【作者】 韩经丹；

【导师】 范吉平；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2012， 博士

【摘要】 缺血脑组织在一定条件下恢复血液供应后,出现了更加严重的脑功能障碍和结构损伤,称之为脑缺血-再灌注损伤。常见于休克、DIC微循环再通、脑血管栓塞再通、心肺脑复苏等。缺血性脑血管疾病是神经科常见疾病,其致残、致死率高,治疗原则是及时恢复缺血区的血液供应,但同时也带来了再灌注损伤,抑制再灌注损伤已成为治疗缺血性脑血管疾病的重要环节。随着现代医学对脑缺血-再灌注损伤病理生理机制认识的不断深化,近些年来中医药防治脑缺血-再灌注损伤的研究发展迅速,涉及到中药药理、方剂学、针刺作用机理等多方面内容。许多研究业已证实中医药对脑缺血-再灌注损伤有保护作用通络化痰胶囊由范吉平教授根据《中风病诊断、疗效评定标准》确定的中风病的病类、证候分类方法,选取中风病最常见的风痰瘀血痹阻脉络证,结合导师王永炎院士“毒损脑络”的学术思想,以及自己治疗中风病临床经验研发而成,由熊胆粉、天麻、三七、丹参、天竺黄、大黄等药物组成,具有活血通络、化痰熄风的功效,主治中风病中经络、痰瘀阻络证,症见半身不遂,口眼歪斜,舌强语謇,或不语,偏身麻木,口角流涎,唇甲色暗,舌苔厚腻,舌质暗,或有瘀点,瘀斑,舌底脉络瘀暗,脉涩或弦滑。通络化痰前期药效学实验研究表明通络化痰胶囊能明显改善脑缺血所致的大鼠的偏瘫体征及神经系统症状,缩小脑梗塞灶面积,能明显降低脑缺血时脑血管的通透性、降低脑组织的含水量,减轻脑水肿和神经元的继发性损害,明显减轻大鼠局灶性脑缺血的病理形态学改变,降低大鼠局灶性脑缺血时血小板聚集率,能明显延缓大鼠血栓的形成时间,抑制血栓形成,另外还能增强小鼠抗缺氧的能力。本实验试图研究通络化痰胶囊及其有效成分熊胆的神经保护作用及机制,为通络化痰胶囊防治脑缺血-再灌注损伤的治疗作用提供客观的实验依据。研究目的：1.探讨通络化痰胶囊的神经保护作用及机制,为通络化痰胶囊防治脑缺血-再灌注损伤的治疗作用提供客观的实验依据。2.探讨通络化痰胶囊有效成分熊胆粉的神经保护作用及机制。研究方法：采用具有线栓法模拟局灶性脑缺血-再灌注损伤大鼠模型,根据神经功能评分和行为学测试方法检测实验动物的神经功能恢复程度、利用HE染色和尼氏染色的方法检测脑组织的病理学改变、采用免疫组化方法检测脑组织中GFAP、 NF-kB、TNF-a、ICAM-1、IL-1β、IL-8的表达,采用westernblot技术检测脑组织中GFAP、NF-kB的蛋白表达；采用RT-PCR方法检测脑组织中GFAP、NF-kB的基因表达。研究结果：1.圆柱实验神经行为学结果假手术组手术后第1天不对称分值较空白组增高(P<0.05),之后恢复至正常水平(与空白对照组比较P>0.05)。模型组手术后第1天不对称分值较空白对照组明显增高(P<0.05),第7天时未恢复至正常水平(P<0.05)。阳性药组手术后第1天不对称分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天不对称分值与模型组相比降低(P<0.05)。低剂量组手术后第1天不对称分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天不对称分值与模型组相比无统计学差异(P>0.05)。中剂量组手术后第1天不对称分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天不对称分值与模型组、低剂量组相比降低(P<0.05),与阳性药组比较无统计学差异(P>0.05)。高剂量组手术后第1天不对称分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天不对称分值与模型组、低剂量组相比降低(P<0.05),与阳性药组、中剂量组比较无统计学差异(P>0.05)。熊胆粉组手术后第1天不对称分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天不对称分值与模型组、低剂量组相比降低(P<0.05),与阳性药组、中剂量组、高剂量组比较无统计学差异(P>0.05)。2.窄通道-夹角神经行为学结果假手术组：手术后第1天转身分值较空白对照组增高(P<0.05)。第7天时未恢复至正常水平(P<0.05)。模型组：手术后第1天转身分值较空白对照组明显增高(P<0.05),第7天时未恢复至正常水平(P<0.05)。阳性药组：手术后第1天转身分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天转身分值与模型组相比降低(P<0.05)。低剂量组：手术后第1天转身分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天转身分值与模型组相比无统计学差异(P>0.05)。中剂量组：手术后第1天转身分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天转身分值与模型组、低剂量组相比降低(P<0.05),与阳性药组比较无统计学差异(P>0.05)。高剂量组：手术后第1天转身分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天转身分值与模型组、低剂量组相比降低(P<0.05),与阳性药组、中剂量组比较无统计学差异(P>0.05)。熊胆粉组：手术后第1天转身分值较空白对照组明显增高(P<0.05),第7天未恢复至正常水平(P<0.05),第7天转身分值与模型组、低剂量组相比降低(P<0.05),与阳性药组、中剂量组、高剂量组比较无统计学差异(P>0.05)。3.大鼠脑组织病理形态学改变肉眼观察空白组、假手术组、阳性药组及给药高剂量组、中剂量组、低剂量组和模型组共八组大鼠脑组织未见明显病变。镜下HE染色观察示空白组大鼠脑组织大脑、小脑各级神经细胞未见有病理形态学改变,小脑浦肯野细胞未见有退行性改变,结构正常。假手术组大鼠脑组织大脑、小脑各级神经细胞未见变形,小血管有轻度充血,未见明显病变。模型组大鼠脑组织大脑、小脑神经细胞有缺血性改变,细胞尼氏小体消失、胞核固缩、核仁消失,部分细胞染色灰色,有退行性变；有的细胞膜与周围分界清楚,染色布均匀。小脑的浦肯野细胞及海马区细胞未见明显改变。给药三个剂量组、阳性药组和熊胆粉组所送检大鼠脑组织：与模型组相比,大脑神经细胞的病变有不同程度改变。尼氏染色镜下观察示空白对照组大鼠脑细胞内尼氏小体显示多见。假手术组大鼠脑细胞内尼氏小体显示多见。模型组大鼠脑细胞内尼氏小体明显减少。给药三个剂量组、阳性药组和熊胆粉组所送检大鼠脑组织细胞各组尼氏小体与模型组相比有不同程度增加4.大鼠缺血再灌注7天后免疫组化法测定大鼠脑组织中GFAP.NF-Kb、TNF-a、ICAM-1、IL-1β、IL-8含量结果显示：通络化痰高、中、低剂量组、熊胆组、阳性药组海马CA1区GFAP表达均显著低于模型组(P<0.05),阳性药组皮质区GFAP表达低于模型组(P<0.05),通络化痰高、中、低剂量组、熊胆组与模型组相比,无显著性差异(P>0.05)阳性药组、低剂量组海马区和皮层区NF-Kb表达低于模型组(P<0.05)。通络化痰高、中剂量组、熊胆组NF-Kb表达均显著低于阳性药组(P<0.05)；模型组脑组织皮层区及海马区TNF-α、ICAM-1、 IL-1β、IL-8水平高于空白对照组及假手术组(P<0.05),与模型组相比,通络化痰各组、阳性药组、熊胆组ICAM-1水平降低(P<0.05),通络化痰高剂量组、熊胆组ICAM-1降低水平高于阳性药组、低剂量组(P<0.05)。5.大鼠缺血再灌注7天后Western-b1ot法测定大鼠脑组织中GFAP、NF-Kb蛋白含量结果显示：通络化痰各组、熊胆组、阳性药组GFAP蛋白表达均低于模型组(P<0.05),用药各组间GFAP蛋白表达无差异(P>0.05)。通络化痰各组、熊胆组、阳性药组NF-K蛋白表达均低于模型组(P<0.05)。通络化痰高、中剂量组、熊胆组NF-Kb蛋白表达均显著低于通络化痰低剂量组(P<0.05)。6.大鼠缺血再灌注7天后测定大鼠脑组织中GFAP、NF-KbmRNA含量测定结果显示：通络化痰各组、熊胆组、阳性药组NF-KbmRNA表达均低于模型组(P<0.05),用药各组之间NF-Kb表达无差异(P>0.05)；通络化痰各组、熊胆组、阳性药组GFAP mRNA表达均低于模型组(P<0.05)。用药各组之间GFAP mRNA表达无差异(P>0.05)。结论：通络化痰胶囊及其有效成分熊胆粉对神经系统具有保护作用,可减轻脑缺血-再灌注损伤大鼠的脑组织损伤,其作用的发挥可能与其可以降低缺血再灌注损伤后脑组织TNF-α、NF-Kb、ICAM-1、IL-1β、IL-8、GFAP等相关因子的表达相关。更多还原

【Abstract】 After Ischemic brain tissue restoring blood supply in certain conditions, there was a more serious brain dysfunction and structural damage, known as cerebral ischemia-repe-rfusion injury, which is Common in shock, DIC reperfusion of microcirculation and Cerebrovascular embolization through again, cardiopulmonary cerebral resuscitation etc. Ischemic cerebral vascular disease is a neurological disease, which has a high rates of disability and death. The treatment principle is to restore a blood supply, but which also brought a reperfusion injury. Inhibiting reperfusion injury have become a important part of treatment of ischemic cerebral vascular diseases. With modern medicine on cerebral ischemia-reperfusion injury in deepening understanding of pathophysiological mechanisms, in recent years, Chinese medicine in preventing and treating cerebral ischemia-reperfusion injury of rapid development, related to Pharmacology, pharmacology, mechanism of acupuncture, and other content. Many studies have confirmed that Chinese medicine has a protective effect on cerebral ischemia-reperfusion injury.TongLuoHuaTan capsule was invented by Professor Fan Jiping according to the Antihypertensive and Syndrome classification method of stroke disease determined by the book "Diagnosis and clinical efficacy evaluation standards of stroke", and selecting the most common stroke type of wind phlegm blood stasis bi resistance thread card, combinating mentor Wang yongyan fellow’s academic thought "heat-toxic affecting the brain collaterals", and with himself s clinical treatment experience of stroke disease. The capsule is made by gall powder, Gastrodia elata, Sanqi, Salvia miltiorrhiza, Tianzhu yellow, and rhubarb, which has a effect of promoting blood circulation, removing obstruction in channels, peliminating phlegm, calming wind. Its major effects are treating stroke precursor caused by phlegm-stasis in channels, syndrome of intermingled phlegm and blood stasis,which show symptoms of hemiplegia, facial paralysis, stiff tongue and impeded speech, or aphasia, hemianesthesia, slobbering, lip color a dark, tongue thick and greasy, tongue qualitative dark, petechia or bruise, tongue base vein stasis and dark, pulse astringent or wiry slide.Early effect experimental research of TongLuoHuaTan capsule indicates that TongLuoHuaTan capsule can clearly improve hemiplegia signs and the nervous system symptoms of large rat due to brain deficiency blood, narrow brain infarction focal area, clearly reduce the permeability of blood vessel of brain when it is cerebral ischemia, and reduce the moisture of brain organization, reduce brain edema and secondary lesion of neurons, significantly reduce large rat Council focal sexual brain deficiency blood of pathology morphology change, reduced pathomorphology change in focal cerebral ischemia of rat, clearly reduce platelet aggregation rate in focal cerebral ischemia of rat, significantly delay rats blood clots forming time, suppression thrombosis formed, Also can enhance the ability of hypoxia in mice.This experiment tried to study the neuroprotective effects and mechanism of TongLuoHuaTan capsule and its effective components of bear gall, provide objective experiment basis of TongLuoHuaTan capsule in preventing and treating cerebral ischemia-reperfusion injury.Research objectives:1. Discuss on neuroprotective effect of TongLuoHuaTan capsule and provide objective experiment basis of TongLuoHuaTan capsule in preventing and treating cerebral ischemia-reperfusion injury.2. Discussion on neuro-protective effect and mechanism of the effective components of bear gall of TongLuoHuaTan capsule.Research methods:Using Suture-occluded method to simulation focal cerebral ischemia-reperfusion injury rat model, test experimental animals’neurological recovery degree According to the nerve functional score and behavioral testing methods, test brain’s pathology change by using HE dyeing and Nepal’s dyeing method, and using immunohistochemical staining to detect the expression of GFAP, NF-kB, TNF-a, ICAM-1, IL-1beta, IL-8in brain organization, using westernblot technology to detect the protein expression of GFAP, and NF-kB in the brain organization, using RT-PCR method to detect the gene expression of GFAP and NF-kB in the brain tissue.Research results:1. Nerve behavior results of Cylinder Test1day after surgery, asymmetric score of sham-operation group is increased compared with the blank Group (P<0.05), and then back to normal level (compared with the blank control group P>0.05).1day after surgery, asymmetric score of the model group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05).1day after surgery, asymmetric score of the positive drug group is significantly increased compared with the blank control group (P<0.05), which did not return to normallevels on the7th day (P<0.05), asymmetrical score of the7th day is reduced compared to the model group (P<0.05).1day after surgery, asymmetric score of the low dose group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day was no statistical difference compared to the model group (P>0.05).1day after surgery, asymmetric score of the medial-dose group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day is lower compared to the model group and low dose group (P<0.05), which has no statistically significant difference compared with positive Group (P>0.05).1day after surgery, asymmetric score of the high-dose group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day is lower compared to the model group and low dose group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group(P>0.05).1day after surgery, asymmetric score of the bear bile powder group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day is lower compared to the model group and low dose group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group and high doze group (P>0.05).2. Nerve behavior results of Narrow-alley Corner TestSham-operation group:1day after surgery, turning score is increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05). Model group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normallevels on the7th day (P<0.05). Positive groups:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05). Turning score of the7th day is lower compared to the model group (P<0.05). Low dose group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is no statistical difference compared to the model group (P>0.05).Middle-dose group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is reduced compared to the model group and low-doze group (P>0.05). High dose group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is reduced compared to the model group and low-doze group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group (P>0.05). Group of bear bile powder:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is reduced compared to the model group and low-doze group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group and high doze group (P>0.05).3. Pathological morphological changes in brain tissues of ratsMacroscopic observation shows that blank group, Sham-operation group, positive drug group and the high-dose group, middle-dose groups and low dose groups and model group for a total of eight group no significant pathological changes of rat brain.Microscopically HE dyeing shows the blank set of brain tissue in rats, cerebellar neuronal has no pathological changes at all levels, no degeneration in cerebellar Purkinje cells, and structural is normal. Sham-operation set of brain tissue of rat has no deformation on cerebellar neurons at all levels in the brain,, small vessels have mild congestion, no significant disease. Model set of brain tissue in rats has ischemic change on cerebrum and cerebellum, cell’s nissl bodies disappears, cell nuclear pyknosis, nucleoli disappear, some staining to be gray, degeneration; some boundaries around the cell membrane are clear, stained uniform. Purkinje cells of the cerebellum and hippocampal cells do not see obvious changes. Three dose groups, drug-positive Group and by the Group of bear bile powder in rat brain: compared with the model group, all have different degree change of nerve cells of the brain.Nissl staining microscopic observation shows in the blank control group Nissl’s body shows more in brain cell of rat. Sham-operation group shows more Nissl’s body in the brain cells of rats. Model groups of brain cells of rats have a marked decline in Nepal’s small body. Three dose groups, drug-positive Group and bear bile powder group, compared to the model Group’s smaller body, have increased in different levels.4.7days after Rats’ischemia-reperfusion, using immunohistochemistry to determine deficiency blood then perfusion using Immunohistochemical method to detect the expression of GFAP, and NF-k b, and TNF-a, and ICAM-1, and IL-1β in thel brain tissue of rats, the results display that the expression of GFAP in Hippocampus CA1area of high doze group, middle doze group, low-doze group, bear gall group are significantly lower than that of model group (P<0.05), the expression of GFAP in cortex area of positive drug group is lower than than of model group (P<0.05), that of high doze group, middle doze group, low-doze group, bear gall group and positive drug group have no significant differences compared to model group (P>0.05); The expression of NF-Kb of drug-positive Group, and the low-dose group are lower than that of the model group (P<0.05). The expression of TNF-a, ICAM-1, IL-1β and IL-8in hippocampus and cortex area of brain tissue of model group are higher than than of the blank controller group and sham-operation group (P<0.05); The expression of NF-Kb of high doze group, middle doze group and bear gall group are all significantly lower than that of positive drug group (P<0.05), compared with the model group, the expression of ICAM-1of doze groups, positive drug group and bear gall group are lower (P<0.05), the reduce level of the expression of ICAM-1of the high doze group and bear gall group are higher than that of positive drug group and low doze group (P<0.05).5.7days after Rats’ischemia-reperfusion, using westernblot technology to detect the protein expression of GFAP and NF-kB in the brain organization, results shows:the protein expression of GFAP of doze groups, bear gall group and positive drug group are all lower than that of model group (P<0.05), and the protein expression of GFAP of doze groups have no difference (P>0.05). The protein expression of NF-kb in doze groups, bear bile group, positive drug group are less than that of the model group (P<0.05). The protein expression of NF-Kb in high doze group, middle doze group and bear bile group are significantly less than that of the low dose group (P<0.05).6.7days after Rats、ischemia-reperfusion, to detect the mRNA expression of GFAP and NF-kB in the brain organization, results shows:the mRNA expression of NF-kB of doze groups, bear gall group and positive drug group are all lower than that of model group (P<0.05), the mRNA expression of NF-kB of doze groups have no difference (P>0.05); the mRNA expression of GFAP of doze groups, bear gall group and positive drug group are all lower than that of model group (P<0.05), the mRNA expression of GFAP of doze groups have no difference (P>0.05).Conclusions:TongLuoHuaTan capsule and its active ingredients bear bile powder have a Significant neural protection, can reduce cerebral ischemia-reperfusion injury in rats with Brain tissue damage, which may due to its reduce the expression of TNF-alpha, NF-k b, ICAM-1, IL-1, R, IL-8, of GFAP and other related factors after ischemia-reperfusion injury in cerebral organization.更多还原