节点文献
四逆汤对内毒素休克大鼠脑损伤保护的分子机制研究
【作者】 裴卉；
【导师】 陈明；
【作者基本信息】 北京中医药大学 ， 中医临床基础， 2009， 博士
【摘要】 感染性休克(Septic Shock)是现代危重病医学面临的普遍存在而又十分复杂的问题,成为直接影响患者预后,阻碍进一步提高救治成功率的突出难题。国外一组临床流行病学资料表明,2527例全身炎症反应患者中,感染性休克发生率为4%,其病死率为46%。我国的一项研究表明,烧伤面积大于30%者约40%并发感染性休克。对危重病患者的研究显示感染性休克发病率为15.7%,其中61.1%进一步发展为多器官功能障碍综合征(MODS),病死率为30.6%。由此可见,揭示感染性休克发病机理、探寻临床防治方法具有十分重要的理论价值和临床意义。在单纯西医治疗感染性休克难以取得进展性疗效时,积极地寻找中西医结合治疗感染性休克的方法就显得更为迫切,很多学者希望能从中医药方面对感染性休克治疗有所突破。根据其临床表现,感染性休克属厥证范围,多为脱证,故名之“厥脱”。中医数千年的发展在很大程度上是研究感染性休克的过程,在这方面积累了很多经验,但由于作用机制不明确,使得临床治疗缺乏依据。研究目的:1.明确内毒素休克后脑损伤组织中炎症介质水平,中性粒细胞和脑血管内皮细胞等细胞表面黏附分子表达情况,脑血管平滑肌细胞、神经胶质细胞和神经细胞对细胞外炎症介质刺激产生的功能代谢变化以及结构改变等三者之间的规律性关系。2.明确内毒素休克脑损伤中起作用的白细胞介素-1α(IL-1α)、肿瘤坏死因子-α(TNF-α)增加等信号异常是否可以上调中性粒细胞和脑血管内皮细胞等细胞表面黏附分子表达,脑血管平滑肌细胞、神经胶质细胞和神经细胞细胞内气体信使一氧化氮(NO)数量的异常等受体后信号转导通路的变化与脑部不同区域血管反应性及神经元损伤修复的关系。3.明确四逆汤对以上指标有何作用,从而说明回阳救逆法改善感染性休克后脑微循环障碍和神经元损伤的分子细胞机制,发展中医急症理论。研究方法:1.以大鼠内毒素休克模型模拟感染性休克,首先运用基因芯片技术筛选出对感染性休克作用明显的受体前信号物质炎症细胞因子。2.探讨四逆汤方对内毒素引起的受体前信号物质的作用,如组织中炎症介质IL-1α、TNF-α水平的变化。具体方法采用ELISA方法测定大鼠脑组织IL-1α、TNF-α的含量。3.探讨四逆汤方对IL-1α、TNF-α引起的受体信号物质的作用,如中性粒细胞和脑血管内皮细胞等细胞表面黏附分子如细胞间黏附分子(ICAM-1,CD54)、神经细胞黏附分子(NCAM,CD56)、血管细胞黏附分子(VCAM-1,CD106)表达情况。具体方法采用免疫组化方法测定大鼠脑组织CD54、CD56、CD106分布与数量变化。4.探讨四逆汤方对脑血管平滑肌细胞、神经胶质细胞和神经细胞接受细胞膜上受体刺激后细胞内信号物质的变化,如脑组织NO产量、超氧化物歧化酶(SOD)与丙二醛(MDA)含量等;用生化法测SOD与MDA含量,免疫组化法测诱导型一氧化氮合酶(iNOS)分布与数量变化。5.最后探讨四逆汤方对内毒素休克脑损伤组织结构的改善作用。用TUNEL法测大鼠脑组织细胞凋亡,用电镜观察脑组织内各细胞的超微结构,用光镜观察脑组织结构变化。研究结果:1.假手术组大鼠平均动脉血压在实验过程于1h开始有轻度下降;在注射内毒素后,模型组从1h开始与假手术组相比有极显著差别(p＜0.01),至6h时血压降至初始值的40.75%。地塞米松组大鼠至6h时血压降至初始值的45.32%,与模型组相比血压有所升高(p＜0.05)。四逆汤组大鼠至6h时血压降至初始值的67.68%,与模型组相比血压升高极显著(p＜0.01)。2.基因芯片筛查出Ccl2,4,7,20、Cxcl1,2,9,10,11、Ccr2,3,9;TNF-α、IL-1α、IL-1β等在内毒素休克大鼠脑组织出现非常显著的高表达;还有很少报道的IL-13、IL-15、I15ra也有差异表达。其中IL-13于1h无变化,于2h、3h、6h均上调,IL-15于1h、2h均下调,I15ra于1h、2h、3h、6h均下调。3.内毒素休克大鼠脑组织中TNF-α与IL-1α从1h至6h的浓度变化规律与基因芯片技术检测出的mRNA水平变化规律基本相同。①模型组大鼠脑组织中IL-1α含量在1h较假手术组有所降低(p＜0.01),2h无显著差异(P＞0.05),3h(p＜0.05)、6h(p＜0.01)有所升高,并于6h达峰值。地塞米松组与四逆汤组大鼠脑组织中IL-1α含量在2h、3h、6h均较模型组有所下降(p＜0.01)。②模型组大鼠脑组织中TNF-α含量在四个时间段较假手术组均有所升高(p＜0.01),并于6h达峰值。地塞米松组大鼠脑组织中TNF-α含量在1h、2h、3h、6h均较模型组有所下降(p＜0.01)。四逆汤组大鼠脑组织中TNF-α含量在3h、6h时间段较模型组有所下降(p＜0.01)。4.正常组、假手术组大鼠脑组织中CD54无表达,CD56、CD106有微弱表达。内毒素休克模型组大鼠脑组织中CD54、CD56、CD106于1h、2h、3h、6h等不同时间段均高于空白对照组与假手术组(p＜0.01),CD54、CD106于1h表达达峰值,CD56于6h表达达峰值。地塞米松组于1h、2h下调CD54的表达(p＜0.01),于1h上调CD56的表达(p＜0.01),于1h、2h、6h下调CD106的表达(p＜0.05)。四逆汤组于1h和2h(p＜0.01)、3h(p＜0.05)均下调CD54的表达,于3h(p＜0.05)、6h(p＜0.01)均上调CD56的表达,于1h、2h和3h(p＜0.01)、6h(p＜0.05)均下调CD106的表达。5.正常组、假手术组大鼠脑组织iNOS无表达,模型组大鼠脑组织发现iNOS在1h表达量最高,2h、3h有所降低,6h时又有所回升。地塞米松组于1h(p＜0.01)、6h(p＜0.05)均下调iNOS的表达。四逆汤组仅于6h下调iNOS的表达(p＜0.01)。SOD在模型组大鼠脑组织中含量于四个时间段较假手术组均有所降低(p＜0.01),并于1h降至最低。地塞米松组与四逆汤组大鼠脑组织中SOD含量在1h、2h时间段较模型组有所升高(p＜0.01)。MDA在模型组大鼠脑组织中含量于四个时间段较假手术组均有所升高(p＜0.01),并于6h达峰值,其变化规律与SOD基本相同,地塞米松组大鼠脑组织中MDA含量在1h、2h、3h、6h时间段较模型组有所降低(p＜0.01),四逆汤组大鼠脑组织中MDA含量在2h、3h、6h时间段较模型组有所降低(p＜0.01)。6.电镜与光镜观察结果显示:内毒素休克模型组大鼠于1h有反应性损伤,2h、3h损伤则有所恢复,6h损伤最严重;地塞米松组于1h较模型组损伤减轻;四逆汤组于6h较模型组损伤减轻。TUNEL法检测结果为:正常组与假手术组大鼠脑组织有极少量的凋亡细胞,内毒素休克模型组大鼠脑组织中的凋亡细胞较假手术组明显升高(p＜0.01),并于6h达峰值。地塞米松组于1h、2h较模型组凋亡减少(p＜0.01)。四逆汤组于2h、3h、6h较模型组凋亡减少(p＜0.01)。研究结论:1.内毒素休克后大鼠脑组织出现91个炎症细胞因子差异表达,其中36种细胞因子参与炎症应答,有较多报道的TNF-α、IL-1β、IL-10,还有很少报道的IL-1α、IL-13、IL-15、I15ra。2.内毒素休克可以通过两条途径造成器官损伤:①LPS引起的受体前信号物质的作用,如组织中炎症介质TNF-α、IL-1α水平的升高→TNF-α、IL-1α引起的受体信号物质如中性粒细胞、脑血管内皮细胞等细胞表面黏附分子CD54表达→脑血管内皮细胞、神经胶质细胞和神经细胞接受细胞膜上受体刺激后细胞内信号物质的NO、MDA增加→脑组织结构损伤。②LPS直接引起受体信号物质CD54表达→CD54诱导TNF-α、IL-1α水平的变化→引发炎性介质的瀑布样效应,导致大量介质的激活→引起多种受体信号物质CD54、CD56、CD106表达→脑血管内皮细胞、神经胶质细胞和神经细胞接受细胞膜上受体刺激后细胞内信号物质的NO、MDA增加→脑组织结构损伤。3.四逆注射液抢救休克是确有疗效的,而且其抗休克作用是多方面的,既可以升高血压、脑组织SOD含量、CD56的表达,又可以降低TNF-α、IL-1α、MDA的含量与CD54、CD106、iNOS、凋亡细胞的表达。但四逆汤的起效时间比较晚,作用效果稳定持久;地塞米松的起效时间比较早,但是作用效果不持久。
【Abstract】 Septic shock is the widespread and complex problem concerned in modern Critical Care Medicine.It becomes the prominent issue to seriously impact on prognosis of patients and further improvement of successful treatment rate.One group of abroad clinical epidemiological research papers indicated that septic shock occurred at a rate of 4%among 2527 cases of patients with systemic inflammatory response,and 46%of septic shock patients were died.A study in China showed that 40%patients whose body surface areas are burned more than 30 percent had concurrently septic shock.Study on critically ill patients also showed that incidence of septic shock was 15.7%,61.1%of septic shock further developed to multiple organ dysfunction syndrome(MODS),fatality rate was 30.6%.Based on above studies,it has very important theoretical value and clinical significance to reveal septic shock pathogenesis and explore clinical prevention and treatment methods.When simplex western medicine treatment hardly makes progress in effect of septic shock,it appears urgently to look for Western & Chinese Traditional Medicine method to treat septic shock.Many medical scholars hope to find a breakthrough for septic shock with Chinese traditional medicine treatment.According to clinical manifestations, the septic shock is the scope of syncope.As most clinical manifestations are also collapse,it’s also named as syncope and collapse syndrome.The development of traditional Chinese medicine in thousands of years was a research on septic shock and accumulated much experience,but the treatment is lack of evidence because of the unclear mechanism of septic shock.Much progress has been made in recent years through studying septic shock,however brain injury is rarely involved.This study takes the models of endotoxin-shock rats as research object to simulate septic shock.The study aims are to explore anomalous signal transduction in brain injury caused by septic shock and the effect of Sini Decoction in the process;define the molecular and cellular mechanisms of reviving yang for resuscitation method used to prevent and treat brain injury in septic shock and establish the scientific foundation for modernizing traditional Chinese medicine.Objective1.Clear level of inflammatory mediators,the expression of cell-surface adhesion molecules of neutrophil and brain vascular endothelial cells,the changes of function metabolism and structural of brain vascular smooth muscle cells,glial cells,nerve cells stimulated by the extracellular inflammatory mediator of brain injury in septic shock.Then sum up regular relationship among them.2.Clear if the role of increasing of IL-1α、TNF-αcan upregulate the expression of cell-surface adhesion molecules of neutrophil and brain vascular endothelial cells,the relationship between the abnormal quantity of gas messenger-NO in brain vascular smooth muscle cells,glial cells and nerve cells and different brain regions vascular response and neuronal damage and repair.3.Clear how Sini decoction act on above indicators in order to descript the method of reviving yang for resuscitation how improve molecular mechanisms of brain microcirculatory disturbances and neuronal damage.Develop Chinese medicine A&E theory.Methods1.This study takes the models of endotoxin-shock rats simulating septic shock for research object.First using technology of gene chip to screen prereceptor signal substances, inflammatory cytokines,which have an obvious role in septic shock.2.To explore the role of Sini decoction on prereceptor signal substances induced by septic shock.Such as levels of tissue inflammatory mediators,IL-1α、TNF-α.The specific methods uses ELISA to measure IL-1α、TNF-αcontent in septic rat brain tissue.3.To explore how Sini decoction act on receptor signal substances induced by increasing IL-1α、TNF-αcontent.Such as the expression of cell-surface adhesion molecules of neutrophil and brain vascular endothelial cells,ICAM-1(CD54)、NCAM(CD56)、VCAM-1(CD106). The specific methods uses SABC to determine distribution and quantity of ICAM-1(CD54)、NCAM(CD56)、VCAM-1(CD106) in rat brain tissue.4.To explore how Sini decoction act on the changes of signal substances after brain vascular smooth muscle cells,glial cells and nerve cells receiving membrane receptor stimulation. Such as NO production and SOD,MDA content in rat brain.The specific methods is to use biochemical methods to determine SOD,MDA content and SABC to determine distribution and quantity of iNOS rat brain tissue.5.At last explore how Sini decoction improve tissue structure after septic brain injury. Using TUNEL to determine apoptosis in rat brain,ultrastructural changes of the cells with electron microscopy,structural changes of brain tissue with microscopy.Results1.Mean arterial blood pressure of sham-operated rats started to decline slightly at 1 hour. After i.v.injection of lipopolysaccharide,significant difference between model group and sham operated group at 1 hour(p<0.01),blood pressure fell to 40.75%of the initial values at 6 hour. Blood pressure of dexamethasone group fell to 45.32%of the initial values at 6 hour and had higher blood pressure compared with model group(p<0.05).Blood pressure of Sini decoction group fell to 67.68%of the initial values at 6 hour had higher blood pressure compared with model group(p<0.01).2.Screening Ccl2,4,7,20、Cxcll,2,9,10,11、Ccr2,3,9;TNF-α、IL-1α、IL-1βwith very significant high expression in endotoxic rat brain.And some inflammatory cytokines such asIL-13、IL- 15、I15ra which were very few reports had differentially expressed. IL-13 has no changes at 1 hour,up-regulation at2,3,6 hour;IL-15 has down-regulation at 1,2 hour and I15ra has down-regulation at 1,2,3,6 hour. 3.Concentration changes of TNF-α、IL-1αin endotoxic rat brain were same as mRNA content changes detected by gene chip technology from 1 hour to 6hour.①Concentration of TNF-αin model group were higher than sham operated group on four time periods(p<0.01) and reached peak at 6 hour.Concentration of TNF-αof dexamethasone group were lower than model group from 1 hour to 6 hour(p<0.01).Concentration of TNF-αof Sini decoction group were lower than model group at 3,6 hour(p<0.01).②Concentration of IL-1αin model group were lower than sham operated group at 1 hour(p<0.01),no significant difference at 2 hour(P>0.05),higher at 3(p<0.05),6(p<0.01)hour and reached peak at 6 hour.Concentration of IL-1αof dexamethasone group were lower than model group from 1 hour to 6hour(p<0.01). Concentration of IL-1αof Sini decoction group were lower than model group at 2,3,6 hour(p<0.01).4.No expression of CD54 and weak expression of CD56,CD106 in normal rat brain.The expression of CD54,CD56,CD106 in model group was higher than sham operated group and control group on four time periods(p<0.01).CD54,CD106 reached peak at 1 hour and CD56 reached peak at 6hour.The CD54 expressions of dexamethasone group were lower than model group at 1,2 hour(p<0.01).The CD56 expressions of dexamethasone group were higher than model group at 1 hour(p<0.01).The CD106 expressions of dexamethasone group were lower than model group at 1,2,6 hour(p<0.05).The CD54 expressions of Sini decoction group were lower than model group at 1,2(p<0.01),3(p<0.05) hour.The CD56 expressions of Sini decoction group were higher than model group at 3(p<0.05),6(p<0.01) hour.The CD106 expressions of Sini decoction group were lower than model group at 1,2,3(p<0.01),6(p<0.05) hour.5.No expression of iNOS in normal rat brain.The expression of iNOS in model group was highest at 1 hour,decreased at 2,3 hour and rebounded at 6 hour.The iNOS expressions of dexamethasone group were lower than model group at 1(p<0.01),6(p<0.05) hour.The iNOS expression of Sini decoction group was lower than model group at 6 hour(p<0.01). Concentration of SOD in model group was lower than sham operated group on four time periods (p<0.01) and reached lowest valley at 1 hour.Concentration of SOD of dexamethasone group and Sini decoction group were higher than model group at 1,2 hour(p<0.01).Concentration of MDA in model group was higher than sham operated group on four time periods(p<0.01) and reached peak at 6 hour.It’s changes regularities are same as SOD.Concentration of MDA of dexamethasone group was lower than model group at 1,2,3,6 hour(p<0.01).Concentration of MDA of Sini decoction group was lower than model group at 2,3,6 hour(p<0.01).6.Electron microscopy and light microscopy showed:There was reactive injury at 1 hour, injury restoration at 2,3 hour,serious damage at 6 hour in endotoxic shock rat brain;rat brain damage of dexamethasone group reduced at 1 hour;rat brain damage of Sini decoction group reduced at 6 hour.TUNEL showed:There was a very small number of apoptotic cells in normal rat brain.Apoptotic cells of model group were less than sham operated group and reached peak at 6 hour.Apoptotic cells of dexamethasone group were less than model group atl,2 hour(p<0.01).Apoptotic cells of Sini decoction group were less than model group at2,3, 6hour(p<0.01).Conclusion1.There were 91 differentially expressed inflammatory cytokines in rat brain after endotoxic shock.There were 36 inflammatory cytokines involved with inflammatory response among them.TNF-α、IL-1β、IL-10 have been reported more and IL-1α、IL-13、IL-15、I15ra have been reported less.2.Organ damage induced by two ways in endotoxic shock:①Increasing prereceptor signal substances such as TNF-α,IL-1αinduced by LPS.→The expression of cell-surface adhesion molecules of neutrophil and brain vascular endothelial cells,CD54.→Increasing signal substances such as NO production and SOD,MDA content after brain vascular smooth muscle cells,glial cells and nerve cells receiving membrane receptor stimulation.→Rat brain tissue structure damage.②The expression of cell-surface adhesion molecules,CD54,induced by LPS.→CD54 increased content of TNF-α,IL-1α.→Lead to inflammatory mediator waterfall cascade reactions and a large number of mediator was activated.→The expression of many cell-surface adhesion molecules,CD54,CD56,CD106.→Increasing signal substances such as NO production and SOD,MDA content after brain vascular smooth muscle cells and nerve cells receiving membrane receptor stimulation.→Rat brain tissue structure damage.3.Sini Tang Injection has efficacy at treating shock and multifaceted roles.It can up-regulate blood pressure,SOD content,CD56 expression and down-regulate content of TNF-α, IL-1α,MDA and CD54,CD106,iNOS,apoptotic cells expression.Sini decoction took effect relatively late but lasting stability and dexamethasone relatively early but unstability.