化浊解毒活血通络方通过调控核因子E2相关因子2/Kelch样环氧氯丙烷相关蛋白-1通路发挥对脑缺血再灌注损伤大鼠脑组织抗氧化作用的机制研究

Antioxidant effect of Huazhuo Jiedu Huoxue Tongluo Prescription in rats with cerebral ischemia-reperfusion injury by regulating nuclear factor erythroid 2-related factor 2/Kelch-like epichlorohydrin-associated protein 1 pathway

目的 观察化浊解毒活血通络方通过调控核因子E2相关因子2(Nrf2)/Kelch样环氧氯丙烷相关蛋白-1(Keap1)通路对脑缺血再灌注损伤大鼠的抗氧化作用,探讨其可能的神经保护机制。方法 将80只SD雄性大鼠随机分为假手术组、模型组、尼莫地平组及化浊解毒活血通络方高、低剂量组,每组16只。除假手术组外,其余组采用Longa法制备大脑中动脉闭塞模型大鼠。造模成功后,化浊解毒活血通络方高、低剂量组给分别予化浊解毒活血通络方25、6.25 g/(kg·d)灌胃,尼莫地平组予尼莫地平混悬液9.375 mg/(kg·d)灌胃,模型组及假手术组均予等容积0.9%氯化钠注射液灌胃。灌胃3 d后比较各组大鼠改良神经功能缺损评分(mNSS),评分后处死取脑,2,3,5-三苯基氯化四氮唑(TTC)染色比较各组大鼠脑梗死体积;制作苏木精-伊红(HE)染色切片观察脑组织病理变化;干湿法测量脑组织含水量;检测脑组织丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性;蛋白免疫印迹法检测脑组织Nrf2、Keap1蛋白表达量;逆转录聚合酶链式反应检测脑组织Nrf2、Keap1基因表达。结果 与假手术组比较,模型组、尼莫地平组及化浊解毒活血通络方高、低剂量组mNSS均升高(P<0.05);与模型组比较,尼莫地平组及化浊解毒活血通络方高、低剂量组mNSS均降低(P<0.05);与尼莫地平组比较,化浊解毒活血通络方高、低剂量组mNSS均升高(P<0.05);与化浊解毒活血通络方高剂量组比较,化浊解毒活血通络方低剂量组mNSS升高(P<0.05)。假手术未见明显脑梗死灶,其余组脑组织均见白色梗死灶。与假手术组比较,模型组、尼莫地平组及化浊解毒活血通络方高、低剂量组脑梗死体积均升高(P<0.05);与模型组比较,尼莫地平组及化浊解毒活血通络方高、低剂量组脑梗死体积均降低(P<0.05);与尼莫地平组比较,化浊解毒活血通络方高、低剂量组脑梗死体积均升高(P<0.05);与化浊解毒活血通络方高剂量组比较,化浊解毒活血通络方低剂量组脑梗死体积升高(P<0.05)。光镜下可见假手术组大鼠脑组织神经元细胞密集,排列整齐,胞浆丰富,染色均匀,胞核居中,核仁清楚,未见明显异常;模型组缺血区脑组织神经元细胞排列紊乱,水肿明显,胞浆染色变淡,出现核固缩、碎裂、溶解现象。与模型组比较,尼莫地平组及化浊解毒活血通络方高、低剂量组缺血坏死区有所减少,细胞排列紊乱、水肿、胞浆淡染色变淡、细胞核固缩、碎裂、溶解现象均有减轻。尼莫地平组、化浊解毒活血通络方高剂量组缺血坏死区减少更为明显。与假手术组比较,模型组、尼莫地平组及化浊解毒活血通络方高、低剂量组脑组织含水量均升高(P<0.05);与模型组比较,尼莫地平组及化浊解毒活血通络方高、低剂量组脑组织含水量均降低(P<0.05);与尼莫地平组比较,化浊解毒活血通络方低剂量组脑组织含水量均升高(P<0.05);与化浊解毒活血通络方高剂量组比较,化浊解毒活血通络方低剂量组脑组织含水量升高(P<0.05)。尼莫地平组与化浊解毒活血通络方高剂量组脑组织含水量比较差异无统计学意义(P>0.05)。与假手术组比较,模型组、尼莫地平组及化浊解毒活血通络方高、低剂量组脑组织MDA含量均升高(P<0.05),SOD、CAT活性均降低(P<0.05);与模型组比较,尼莫地平组及化浊解毒活血通络方高、低剂量组脑组织MDA含量均降低(P<0.05),SOD、CAT活性均增高(P<0.05);与尼莫地平组比较,化浊解毒活血通络方低剂量组脑组织MDA含量升高(P<0.05),SOD、CAT活性降低(P<0.05);与化浊解毒活血通络方高剂量组比较,化浊解毒活血通络方低剂量组脑组织MDA含量升高(P<0.05),SOD、CAT活性降低(P<0.05)。尼莫地平组与化浊解毒活血通络方高剂量组脑组织MDA含量及SOD、CAT活性比较差异无统计学意义(P>0.05)。与假手术组比较,模型组、尼莫地平组及化浊解毒活血通络方高、低剂量组脑组织Nrf2蛋白表达水平及mRNA表达水平均降低(P<0.05),Keap1蛋白表达水平及mRNA表达水平均升高(P<0.05);与模型组比较,尼莫地平组及化浊解毒活血通络方高、低剂量组脑组织Nrf2蛋白表达水平及mRNA表达水平均升高(P<0.05),Keap1蛋白表达水平及mRNA表达水平均降低(P<0.05);与尼莫地平组比较,化浊解毒活血通络方低剂量组脑组织Nrf2蛋白表达水平及mRNA表达水平均降低(P<0.05),Keap1蛋白表达水平及mRNA表达水平均升高(P<0.05);与化浊解毒活血通络方高剂量组比较,化浊解毒活血通络方低剂量组脑组织Nrf2蛋白表达水平及mRNA表达水平均降低(P<0.05),Keap1蛋白表达水平及mRNA表达水平均升高(P<0.05)。尼莫地平组与化浊解毒活血通络方高剂量组脑组织Nrf2、Keap1的蛋白表达水平及mRNA表达水平比较差异无统计学意义(P>0.05)。结论 化浊解毒活血通络方具有减轻脑缺血再灌注损伤的作用,其作用机制可能与其通过调控Nrf2/Keap1通路减轻氧化应激反应对机体的损伤作用有关。

Objective To observe the antioxidant effect of Huazhuo Jiedu Huoxue Tongluo Prescription(HJHTP) for rats with cerebral ischemia-reperfusion injury(CIRI) by regulating nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like epichlorohydrin-associated protein 1(Keap1) pathway, and explore its possible neuroprotective mechanism. Methods Totally 80 SD male rats were randomized 1∶1∶1∶1∶1 assigned to the sham-operated group(0.9% sodium chloride), the model group(0.9% sodium chloride), the nimodipine(NMD) group(NMD suspension 9.375 mg/[kg·d]), HJHTP high-dose group(HJHTP 25 g/[kg·d]), and HJHTP low-dose group(HJHTP 6.25 g/[kg·d]). The middle cerebral artery occlusion(MCAO) model rats were prepared by the Longa method in the other groups except for sham-operated group. After 3 days of treatment, the modified neurological severity score(mNSS) was recorded, then the rats were killed and the brains were taken.The cerebral infarct volume was compared using 2,3,5-Triphenyltetrazolium chloride(TTC). The hematoxylin and eosin(H&E) staining was performed to observe the pathological change of brain tissue. The dry-wet way was conducted to measure water content, malonaldehyde(MDA), superoxide dismutase(SOD) and catalase(CAT) of brain tissue were detected. The Western blot was used to detect protein expression of Nrf2, Keap1.The Reverse transcription coupled to the polymerase chain reaction(RT-PCR)was applied to detect gene expression of Nrf2, Keap1 in brain tissue.Results The mNSS and the cerebral infarct volume in other groups increased than those in the sham-operated group(P<0.05), the mNSS and the cerebral infarct volume in the NMD and HJHTP groups decreased than those in the model group(P<0.05). The mNSS and the cerebral infarct volume appeared higher with HJHTP groups versus NMD group(P<0.05), and the similar result was obtained HJHTP low-dose group versus HJHTP high-dose group(P<0.05).Through light microscopy, it was found that the neurons in the rat brain tissue were densely arranged, neatly arranged, rich in cytoplasm, uniformly stained, centered in the nucleus, clear in the nucleolus, and no obvious abnormalities were observed in the sham-operated group;in the model group, the arrangement of neurons in the ischemic brain tissue was disordered, with obvious edema, light cytoplasmic staining, and nuclear pyknosis, fragmentation, and dissolution. The ischemic necrosis in the NMD group, and the HJHTP high-dose and low-dose groups was decreased compared with the model group;the disordered cell arrangement, edema, light cytoplasmic staining, nuclear pyknosis, fragmentation, and dissolution were all alleviated. The ischemic necrosis in the NMD group and the HJHTP high-dose group was significantly decreased. Compared with the sham-operated group, the levels of brain water content, MDA, the protein and mRNA expressions of Keap-1 in the other groups were significantly increased(all P<0.05), the activities of SOD and CAT, the protein and mRNA expressions of Nrf2 were significantly decreased(all P<0.05). Compared with the model group, the levels of brain water content, MDA, the protein and mRNA expressions of Keap-1 in the NMD and the HJHTP groups were significantly decreased(all P<0.05), the activities of SOD and CAT, the protein and mRNA expressions of Nrf2 were significantly increased(all P<0.05). Compared with the NMD group and the HJHTP high-dose group, the levels of brain water content, MDA, the protein and mRNA expressions of Keap-1 in the HJHTP low-dose group were significantly increased(all P<0.05), the activities of SOD and CAT, the protein and mRNA expressions of Nrf2 were significantly decreased(all P<0.05). There was no significant difference in the above indexes between the NMD group and the HJHTP high-dose group(P>0.05). Conclusion For rats with CIRI, HJHTP can reduce the injury effects of oxidative stress on the body by regulating the Nrf2/Keap1 pathway.