目的:研究川芎嗪通过核转录因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)通路抑制脊髓损伤大鼠氧化应激损伤的作用机制.方法:雌性SD大鼠60只,随机分为假手术组,模型组,川芎嗪高、中、低剂量组.假手术组大鼠切除椎板后,不进行脊髓打击,每日予腹腔注射等量0.9％氯化钠溶液;模型组大鼠切除椎板后,打击脊髓制备脊髓损伤模型,术后每日给予腹腔注射0.9％氯化钠溶液;川芎嗪组大鼠切除椎板后,打击脊髓制备脊髓损伤模型,术后每日给予川芎嗪腹腔注射.评估大鼠造模前、造模后1、3、7、14、21、28天BBB后肢运动功能评分,尼氏染色观察脊髓神经元形态,ELISA检测超氧化物歧化酶(SOD)、丙二醛(MDA)表达水平,Western Blot检测Nrf2、HO-1表达水平,qRT-PCR检测Nrf2、HO-1 mRNA表达水平,免疫组化法检测Keap1、P62表达水平,荧光染色检测活性氧(ROS)的表达量.结果:造模后14、21、28天各剂量川芎嗪组大鼠BBB评分高于模型组(P＜0.05).尼氏染色结果显示:假手术组脊髓灰质内可见均匀分布的深蓝色斑块状或颗粒状尼氏体;模型组脊髓组织灰质及白质区域见尼氏体解体、减少;各剂量川芎嗪组脊髓组织内可见少量存活的斑块状尼氏体.ELISA检测显示:各剂量川芎嗪组大鼠MDA表达低于模型组(P＜0.01);SOD表达量高于模型组(P＜0.01)o Western Blot结果显示:川芎嗪组大鼠脊髓组织Nrf2、HO-1蛋白表达量高于模型组(P＜0.01,P＜0.05)和假手术组(P＜0.05).qRT-PCR结果显示:川芎嗪组大鼠脊髓组织Nrf2、HO-1 mRNA表达量高于模型组和假手术组(P＜0.01,P＜0.05).免疫组化染色结果显示:川芎嗪组Keap1表达显著低于模型组和假手术组(P＜0.05),P62表达显著高于模型组与假手术组(P＜0.05).ROS荧光染色结果显示:川芎嗪组ROS荧光强度低于模型组(P＜0.01),高于假手术组(P＜0.01).结论:川芎嗪能够通过NRF2/HO-1信号通路调控抗氧化作用发挥神经保护作用.
Effects of tetramethylpyrazine on Nrf2/HO-1 pathway and oxidative stress in spinal cord injury rats
Objective:To investigate the mechanism of action of tetramethylpyrazine(TMP)in inhibiting oxidative stress injury via Nrf2/HO-1 pathway in spinal cord injured rats.Methods:Thirty-six female SD rats were randomly divided into sham-operated group,model group,and high-dose,middle-dose,low-dose TMP group.In the sham-operated group,the rats were excised from the vertebral plate,no spinal cord blow was performed,and equal amount of saline was injected intraperitoneally every day.In the model group,the rats were excised from the vertebral plate,and the spinal cord was perturbed to prepare a spinal cord injury model,and saline was injected intraperitoneally every day after surgery.In the TMP group,the rats were excised from the vertebral plate,and the spinal cord was perturbed to prepare a spinal cord injury model.The motor function scores of BBB hind limbs before and 1,3,7,14,21 and 28 days after modeling were evaluated,the morphology of spinal cord neurons was observed by Nissl staining,the expression levels of SOD and MDA were detected by ELISA,the expression levels of Nrf2 and HO-1 were detected by Western Blot,the mRNA expression levels of Nrf2 and HO-1 were detected by qRT-PCR,and the immunohistochemical.The expression levels of Keap1 and P62 were detected by Western Blot,Nrf2 and HO-1 by qRT-PCR,and ROS by immunohistochemistry.Results:The BBB scores of rats in the tetramethylpyrazine group were higher than those in the model group on 14 d,21 d and 28 d after sham operation(P＜0.05).Nissel staining showed that the spinal cord of rats in the sham-operated group showed uniformly distributed dark blue plaques or granules of Nissl bodies in the gray matter;in the model group,the disintegration and reduction of Nissl bodies were seen in the gray matter and white matter areas of the spinal cord;in the tetramethylpyrazine group,a small number of surviving plaques of Nissl bodies were seen in the spinal cord.ELISA showed that MDA expression in tetramethylpyrazine group was significantly lower than that in model group(P＜0.0l).SOD expression was significantly higher than that in model group(P＜0.01).Western Blot showed that the expression of Nrf2 and HO-1 protein in the spinal cord of rats in the sham-operated group was the lowest,and the expression of SOD in the model group was the lowest,while the expression of SOD in the tetramethylpyrazine group was lower than that in the sham-operated group and higher than that in the model group(P＜0.0l,P＜0.05).The qRT-PCR results showed that the mRNA expression of Nrf2 and HO-1 in the spinal cord tissues of rats in the sham-operated group was the lowest,while the mRNA expression of Nrf2 and HO-1 in the spinal cord tissues of rats in the tetramethylpyrazine group was higher than that in the model group and the sham-operated group(P＜0.01,P＜0.05).Immunohistochemical staining showed that Keapl expression in the model group and sham-operated group was significantly higher than that in the tetramethylpyrazine group(P＜0.05),the P62 expression in tetramethylpyrazine group was significantly higher than that in the model group and sham-operated group(P＜0.05).ROS fluorescence staining showed that the ROS fluorescence intensity was lowest in the sham-operated group and highest in the model group,and the ROS fluorescence intensity in the tetramethylpyrazine group was lower than that in the model group and higher than that in the sham-operated group(P＜0.01).Conclusion:TMP can exert neuroprotective effects by regulating the antioxidant effect through Nrf2/HO-1 signaling pathway.
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