滋阴填髓益脑法对大鼠局灶性脑缺血—再灌注慢性损伤保护机理的研究
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摘要
目的：通过滋阴填髓益脑法对局灶性脑缺血大鼠行为、脑组织形态学和神经生化标志物的实验研究,揭示滋阴填髓益脑法对大鼠局灶性脑缺血-再灌注的作用及其机理。
方法：1.选用雄性Sprague-Dawley大鼠,SPF级,体重250g-280g,按随机化原则,分为5组,分别为假手术组、模型组、滋阴填髓益脑方组、补阳还五汤组、尼莫地平组,每组15只。假手术组做动脉分离手术,不栓塞大脑中动脉。模型组,滋阴填髓益脑方组,补阳还五汤组,尼莫地平组均行大脑中动脉栓塞术。滋阴填髓益脑方、补阳还五汤、尼莫地平分别按照每日70kg成人121g、80g、90mg药量计算。大鼠大脑中动脉栓塞术后24小时开始灌胃对应组药物,连续灌胃7天,每日一次,给药量分别为滋阴填髓益脑方10.8g/kg,补阳还五汤7.15g/kg,尼莫地平8.0mg/kg(均以生药量计算),假手术组和造模组分别以等量生理盐水10ml/kg灌胃。最后一次给药后30min断头取脑。
2.结果观察,1)脑梗死体积的测定；2)组织病理学观察；3)血清T-SOD及Cu-Zn-SOD测定；4)血清丙二醛(MDA)；5)Bcl-2、Bax免疫组化染色；6)神经细胞凋亡检测(TUNEL法)。
结果：1：显示滋阴填髓益脑方,补阳还五汤,尼莫地平均有减小梗死体积的作用,但尼莫地平该作用弱于滋阴填髓益脑方与补阳还五汤。2：脑组织病理学检查：滋阴填髓益脑方,补阳还五汤组明显优于模型组；3：滋阴填髓益脑方组明显增加T-SOD活性,降低MDA含量；4：对损伤后脑组织Bcl-2、Bax表达影响：滋阴填髓益脑方组、补阳还五汤组、尼莫地平组Bcl-2表达上升明显,Bax表达下降,Bax/Bcl-2明显低于缺血再灌组(P均<0.01)。其中滋阴填髓益脑方组Bax/Bcl-2下降较补阳还五汤组、尼莫地平组更为明显(p<0.01)。5：与模型组相比,填髓益脑方组、补阳还五汤组、尼莫地平组大鼠缺血侧脑组织凋亡神经细胞数目显著减少(P<0.01),三组间凋亡细胞数目无显著性差异(P>0.05)。
结论：通过本实验发现显示滋阴填髓益脑方能够有效地减少缺血再灌注后的脑梗死面积,改善梗死区脑组织修复,滋阴填髓益脑方可提高模型动物血清SOD、Cu-Zn-SOD活性,表明滋阴填髓益脑方对脑组织细胞脂质过氧化损伤有显著保护作用,滋阴填髓益脑方可明显促进缺血皮质神经细胞Bcl-2的表达,同时也有抑制Bax表达的作用,即对Bax/Bcl-2有调低作用,因此可以推测滋阴填髓益脑法可以抑制神经细胞凋亡从而起到神经保护作用,而这种抗凋亡作用有可能是通过调低Bax/Bcl-2来实现的。
Objective:To fill in method of nourishing yin, filling pith and benifiting brain of focal cerebral ischemia in rats behavior and brain morphology and neural biochemical markers of experimental research, reveals Ziyin tiansui Yinao France on cerebral missing Blood-The role of reperfusion and its mechanism.
Method:1. Choice of male Sprague-Dawley rats, SPF grade, weighing 250g-280g, according to the principle of randomization, were divided into five groups, Were sham-operated group, model group, nourishing yin, filling pith and benifiting brain side group, Buyang Huanwu Tang group, nimodipine group, n=15. Sham-operated group to do separate artery surgery, not the middle cerebral artery embolization. Model group, nourishing yin,filling pith and benifiting brain side group, Buyang Huanwu Tang group, nimodipine group underwent middle cerebral artery embolization. nourishing yin, filling pith and benifiting brain side Buyang Huanwu Tang, nimodipine and 70kg respectively, according to the daily adult 121g, 80g,90mg dose calculation, Middle cerebral artery occlusion 24 hours after starting the corresponding group of drugs fed continuously fed 7 days, once a day, Dose, respectively nourishing yin, filling pith and benifiting brain side 10.8g/kg, Buyang Huanwu Tang 7.15g/kg, nimodipine 8.0mg/kg (calculations are based on the amount of crude drug), sham operation group and model group were with normal saline 10ml/kg gavage.30min after the last dose were decapitated brain.
2. The results of observation,1) infarct volume determination; 2) The histopathological observation; 3) serum T-SOD, and cu-zn-SOD determination; 4) The serum levels of malondialdehyde (MDA); 5) Bcl-2, Bax immunohistochemical staining; 6) The rieuronal apoptosis detection (TUNEL method).
Results:1:Display nourishing yin, filling pith and benifiting brain side Buyang Huanwu Tang, nimodipine on average, reduce infarct size effect, However, the effect of nimodipine was weaker than nourishing yin, filling pith and benifiting brain and Buyang Huanwu Tang.2:The brain tissue pathological examination: nourishing yin, filling pith and benifiting brain side Buyang Huanwu Tang group was significantly better than the model group; 3:nourishing yin, filling pith and benifiting brain side group was significantly increased T-SOD activity and lower MDA content in; 4:The right brain tissue injury in Bcl-2, Bax expression: nourishing yin, filling pith and benifiting brain side group, Buyang Huanwu Tang group, nimodipine significantly increased bcl-2 expression, Bax expression decreased, Bax/bcl-2 significantly lower than ischemia-reperfusion group (P all "0.01). Which nourishing yin,filling pith and benifiting brain side group decreased compared with Bax/bcl-2 Buyang Huanwu Tang group, nimodipine group even more pronounced (p<0.01).5:Compared with model group, fill the marrow Yinao side group, Buyang Huanwu Tang group, nimodipine rat ischemic brain tissue of the number of apoptotic neurons significantly reduced (P<0.01), among the three groups wither The number of dead cells, no significant difference (P> 0.05)
Conclusion:Through the experimental findings suggest that marrow nourishing yin,filling pith and benifiting brain side to effectively reduce the ischemia-reperfusion of the infarct area after, to improve the infarcted region of brain tissue repair, method of nourishing yin, filling pith and benifiting brain to raise the animal model of serum SOD, Cu-Zn-SOD activity in, indicating that benefits nourishing yin fill in myeloid cells in the brain side of the brain tissue lipid peroxidation damage in a significant protective effect, nourishing yin fill in the brain may benefit significantly promote myeloid ischemic cortical neurons the expression of Bcl-2 also inhibit the expression of the role of Bax, that have reduced the role of Bax/Bcl-2, So you can speculate method of nourishing yin, filling pith and benifiting brain could inhibit neuronal apoptosis and thus play a neuroprotective role, while the anti-apoptotic effect may be achieved through lower Bax/Bcl-2.
方法：1.选用雄性Sprague-Dawley大鼠,SPF级,体重250g-280g,按随机化原则,分为5组,分别为假手术组、模型组、滋阴填髓益脑方组、补阳还五汤组、尼莫地平组,每组15只。假手术组做动脉分离手术,不栓塞大脑中动脉。模型组,滋阴填髓益脑方组,补阳还五汤组,尼莫地平组均行大脑中动脉栓塞术。滋阴填髓益脑方、补阳还五汤、尼莫地平分别按照每日70kg成人121g、80g、90mg药量计算。大鼠大脑中动脉栓塞术后24小时开始灌胃对应组药物,连续灌胃7天,每日一次,给药量分别为滋阴填髓益脑方10.8g/kg,补阳还五汤7.15g/kg,尼莫地平8.0mg/kg(均以生药量计算),假手术组和造模组分别以等量生理盐水10ml/kg灌胃。最后一次给药后30min断头取脑。
2.结果观察,1)脑梗死体积的测定；2)组织病理学观察；3)血清T-SOD及Cu-Zn-SOD测定；4)血清丙二醛(MDA)；5)Bcl-2、Bax免疫组化染色；6)神经细胞凋亡检测(TUNEL法)。
结果：1：显示滋阴填髓益脑方,补阳还五汤,尼莫地平均有减小梗死体积的作用,但尼莫地平该作用弱于滋阴填髓益脑方与补阳还五汤。2：脑组织病理学检查：滋阴填髓益脑方,补阳还五汤组明显优于模型组；3：滋阴填髓益脑方组明显增加T-SOD活性,降低MDA含量；4：对损伤后脑组织Bcl-2、Bax表达影响：滋阴填髓益脑方组、补阳还五汤组、尼莫地平组Bcl-2表达上升明显,Bax表达下降,Bax/Bcl-2明显低于缺血再灌组(P均<0.01)。其中滋阴填髓益脑方组Bax/Bcl-2下降较补阳还五汤组、尼莫地平组更为明显(p<0.01)。5：与模型组相比,填髓益脑方组、补阳还五汤组、尼莫地平组大鼠缺血侧脑组织凋亡神经细胞数目显著减少(P<0.01),三组间凋亡细胞数目无显著性差异(P>0.05)。
结论：通过本实验发现显示滋阴填髓益脑方能够有效地减少缺血再灌注后的脑梗死面积,改善梗死区脑组织修复,滋阴填髓益脑方可提高模型动物血清SOD、Cu-Zn-SOD活性,表明滋阴填髓益脑方对脑组织细胞脂质过氧化损伤有显著保护作用,滋阴填髓益脑方可明显促进缺血皮质神经细胞Bcl-2的表达,同时也有抑制Bax表达的作用,即对Bax/Bcl-2有调低作用,因此可以推测滋阴填髓益脑法可以抑制神经细胞凋亡从而起到神经保护作用,而这种抗凋亡作用有可能是通过调低Bax/Bcl-2来实现的。
Objective:To fill in method of nourishing yin, filling pith and benifiting brain of focal cerebral ischemia in rats behavior and brain morphology and neural biochemical markers of experimental research, reveals Ziyin tiansui Yinao France on cerebral missing Blood-The role of reperfusion and its mechanism.
Method:1. Choice of male Sprague-Dawley rats, SPF grade, weighing 250g-280g, according to the principle of randomization, were divided into five groups, Were sham-operated group, model group, nourishing yin, filling pith and benifiting brain side group, Buyang Huanwu Tang group, nimodipine group, n=15. Sham-operated group to do separate artery surgery, not the middle cerebral artery embolization. Model group, nourishing yin,filling pith and benifiting brain side group, Buyang Huanwu Tang group, nimodipine group underwent middle cerebral artery embolization. nourishing yin, filling pith and benifiting brain side Buyang Huanwu Tang, nimodipine and 70kg respectively, according to the daily adult 121g, 80g,90mg dose calculation, Middle cerebral artery occlusion 24 hours after starting the corresponding group of drugs fed continuously fed 7 days, once a day, Dose, respectively nourishing yin, filling pith and benifiting brain side 10.8g/kg, Buyang Huanwu Tang 7.15g/kg, nimodipine 8.0mg/kg (calculations are based on the amount of crude drug), sham operation group and model group were with normal saline 10ml/kg gavage.30min after the last dose were decapitated brain.
2. The results of observation,1) infarct volume determination; 2) The histopathological observation; 3) serum T-SOD, and cu-zn-SOD determination; 4) The serum levels of malondialdehyde (MDA); 5) Bcl-2, Bax immunohistochemical staining; 6) The rieuronal apoptosis detection (TUNEL method).
Results:1:Display nourishing yin, filling pith and benifiting brain side Buyang Huanwu Tang, nimodipine on average, reduce infarct size effect, However, the effect of nimodipine was weaker than nourishing yin, filling pith and benifiting brain and Buyang Huanwu Tang.2:The brain tissue pathological examination: nourishing yin, filling pith and benifiting brain side Buyang Huanwu Tang group was significantly better than the model group; 3:nourishing yin, filling pith and benifiting brain side group was significantly increased T-SOD activity and lower MDA content in; 4:The right brain tissue injury in Bcl-2, Bax expression: nourishing yin, filling pith and benifiting brain side group, Buyang Huanwu Tang group, nimodipine significantly increased bcl-2 expression, Bax expression decreased, Bax/bcl-2 significantly lower than ischemia-reperfusion group (P all "0.01). Which nourishing yin,filling pith and benifiting brain side group decreased compared with Bax/bcl-2 Buyang Huanwu Tang group, nimodipine group even more pronounced (p<0.01).5:Compared with model group, fill the marrow Yinao side group, Buyang Huanwu Tang group, nimodipine rat ischemic brain tissue of the number of apoptotic neurons significantly reduced (P<0.01), among the three groups wither The number of dead cells, no significant difference (P> 0.05)
Conclusion:Through the experimental findings suggest that marrow nourishing yin,filling pith and benifiting brain side to effectively reduce the ischemia-reperfusion of the infarct area after, to improve the infarcted region of brain tissue repair, method of nourishing yin, filling pith and benifiting brain to raise the animal model of serum SOD, Cu-Zn-SOD activity in, indicating that benefits nourishing yin fill in myeloid cells in the brain side of the brain tissue lipid peroxidation damage in a significant protective effect, nourishing yin fill in the brain may benefit significantly promote myeloid ischemic cortical neurons the expression of Bcl-2 also inhibit the expression of the role of Bax, that have reduced the role of Bax/Bcl-2, So you can speculate method of nourishing yin, filling pith and benifiting brain could inhibit neuronal apoptosis and thus play a neuroprotective role, while the anti-apoptotic effect may be achieved through lower Bax/Bcl-2.
引文
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[23]朱兆洪黄芪益母草注射液对脑缺血再灌注大鼠NMDA受体的影响.浙江中医学院学报,2005；29(6)：47-48.
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[26]杨黎,何世银.葛根素对大鼠脑缺血再灌注后炎性细胞因子变化的影响.中国老年学杂志,2003；23(3)：173—174.