三七总皂苷对脑出血大鼠脑保护的实验研究

【作者】 赵雪松；

【导师】 陈志刚；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2014， 博士

【摘要】 脑出血是一种发病急、变化快、病情危重的疾病,它约占脑血管疾病的10%-25%,致残率,病死率分别为70%-80%,38%-43%,其复发率约为1.8%-11%。本病不仅对患者造成了严重影响,也给社会和家庭带来沉重负担,临床必须紧急救治,挽救患者生命,提高患者生存质量。脑出血后,脑组织的直接损伤是来源于血肿块的机械压迫,而最主要的损伤则是继发性脑损伤,脑水肿和炎症反应是造成继发性脑损伤的主要元凶。凝血酶在继发性脑损伤中占有重要地位,NF-KB、MMP-9以及NMDA受体在继发性脑组织损害中扮演重要角色。目前对于脑出血神经损伤机制尚未完全阐明,治疗尚无突破性进展,多数患者留下神经功能缺损。中医药有其优势,我们从瘀毒入手,探讨其机制,发挥中医药的治疗价值。三七总皂苷是中药三七的提取物,可减轻脑水肿和炎症反应,促进脑出血后颅内血肿的吸收,血栓通冻干粉是三七总皂苷针剂的一种。目的：本研究运用实验大鼠制造脑出血模型,实验用药选取中药三七提取物三七总皂苷,通过行为学实验观察神经功能缺损,通过检测脑含水量和免疫组化实验观察脑组织形态学改变,通过蛋白质印迹法观察蛋白表达情况,通过逆转录-聚合酶链反应观察基因表达情况,探讨其对继发性脑损伤的影响和脑保护机制。方法：健康同系雄性SD大鼠,体重230-250g,随机分为假手术组,模型组和用药组三个组,每组又分为6h,24h,48h,72h和7天五个时间点。采用大鼠脑立体定位仪和微量进样器把2ulⅣ型胶原酶匀速缓慢打入尾壳核(模型组和用药组大鼠),假手术组则用微量进样器打入2ul灭菌生理盐水,留针5分钟,缓慢退针。生理盐水和血栓通针粉都通过腹腔注射给药,6h时间点的假手术组和模型组大鼠于造模完成后立即按体重腹腔注射生理盐水,用药组按体重立即腹腔注射血栓通,其余各时间点大鼠在相应时间点给药,每天一次,在相应时间点测神经功能评分,然后取材,采用Billiot公式检测脑组织含水量,采用免疫组化、蛋白质印迹法和逆转录一聚合酶链反应检测相关指标。实验数据采用SPSS17.0统计软件包进行数据统计分析,多组间比较采用单因素方差分析(One-Way ANOVA)进行统计处理,P<0.05作为差异有统计学意义的界限。结果：1.神经功能缺损评分：模型组和假手术组相比,都具有统计学差异。模型组mNSS评分明显升高,6h开始到48h逐渐升高,在48h时达到峰值,神经功能缺损最重,以后缓慢下降,药物干预后,用药组运动、感觉、平衡以及反射四个方面神经功能缺失程度减轻,用药组与模型组相比,神经缺损评分比相应时间点模型组都下降,除6h组下降较小,没有统计学差异外(P=0.228),其余各组神经缺损评分较相应时间点模型组都有统计学差异。2.脑含水量：模型组大鼠脑含水量呈先升高后下降趋势,在造模后6h即出现脑水肿,以后急速升高,在48h达到高峰,从72h开始逐渐下降,7天时基本接近正常。假手术组和模型组相比,各时间点都具有统计学差异(P<0.05)。用药治疗后,各时间点脑水肿都减轻,用药组与模型组相比,6h时间点虽然有下降,但是没有统计学差异(p=0.062),24h、48h、72h三个时间点的模型组和用药组相比,差异都具有统计学意义(P<0.05),7天组用药组与模型组相比,虽然脑水肿有所减轻,但是没有统计学差异(p=0.41)3.HE和免疫组化检测结果：HE染色显示假手术组尾壳核细胞形态结构正常,排列整齐,无水肿及炎性细胞浸润。模型组见神经细胞肿胀,细胞周围间隙增大,细胞固缩,大量炎症细胞浸润,神经细胞坏死消失,在6h出现,48h达高峰,以后逐渐下降,治疗组明显好转。免疫组化检测结果：PAR-1、NMDA、NF-KB和MMP-9四个指标,模型组的表达呈现了先升后降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比,差异都具有统计学意义。用药治疗后,相应各时间点各指标都下降,模型组和治疗组相比,差异都具有统计学差异。4. Western Blot检测结果：PAR-1模型组的表达呈现了先升后降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比,6h时间点没有统计学差异(p=0.074),其余时间点模型组和假手术组相比,差异都具有统计学意义。用药治疗后,相应各时间点PAR-1都下降,模型组和治疗组相比,6h时间点没有统计学差异(p=0.131),其余时间点都具有统计学差异；NMDA模型组的表达呈现了先升后降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比,各时间点都有统计学差异。用药治疗后,相应各时间点表达都下降,模型组和治疗组相比,6h时间点没有统计学差异(p=0.072),7天这个时间点虽然用药后表达下调,但是差异没有统计学意义(p=0.145),其余时间点模型组和用药组相比,都具有统计学差异。5. RT-PCR检测结果：PAR-1模型组的表达呈现了先升后降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比,各时间点都有统计学差异。用药治疗后,相应各时间点PAR-1都下降,模型组和治疗组相比,各时间点都有统计学差异；NMDA模型组的表达呈现了先升后降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比,各时间点都有统计学差异。用药治疗后,相应各时间点都具有统计学差异；NF-KB模型组的表达呈现了先升高后下降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比表达显著升高,在各时间点都有统计学差异。用药治疗后,相应各时间点表达都下降,模型组和治疗组相比,各时间点都有统计学差异；MMP-9模型组的表达呈现了先升后降的趋势,从6h开始逐渐升高,在48h达高峰,而后逐渐下降。模型组与假手术组相比,各时间点都有统计学差异。用药治疗后,相应各时间点MMP-9都下降,模型组和治疗组相比,各时间点都有统计学差异。结论：1.三七总皂苷对脑出血后大鼠神经功能缺损具有促恢复作用。2.指标PAR-1、NMDA、NK-KB和MMP-9的异常表达在脑出血后继发性脑损伤中起重要作用。3.三七总皂苷对PAR-1、NMDA、NF-KB和MMP-9的表达具有下调作用。4.三七总皂苷对脑出血大鼠具有脑保护作用。更多还原

【Abstract】 Intracerebral hemorrhage is a complex disease which has the features of urgent attaching, fast change, critical condition, and it accounts for about10%-25%of the cerebrovascular disease,70%-80%of the morbidity,38%-43%of the mortality,1.8%-11%of the recurrence rate. Intracerebral hemorrhage not only causes serious influence on the patients, and also brings heavy burden to the society and family, which must be treated emergently to save lives and improve the quality of life. After Intracerebral hemorrhage, the brain tissue damage is directly derived from the hematoma mechanical oppression, and the main damage is secondary brain injury, brain edema and inflammatory reaction which are the main culprit of secondary brain injury. Thrombin occupies an important position in the secondary injury, and NF-KB, MMP-9and NMDA receptors play an important role in the secondary brain damage. The mechanism of brain nerve damage in this disease has not been fully elucidated, and the treatment also lack of breakthrough. Most patients has neurological functional defect. TCM has its advantages and from the perspective of the poison and blood stasis to explore its mechanism. Panax Notoginseng Saponins is extracted from Chinese herb pseudo-ginseng, which has the effect of reducing brain edema and inflammatory reaction, promoting the absorption of cerebral hemorrhage of intracranial hematoma.Objective:This study used experimental animals to create intracerebral hemorrhage model, and selected Chinese medicine notoginseng extracts Panax Notoginseng Saponins to observe the nerve function defect change, brain tissue morphology changes by examining the brain water content and immunohistochemical experimental and the protein and mRNA expression by the western blot and PCR methods, to explore the mechanism of Panax Notoginseng Saponins on the effects of secondary brain injury and brain protection.Methods:Healthy homologous male SD rats,230-250g, were randomly divided into control group, model group and treatment group, and observe6h,24h,48h,72h and7days respectively. Using rat stereotaxic instrument and microsyringe to inject2uL Ⅳ collagenase into the caudate putamen (model group and treatment group), while the control group was injected saline instead. Saline and Panax Notoginseng Saponins were treated by intraperitoneal injection.6hours time points, All groups were treated immediately after surgery and the dose depend on the weight. The rats were treated everyday and acess the nerve function score, and the brain water content by Billiot formula, and the protein and mRNA expression by the western blot and PCR methods at each time point. Experimental data were analyzed by the single factor analysis of variance (One-Way ANOVA) in SPSS17.0statistical software package, and P<0.05as the boundaries that the difference was significant.Results:1. Neural function defect scale:there is a significantly difference between model group and control group. mNSS score in the model group was significantly higher, from6h to48h, and increased to peak at48h and then began to decrease slowly. In the treatment group, the score was significantly decreased comparing with the model group at movement, sensation, balance and reflect nerve function at each time point except at6h time point(P=0.228).2. Brain water content:the brain water content in the model group showed a trend of increase first at6time point, and peaked at48h, and declined from72h and then back to the normal level and there is significantly difference at each time point(P<0.05). Cerebral edema has been reduced in the treatment group at each time point and there is a significant difference at24h,48h, and72h time points comparing with the model group,(p<0.05). At6h and7days time points, the difference are not significant, medication group compared with model group, while reduced cerebral edema, but there was no statistical difference (p=0.062and p=0.41respectively).3. HE and immunohistochemitry:It showed that the cell morphology is normal, neatly, without edema and inflammatory cells in caudate putamen of the sham group by the HE staining. In the model group, the nerve cells are swelling; the gap between the cells increases, and the cell begin pycnosis and necrosis, beginning from6h, and peaking at48h and then gradually decrease. Immunohistochemical results:the expression of PAR-1, NMDA, NF-KB and MMP-9in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point(P<0.05). the expression of PAR-1, NMDA, NF-KB and MMP-9reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group (p<0.05).4. Western Blot test:the expression of PAR-1in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point except at6h time point(p=0.074). The expression of NMDA reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group except at6h(p=0.131). The expression of NMDA in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point. The expression of NMDA reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group except at6h(p=0.072) and7d time point(p=0.145).5. Rt-pcr test:the expression of PAR-1in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point. The expression of PAR-1reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group. The expression of NMDA in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point. The expression of NMDA reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group. The expression of NF-KB in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point. The expression of NF-KB reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group. The expression of MMP-9in the model group showed a trend of increase first at6time point, and peaked at48h, and then declined and there is significantly difference between model group and sham group at each time point. The expression of MMP-9reduced in the treatment group at each time point and there is a significant difference at each time points comparing with the model group.Conclusion:1. Panax Notoginseng Saponins promotes the recovery of neurologic deficits in rats after cerebral hemorrhage.2. The abnormal expression of NMD A, PAR-1, NF-KB and MMP-9play important roles in secondary brain injury after intracerebral hemorrhage.3. Panax Notoginseng Saponins has a decrease effect on the expression of PAR-1, NMDA, NF-KB and MMP-9.4. Panax Notoginseng Saponins has a protection effect after cerebral hemorrhage in rats.更多还原