益脑复健方对大脑中动脉闭塞大鼠及氯化钴诱导的PC12细胞氧化应激损伤的影响

Effects of Yinao Fujian Formula on oxidative stress injury in MCAO rats and CoCl2 induced PC12 cells

目的:研究益脑复健方对大脑中动脉闭塞(MCAO)模型大鼠和氯化钴(CoCl)诱导的PC12细胞氧化应激损伤的影响及作用机制。方法:分别应用MCAO模型大鼠和神经生长因子(NGF)诱导分化的PC12细胞进行实验。利用HE染色观察脑组织病理学变化,Longa评分和平衡木行走实验对大鼠脑神经功能损伤程度进行评估,利用MTT法检测细胞存活率,通过乳酸脱氢酶(LDH)释放率测定细胞活性,采用流式细胞术检测细胞内活性氧(ROS)产生,通过测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)含量检测细胞氧化还原状态。Western Blot检测益脑复健方对核因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)信号通路的调控作用。结果:模型组神经功能缺损评分显著高于假手术组(P<0.01);与模型组比较,益脑复健方治疗后神经功能缺损评分显著降低(P<0.05)。浓度为250μmol/L的CoCl诱导PC12细胞24 h后,细胞存活率显著下降(P<0.01),且LDH释放和ROS产生显著升高(P<0.01)。与模型组比较,益脑复健方(100、200μg/mL)显著增加细胞存活率(P<0.05),降低LDH释放和ROS产生(P<0.01)。体内外实验表明,益脑复健方显著促进SOD活性(P<0.01)、增加GSH-Px含量(P<0.05,P<0.01)、降低MDA含量(P<0.05,P<0.01)、促进CAT活性(P<0.05,P<0.01)。不同浓度益脑复健方显著上调细胞核内Nrf2的表达(P<0.05,P<0.01),并显著上调HO-1的表达(P<0.01)。结论:益脑复健方能够通过Nrf2/HO-1信号通路减轻MCAO大鼠和CoCl诱导的PC12细胞氧化应激损伤,从而发挥神经保护作用。

Objective:To investigate the effect and mechanism of Yinao Fujian Formula on middle cerebral artery occlusion (MCAO) model rats and cobalt chloride (CoCl)-induced oxidative stress injury in PC12 cells.Methods:MCAO model rats and NGF-induced differentiation of PC12 cells were used for experiments.Hematoxylin-eosin staining (HE) was used to observe the pathological changes of brain tissue.Longa score and balance beam walking test were used to evaluate the degree of brain nerve damage in rats.Cell viability was detected by MTT assay.LDH release rate was used to determine cell activity.Flow cytometry was used to detect intracellular ROS production.The cellular redox status was indicated by detecting SOD activity,CAT activity,GSH-Px content and MDA content.Western Blot was used to detect the regulatory effect of Yinao Fujian Formula on Nrf2/HO-1 signaling pathway.Results:There was no behavioral change in the sham operation group,and the neurological deficit score in model group was significantly higher than that in sham operation group (P<0.01).However,the neurological deficit score of Yinao Fujian Formula group was significantly lower than that in the model group (P<0.05).After PC12 cells were induced by 250μmol/L CoClfor 24 h,the cell viability was significantly decreased,and the ROS production was significantly increased(P<0.01).Compared with the model group,Yinao Fujian Formula (100,200μg/mL) significantly increased cell survival rate (P<0.05),reduced LDH release and ROS production (P<0.01).In vivo and in vitro experiments show that Yinao Fujian Formula significantly promoted SOD activity (P<0.01),GSH-Px content (P<0.05,P<0.01),reduced the content of MDA (P<0.05,P<0.01),and promoted CAT activity (P<0.05,P<0.01).Different concentrations of Yinao Fujian Formula significantly increased the expression of Nrf2 in the nucleus (P<0.05,P<0.01),and significantly increased the expression of HO-1 (P<0.01).Conclusion:The neuroprotective effect of Yinao Fujian Formula is through regulating the Nrf2/HO-1 signaling pathway attenuating oxidative stress injury in MCAO rats and CoCl-induced PC12 cells.