艾片与合成冰片脑保护及其对血脑屏障影响机制的研究
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摘要
目的：比较艾片与合成冰片的脑保护及其对血脑屏障影响机制,为阐释冰片“辛开入脑”,“开窍醒神”的科学内涵,并为临床合理用药提供参考。
方法：本研究从整体-细胞-分子水平探讨并比较艾片与合成冰片脑保护及其对血脑屏障影响的机制。①采用小鼠断头、腹腔注射亚硝酸钠、尾静脉注射氯化镁、双侧颈总动脉伴迷走神经结扎脑缺血模型,观察艾片与合成冰片对脑缺血缺氧小鼠的存活时间的影响。②线栓法制备局灶性MCAO大鼠模型,测定各组给药前后大鼠肛温,测定缺血侧脑水肿率。③采用GC法测定灌胃给予艾片和合成冰片后小鼠脑组织中龙脑透过率。④采用比色法测定MCAO大鼠血清与缺血侧脑组织中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,血清与缺血侧脑组织中NO含量、tNOs及iNOs活性,缺血侧中Ca2+含量、T-ATPase、Na+-K+-ATPase、 Ca2+-Mg2+-ATPase活性。⑤采用real-time PCR法测定MCAO大鼠模型缺血侧脑水肿中MDR1mRNA的表达量；western-blot法测定缺血侧脑水肿P-GP灰度值/β-actin灰度值。⑥采用透射电镜观察MCAO大鼠缺血侧半球额顶区皮质血脑屏障的超微结构。⑦利用PC12细胞(大鼠肾上腺髓质嗜铬瘤细胞株),用MTT法测定缺血缺氧细胞的生长；分光光度法测定直接加药缺血缺氧细胞Ca2+含量。
结果：
(1)艾片与合成冰片抗脑缺血缺氧：①艾片200、133.33、66.67mg/kg剂量组及合成冰片200mg/kg剂量组显著增加小鼠张口次数(P<0.01,P<0.05)；艾片和合成冰片200mg/kg剂量组均能显著延长断头、腹腔注射亚硝酸钠、尾静脉注射氯化镁、双侧劲总动脉结扎小鼠的存活时间(P<0.01,P<0.05)。②艾片200mg/kg组能显著抑制脑缺血再灌注4h的模型大鼠体温(P<0.05)；艾片200、133.33mg/kg和合成冰片200mg/kg均能显著抑制再灌注22h的模型大鼠体温(P<0.01,P<0.05);艾片和合成冰片200、133.33mg/kg组均能显著降低模型大鼠脑水肿率(P<0.01,P<0.05)。
(2)艾片与合成冰片对血脑屏障的影响机制：①艾片灌胃5min的龙脑透过率即可达0.174%,15min达高峰,为0.225%;合成冰片灌胃5min的龙脑透过率为0.113%,20mmin达高峰,为0.190%。②艾片与合成冰片200mg/kg组均能显著降低大鼠缺血侧脑组织及血清MDA含量(P<0.01,P<0.05)；极显著增强脑组织及血清SOD活力(P<0.01)。③艾片与合成冰片200mg/kg组均能显著降低大鼠缺血侧脑组织及血清NO含量、tNOs及iNOs活性(P<0.01,P<0.05)。④艾片与合成冰片200mg/kg组均能显著降低大鼠缺血侧脑组织Ca2+含量、T-ATPase、Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性(P<0.01,P<0.05)。⑤艾片和合成冰片200mg/kg组均能显著降低模型大鼠缺血侧脑组织P-GP蛋白表达及MDR1mRNA表达量(P<0.01)。⑥与假手术组比较,模型组血脑屏障结构受到破坏,线粒体肿胀,粗面内质网扩张：而艾片组及合成冰片200mg/kg组能不同程度改善血脑屏障超微结构。⑦艾片与合成冰片的1.6、0.8、0.4μg/100μL均能极显著增加PC12细胞OD值(P<0.01)；极显著地降低细胞内的Ca2+含量(P<0.01)，且艾片Ca2+含量显著低于同剂量合成冰片(P<0.01)。
结论：
(1)药效：艾片与人工合成冰片均有显著改善生理和病理状态下的脑缺血缺氧而发挥脑保护作用,可能是发挥“开窍醒神”的主要药效学基础。
(2)机制：①调节血脑屏障：可能与艾片和人工合成冰片所含龙脑迅速透过血脑屏障,分布于脑组织中,改善血脑屏障紧密连接的破坏程度有关；②抗自由基损伤：均能提高脑内及血清SOD活性,并降低MDA含量：③NO通路：能降低脑内及血清NO水平,降低tNs、iNOs活性；④钙通路：均能降低细胞内钙水平,降低影响钙水平的T-ATPase、Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性；⑤P-GP通路：降低MDR1mRNA表达量,降低P-GP水平。其通过多环节、多路径发挥脑保护作用。
综合分析提示：艾片及合成冰片脑保护的关键机制与调节P-GP通路、脂质过氧化反应及NO通路最为密切；其次能调节钙通路,这可能是冰片表达“辛-浮-心”药性与入脑“开窍醒神”的主要生物学机制。
(3)艾片与合成冰片比较优势：依据强度积分规则的统计结果,艾片的脑保护及其对血脑屏障调节机制等各方面均有优于合成冰片的趋势。提示治疗脑血管疾病,以选择艾片为妥。
Objective:To compare the mechanism of neuroprotective effect and influence of blood-brain barrier between L-borneolum and borneolum syntheticum. It provides experimental basis to explain the scientific connotation that borneol with the effect of inducing resuscitation and restoring consciousness can be distributed in brain for flavour of pungent.
Methods:In this study, mechanisms of neuroprotective effect and influence of blood-brain barrier were explained from whole-cell-molecular level.①The mouse models of cerebral anoxia and ischemia were established by sodium nitrite, decollation, intravenous injection of magnesium chloride and the occlusion of bilateral common carotid arteries with vague nerves. The survival times were detected to observe the influence of the mouse models after drugs.②The focal middle cerebral artery occlusion (MCAO) model was made by suture-occluded method. The rectal temperature of rats was observed on preoperative and reperfusion4h,22h. Rates of cerebral edema were examined after22h reperfusion.③Borneol in mice brain was determined by GC, and compare borneol transmittance in mice brain between L-borneolum and borneolum syntheticum after intragastric administration.④superoxide dismutase(SOD) activity and the content of malondialdehyde(MDA) both in serum and ischemia side brain tissue, the level of nitrogen monoxidum (NO) and nitricoxide synthase (tNOs and iNOs) activity both in serum and ischemia side brain tissue, calcium ion(Ca2+) content and ATPase(T-ATPase, Na+-K+-ATPase, Ca2+-Mg2+-ATPase) activity in ischemia side brain tissue were measured by spectrophotometry.⑤The expression of MDR1mRNA and P-GP in ischemia side brain tissue were measured by real-time PCR and western-blot.⑥the ultra micro structure of blood brain barrier of ischemia hemisphere side in fronto-parietal cortex region was observed by transmission electron microscope.⑦Growth of Hypoxic ischemic PCl2(rat pheochromocytoma cell) was measured by MTT, and Calcium ion (Ca2+) content in cell was measured by spectrophotometry
Result:L-borneolum and borneolum syntheticum showed the ability of anti-cerebral ischemia and hypoxia against brain ischemia and hypoxia:
①Compared with the model group, opening times of L-borneolum (200,133.33,66.67mg/kg) and borneolum syntheticum(200mg/kg) increased significantly (P<0.05,P<0.01).;Compared with the control group, survival time of the mouse which were established by sodium nitrite, decollation, intravenous injection of magnesium chloride and the occlusion of bilateral common carotid arteries with vague nerves in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) increased significantly (P<0.05,P<0.01).②Compared with the model group, increases of rectal temperature were inhibited significantly in groups of L-borneolum (200mg/kg) after reperfusion4h(P<0.05). Compared with the model group, increases of rectal temperature were inhibited significantly in groups of L-borneolum (200,133.33mg/kg) and borneolum syntheticum (200mg/kg) after reperfusion22h (P <0.01, P<0.05); Compared with the model group, rates of cerebral edema in groups of L-borneolum(200,133.33mg/kg) and borneolum syntheticum(200mg/kg,133.33mg/kg) were reduced significantly (P<0.05,P<0.01).
Mechanism of neuroprotective effect and influence of blood-brain barrier:
①After oral administration of L-borneolum, borneol transmittance was0.174%at5min, reached the maxima of0.225%at15min, and then reduced gradually. After oral administration of borneolum syntheticum, borneol transmittance was0.112%at5min, reached the maxima of0.190%at20min, and then reduced rapidly.②Compared with the model group, content of malondialdehyde(MDA) both in serum and ischemia side brain tissue in groups of L-borneolum(200mg/kg) and bomeolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01),and superoxide dismutase(SOD) activity in serum and ischemia side brain tissue increased extremely significantly (P<0.01).③Compared with the model group, the content of nitrogen monoxidum(NO) and the activity of nitricoxide synthase(tNOs and iNOs) both in serum and ischemia side brain tissue in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01).④Compared with the model group, content of calcium ion(Ca2+) and activities of T-ATPase,Na+-K+-ATPase and Ca2+-Mg2+-ATPase in ischemia side brain tissue in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01).⑤Compared with the model group, P-GP protein expression levels and MDR1mRNA gene expression level in ischemia side brain tissue in groups of L-borneolum and borneolum syntheticum (200mg/kg) decreased extremely significantly (P<0.01).⑥Structure of blood-brain barrier in modle group was damaged compared with sham-operated groups, which exhibited tight junction between endothelial cells being opened, swelling of mitochondria and rough endoplasmic reticulum expansion.Damage of structure of blood-brain barrier in groups of L-borneolum and borneolum syntheticum was improved to different extent.⑦Compared with the model group, OD values in Hypoxia-ischemia PC12in groups of L-borneolum (1.6、0.8、0.4μg/100μL) and borneolum syntheticum (1.6%0.8%0.4μg/100μL) increased extremely significantly (P<0.01).content of calcium ion (Ca2+) in Hypoxia-ischemia PC12in groups of L-borneolum(1.6、0.8、0.4μg/100μL) and borneolum syntheticum(200mg/kg)(1.6、0.8、0.4μg/100μL) decreased extremely significantly (P<0.01).Compared with the groups of borneolum syntheticum in same dosage, content of calcium ion (Ca2+) in groups of L-borneolum decreased extremely significantly (P<0.01).
Conclusion:
1. Neuroprotective effect:L-borneolum and borneolum syntheticum have protective effect obviously on cerebral ischemia and anoxia. It may be the pharmacodynamics basis for explaining borneol can induce resuscitationthe and restore consciousness
2. Mechanism:①L-borneolum and borneolum syntheticum can distribute in the brain tissue quickly and stabilize BBB structure and reduce the damage of tight junction.②They can affect lipid peroxidation. It manifested as increasing the activity of SOD and decreasing the content of MDA in serum and tissue.③They can affect NO pathway. It manifested as decreasing the content of NO and decreasing activities of NOs (tNOs and iNOs) in serum and tissue.④They can affect Ca2+pathway. It manifested as decreasing the content of Ca2+in cell and tissue. Also they can decreasing the activities of T-ATPase,Na+-K+-ATPase and Ca2+-Mg2+-ATPase.⑤They can affect P-GP pathway. It manifested as decreasing MDR1gene expression levels and P-GP protein expression level. There are multiple links and multiple paths in mechanisms of neuroprotective effect
Through comprehensive analysis, it can be seen that neuroprotective effect of L-borneolum and borneolum syntheticum may be most closely with the P-GP pathway, lipid oxidation reaction and NO pathway. It is associated with Ca2+pathway secondly. It may be the main biological mechanism of borneol that show the property of "pungent-float-heart" and the effect of inducing resuscitation and restoring consciousness
3. Comparison between L-borneolum and borneolum syntheticum:Based on the statistical results of the intensity integral rules, L-borneolum showed a superior trend than borneolum syntheticum in neuroprotective effect and mechanism of influence of blood-brain barrier. Tip:to select L-borneolum properly when cerebrovascular disease.
方法：本研究从整体-细胞-分子水平探讨并比较艾片与合成冰片脑保护及其对血脑屏障影响的机制。①采用小鼠断头、腹腔注射亚硝酸钠、尾静脉注射氯化镁、双侧颈总动脉伴迷走神经结扎脑缺血模型,观察艾片与合成冰片对脑缺血缺氧小鼠的存活时间的影响。②线栓法制备局灶性MCAO大鼠模型,测定各组给药前后大鼠肛温,测定缺血侧脑水肿率。③采用GC法测定灌胃给予艾片和合成冰片后小鼠脑组织中龙脑透过率。④采用比色法测定MCAO大鼠血清与缺血侧脑组织中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,血清与缺血侧脑组织中NO含量、tNOs及iNOs活性,缺血侧中Ca2+含量、T-ATPase、Na+-K+-ATPase、 Ca2+-Mg2+-ATPase活性。⑤采用real-time PCR法测定MCAO大鼠模型缺血侧脑水肿中MDR1mRNA的表达量；western-blot法测定缺血侧脑水肿P-GP灰度值/β-actin灰度值。⑥采用透射电镜观察MCAO大鼠缺血侧半球额顶区皮质血脑屏障的超微结构。⑦利用PC12细胞(大鼠肾上腺髓质嗜铬瘤细胞株),用MTT法测定缺血缺氧细胞的生长；分光光度法测定直接加药缺血缺氧细胞Ca2+含量。
结果：
(1)艾片与合成冰片抗脑缺血缺氧：①艾片200、133.33、66.67mg/kg剂量组及合成冰片200mg/kg剂量组显著增加小鼠张口次数(P<0.01,P<0.05)；艾片和合成冰片200mg/kg剂量组均能显著延长断头、腹腔注射亚硝酸钠、尾静脉注射氯化镁、双侧劲总动脉结扎小鼠的存活时间(P<0.01,P<0.05)。②艾片200mg/kg组能显著抑制脑缺血再灌注4h的模型大鼠体温(P<0.05)；艾片200、133.33mg/kg和合成冰片200mg/kg均能显著抑制再灌注22h的模型大鼠体温(P<0.01,P<0.05);艾片和合成冰片200、133.33mg/kg组均能显著降低模型大鼠脑水肿率(P<0.01,P<0.05)。
(2)艾片与合成冰片对血脑屏障的影响机制：①艾片灌胃5min的龙脑透过率即可达0.174%,15min达高峰,为0.225%;合成冰片灌胃5min的龙脑透过率为0.113%,20mmin达高峰,为0.190%。②艾片与合成冰片200mg/kg组均能显著降低大鼠缺血侧脑组织及血清MDA含量(P<0.01,P<0.05)；极显著增强脑组织及血清SOD活力(P<0.01)。③艾片与合成冰片200mg/kg组均能显著降低大鼠缺血侧脑组织及血清NO含量、tNOs及iNOs活性(P<0.01,P<0.05)。④艾片与合成冰片200mg/kg组均能显著降低大鼠缺血侧脑组织Ca2+含量、T-ATPase、Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性(P<0.01,P<0.05)。⑤艾片和合成冰片200mg/kg组均能显著降低模型大鼠缺血侧脑组织P-GP蛋白表达及MDR1mRNA表达量(P<0.01)。⑥与假手术组比较,模型组血脑屏障结构受到破坏,线粒体肿胀,粗面内质网扩张：而艾片组及合成冰片200mg/kg组能不同程度改善血脑屏障超微结构。⑦艾片与合成冰片的1.6、0.8、0.4μg/100μL均能极显著增加PC12细胞OD值(P<0.01)；极显著地降低细胞内的Ca2+含量(P<0.01)，且艾片Ca2+含量显著低于同剂量合成冰片(P<0.01)。
结论：
(1)药效：艾片与人工合成冰片均有显著改善生理和病理状态下的脑缺血缺氧而发挥脑保护作用,可能是发挥“开窍醒神”的主要药效学基础。
(2)机制：①调节血脑屏障：可能与艾片和人工合成冰片所含龙脑迅速透过血脑屏障,分布于脑组织中,改善血脑屏障紧密连接的破坏程度有关；②抗自由基损伤：均能提高脑内及血清SOD活性,并降低MDA含量：③NO通路：能降低脑内及血清NO水平,降低tNs、iNOs活性；④钙通路：均能降低细胞内钙水平,降低影响钙水平的T-ATPase、Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性；⑤P-GP通路：降低MDR1mRNA表达量,降低P-GP水平。其通过多环节、多路径发挥脑保护作用。
综合分析提示：艾片及合成冰片脑保护的关键机制与调节P-GP通路、脂质过氧化反应及NO通路最为密切；其次能调节钙通路,这可能是冰片表达“辛-浮-心”药性与入脑“开窍醒神”的主要生物学机制。
(3)艾片与合成冰片比较优势：依据强度积分规则的统计结果,艾片的脑保护及其对血脑屏障调节机制等各方面均有优于合成冰片的趋势。提示治疗脑血管疾病,以选择艾片为妥。
Objective:To compare the mechanism of neuroprotective effect and influence of blood-brain barrier between L-borneolum and borneolum syntheticum. It provides experimental basis to explain the scientific connotation that borneol with the effect of inducing resuscitation and restoring consciousness can be distributed in brain for flavour of pungent.
Methods:In this study, mechanisms of neuroprotective effect and influence of blood-brain barrier were explained from whole-cell-molecular level.①The mouse models of cerebral anoxia and ischemia were established by sodium nitrite, decollation, intravenous injection of magnesium chloride and the occlusion of bilateral common carotid arteries with vague nerves. The survival times were detected to observe the influence of the mouse models after drugs.②The focal middle cerebral artery occlusion (MCAO) model was made by suture-occluded method. The rectal temperature of rats was observed on preoperative and reperfusion4h,22h. Rates of cerebral edema were examined after22h reperfusion.③Borneol in mice brain was determined by GC, and compare borneol transmittance in mice brain between L-borneolum and borneolum syntheticum after intragastric administration.④superoxide dismutase(SOD) activity and the content of malondialdehyde(MDA) both in serum and ischemia side brain tissue, the level of nitrogen monoxidum (NO) and nitricoxide synthase (tNOs and iNOs) activity both in serum and ischemia side brain tissue, calcium ion(Ca2+) content and ATPase(T-ATPase, Na+-K+-ATPase, Ca2+-Mg2+-ATPase) activity in ischemia side brain tissue were measured by spectrophotometry.⑤The expression of MDR1mRNA and P-GP in ischemia side brain tissue were measured by real-time PCR and western-blot.⑥the ultra micro structure of blood brain barrier of ischemia hemisphere side in fronto-parietal cortex region was observed by transmission electron microscope.⑦Growth of Hypoxic ischemic PCl2(rat pheochromocytoma cell) was measured by MTT, and Calcium ion (Ca2+) content in cell was measured by spectrophotometry
Result:L-borneolum and borneolum syntheticum showed the ability of anti-cerebral ischemia and hypoxia against brain ischemia and hypoxia:
①Compared with the model group, opening times of L-borneolum (200,133.33,66.67mg/kg) and borneolum syntheticum(200mg/kg) increased significantly (P<0.05,P<0.01).;Compared with the control group, survival time of the mouse which were established by sodium nitrite, decollation, intravenous injection of magnesium chloride and the occlusion of bilateral common carotid arteries with vague nerves in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) increased significantly (P<0.05,P<0.01).②Compared with the model group, increases of rectal temperature were inhibited significantly in groups of L-borneolum (200mg/kg) after reperfusion4h(P<0.05). Compared with the model group, increases of rectal temperature were inhibited significantly in groups of L-borneolum (200,133.33mg/kg) and borneolum syntheticum (200mg/kg) after reperfusion22h (P <0.01, P<0.05); Compared with the model group, rates of cerebral edema in groups of L-borneolum(200,133.33mg/kg) and borneolum syntheticum(200mg/kg,133.33mg/kg) were reduced significantly (P<0.05,P<0.01).
Mechanism of neuroprotective effect and influence of blood-brain barrier:
①After oral administration of L-borneolum, borneol transmittance was0.174%at5min, reached the maxima of0.225%at15min, and then reduced gradually. After oral administration of borneolum syntheticum, borneol transmittance was0.112%at5min, reached the maxima of0.190%at20min, and then reduced rapidly.②Compared with the model group, content of malondialdehyde(MDA) both in serum and ischemia side brain tissue in groups of L-borneolum(200mg/kg) and bomeolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01),and superoxide dismutase(SOD) activity in serum and ischemia side brain tissue increased extremely significantly (P<0.01).③Compared with the model group, the content of nitrogen monoxidum(NO) and the activity of nitricoxide synthase(tNOs and iNOs) both in serum and ischemia side brain tissue in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01).④Compared with the model group, content of calcium ion(Ca2+) and activities of T-ATPase,Na+-K+-ATPase and Ca2+-Mg2+-ATPase in ischemia side brain tissue in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01).⑤Compared with the model group, P-GP protein expression levels and MDR1mRNA gene expression level in ischemia side brain tissue in groups of L-borneolum and borneolum syntheticum (200mg/kg) decreased extremely significantly (P<0.01).⑥Structure of blood-brain barrier in modle group was damaged compared with sham-operated groups, which exhibited tight junction between endothelial cells being opened, swelling of mitochondria and rough endoplasmic reticulum expansion.Damage of structure of blood-brain barrier in groups of L-borneolum and borneolum syntheticum was improved to different extent.⑦Compared with the model group, OD values in Hypoxia-ischemia PC12in groups of L-borneolum (1.6、0.8、0.4μg/100μL) and borneolum syntheticum (1.6%0.8%0.4μg/100μL) increased extremely significantly (P<0.01).content of calcium ion (Ca2+) in Hypoxia-ischemia PC12in groups of L-borneolum(1.6、0.8、0.4μg/100μL) and borneolum syntheticum(200mg/kg)(1.6、0.8、0.4μg/100μL) decreased extremely significantly (P<0.01).Compared with the groups of borneolum syntheticum in same dosage, content of calcium ion (Ca2+) in groups of L-borneolum decreased extremely significantly (P<0.01).
Conclusion:
1. Neuroprotective effect:L-borneolum and borneolum syntheticum have protective effect obviously on cerebral ischemia and anoxia. It may be the pharmacodynamics basis for explaining borneol can induce resuscitationthe and restore consciousness
2. Mechanism:①L-borneolum and borneolum syntheticum can distribute in the brain tissue quickly and stabilize BBB structure and reduce the damage of tight junction.②They can affect lipid peroxidation. It manifested as increasing the activity of SOD and decreasing the content of MDA in serum and tissue.③They can affect NO pathway. It manifested as decreasing the content of NO and decreasing activities of NOs (tNOs and iNOs) in serum and tissue.④They can affect Ca2+pathway. It manifested as decreasing the content of Ca2+in cell and tissue. Also they can decreasing the activities of T-ATPase,Na+-K+-ATPase and Ca2+-Mg2+-ATPase.⑤They can affect P-GP pathway. It manifested as decreasing MDR1gene expression levels and P-GP protein expression level. There are multiple links and multiple paths in mechanisms of neuroprotective effect
Through comprehensive analysis, it can be seen that neuroprotective effect of L-borneolum and borneolum syntheticum may be most closely with the P-GP pathway, lipid oxidation reaction and NO pathway. It is associated with Ca2+pathway secondly. It may be the main biological mechanism of borneol that show the property of "pungent-float-heart" and the effect of inducing resuscitation and restoring consciousness
3. Comparison between L-borneolum and borneolum syntheticum:Based on the statistical results of the intensity integral rules, L-borneolum showed a superior trend than borneolum syntheticum in neuroprotective effect and mechanism of influence of blood-brain barrier. Tip:to select L-borneolum properly when cerebrovascular disease.
引文
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