清脑宣窍滴丸调控大鼠急性脑缺血损伤炎性代谢网络紊乱的研究
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摘要
缺血性脑血管病是威胁人类健康与生存的主要疾病之一,具有高发病率、高患病率、高死亡率、高致残率、高复发率的特点。近年来研究表明脑缺血促发的炎性反应是导致脑缺血性损伤的重要因素。脑缺血促发的炎症反应是多途径、多步骤、多因素相互作用的复杂网络过程,其主要涉及炎性细胞的浸润、黏附分子及趋化因子的表达、核转录因子的活化等病理环节。体内炎性代谢网络存在复杂的动态调控机制,有多种致炎因子、细胞因子、环氧化酶和5-脂氧酶(5-LOX)两条通路参与调控。其中,5-LOX通路中半胱氨酰白三烯(CysLTs)及其受体(CysLT1和CysLT2)在急性脑缺血中的作用及意义研究较少。因此深入探讨缺血性脑血管病的炎性网络紊乱,明确其在脑缺血病变过程中的作用机制,具有十分重要的医学价值。
清脑宣窍滴丸(QNXQDP)来源于王永炎院士的临床经验方,由三七、栀子、冰片组成,功效为醒神开窍、解毒通络,本校中药化学教研室经工艺优化提取和大孔树脂分离富集后,运用现代制剂技术制成QNXQDP,拟用于缺血性脑中风急性期和恢复早期阶段的防治。
基于脑缺血疾病和药物干预下的系统代谢网络的整体性和动态性的变化,系统生物学的策略和方法适用于评价中药整体效应,从而解释中药复方对疾病的治疗作用及其机制。
本课题在前期QNXQDP防治脑缺血药效学研究基础上,结合缺血性损伤炎症反应的研究现状,运用药理学、分子生物学等技术对QNXQDP调控大鼠急性脑缺血损伤炎性代谢网络紊乱进行了深入探讨。主要包括三部分研究内容:一QNXQDP对急性脑缺血再灌注损伤大鼠脑损伤的保护作用。二线散QNXQDP对急性脑缺血再灌注损伤大鼠炎症级联反应的阻抑作用。三QNXQDP对急性脑缺血再灌注损伤大鼠脑组织5-LOX/白三烯通路的干预。重点探讨脑缺血损伤炎症级联反应和白三烯类物质的变化及药物对其的影响,探索性研究CysLTs受体在脑部的分布及功能,为QNXQDP防治脑缺血损伤提供依据,以期发现可能调控脑缺血炎性代谢网络的作用靶点。
1清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠的脑保护作用
1.1清脑宣窍滴丸对MCAO大鼠神经症状评分及脑梗死范围的影响
目的:通过观察神经缺失症状、大鼠脑梗塞范围,评价QNXQDP对急性脑缺血再灌注损伤的保护作用。方法:线拴法复制急性脑缺血再灌注损伤模型(MCAO),采用Zea Longa(1989)5级评分法评价神经缺失症状,染色称重法,计算大鼠脑梗塞百分比。结果:①缺血2h再灌4h和22h,假手术组大鼠没有神经功能缺损表现,其他组大鼠再灌4h出现了不同程度的神经功能缺损症状,22h有所加重。各给药组可不同程度改善损伤模型大鼠再灌4h和22h的神经缺损症状,QNXQDP180mg/kg组和AGNHW300mg/kg组与模型组比较有显著差异(P＜0.05)。缺血2h再灌注22h,模型组大鼠脑梗塞范围明显增大,QNXQDP 90、180mg/kg可明显减小脑梗塞范围。结论:急性脑缺血再灌注4h和22h,模型组具有明显的神经缺失症状,脑缺血再灌注22h,梗死范围明显增大;QNXQDP各剂量组可不同程度改善神经缺失症状,减小梗死范围。
1.2清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织病理形态学的影响
目的:通过检测脑组织的病理形态改变,评价QNXQDP对急性脑缺血再灌注损伤的保护作用。方法:复制MCAC模型,采用HE染色观察脑缺血再灌注后脑组织的病理变化和神经元形态、结构变化;采用尼氏体染色方法检测海马锥体细胞尼氏体的变化;应用免疫组织化学技术检测MCAO致急性脑缺血损伤大鼠皮层、海马CA1和CA3区胶质纤维酸性蛋白(GFAP)的表达。结果:缺血2h再灌22h后,模型组大鼠可见皮层、海马组织形态学异常,损伤皮层、海马CA1、CA3区锥体细胞排列紊乱,部分神经元脱失,尼氏小体含量明显减少,部分神经元变性、坏死,完整的锥体细胞数明显减少;QNXQDP各剂量组可不同程度改善MCAO大鼠术后24h的病理形态改变;明显减轻模型大鼠皮层、海马神经元的变性程度,海马CA1区完整锥体细胞数明显增多,皮质部和海马区星形胶质细胞明显增多,GFAP表达上调。结论:急性脑缺血再灌注22h后,模型具有明显的缺血损伤,皮层、海马区神经细胞有明显损伤,GFAP蛋白表达明显减少;QNXQDP可以保护皮层、海马神经细胞,减弱海马脑区星形胶质细胞的反应性。
1.3清脑宣窍滴丸对急性脑缺血损伤大鼠神经元特异性损伤标志物NSE含量的影响
目的:检测急性脑缺血损伤模型大鼠神经元损伤标志酶特异性烯醇化酶(NSE)的含量,研究急性脑缺血损伤后神经元的损害程度及药物对神经元的影响。方法:复制MCAO模型,缺血2h再灌22h后处死,放射免疫法测血清中神经元特异性烯醇化酶(NSE)的含量。结果:缺血2h再灌22h后,大鼠血清中NSE含量明显增加,与假手术组相比,有显著差异((P＜0.05);QNXQDP各剂量组和CDSDP 81mg/kg组大鼠血清NSE含量显著降低,与模型组比有明显差异(p＜0.05)。结论:急性脑缺血损伤后,大鼠血清中NSE含量增加,QNXQDP各剂量组大鼠血清中NSE含量降低,表明QNXQDP可能通过阻抑大脑NSE释放减轻脑缺血损伤。
1.4清脑宣窍滴丸对急性脑缺血损伤大鼠脑细胞钙离子含量的影响
目的:检测急性脑缺血损伤模型大鼠脑细胞钙离子的含量,研究急性脑缺血损伤后炎症级联反应前期钙离子超载及药物对其的阻抑作用。方法:SD大鼠连续给药三天后复制MACO模型,缺血2h再灌22h后处死,流式细胞仪测大鼠患侧脑细胞内钙离子的含量。结果:缺血2h再灌22h后,大鼠患侧脑细胞钙离子含量明显增加,与假手术组相比,有显著差异((P＜0.001);QNXQDP45mg/kg组大鼠脑细胞钙离子含量显著降低,与模型组比较,具有统计学意义(p＜0.01);QNXQDP90、180mg/kg组大鼠脑细胞钙离子含量显著降低,与模型组比有极显著性差异(p＜0.001)。结论:急性脑缺血损伤后,大鼠脑细胞内钙离子含量增加,QNXQDP各剂量组大鼠脑细胞钙离子含量降低,表明QNXQDP可能通过阻抑大脑钙离子含量减轻脑缺血损伤。
2清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠炎症级联反应的阻抑作用
2.1清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织炎性细胞因子的影响
目的:检测急性脑缺血损伤大鼠脑组织中炎性细胞因子含量,观察急性脑缺血损伤后细胞因子的变化,探讨QNXQDP对其的阻抑作用。方法:SD大鼠连续给药三天后复制MACO模型,缺血2h再灌22h后,采用ELISA法检测脑组织中促炎细胞因子、抗炎细胞因子含量。结果:①缺血2h再灌22h后,模型组大鼠患侧脑组织促炎细胞因子IL-1β、IL-6、TNF-a和IL-8均明显升高,与假手术组相比有显著差异((P＜0.05或P＜0.01)。QNXQDP45、90、180mg/kg组患侧脑组织中的IL-1β含量降低,与模型组相比具有显著差异(P＜0.01);QNXQDP180mg/kg组患侧脑组织中的IL-6含量降低,与模型组相比具有显著差异(P＜0.05);QNXQDP 45、180 mg/kg组患侧脑组织中的TNF-a含量降低,与模型组相比具有显著差异(P＜0.01,P＜0.001);45、90mg/kg组患侧脑组织中的IL-8含量降低,与模型组相比具有显著差异(P＜0.001)。②缺血2h再灌22h后,模型组大鼠患侧脑组织抗炎细胞因子IL-4、IL-10、TGF-β含量有明显升高,与假手术组相比有显著差异((P＜0.001)。QNXQDP45、180mg/kg组患侧脑组织中的IL-4、IL-10、TGF-β含量降低,与模型组相比具有显著差异(P＜0.001);QNXQDP90mg/kg组患侧脑组织中的IL-10含量降低,与模型组相比具有显著差异(P＜0.05);QNXQDP90mg/kg组患侧脑组织中的TGF-β含量显著降低,与模型组相比具有显著差异(P＜0.001)。结论:急性脑缺血损伤后,大鼠患侧脑组织中炎性细胞因子含量明显升高,QNXQDP组炎性细胞因子含量均明显降低,表明QNXQDP可能通过调节炎性因子释放减轻脑缺血损伤。
2.2清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠一氧化氮及其合酶和前列腺素类物质的阻抑作用
目的:检测急性脑缺血损伤大鼠一氧化氮及其合酶和前列腺素类物质的变化,探讨QNXQDP对其的阻抑作用。方法:SD大鼠连续给药三天后造MACO模型,缺血2h再灌22h后,硝酸还原酶法测定脑匀浆NO含量、cNO和iNO活性;RIA法测血浆中TXB2和6-Keto-PGF1α的含量。结果:①缺血2h再灌22h后,其NO、TNO、nNO、iNO均有变化。模型组大鼠NO含量和TNO、nNO、iNO活力明显高于假手术组。QNXQDP45、90、180mg/kg可明显降低大鼠手术侧大脑NO含量(P＜0.001),180mg/kg可明显降低升高的TNO、nNO和iNO活力(P＜0.05);90mg/kg也可明显降低升高的TNO活力(P＜0.01)。45mg/kg明显降低升高的TNO活力(P＜0.05)。②缺血2h再灌22h后,大鼠血浆中炎性介质TXB_2和6-Keto-PGF1α含量均明显升高。QNXQDP180mg/kg和90mg/kg组血浆中TXB_2和6-Keto-PGF1α含量降低,与模型组相比具有显著差异(P＜0.05);45mg/kg组血浆中6-Keto-PGF1α含量明显降低,与模型组相比具有显著差异(P＜0.01)。结论:急性脑缺血损伤后,大鼠患侧脑组织NO含量及其合酶活性均增加,QNXQDP组NO含量及其合酶活性均明显降低,表明QNXQDP可能通过降低NO含量,抑制NOS活性减轻脑缺血损伤,调节TXB_2和6-Keto-PGF1α改善脑缺血损伤。
2.3清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织黏附、趋化及浸润的影响
目的:通过检测急性脑缺血损伤大鼠脑组织细胞黏附分子(ICAM-1)和巨嗜炎性细胞蛋白(MIP-a)的阳性表达,患侧脑匀浆中MPO含量的变化,探讨药物对急性脑缺血损伤大鼠脑组织白细胞和内皮细胞的黏附、趋化及浸润情况。方法:复制MCAO,采用免疫组织化学技术检测大鼠皮层、海马CA1区、CA3区ICAM-1和MIP-a的阳性表达面积和积分光密度值;采用生化法检测大鼠脑匀浆中MPO的含量。结果:①假手术组大鼠脑组织皮层、海马CA1、CA3区ICAM-1均有少量表达;缺血2h再灌22h后,大鼠患侧脑组织皮层ICAM-1阳性表达面积和积分光密度值明显升高,与假手术组比具有明显差异(P＜0.05);QNXQDP180mg/kg可明显降低MCAO大鼠皮层的ICAM-1积分光密度值,与模型组比较有明显差异(P＜0.05)。QNXQDP各剂量组及CDSDP组大鼠海马CA1、CA3区ICAM-1阳性表达面积和积分光密度值有降低趋势,但无统计学意义。②假手术组大鼠脑组织皮层、海马CA1、CA3区MIP-1α有少量表达;缺血2h再灌22h后,大鼠患侧脑组织皮层、海马CA1、CA3区MIP-1α阳性表达面积和积分光密度值明显升高;QNXQDP45、90mg/kg可明显提高降低MCAO大鼠海马CA3区的MIP-1α阳性表达,与模型组比较有明显差异(P＜0.05);QNXQDP180mg/kg组可明显提高降低MCAO大鼠皮层、海马CA1、CA3区的MIP-1α表达,与模型组比较有明显差异(P＜0.01)。③缺血2h再灌22h后,大鼠患侧脑匀浆中MPO含量明显升高。QNXQDP各剂量组患侧脑匀浆中MPO含量均降低,与模型组相比具有显著差异(P＜0.05)。结论:急性脑缺血损伤后,大鼠患侧脑组织海马区ICAM-1和MIP-1α阳性表达明显升高,患侧脑匀浆中MPO含量明显升高;QNXQDP不同剂量组ICAM-1、MIP-1α阳性表达均明显下调,患侧脑匀浆中MPO含量明显降低,表明QNXQDP可能通过抑制脑组织白细胞和内皮细胞的黏附、趋化及浸润减轻急性脑缺血损伤。
2.4清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织核转录因子的干预
目的:研究急性脑缺血损伤大鼠脑组织核转录因子(NF-KBP65)的变化,探讨QNXQDP对核转录因子的阻抑作用。方法:复制MCAO模型,脑缺血2h再灌注22h,采用免疫组化技术检测皮层、海马CA1区、CA3区NF-KBP65的阳性表达面积和积分光密度值。结果:假手术组患侧大脑半球中,NF-κB免疫反应阳性表达物多数位于细胞浆,缺血再灌模型大鼠缺血侧脑皮质NF-κB免疫反应阳性细胞明显发生核易位,棕黄色沉积物多表达于细胞核中,且多数神经元变性坏死。缺血2h再灌22h后,大鼠患侧脑组织皮层、海马CA1区、CA3区NF-KBP65阳性表达明显升高;QNXQDP90 mg/kg可明显下调MCAO大鼠海马CA3区的NF-KBP65阳性表达,与模型组比较有明显差异(P＜0.05);QNXQDP90、180mg/kg组可明显下调急性脑缺血损伤大鼠皮层、CA1、CA3区NF-KBP65蛋白的表达,与模型组比较有明显差异((P＜0.05))。结论:急性脑缺血损伤后,大鼠患侧脑组织海马区NF-KBP65蛋白阳性表达明显升高;QNXQDP不同剂量组NF-KBP65蛋白阳性表达均明显下调,表明QNXQDP可能通过阻抑海马区NF-KBP65蛋白表达减轻急性脑缺血损伤。
3清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织5-脂氧酶/白三烯通路的干预
3.1急性脑缺血损伤大鼠脑组织5-LOX、LTB_4、CysLTs含量的动态变化规律及药物对其的影响
目的:通过检测缺血12h再灌48h内急性脑缺血损伤大鼠脑组织5-LOX、LTB_4和CysLTs的含量变化,以期发现5-LOX、LTB_4和CysLTs的动态变化规律及在急性脑缺血损伤的作用,评价药物对其的影响。方法:SD大鼠连续给QNXQDP45、180mg/kg三天后复制MCAO模型,缺血2h,再灌注6、12、24、36、48h,用ELISA法检测上述各时间点患侧脑匀浆中5-LOX、LTB_4和CysLTs的含量。结果:①模型组大鼠脑缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中5-LOX的活力均明显增强,与假手术组相比有显著性差异(P＜0.001),再灌注后6h开始5-LOX的活力即明显增强,再灌注后6h至48h持续维持此高活力,最高峰值在缺血后24h,活力为假手术组的25.4倍。脑缺血2h再灌注不同时段,QNXQDP不同剂量组和Zileuton组大鼠患侧脑匀浆中5-LOX活力均有不同程度减弱。②模型组大鼠脑缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中LTB_4含量均明显升高,与假手术组相比有显著性差异(P＜0.01或P＜0.001),再灌注后6h开始LTB_4含量即明显升高,再灌注后6h至48h持续维持此高水平,最高峰值在缺血后12h,含量为假手术组的4.6倍。与模型组比较,脑缺血2h再灌注不同时段,QNXQDP不同剂量组和Zileuton组大鼠患侧脑匀浆中LTB_4含量均有不同程度降低。③模型组大鼠脑缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中CysLTs含量均明显升高,与假手术组相比有显著性差异(P＜0.01或P＜0.001),再灌注后6h开始CysLTs含量即有所升高,12h明显升高,再灌注后6h至48h持续维持高水平,最高峰值在缺血后36h,含量为假手术组的10.75倍。与模型组比较,QNXQDP各组和Zileuton组缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中CysLTs含量均有不同程度降低。结论:急性脑缺血损伤后,5-LOX在缺血后脑组织内的含量变化高峰在脑缺血后24h;LTB_4在缺血后脑组织内的含量变化高峰在脑缺血后12h;CysLTs在缺血后脑组织内的含量变化高峰在脑缺血后36h。与模型组比较,QNXQDP不同剂量组大鼠在缺血后不同时段脑组织内5-LOX、LTB_4和CysLTs的含量变化均有不同程度降低。表明QNXQDP可能通过抑制脑组织5-LOX活性、降低LTB_4及CysLTs的含量减轻急性脑缺血损伤。
3.2清脑宣窍滴丸对急性脑缺血损伤大鼠脑组织半胱氨酰白三烯受体(CysLT1及CysLT2)蛋白表达的干预
目的:检测急性脑缺血损伤大鼠脑组织皮层和海马CysLT1及CysLT2蛋白表达情况,研究急性脑缺血再灌注后5-LOX、LTB_4及CysLTs含量变化的分子机制,以期发现QNXQDP调控炎性代谢网络的分子靶点。方法:SD大鼠连续给QNXQDP45、180mg/kg三天后采用MCAO制备大鼠急性脑缺血再灌注模型,缺血2h再灌注12h,Western blot方法检测脑组织皮层和海马CysLT1及CysLT2蛋白的表达变化,Real time RT-PCR方法检测脑组织皮层和海马CYSLT1mRNA及CysLT2mRNA表达的变化。结果:缺血2h再灌12h后,模型组大鼠CysLT1和CysLT2蛋白在皮层和海马表达明显比假手术组升高(P＜0.05或P＜0.01),QNXQDP180mg/kg大鼠皮层和海马CysLT1和CysLT2蛋白表达量较模型组明显降低(P＜0.05);模型组大鼠CysLT1mRNA和CysLT2mRNA表达明显比假手术组升高(P＜0.05),QNXQDP180mg/kg大鼠皮层和海马CysLT1mRNA和CysLT2mRNA蛋白表达量较模型组明显降低(P＜0.01或P＜0.05)。结论:急性脑缺血损伤后,大鼠患侧脑组织皮层和海马CysLT1和CysLT2表达明显升高;QNXQDP180mg/kg组CysLT1和CysLT2表达均明显下调;大鼠患侧脑组织海马CysLT1mRNA和CysLT2表达明显升高;QNXQDP180mg/kg组CysLT1mRNA和CysLT2mRNA表达均明显下调,表明QNXQDP可能通过抑制脑组织CysLTs受体表达减轻脑损伤炎症,CysLT1及CysLT2可能是QNXQDP调控炎性代谢网络的分子靶点。
结论
QNXQDP可以减轻急性脑缺血再灌注损伤大鼠的神经病理损伤。其可能的作用环节包括:①抑制神经元特异性烯醇化酶(NSE)的含量,抑制钙超载;②调节炎症因子和介质,减少NO含量及抑制NOS的活性;③抑制脑组织白细胞和内皮细胞的黏附、趋化及浸润;④减少5-LOX、LTB_4及CysLTs含量及抑制CysLTs受体(CysLT1和CysLT2)表达。其防治缺血再灌注脑损伤,可能通过作用靶点CysLT1和CysLT2来调控炎性代谢网络紊乱。
本研究的创新点
1.基于系统生物学观点,以成分明确的小复方为研究对象,从急性脑缺血损伤后机体内炎性代谢网络研究思路入手,探讨QNXQDP调控炎性代谢网络紊乱的可能靶点,为脑缺血损伤炎症机制研究提供新思路。
2.动态观察48h内急性脑缺血再灌注损伤大鼠脑组织5-LOX、LTB_4及CysLTs的动态变化规律,探索性研究了CysLT1和CysLT2在脑缺血损伤后皮层和海马部位的表达及功能。
Ischemic cerebral vascular disease(ICVD) is well known with its high incidence,high death rate,high relapse rate and high mutilation rate.In recent years,it is proved that inflammatory reaction plays an important role in ischemic damage.It has been recognized that cytokines, adhesion molecules,inflammatory mediums,transcription factors and leukocyte infiltration are associated with ICVD and intimately related to each other,moreover,5-LOX,CysLT1,CysLT2 and their receptors which are reported to express in the brain,may be involved in the inflammatory pathogenesis of cerebral ischemia.All the factors mentionded above and their effect is the so-called inflammatory reaction cascade.Therefore,further research about the mechanism of ischemic cerebrovascular disease and the action targets of the pathological Changes have profound significance in modern medicine.
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In this study,by means of biochemical assay,radioimmunoassay(RIA),enzyme-linked immunosorbent assay(ELISA),immunohistochemistry(IHC) and molecular biology,the QNXQDP on the inflammatory reaction in rats with middle cerebral artery occlusion(MCAO) are investigated.
Meanwhile the mechanisms are further investigated from following aspects:cytokine production,leukocyte infiltrate,adhesion molecules and activation from nuclear transcription factors to the t expression level of CysLTs and CysLTs receptors
The chief results are as follows.
1.Protective effects of QNXQDP on cerebral ischemic injury in rats with MCAO
OBJECTIVE:To study the effects of QNXQDP on nerve symptoms,infarction size, pathomorphology changes,content of neurone specific enolase(NSE) in serum and Ca~(2+) in brain cell.METHODS:nerve symptoms were scored at different time-points(ischemia 2 hours, reperfusion 4 hours and reperfusion 22 hours),After 22 hours of reperfusion,TTC staining of brain tissue was made to determine the size of cerebral infarction,HE staining of brain was also performed to observe morphological changes,the content of NSE was assayed by RIA,the content of Ca~(2+) was assayed using Flow Cytometry.RESULTS:After MCAO,nerve symptoms, the size of cerebral infarction,the.content of NSE and Ca~(2+) rose greatly,QNXQDP decreased significantly.In model group,a number of neurons appeared degeneration,necrosis or disappearance,with leukocyte infiltration.In QNXQDP group,the status of pathomorphology was improved.CONCLUSION:QNXQDP can treat cerebral ischemic injury.
2.Effects of QNXQDP on inflammatory cascade in rats with MCAO
2.1 Effects of QNXQDP on content of inflammatory cytokines in rats with MCAO
OBJECTIVE:To research the effects of QNXQDP on the content of inflammatory cytokines after ischemia-reperfusion in rats with MCAO.METHODS:After ischemia 2 hours and reperfusion 22 hours,the contents of IL-1β、IL-8、IL-6、TNF-α、IL-4、IL-10 and TGF-βwere asssyed by ELISA.RESULTS:After MCAO,the contents of IL-1β、IL-8、IL-6、TNF-α、IL-4、IL-10 and TGF-βincreased significantly in contrast to sham group,QNXQDP can decrease them remarkably.CONCLUSION:QNXQDP can reduce the contents of cytokines in rats with MCAO to alleviate cerebral ischemic injury.
2.2 Effects of QNXQDP on content of nitric oxide(NO) and prostaglandin analogs,Activity of nitric oxide synthase(NOS) in rats with MCAO
OBJECTIVE:To research the effects of QNXQDP on the nitric oxide、nitric oxide synthase and prostaglandin analogs after ischemia-reperfusion in rats with MCAO.METHODS: ischemia 2 hours and reperfusion 22 hours,determining of nitrate reductase NO content in brain homogenate,cNO and iNO activity;measuring of plasma TXB2 and 6 - keto -PGF1αcontent with RIA.RESULTS:①After MCAO,content of NO and activity of TNO,nNO, iNO were significantly higher than sham group in brain.②After MCAO,QNXQDP decreasing content of NO and activity of TNO,nNO and iNO,also decreasing contents of TXB2 and 6 - keto-PGF1αin plasma.CONCLUSION:QNXQDP can decreased content of NO and activity of TNO,nNO and iNO,regulating TXB2 and 6 - keto-PGF1αto relieve cerebral ischemic injury.
2.3 Effects of QNXQDP on leukocyte and endothelial cells adhesion chemotaxis and infiltration in rats with MCAO
OBJECTIVE:To research the effects of QNXQDP on the expression of cell adhesion molecules(ICAM-I) and Macrophage inflammatory protein-1a(MIP-1a),changes of myoloperoxidase(MPO) content in brain tissue.METHODS:The expression of ICAM-Ⅰand MIP-1a protein was assayed by IHC.MPO was assayed by biochemisty.RESULTS:After MCAO,the expressing positive area and integral optical density of ICAM-Ⅰand MIP-1a, content of MPO increased significantly in model group,QNXQDP 180mg/kg and 90 mg/kg can inhibit the positive area and integral optical density of ICAM-Ⅰand MIP-1a on cortex and hippocampus,reduce the content of MPO in brain tissue.CONCLUSION:QNXQDP can decrease leukocyte and endothelial cells adhesion、chemotaxis and infiltration in rats with MCAO.
2.4 Effects of QNXQDP on nuclear transcription factors in rats with MCAO OBJECTIVE:To investigate the effects of QNXQDP on the expression of nuclear factor-κB (NF-κB) in brain tissue.METHODS:The expression of NF-κB protein was assayed by IHC. RESULTS:After MCAO,the expression of NF-κB increased significantly in model group, and cortex and hippocampus showed nuclear translocation.QNXQDP 180mg/kg and 90 mg/kg could inhibit the nuclear translocation and decreace the positive area and integral optical density of NF-κB in cortex and hippocampus.CONCLUSION:QNXQDP could inhibit the expression of transcription factors in brain.
3.Effects of QNXQDP on 5 - lipoxygenase / leukotriene pathway in rats with MCAO
OBJECTIVE:To investigate the effects of QNXQDP on the content of 5-LOX、LTB4 and CysLTs,CysLT1 and CysLT2 expression in brain tissue.METHODS:ischemia 2 hours and reperfusion 6、12、24、36、48h hours,the content of 5-LOX、LTB4 and CysLTs was assayed by ELISA.RESULTS:ischemia 2 hours and reperfusion 6、12、24、36、48h hours,the content of 5-LOX、LTB4 and CysLTs were increased in model group compare with shame group, expression of CysLT1 and CysLT2 in cortex and hippocampus also increased in cortex and hippocampus.QNXQDP can decrease the content of 5-LOX、LTB4 and CysLTs in brain tissue,CONCLUSION:QNXQDP could decrease the contents of 5-LOX、LTB4 and CysLTs in brain,also could inhibit the expression of CysLT1 and CysLT2 in cortex and hippocampus.
CONCLUSION
QNXQDP can alleviate neuronal injury and the inflammatory reaction after of ischemia-reperfusion in rats,whose its main mechanisms of action may be associated with:①inhibition of neuron-specific enolase(NSE) levels and calcium overload;②regulation of inflammatory cytokines,reducing the NO content and inhibiting NOS activity;③inhibiting leukocyte and endothelial cells adhesion、chemotaxis and infiltration in brain tissue;④reducing the content of 5-LOX,LTB4 and CysLTs,inhibiting the expression of cysteinyl leukotriene receptor(CysLT1 and CysLT2) in cortex and hippocampus.Its prevention and treatment inflammatory injury after cerebral ischemia-reperfusion.In addition,CysLT1 and CysLT2may be targets for inhibition.
In this experiment study,we observed the effects of QNXQDP on the expression of protein CysLT1 and CysLT2 in cortex and hippocampus of ischemia-reperfusion rats,which has not been reported by now.The reports about the dynamic changes of the content 5-LOX, LTB4 and CysLTs after cerebral ischemia-reperfusion within 48 hours are also very few. Therefore,our study is innovative to a certain some extent.
清脑宣窍滴丸(QNXQDP)来源于王永炎院士的临床经验方,由三七、栀子、冰片组成,功效为醒神开窍、解毒通络,本校中药化学教研室经工艺优化提取和大孔树脂分离富集后,运用现代制剂技术制成QNXQDP,拟用于缺血性脑中风急性期和恢复早期阶段的防治。
基于脑缺血疾病和药物干预下的系统代谢网络的整体性和动态性的变化,系统生物学的策略和方法适用于评价中药整体效应,从而解释中药复方对疾病的治疗作用及其机制。
本课题在前期QNXQDP防治脑缺血药效学研究基础上,结合缺血性损伤炎症反应的研究现状,运用药理学、分子生物学等技术对QNXQDP调控大鼠急性脑缺血损伤炎性代谢网络紊乱进行了深入探讨。主要包括三部分研究内容:一QNXQDP对急性脑缺血再灌注损伤大鼠脑损伤的保护作用。二线散QNXQDP对急性脑缺血再灌注损伤大鼠炎症级联反应的阻抑作用。三QNXQDP对急性脑缺血再灌注损伤大鼠脑组织5-LOX/白三烯通路的干预。重点探讨脑缺血损伤炎症级联反应和白三烯类物质的变化及药物对其的影响,探索性研究CysLTs受体在脑部的分布及功能,为QNXQDP防治脑缺血损伤提供依据,以期发现可能调控脑缺血炎性代谢网络的作用靶点。
1清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠的脑保护作用
1.1清脑宣窍滴丸对MCAO大鼠神经症状评分及脑梗死范围的影响
目的:通过观察神经缺失症状、大鼠脑梗塞范围,评价QNXQDP对急性脑缺血再灌注损伤的保护作用。方法:线拴法复制急性脑缺血再灌注损伤模型(MCAO),采用Zea Longa(1989)5级评分法评价神经缺失症状,染色称重法,计算大鼠脑梗塞百分比。结果:①缺血2h再灌4h和22h,假手术组大鼠没有神经功能缺损表现,其他组大鼠再灌4h出现了不同程度的神经功能缺损症状,22h有所加重。各给药组可不同程度改善损伤模型大鼠再灌4h和22h的神经缺损症状,QNXQDP180mg/kg组和AGNHW300mg/kg组与模型组比较有显著差异(P＜0.05)。缺血2h再灌注22h,模型组大鼠脑梗塞范围明显增大,QNXQDP 90、180mg/kg可明显减小脑梗塞范围。结论:急性脑缺血再灌注4h和22h,模型组具有明显的神经缺失症状,脑缺血再灌注22h,梗死范围明显增大;QNXQDP各剂量组可不同程度改善神经缺失症状,减小梗死范围。
1.2清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织病理形态学的影响
目的:通过检测脑组织的病理形态改变,评价QNXQDP对急性脑缺血再灌注损伤的保护作用。方法:复制MCAC模型,采用HE染色观察脑缺血再灌注后脑组织的病理变化和神经元形态、结构变化;采用尼氏体染色方法检测海马锥体细胞尼氏体的变化;应用免疫组织化学技术检测MCAO致急性脑缺血损伤大鼠皮层、海马CA1和CA3区胶质纤维酸性蛋白(GFAP)的表达。结果:缺血2h再灌22h后,模型组大鼠可见皮层、海马组织形态学异常,损伤皮层、海马CA1、CA3区锥体细胞排列紊乱,部分神经元脱失,尼氏小体含量明显减少,部分神经元变性、坏死,完整的锥体细胞数明显减少;QNXQDP各剂量组可不同程度改善MCAO大鼠术后24h的病理形态改变;明显减轻模型大鼠皮层、海马神经元的变性程度,海马CA1区完整锥体细胞数明显增多,皮质部和海马区星形胶质细胞明显增多,GFAP表达上调。结论:急性脑缺血再灌注22h后,模型具有明显的缺血损伤,皮层、海马区神经细胞有明显损伤,GFAP蛋白表达明显减少;QNXQDP可以保护皮层、海马神经细胞,减弱海马脑区星形胶质细胞的反应性。
1.3清脑宣窍滴丸对急性脑缺血损伤大鼠神经元特异性损伤标志物NSE含量的影响
目的:检测急性脑缺血损伤模型大鼠神经元损伤标志酶特异性烯醇化酶(NSE)的含量,研究急性脑缺血损伤后神经元的损害程度及药物对神经元的影响。方法:复制MCAO模型,缺血2h再灌22h后处死,放射免疫法测血清中神经元特异性烯醇化酶(NSE)的含量。结果:缺血2h再灌22h后,大鼠血清中NSE含量明显增加,与假手术组相比,有显著差异((P＜0.05);QNXQDP各剂量组和CDSDP 81mg/kg组大鼠血清NSE含量显著降低,与模型组比有明显差异(p＜0.05)。结论:急性脑缺血损伤后,大鼠血清中NSE含量增加,QNXQDP各剂量组大鼠血清中NSE含量降低,表明QNXQDP可能通过阻抑大脑NSE释放减轻脑缺血损伤。
1.4清脑宣窍滴丸对急性脑缺血损伤大鼠脑细胞钙离子含量的影响
目的:检测急性脑缺血损伤模型大鼠脑细胞钙离子的含量,研究急性脑缺血损伤后炎症级联反应前期钙离子超载及药物对其的阻抑作用。方法:SD大鼠连续给药三天后复制MACO模型,缺血2h再灌22h后处死,流式细胞仪测大鼠患侧脑细胞内钙离子的含量。结果:缺血2h再灌22h后,大鼠患侧脑细胞钙离子含量明显增加,与假手术组相比,有显著差异((P＜0.001);QNXQDP45mg/kg组大鼠脑细胞钙离子含量显著降低,与模型组比较,具有统计学意义(p＜0.01);QNXQDP90、180mg/kg组大鼠脑细胞钙离子含量显著降低,与模型组比有极显著性差异(p＜0.001)。结论:急性脑缺血损伤后,大鼠脑细胞内钙离子含量增加,QNXQDP各剂量组大鼠脑细胞钙离子含量降低,表明QNXQDP可能通过阻抑大脑钙离子含量减轻脑缺血损伤。
2清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠炎症级联反应的阻抑作用
2.1清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织炎性细胞因子的影响
目的:检测急性脑缺血损伤大鼠脑组织中炎性细胞因子含量,观察急性脑缺血损伤后细胞因子的变化,探讨QNXQDP对其的阻抑作用。方法:SD大鼠连续给药三天后复制MACO模型,缺血2h再灌22h后,采用ELISA法检测脑组织中促炎细胞因子、抗炎细胞因子含量。结果:①缺血2h再灌22h后,模型组大鼠患侧脑组织促炎细胞因子IL-1β、IL-6、TNF-a和IL-8均明显升高,与假手术组相比有显著差异((P＜0.05或P＜0.01)。QNXQDP45、90、180mg/kg组患侧脑组织中的IL-1β含量降低,与模型组相比具有显著差异(P＜0.01);QNXQDP180mg/kg组患侧脑组织中的IL-6含量降低,与模型组相比具有显著差异(P＜0.05);QNXQDP 45、180 mg/kg组患侧脑组织中的TNF-a含量降低,与模型组相比具有显著差异(P＜0.01,P＜0.001);45、90mg/kg组患侧脑组织中的IL-8含量降低,与模型组相比具有显著差异(P＜0.001)。②缺血2h再灌22h后,模型组大鼠患侧脑组织抗炎细胞因子IL-4、IL-10、TGF-β含量有明显升高,与假手术组相比有显著差异((P＜0.001)。QNXQDP45、180mg/kg组患侧脑组织中的IL-4、IL-10、TGF-β含量降低,与模型组相比具有显著差异(P＜0.001);QNXQDP90mg/kg组患侧脑组织中的IL-10含量降低,与模型组相比具有显著差异(P＜0.05);QNXQDP90mg/kg组患侧脑组织中的TGF-β含量显著降低,与模型组相比具有显著差异(P＜0.001)。结论:急性脑缺血损伤后,大鼠患侧脑组织中炎性细胞因子含量明显升高,QNXQDP组炎性细胞因子含量均明显降低,表明QNXQDP可能通过调节炎性因子释放减轻脑缺血损伤。
2.2清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠一氧化氮及其合酶和前列腺素类物质的阻抑作用
目的:检测急性脑缺血损伤大鼠一氧化氮及其合酶和前列腺素类物质的变化,探讨QNXQDP对其的阻抑作用。方法:SD大鼠连续给药三天后造MACO模型,缺血2h再灌22h后,硝酸还原酶法测定脑匀浆NO含量、cNO和iNO活性;RIA法测血浆中TXB2和6-Keto-PGF1α的含量。结果:①缺血2h再灌22h后,其NO、TNO、nNO、iNO均有变化。模型组大鼠NO含量和TNO、nNO、iNO活力明显高于假手术组。QNXQDP45、90、180mg/kg可明显降低大鼠手术侧大脑NO含量(P＜0.001),180mg/kg可明显降低升高的TNO、nNO和iNO活力(P＜0.05);90mg/kg也可明显降低升高的TNO活力(P＜0.01)。45mg/kg明显降低升高的TNO活力(P＜0.05)。②缺血2h再灌22h后,大鼠血浆中炎性介质TXB_2和6-Keto-PGF1α含量均明显升高。QNXQDP180mg/kg和90mg/kg组血浆中TXB_2和6-Keto-PGF1α含量降低,与模型组相比具有显著差异(P＜0.05);45mg/kg组血浆中6-Keto-PGF1α含量明显降低,与模型组相比具有显著差异(P＜0.01)。结论:急性脑缺血损伤后,大鼠患侧脑组织NO含量及其合酶活性均增加,QNXQDP组NO含量及其合酶活性均明显降低,表明QNXQDP可能通过降低NO含量,抑制NOS活性减轻脑缺血损伤,调节TXB_2和6-Keto-PGF1α改善脑缺血损伤。
2.3清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织黏附、趋化及浸润的影响
目的:通过检测急性脑缺血损伤大鼠脑组织细胞黏附分子(ICAM-1)和巨嗜炎性细胞蛋白(MIP-a)的阳性表达,患侧脑匀浆中MPO含量的变化,探讨药物对急性脑缺血损伤大鼠脑组织白细胞和内皮细胞的黏附、趋化及浸润情况。方法:复制MCAO,采用免疫组织化学技术检测大鼠皮层、海马CA1区、CA3区ICAM-1和MIP-a的阳性表达面积和积分光密度值;采用生化法检测大鼠脑匀浆中MPO的含量。结果:①假手术组大鼠脑组织皮层、海马CA1、CA3区ICAM-1均有少量表达;缺血2h再灌22h后,大鼠患侧脑组织皮层ICAM-1阳性表达面积和积分光密度值明显升高,与假手术组比具有明显差异(P＜0.05);QNXQDP180mg/kg可明显降低MCAO大鼠皮层的ICAM-1积分光密度值,与模型组比较有明显差异(P＜0.05)。QNXQDP各剂量组及CDSDP组大鼠海马CA1、CA3区ICAM-1阳性表达面积和积分光密度值有降低趋势,但无统计学意义。②假手术组大鼠脑组织皮层、海马CA1、CA3区MIP-1α有少量表达;缺血2h再灌22h后,大鼠患侧脑组织皮层、海马CA1、CA3区MIP-1α阳性表达面积和积分光密度值明显升高;QNXQDP45、90mg/kg可明显提高降低MCAO大鼠海马CA3区的MIP-1α阳性表达,与模型组比较有明显差异(P＜0.05);QNXQDP180mg/kg组可明显提高降低MCAO大鼠皮层、海马CA1、CA3区的MIP-1α表达,与模型组比较有明显差异(P＜0.01)。③缺血2h再灌22h后,大鼠患侧脑匀浆中MPO含量明显升高。QNXQDP各剂量组患侧脑匀浆中MPO含量均降低,与模型组相比具有显著差异(P＜0.05)。结论:急性脑缺血损伤后,大鼠患侧脑组织海马区ICAM-1和MIP-1α阳性表达明显升高,患侧脑匀浆中MPO含量明显升高;QNXQDP不同剂量组ICAM-1、MIP-1α阳性表达均明显下调,患侧脑匀浆中MPO含量明显降低,表明QNXQDP可能通过抑制脑组织白细胞和内皮细胞的黏附、趋化及浸润减轻急性脑缺血损伤。
2.4清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织核转录因子的干预
目的:研究急性脑缺血损伤大鼠脑组织核转录因子(NF-KBP65)的变化,探讨QNXQDP对核转录因子的阻抑作用。方法:复制MCAO模型,脑缺血2h再灌注22h,采用免疫组化技术检测皮层、海马CA1区、CA3区NF-KBP65的阳性表达面积和积分光密度值。结果:假手术组患侧大脑半球中,NF-κB免疫反应阳性表达物多数位于细胞浆,缺血再灌模型大鼠缺血侧脑皮质NF-κB免疫反应阳性细胞明显发生核易位,棕黄色沉积物多表达于细胞核中,且多数神经元变性坏死。缺血2h再灌22h后,大鼠患侧脑组织皮层、海马CA1区、CA3区NF-KBP65阳性表达明显升高;QNXQDP90 mg/kg可明显下调MCAO大鼠海马CA3区的NF-KBP65阳性表达,与模型组比较有明显差异(P＜0.05);QNXQDP90、180mg/kg组可明显下调急性脑缺血损伤大鼠皮层、CA1、CA3区NF-KBP65蛋白的表达,与模型组比较有明显差异((P＜0.05))。结论:急性脑缺血损伤后,大鼠患侧脑组织海马区NF-KBP65蛋白阳性表达明显升高;QNXQDP不同剂量组NF-KBP65蛋白阳性表达均明显下调,表明QNXQDP可能通过阻抑海马区NF-KBP65蛋白表达减轻急性脑缺血损伤。
3清脑宣窍滴丸对急性脑缺血再灌注损伤大鼠脑组织5-脂氧酶/白三烯通路的干预
3.1急性脑缺血损伤大鼠脑组织5-LOX、LTB_4、CysLTs含量的动态变化规律及药物对其的影响
目的:通过检测缺血12h再灌48h内急性脑缺血损伤大鼠脑组织5-LOX、LTB_4和CysLTs的含量变化,以期发现5-LOX、LTB_4和CysLTs的动态变化规律及在急性脑缺血损伤的作用,评价药物对其的影响。方法:SD大鼠连续给QNXQDP45、180mg/kg三天后复制MCAO模型,缺血2h,再灌注6、12、24、36、48h,用ELISA法检测上述各时间点患侧脑匀浆中5-LOX、LTB_4和CysLTs的含量。结果:①模型组大鼠脑缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中5-LOX的活力均明显增强,与假手术组相比有显著性差异(P＜0.001),再灌注后6h开始5-LOX的活力即明显增强,再灌注后6h至48h持续维持此高活力,最高峰值在缺血后24h,活力为假手术组的25.4倍。脑缺血2h再灌注不同时段,QNXQDP不同剂量组和Zileuton组大鼠患侧脑匀浆中5-LOX活力均有不同程度减弱。②模型组大鼠脑缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中LTB_4含量均明显升高,与假手术组相比有显著性差异(P＜0.01或P＜0.001),再灌注后6h开始LTB_4含量即明显升高,再灌注后6h至48h持续维持此高水平,最高峰值在缺血后12h,含量为假手术组的4.6倍。与模型组比较,脑缺血2h再灌注不同时段,QNXQDP不同剂量组和Zileuton组大鼠患侧脑匀浆中LTB_4含量均有不同程度降低。③模型组大鼠脑缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中CysLTs含量均明显升高,与假手术组相比有显著性差异(P＜0.01或P＜0.001),再灌注后6h开始CysLTs含量即有所升高,12h明显升高,再灌注后6h至48h持续维持高水平,最高峰值在缺血后36h,含量为假手术组的10.75倍。与模型组比较,QNXQDP各组和Zileuton组缺血2h再灌注6h-48h时段,大鼠患侧脑匀浆中CysLTs含量均有不同程度降低。结论:急性脑缺血损伤后,5-LOX在缺血后脑组织内的含量变化高峰在脑缺血后24h;LTB_4在缺血后脑组织内的含量变化高峰在脑缺血后12h;CysLTs在缺血后脑组织内的含量变化高峰在脑缺血后36h。与模型组比较,QNXQDP不同剂量组大鼠在缺血后不同时段脑组织内5-LOX、LTB_4和CysLTs的含量变化均有不同程度降低。表明QNXQDP可能通过抑制脑组织5-LOX活性、降低LTB_4及CysLTs的含量减轻急性脑缺血损伤。
3.2清脑宣窍滴丸对急性脑缺血损伤大鼠脑组织半胱氨酰白三烯受体(CysLT1及CysLT2)蛋白表达的干预
目的:检测急性脑缺血损伤大鼠脑组织皮层和海马CysLT1及CysLT2蛋白表达情况,研究急性脑缺血再灌注后5-LOX、LTB_4及CysLTs含量变化的分子机制,以期发现QNXQDP调控炎性代谢网络的分子靶点。方法:SD大鼠连续给QNXQDP45、180mg/kg三天后采用MCAO制备大鼠急性脑缺血再灌注模型,缺血2h再灌注12h,Western blot方法检测脑组织皮层和海马CysLT1及CysLT2蛋白的表达变化,Real time RT-PCR方法检测脑组织皮层和海马CYSLT1mRNA及CysLT2mRNA表达的变化。结果:缺血2h再灌12h后,模型组大鼠CysLT1和CysLT2蛋白在皮层和海马表达明显比假手术组升高(P＜0.05或P＜0.01),QNXQDP180mg/kg大鼠皮层和海马CysLT1和CysLT2蛋白表达量较模型组明显降低(P＜0.05);模型组大鼠CysLT1mRNA和CysLT2mRNA表达明显比假手术组升高(P＜0.05),QNXQDP180mg/kg大鼠皮层和海马CysLT1mRNA和CysLT2mRNA蛋白表达量较模型组明显降低(P＜0.01或P＜0.05)。结论:急性脑缺血损伤后,大鼠患侧脑组织皮层和海马CysLT1和CysLT2表达明显升高;QNXQDP180mg/kg组CysLT1和CysLT2表达均明显下调;大鼠患侧脑组织海马CysLT1mRNA和CysLT2表达明显升高;QNXQDP180mg/kg组CysLT1mRNA和CysLT2mRNA表达均明显下调,表明QNXQDP可能通过抑制脑组织CysLTs受体表达减轻脑损伤炎症,CysLT1及CysLT2可能是QNXQDP调控炎性代谢网络的分子靶点。
结论
QNXQDP可以减轻急性脑缺血再灌注损伤大鼠的神经病理损伤。其可能的作用环节包括:①抑制神经元特异性烯醇化酶(NSE)的含量,抑制钙超载;②调节炎症因子和介质,减少NO含量及抑制NOS的活性;③抑制脑组织白细胞和内皮细胞的黏附、趋化及浸润;④减少5-LOX、LTB_4及CysLTs含量及抑制CysLTs受体(CysLT1和CysLT2)表达。其防治缺血再灌注脑损伤,可能通过作用靶点CysLT1和CysLT2来调控炎性代谢网络紊乱。
本研究的创新点
1.基于系统生物学观点,以成分明确的小复方为研究对象,从急性脑缺血损伤后机体内炎性代谢网络研究思路入手,探讨QNXQDP调控炎性代谢网络紊乱的可能靶点,为脑缺血损伤炎症机制研究提供新思路。
2.动态观察48h内急性脑缺血再灌注损伤大鼠脑组织5-LOX、LTB_4及CysLTs的动态变化规律,探索性研究了CysLT1和CysLT2在脑缺血损伤后皮层和海马部位的表达及功能。
Ischemic cerebral vascular disease(ICVD) is well known with its high incidence,high death rate,high relapse rate and high mutilation rate.In recent years,it is proved that inflammatory reaction plays an important role in ischemic damage.It has been recognized that cytokines, adhesion molecules,inflammatory mediums,transcription factors and leukocyte infiltration are associated with ICVD and intimately related to each other,moreover,5-LOX,CysLT1,CysLT2 and their receptors which are reported to express in the brain,may be involved in the inflammatory pathogenesis of cerebral ischemia.All the factors mentionded above and their effect is the so-called inflammatory reaction cascade.Therefore,further research about the mechanism of ischemic cerebrovascular disease and the action targets of the pathological Changes have profound significance in modern medicine.
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In this study,by means of biochemical assay,radioimmunoassay(RIA),enzyme-linked immunosorbent assay(ELISA),immunohistochemistry(IHC) and molecular biology,the QNXQDP on the inflammatory reaction in rats with middle cerebral artery occlusion(MCAO) are investigated.
Meanwhile the mechanisms are further investigated from following aspects:cytokine production,leukocyte infiltrate,adhesion molecules and activation from nuclear transcription factors to the t expression level of CysLTs and CysLTs receptors
The chief results are as follows.
1.Protective effects of QNXQDP on cerebral ischemic injury in rats with MCAO
OBJECTIVE:To study the effects of QNXQDP on nerve symptoms,infarction size, pathomorphology changes,content of neurone specific enolase(NSE) in serum and Ca~(2+) in brain cell.METHODS:nerve symptoms were scored at different time-points(ischemia 2 hours, reperfusion 4 hours and reperfusion 22 hours),After 22 hours of reperfusion,TTC staining of brain tissue was made to determine the size of cerebral infarction,HE staining of brain was also performed to observe morphological changes,the content of NSE was assayed by RIA,the content of Ca~(2+) was assayed using Flow Cytometry.RESULTS:After MCAO,nerve symptoms, the size of cerebral infarction,the.content of NSE and Ca~(2+) rose greatly,QNXQDP decreased significantly.In model group,a number of neurons appeared degeneration,necrosis or disappearance,with leukocyte infiltration.In QNXQDP group,the status of pathomorphology was improved.CONCLUSION:QNXQDP can treat cerebral ischemic injury.
2.Effects of QNXQDP on inflammatory cascade in rats with MCAO
2.1 Effects of QNXQDP on content of inflammatory cytokines in rats with MCAO
OBJECTIVE:To research the effects of QNXQDP on the content of inflammatory cytokines after ischemia-reperfusion in rats with MCAO.METHODS:After ischemia 2 hours and reperfusion 22 hours,the contents of IL-1β、IL-8、IL-6、TNF-α、IL-4、IL-10 and TGF-βwere asssyed by ELISA.RESULTS:After MCAO,the contents of IL-1β、IL-8、IL-6、TNF-α、IL-4、IL-10 and TGF-βincreased significantly in contrast to sham group,QNXQDP can decrease them remarkably.CONCLUSION:QNXQDP can reduce the contents of cytokines in rats with MCAO to alleviate cerebral ischemic injury.
2.2 Effects of QNXQDP on content of nitric oxide(NO) and prostaglandin analogs,Activity of nitric oxide synthase(NOS) in rats with MCAO
OBJECTIVE:To research the effects of QNXQDP on the nitric oxide、nitric oxide synthase and prostaglandin analogs after ischemia-reperfusion in rats with MCAO.METHODS: ischemia 2 hours and reperfusion 22 hours,determining of nitrate reductase NO content in brain homogenate,cNO and iNO activity;measuring of plasma TXB2 and 6 - keto -PGF1αcontent with RIA.RESULTS:①After MCAO,content of NO and activity of TNO,nNO, iNO were significantly higher than sham group in brain.②After MCAO,QNXQDP decreasing content of NO and activity of TNO,nNO and iNO,also decreasing contents of TXB2 and 6 - keto-PGF1αin plasma.CONCLUSION:QNXQDP can decreased content of NO and activity of TNO,nNO and iNO,regulating TXB2 and 6 - keto-PGF1αto relieve cerebral ischemic injury.
2.3 Effects of QNXQDP on leukocyte and endothelial cells adhesion chemotaxis and infiltration in rats with MCAO
OBJECTIVE:To research the effects of QNXQDP on the expression of cell adhesion molecules(ICAM-I) and Macrophage inflammatory protein-1a(MIP-1a),changes of myoloperoxidase(MPO) content in brain tissue.METHODS:The expression of ICAM-Ⅰand MIP-1a protein was assayed by IHC.MPO was assayed by biochemisty.RESULTS:After MCAO,the expressing positive area and integral optical density of ICAM-Ⅰand MIP-1a, content of MPO increased significantly in model group,QNXQDP 180mg/kg and 90 mg/kg can inhibit the positive area and integral optical density of ICAM-Ⅰand MIP-1a on cortex and hippocampus,reduce the content of MPO in brain tissue.CONCLUSION:QNXQDP can decrease leukocyte and endothelial cells adhesion、chemotaxis and infiltration in rats with MCAO.
2.4 Effects of QNXQDP on nuclear transcription factors in rats with MCAO OBJECTIVE:To investigate the effects of QNXQDP on the expression of nuclear factor-κB (NF-κB) in brain tissue.METHODS:The expression of NF-κB protein was assayed by IHC. RESULTS:After MCAO,the expression of NF-κB increased significantly in model group, and cortex and hippocampus showed nuclear translocation.QNXQDP 180mg/kg and 90 mg/kg could inhibit the nuclear translocation and decreace the positive area and integral optical density of NF-κB in cortex and hippocampus.CONCLUSION:QNXQDP could inhibit the expression of transcription factors in brain.
3.Effects of QNXQDP on 5 - lipoxygenase / leukotriene pathway in rats with MCAO
OBJECTIVE:To investigate the effects of QNXQDP on the content of 5-LOX、LTB4 and CysLTs,CysLT1 and CysLT2 expression in brain tissue.METHODS:ischemia 2 hours and reperfusion 6、12、24、36、48h hours,the content of 5-LOX、LTB4 and CysLTs was assayed by ELISA.RESULTS:ischemia 2 hours and reperfusion 6、12、24、36、48h hours,the content of 5-LOX、LTB4 and CysLTs were increased in model group compare with shame group, expression of CysLT1 and CysLT2 in cortex and hippocampus also increased in cortex and hippocampus.QNXQDP can decrease the content of 5-LOX、LTB4 and CysLTs in brain tissue,CONCLUSION:QNXQDP could decrease the contents of 5-LOX、LTB4 and CysLTs in brain,also could inhibit the expression of CysLT1 and CysLT2 in cortex and hippocampus.
CONCLUSION
QNXQDP can alleviate neuronal injury and the inflammatory reaction after of ischemia-reperfusion in rats,whose its main mechanisms of action may be associated with:①inhibition of neuron-specific enolase(NSE) levels and calcium overload;②regulation of inflammatory cytokines,reducing the NO content and inhibiting NOS activity;③inhibiting leukocyte and endothelial cells adhesion、chemotaxis and infiltration in brain tissue;④reducing the content of 5-LOX,LTB4 and CysLTs,inhibiting the expression of cysteinyl leukotriene receptor(CysLT1 and CysLT2) in cortex and hippocampus.Its prevention and treatment inflammatory injury after cerebral ischemia-reperfusion.In addition,CysLT1 and CysLT2may be targets for inhibition.
In this experiment study,we observed the effects of QNXQDP on the expression of protein CysLT1 and CysLT2 in cortex and hippocampus of ischemia-reperfusion rats,which has not been reported by now.The reports about the dynamic changes of the content 5-LOX, LTB4 and CysLTs after cerebral ischemia-reperfusion within 48 hours are also very few. Therefore,our study is innovative to a certain some extent.
引文
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