朱砂、雄黄与安宫牛黄丸脑缺血损伤保护作用的相关性考察

【作者】 赵雍；

【导师】 孙建宁； 杜贵友；

【作者基本信息】 北京中医药大学 ， 中西医结合基础（药理学）， 2003， 硕士

【摘要】 安宫牛黄丸出自清代吴鞠通《温病条辨》，由牛黄、犀角、麝香、珍珠、朱砂、雄黄、黄连、黄芩、栀子、郁金、冰片等11味药组成。具有清热解毒、豁痰开窍的功效，主治热病、邪入心包、神昏谵语。安宫牛黄丸治疗急性脑血管病，疗效显著，后遗症相对较轻，是临床上治疗高热惊厥、脑卒中的要药。但由于组方中的朱砂、雄黄分别含有HgS和As2S2，汞砷含量超标，其使用的安全性受到质疑，严重影响安宫牛黄丸的临床应用，也成为其对外出口的主要障碍。 脑血管病是神经系统的常见病，其发病率、死亡率和致残率都很高。现代病理生理学研究认为，脑卒中昏迷的重要病理基础是脑水肿及其后的脑疝，并一系列生化紊乱，如能量代谢、电解质平衡、酸碱平衡、酶系统功能、氧自由基和神经递质含量与功能等异常。中医认为，中风其本在肝肾亏虚，其标在风、火、痰、瘀、气，它们在发病过程中相互影响，相互作用，临床出现烦躁神昏谵语，此乃为脑血管意外急性期的关键所在。故用安宫牛黄丸以达清心醒脑开窍、保护脑组织、促其苏醒目的。本研究采用MCAO和双侧颈总动脉结扎再灌两种模型，通过含和不含朱砂、雄黄的安宫牛黄丸对脑缺血模型动物保护作用的比较，分析朱砂、雄黄对安宫牛黄丸脑保护作用的影响。主要结果如下：1.含与不含朱砂、雄黄安宫牛黄丸对MCAO模型大鼠梗塞面积的影响 采用三氯化铁覆着大脑中动脉血管壁形成血栓方法可造成大脑中动脉栓塞模型（MCAO），经TTC染色后脑组织梗塞区为4.51±2.25％，产生明显的脑缺血损伤。安宫牛黄丸3.0g/kg、1.5g/kg、0.75g/kg（分别相当于人临床用量的25倍、12.5倍、6.3倍，以下同）明显减少MCAO模型大鼠脑梗塞面积，提示安宫牛黄丸可明显改善MCAO引起的脑缺血损伤。朱砂、雄黄对安宫牛黄丸减少MCAO模型大鼠脑梗塞面积无显著影响。2.含与不含朱砂、雄黄安宫牛黄丸对脑缺血模型大鼠脑含水量的影响 MCAO模型大鼠，缺血区脑含水量明显提高，与对照组和自身非缺血区脑含水量比较有显著性差异。脑缺血再灌模型大鼠脑含水量显著增加，与对照组比较有显著性差异。安宫牛黄丸3.0g/kg、1.5g/kg、0.75g/kg均能不同程度地降低MCAO模型缺血区脑含水量；安宫牛黄丸2.4g/kg、0.8g/kg（分别相当于人临床用量19倍、6.3倍，以下同）能显著降低脑缺血再灌注大鼠脑含水量，提示安宫牛黄丸通过保护血脑屏障、减少毛细血管通透性、提高脑组织对脑缺血及再灌的耐受性，从而达到对脑细胞的保护作用。朱砂、雄黄对安宫牛黄丸减少脑缺血模型大鼠脑含水量作用有一定的影响。3.含与不含朱砂、雄黄安宫牛黄丸对脑缺血模型大鼠脑组织中SOD、CAT、GPX活力和GSH、LPO含量的影响MCAO模型大鼠脑缺血区海马、皮层组织中CAT和GPX活力明显降低， LPO含量明显增加；脑缺血再灌模型大鼠脑、血、肝、肾组织中SOD、CAT、GPX活力明显下降、GSH含量下降，LPO含量显著升高，导致机体氧自由基清除剂耗竭，造成自由基损伤。安宫牛黄丸3.0g/kg、1.5g/<WP=5>kg、0.75g/kg均能提高MCAO模型大鼠脑缺血区海马、皮层的CAT和GPX活力，降低LPO含量；安宫牛黄丸24g/kg、0.8g/kg均能显著增加脑缺血再灌模型大鼠SOD、CAT、GPX活力和GSH含量，降低LPO含量，表明安宫牛黄丸通过抗自由基损伤作用，减轻脑缺血模型大鼠脑缺血损伤，达到脑保护作用。朱砂、雄黄对对安宫牛黄丸提高MCAO模型大鼠非缺血区皮层GPX活力和增加脑缺血再灌模型大鼠海马中GSH含量的作用有一定影响。同时，朱砂、雄黄对安宫牛黄丸增加脑缺血再灌模型大鼠肾组织中CAT活力和肝、肾组织中GPX活力作用有一定影响。4.含与不含朱砂、雄黄安宫牛黄丸对脑缺血模型大鼠乳酸酸中毒的影响MCAO模型大鼠脑缺血区海马、皮层组织中LD含量明显增加，脑缺血再灌注模型大鼠脑、血、肝、肾组织中LD含量显著升高，引起乳酸酸中毒损害，产生较严重的脑缺血损伤。安宫牛黄丸3.0g/kg、1.5g/ kg、0.75g/kg均能降低MCAO模型大鼠脑缺血区海马、皮层的LD含量；安宫牛黄丸24g/kg、0.8g/kg均能显著降低脑缺血再灌模型大鼠血、脑、肝、肾的LD含量，提示：安宫牛黄丸对脑缺血模型大鼠有抗乳酸酸中毒的作用是安宫牛黄丸防治脑缺血损伤的主要作用之一。朱砂、雄黄对安宫牛黄丸降低MCAO模型大鼠非缺血区海马LD含量和脑缺血再灌模型大鼠海马中LD含量作用有一定的影响。5.安宫牛黄丸中重金属汞、砷在正常大鼠和脑缺血大鼠体内分布采用原子分光光度法测定体内血及各组织中总汞、总砷含量。正常大鼠灌胃给予安宫牛黄丸后，血液中总汞含量在0.5h达到高峰，随后持续下降；血液中总砷含量在1.0h达到高峰，随后迅速下降。正常大鼠灌胃给于安宫牛黄丸后1.0h，总汞、总砷在肾、血、肝、脑组织中含量都显著增加；其中重金属汞在肾脏中分布最高，重金属砷在血中分布最高。脑缺血模型大鼠灌胃给予安宫牛黄丸后1.0h，总汞、总砷在肾、血、肝、脑组织中含量都有所增加；其中重金属汞在脑缺血模型大鼠肾脏中分布最高，重金属砷在脑缺血模型大鼠血中分布最高；HgS组、As2S2组总汞、总砷在肾、血、肝、脑组织中含量均无明显变化，提示纯品的HgS和As2S2在体内几乎不被吸收。安宫牛黄丸中总更多还原

【Abstract】 Angong Niuhuang Wan，which is from Treatise on Differetiation and Treatment of Epidemic Febrile Diseases writtened by Wu Tang in the QIN Dynasty，is consist of Calculus Bovis、Cornu Rhinocorotics、Moschus、Margarita、Cinnabaris、Realgar、Rhizoma Coptidis、Radix Scutellariae、Fructus Gardeniae、 Radix Curcumae、Borneolum Syntheticum. It has functions of clearing away heat and toxic material and eliminating phlegm for resuscitation，which can indicate high fever and delirium due to invasion of pericardium by pathogenic heat and retention of heat and phlegm within。Angong Niuhuang Wan can cure various acute cerebrovascular diseases with good effectiveness and light after-effect，which is the main chinese patent medicine of treating pyrexia with delirium and stroke. But because the components of Cinnabaris and Realgar include HgS and As2s2，the concentration of mercury and arsenic get above the standard，which is critical affect the clinical application and export of Angong Niuhuang Wan.Cerebrovascular disease is common disease of nervous system，which morbidity、mortality and disability is high. The important pathologial base of stroke is cerebral edema and hernia，which can cause a series of biochemical disturbance，such as energe equilibrium、electrolyte balance、enzyme system function、oxygen free radical and nerve transmitter content and function disturbance and so on. Tranditional Chinese Medicine take the view that the cause of stroke is deficiency of liver-yin and kidney-yin，and the symptoms of stroke is consist in wind、fire、phlegm and breathe，which can influence each other in the course of stroke. So Angong Niuhuang Wan can remove heat from the heart to resuscitate and protect brain tissues in order to awake the patients. Using the middle cerebral artery obstruction（MCAO）and bilarteral common carotid artery ligeature model，the present study was disigned to analyse the influence of Cinnabaris and Realgar in the Angong Niuhuang Wan’s protective effects of brain tissues by comparing the effects of Angong Niuhuang Wan with un-including Cinnabaris and Realgar Angong Niuhuang Wan. The main results is as follow：1.Effects of the inflarct size of including and un-including Cinnabaris and Realgar Angong Niuhuang Wan in MCAO model ratsUsing FeCl3，we have made successfully MCAO model rats. The inflarct size of model rats is 4.51±2.25％，showing apparent cerebral ischemia injury. Angong Niuhuang Wan 3.0g/kg、1.5g/kg、0.75g/kg（coinciding with clinial dosage 25、12.5、6.3 times respectively）had obviously lessened the inflarct size in MCAO model rats，which shows Angong Niuhuang Wan can markedly improved cerebral ischemia injury. Cinnabaris and Realgar had not conspicuous influence in this effect.<WP=7>2.Effects of the brain water content of including and un-including Cinnabaris and Realgar Angong Niuhuang Wan in cerebral ischemia model ratsIn MCAO model rats，the brain water content of ischemia field was markedly increased，which had significant difference comparing with non-ischemia and control group. In cerebral ischemia-reinfusion model rats，the brain water content was obviously increased，which had significant difference comparing with control group. Angong Niuhuang Wan 3.0g/kg、1.5g/kg、0.75g/kg had the different degree in decreasing the brain water content of ischemia field in MCAO model rats；Angong Niuhuang Wan 2.4g/kg、0.8g/kg（coinciding with clinial dosage 19、6.3 times respectively） had obviously decreased the brain water content in cerebral ischemia-reinfusion model rats. The results shows that Angong Niuhuang Wan can protect brain tissues by protecting blood-brain barrier、decreasing capillary permeability、rising the brain tissues’stolerance to cerebral ischemia and reinfusion. Cinnabaris and Realgar had a little influence in this effect.3.Effects of the activity of SOD、CAT、GPX and the content of LPO、GSH of including and un-including Cinnabaris and Realgar Angong Niuhuang Wan in cerebral ischemia model ratsIn MCAO model rats，the activity of CAT、GPX was markedly drop更多还原