黄芩苷对缺血性脑损伤的保护作用及作用机理研究
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摘要
缺血性脑血管疾病(ICVD)严重危害人类健康,具有高发病率、高复发率和高致残率的特点。因其发病机制复杂,涉及兴奋性氨基酸损伤、氧自由基学说、炎症反应以及细胞凋亡等多个方面,一直是各国研究的难点。现在临床应用的药物因存在颅内出血、脑水肿等问题,疗效并不理想。因此,深入研究该病的发病机理、发现新的作用靶点以及寻找安全有效的治疗药物将会对缺血性脑血管疾病的防治产生重大意义。
近期研究表明,AngⅡ参与缺血性脑损伤的发生发展,但其具体作用还未明确。炎症在介导缺血再灌后继发性损伤方面起着关键作用。黄芩苷是中药黄芩根部的主要活性成分,临床已用于抗菌、消炎和抗感染治疗。研究发现,黄芩苷对脑缺血再灌注损伤有保护作用。
本实验采用线栓法建立大鼠脑缺血再灌注损伤模型,单次尾静脉注射给予黄芩苷。通过行为学、病理生理学以及分子生物学的方法论证黄芩苷对缺血性脑损伤的保护作用,探讨其作用机制,为缺血再灌注损伤的防治提供新思路。
1黄芩苷对大鼠脑缺血再灌注损伤后脑梗死体积的影响
TTC染色测定脑梗死体积。结果显示,与假手术组比较,模型组大鼠可见大面积脑梗死(p＜0.01)。与模型组比较,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200mg·kg~(-1)均可显著降低脑梗死体积。p值分别为p＜0.01,p＜0.05,p＜0.01,p＜0.01。与尼莫地平组比较,黄芩苷200 mg·kg~(-1)显著降低脑梗死体积(p＜0.01)。
2黄芩苷对大鼠脑缺血再灌注损伤后神经功能学评分的影响
结果显示,与假手术组比较,模型组大鼠出现明显的神经功能缺损体征(p＜0.01)。与模型组比较,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100 mg·kg~(-1)神经功能缺损体征改善不明显。黄芩苷200 mg·kg~(-1)可明显改善神经功能缺损体征(p＜0.05)。
3黄芩苷对脑缺血再灌注损伤大鼠病理形态学的影响
HE染色结果可见,假手术组神经元细胞未见病理学损伤。模型组细胞出现明显病理学损伤。尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)均可减轻脑组织病理学损伤。
4黄芩苷对脑缺血再灌注损伤大鼠脑组织内AngⅡ含量的影响
将大鼠脑组织匀浆,取上清液,按照放免试剂盒说明书操作。结果显示,与假手术组比较,模型组AngⅡ含量降低(p＜0.05)。尼莫地平0.4 mg·kg~(-1),黄芩苷100、200 mg·kg~(-1)可使脑中AngⅡ含量升高(p＜0.05)。
5黄芩苷对脑缺血再灌注损伤大鼠NF-κBp65核转位的影响
免疫组化结果显示,假手术组可见皮层神经元NF-κBp65胞浆表达,胞核表达阴性。与假手术组比较,模型组可见皮层神经元NF-κBp65胞核表达阳性(p＜0.01)。与模型组比较,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)可使胞核内NF-κBp65表达强度明显减弱(p＜0.01),与尼莫地平组相比,黄芩苷200mg·kg~(-1)可明显减弱NF-κBp65在胞核内的表达(p＜0.05)。
6黄芩苷对脑缺血再灌注损伤大鼠NF-κBp65蛋白表达量的影响
Western blotting结果显示,假手术组NF-κBp65蛋白有一定的基础表达。与假手术组比较,模型组表达量明显升高(p＜0.01)。与模型组比较,尼莫地平0.4mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)可显著降低NF-κBp65蛋白的表达(p＜0.01)。与尼莫地平组比较,黄芩苷200 mg·kg~(-1)显著降低蛋白表达量(p＜0.05)。
7黄芩苷对脑缺血再灌注损伤大鼠NF-κBp65 mRNA表达量的影响
以B-actin为内参,RT-PCR半定量检测缺血侧皮层NF-κBp65 mRNA表达。结果显示,模型组NF-κBp65 mRNA较假手术组明显升高,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)可显著降低NF-κBp65 mRNA表达量(p＜0.01)。与尼莫地平组比较,与尼莫地平组比较,黄芩苷200 mg·kg~(-1)可显著降低NF-κBp65mRNA表达量(p＜0.05)。
Ischemic cerebral vascular disease (ICVD), characterized by high incidence, high recurrence and high mutilation rate, does great harm to human health. It is difficult to study the mechanism as its pathogenesis is complicated , involved in excitatory amino acids injury, oxyradical theory, inflammatory reaction, apoptosis and so on. At present, clinical medication tend to lead to intracranial hemorrhage, brain edema and other problems, and therapeutical effect is not ideal. Therefore, searching for safe and effective drugs by further research on pathogenesis of ICVD will contribute to prevention and cure of this disease greatly.
Recent study has revealed that AngⅡis involved in the development of ICVD, but it is still not sure about the mechanism of AngⅡon ischemia reperfusion. Inflammation plays a pivotal role in mediating secondary injury to ischemia reperfusion. Baicalin is the main active component derived from traditional Chinese medicine radix of Scutellariae, which is wildly used for antibacterial treatment, eliminating inflammation and anti-infective therapy in clinical. Studies indicates that Baicalin can protect brain from ischemia-reperfusion injury.
In our present study, the ischemia reperfusion injury model was established by middle cerebral artery occlusion (MCAO) to assess the effect of Baicalin by single vena caudalis injection. The protective effect of Baicalin to CIRI was demonstrated using methods from ethology, pathophysiology and molecular biology, and we explored its mechanism, hoping to provide ideas for protection and treatment of CIRI.
1 Effect of Baicalin on infarct volume of rats subjected to ischemia-reperfusion injury
TTC staining for assessment of volume of infarction. The results indicated that the infarction volume were obvious in the model group compared with sham operation group (p<0.01). After the administration of nimodipine 0.4 mg·kg~(-1), baicalin at the dosage of 50, 100, 200 mg·kg~(-1) respectively, the infarction volume was decreased significantly (p<0.01,p<0.05, p<0.01, p<0.01). Furthermore, 200 mg·kg~(-1)and exhibited the better therapy results compared with nimodipine (p<0.05).
2 Effect of Baicalin on neurological deficits score of rats subjected to ischemia reperfusion injury
The results indicated that the neurological deficit were obvious in the model group compared with sham operation group (p <0.01). After the administration of baicalin at the dosage of 200 mg·kg~(-1) , the neurological deficit was eased obviously compared with the model group (p<0.05). Other groups demonstrated no significant differences.
3 Effect of Baicalin on path morphology of rats subjected to ischemia reperfusion injury
HE staining result showed that neurons in sham group presented normal cell structures. The neurons in the model group showed paramorphia. After the administration of baicalin at the dosage of 50, 100, 200 mg·kg~(-1) and nimodipine 0.4mg·kg~(-1), path morphology injury were markedly ascended.
3 Effect of Baicalin on contents of AngⅡin rats subjected to ischemia reperfusion injury
We collected the supernate of rats' brain and determined the contents of AngⅡby kit. The results showed that after the ischemia-reperfusion injury the contents of AngⅡbecome less in model group compared with sham group (p<0.05). After the administration of baicalin at the dosage of 100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1), the contents of AngⅡwere increased obviously, (p<0.05)
4 Effect of Baicalin on nuclear translocation of NF-κB p65 in rats subjected to ischemia reperfusion injury
NF-κB p65 are detected in cytoplasm and negative expression in nucleus by immunohistochemistry in sham groups. Compared with sham groups, in model group there are many positive NF-κB p65 expressions in cell nucleus (p<0.01). Baicalin at the dosage of 50, 100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1) decreased the NF-κB p65 expression in cell nucleus (p<0.01). Baicalin 200 mg·kg~(-1) inhibited the NF-κB p65 nuclear translocation compared with nimodipine obviously (p<0.05).
5 Effect of Baicalin on protein expression of NF-κB p65 in rats subjected to ischemia-reperfusion injury
The protein expression of the NF-κB p65 is faintly detected in sham groups as indicated by western blot. Whereas, the protein expression of the NF-κB p65 is dramatically increased in ischemia reperfusion injury group. After the administration of baicalin at 50, 100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1), the increased protein expression is markedly down-regulated (p<0.01). Furthermore, 200 mg·kg~(-1) baicalin eased the protein expression obviously compared with nimodipine (p<0.05).
6 Effect of Baicalin on the mRNA levels of NF-κB p65 in rats subjected to ischemia reperfusion injury
Semiquantative RT-PCR withβ-actin as an internal control was used to detect the NF-κBp65 mRNA expression in the ischemic cortex. The NF-κB p65 mRNA expression were increased obviously in model group compared with sham group. The NF-κB p65 mRNA expression were significantly diminished after the administration of baicalin at 50,100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1) (p<0.01). Furthermore, 200 mg·kg~(-1) baicalin decreased the mRNA levels compared with nimodipine markedly (p<0.05).
近期研究表明,AngⅡ参与缺血性脑损伤的发生发展,但其具体作用还未明确。炎症在介导缺血再灌后继发性损伤方面起着关键作用。黄芩苷是中药黄芩根部的主要活性成分,临床已用于抗菌、消炎和抗感染治疗。研究发现,黄芩苷对脑缺血再灌注损伤有保护作用。
本实验采用线栓法建立大鼠脑缺血再灌注损伤模型,单次尾静脉注射给予黄芩苷。通过行为学、病理生理学以及分子生物学的方法论证黄芩苷对缺血性脑损伤的保护作用,探讨其作用机制,为缺血再灌注损伤的防治提供新思路。
1黄芩苷对大鼠脑缺血再灌注损伤后脑梗死体积的影响
TTC染色测定脑梗死体积。结果显示,与假手术组比较,模型组大鼠可见大面积脑梗死(p＜0.01)。与模型组比较,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200mg·kg~(-1)均可显著降低脑梗死体积。p值分别为p＜0.01,p＜0.05,p＜0.01,p＜0.01。与尼莫地平组比较,黄芩苷200 mg·kg~(-1)显著降低脑梗死体积(p＜0.01)。
2黄芩苷对大鼠脑缺血再灌注损伤后神经功能学评分的影响
结果显示,与假手术组比较,模型组大鼠出现明显的神经功能缺损体征(p＜0.01)。与模型组比较,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100 mg·kg~(-1)神经功能缺损体征改善不明显。黄芩苷200 mg·kg~(-1)可明显改善神经功能缺损体征(p＜0.05)。
3黄芩苷对脑缺血再灌注损伤大鼠病理形态学的影响
HE染色结果可见,假手术组神经元细胞未见病理学损伤。模型组细胞出现明显病理学损伤。尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)均可减轻脑组织病理学损伤。
4黄芩苷对脑缺血再灌注损伤大鼠脑组织内AngⅡ含量的影响
将大鼠脑组织匀浆,取上清液,按照放免试剂盒说明书操作。结果显示,与假手术组比较,模型组AngⅡ含量降低(p＜0.05)。尼莫地平0.4 mg·kg~(-1),黄芩苷100、200 mg·kg~(-1)可使脑中AngⅡ含量升高(p＜0.05)。
5黄芩苷对脑缺血再灌注损伤大鼠NF-κBp65核转位的影响
免疫组化结果显示,假手术组可见皮层神经元NF-κBp65胞浆表达,胞核表达阴性。与假手术组比较,模型组可见皮层神经元NF-κBp65胞核表达阳性(p＜0.01)。与模型组比较,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)可使胞核内NF-κBp65表达强度明显减弱(p＜0.01),与尼莫地平组相比,黄芩苷200mg·kg~(-1)可明显减弱NF-κBp65在胞核内的表达(p＜0.05)。
6黄芩苷对脑缺血再灌注损伤大鼠NF-κBp65蛋白表达量的影响
Western blotting结果显示,假手术组NF-κBp65蛋白有一定的基础表达。与假手术组比较,模型组表达量明显升高(p＜0.01)。与模型组比较,尼莫地平0.4mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)可显著降低NF-κBp65蛋白的表达(p＜0.01)。与尼莫地平组比较,黄芩苷200 mg·kg~(-1)显著降低蛋白表达量(p＜0.05)。
7黄芩苷对脑缺血再灌注损伤大鼠NF-κBp65 mRNA表达量的影响
以B-actin为内参,RT-PCR半定量检测缺血侧皮层NF-κBp65 mRNA表达。结果显示,模型组NF-κBp65 mRNA较假手术组明显升高,尼莫地平0.4 mg·kg~(-1),黄芩苷50、100、200 mg·kg~(-1)可显著降低NF-κBp65 mRNA表达量(p＜0.01)。与尼莫地平组比较,与尼莫地平组比较,黄芩苷200 mg·kg~(-1)可显著降低NF-κBp65mRNA表达量(p＜0.05)。
Ischemic cerebral vascular disease (ICVD), characterized by high incidence, high recurrence and high mutilation rate, does great harm to human health. It is difficult to study the mechanism as its pathogenesis is complicated , involved in excitatory amino acids injury, oxyradical theory, inflammatory reaction, apoptosis and so on. At present, clinical medication tend to lead to intracranial hemorrhage, brain edema and other problems, and therapeutical effect is not ideal. Therefore, searching for safe and effective drugs by further research on pathogenesis of ICVD will contribute to prevention and cure of this disease greatly.
Recent study has revealed that AngⅡis involved in the development of ICVD, but it is still not sure about the mechanism of AngⅡon ischemia reperfusion. Inflammation plays a pivotal role in mediating secondary injury to ischemia reperfusion. Baicalin is the main active component derived from traditional Chinese medicine radix of Scutellariae, which is wildly used for antibacterial treatment, eliminating inflammation and anti-infective therapy in clinical. Studies indicates that Baicalin can protect brain from ischemia-reperfusion injury.
In our present study, the ischemia reperfusion injury model was established by middle cerebral artery occlusion (MCAO) to assess the effect of Baicalin by single vena caudalis injection. The protective effect of Baicalin to CIRI was demonstrated using methods from ethology, pathophysiology and molecular biology, and we explored its mechanism, hoping to provide ideas for protection and treatment of CIRI.
1 Effect of Baicalin on infarct volume of rats subjected to ischemia-reperfusion injury
TTC staining for assessment of volume of infarction. The results indicated that the infarction volume were obvious in the model group compared with sham operation group (p<0.01). After the administration of nimodipine 0.4 mg·kg~(-1), baicalin at the dosage of 50, 100, 200 mg·kg~(-1) respectively, the infarction volume was decreased significantly (p<0.01,p<0.05, p<0.01, p<0.01). Furthermore, 200 mg·kg~(-1)and exhibited the better therapy results compared with nimodipine (p<0.05).
2 Effect of Baicalin on neurological deficits score of rats subjected to ischemia reperfusion injury
The results indicated that the neurological deficit were obvious in the model group compared with sham operation group (p <0.01). After the administration of baicalin at the dosage of 200 mg·kg~(-1) , the neurological deficit was eased obviously compared with the model group (p<0.05). Other groups demonstrated no significant differences.
3 Effect of Baicalin on path morphology of rats subjected to ischemia reperfusion injury
HE staining result showed that neurons in sham group presented normal cell structures. The neurons in the model group showed paramorphia. After the administration of baicalin at the dosage of 50, 100, 200 mg·kg~(-1) and nimodipine 0.4mg·kg~(-1), path morphology injury were markedly ascended.
3 Effect of Baicalin on contents of AngⅡin rats subjected to ischemia reperfusion injury
We collected the supernate of rats' brain and determined the contents of AngⅡby kit. The results showed that after the ischemia-reperfusion injury the contents of AngⅡbecome less in model group compared with sham group (p<0.05). After the administration of baicalin at the dosage of 100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1), the contents of AngⅡwere increased obviously, (p<0.05)
4 Effect of Baicalin on nuclear translocation of NF-κB p65 in rats subjected to ischemia reperfusion injury
NF-κB p65 are detected in cytoplasm and negative expression in nucleus by immunohistochemistry in sham groups. Compared with sham groups, in model group there are many positive NF-κB p65 expressions in cell nucleus (p<0.01). Baicalin at the dosage of 50, 100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1) decreased the NF-κB p65 expression in cell nucleus (p<0.01). Baicalin 200 mg·kg~(-1) inhibited the NF-κB p65 nuclear translocation compared with nimodipine obviously (p<0.05).
5 Effect of Baicalin on protein expression of NF-κB p65 in rats subjected to ischemia-reperfusion injury
The protein expression of the NF-κB p65 is faintly detected in sham groups as indicated by western blot. Whereas, the protein expression of the NF-κB p65 is dramatically increased in ischemia reperfusion injury group. After the administration of baicalin at 50, 100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1), the increased protein expression is markedly down-regulated (p<0.01). Furthermore, 200 mg·kg~(-1) baicalin eased the protein expression obviously compared with nimodipine (p<0.05).
6 Effect of Baicalin on the mRNA levels of NF-κB p65 in rats subjected to ischemia reperfusion injury
Semiquantative RT-PCR withβ-actin as an internal control was used to detect the NF-κBp65 mRNA expression in the ischemic cortex. The NF-κB p65 mRNA expression were increased obviously in model group compared with sham group. The NF-κB p65 mRNA expression were significantly diminished after the administration of baicalin at 50,100, 200 mg·kg~(-1) and nimodipine 0.4 mg·kg~(-1) (p<0.01). Furthermore, 200 mg·kg~(-1) baicalin decreased the mRNA levels compared with nimodipine markedly (p<0.05).
引文
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