediated ferroptosis

目的基于姜黄素(curcumin,Cur)对炎症反应和铁死亡的调控作用,探讨Cur保护脑缺血-再灌注损伤(cerebral ischemia reperfusion injury,CIRI)机制。方法采用改良式线栓法制备大鼠大脑中动脉栓塞(middle cerebral artey occlusion,MCAO)模型。将SD大鼠随机分成:假手术(Sham)组、CIRI组、Cur组。采用Longa法对大鼠神经行为学评分;苏木精-伊红染色观察脑组织形态学变化;经试剂盒检测缺血侧大脑皮层组织中谷胱甘肽、丙二醛、Fe^(2+)含量以及炎症因子肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)、白介素(interleukin,IL)-1β、IL-6水平;Western blot检测脑皮层组织中铁死亡关键调控蛋白谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)的表达;通过TUNEL法检测脑神经细胞凋亡情况;透射电镜下观察脑皮层神经细胞超微结构改变。结果与CIRI组比较,Cur组大鼠神经行为学评分降低,脑皮层中丙二醛、Fe^(2+)和TNF-α、IL-1β、IL-6含量显著降低(均P<0.05),而谷胱甘肽含量和GPX4蛋白表达显著升高(均P<0.05)。病理学检查可见CIRI组神经细胞水肿、破裂和坏死,Cur组仅有轻度水肿,少量坏死细胞。TUNEL染色可见Cur组神经细胞凋亡低于CIRI组[(23.6±3.5)%vs.(36.8±4.2)%,P<0.05]。透射电镜下CIRI组线粒体体积缩小、双层膜结构增厚、嵴减少或断裂,Cur组细胞核染色质部分边集,线粒体破坏减少。结论Cur可以减轻CIRI,其机制与抑制炎症反应和GPX4介导的铁死亡有关。

Objective Based on the regulatory effect of curcumin(Cur)on inflammation and iron death,to explore the mechanism of Cur protecting against cerebral ischemia-reperfusion injury(CIRI).Methods A rat model of middle cerebral artery occlusion(MCAO)was established by the modified suture-occluded method.The modeled SD rats were randomly divided into the Sham group,CIRI group and Cur group.The neurobehavioral score of rats was measured by the Longa method.Hematoxylin-eosin(HE)staining was used to observe the pathological changes in the brain tissue of rats in each group.Furthermore,the contents of glutathione(GSH),malondialdehyde(MDA)and Fe^(2+),as well as the levels of the inflammatory factors tumor necrosis factor(TNF)-α,interleukin(IL)-1βand IL-6 in the ischemic cerebral cortex,were detected by corresponding testing kits.Western blotting was applied to detect the expression of glutathione peroxidase 4(GPX4),a key regulatory protein of ferroptosis in the cerebral cortex.In addition,neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay,and ultrastructural changes in neurons in the cerebral cortex were observed under a transmission electron microscope.Results Compared with the CIRI group,the Cur group showed decreased neurobehavioral scores,significantly reduced contents of MDA,Fe^(2+),TNF-α,IL-1βand IL-6(all P<0.05),but obviously increased content of GSH and protein expression of GPX4(both P<0.05).Further pathological examination revealed edema,rupture and necrosis of neurons in the CIRI group,while mild edema and a small number of necrotic cells were observed in the Cur group only.The results of TUNEL staining indicated that the rate of neuronal apoptosis in the Cur group was lower than that in the CIRI group,with a statistically significant difference between groups[(23.6±3.5)%vs.(36.8±4.2)%;P<0.05].In addition,under the transmission electron microscope,the CIRI group had a reduced volume of mitochondria,thickened double-layer membrane structure,and decreased or disappeared mitochondrial cristae,while the Cur group showed partial margination of nuclear chromatin and alleviated damage to mitochondria.Conclusions Cur could attenuate CIRI,and its neuroprotective mechanism may be related to the inhibition of the inflammatory response and GPX4-mediated ferroptosis.