梓葛冻干粉针对大鼠脑微循环障碍的改善作用及其机制研究
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摘要
背景
“梓葛冻干粉针”为本实验室自主创制的抗脑缺血卒中的中药单体复方新药,由梓醇和葛根素组成,已经申请技术发明专利。课题组前期已经进行了该制剂对大鼠永久性脑缺血后神经功能恢复,对脑梗死面积、脑血管新生和改善气虚血瘀证等方面的研究。但该制剂对脑部微循环是否有影响,尚不确定,有待研究。目的
在整体动物与离体细胞实验中,观察梓葛冻干粉针对脑缺血再灌注损伤的保护作用效果,并从调节微血管的舒缩功能、通透性和血液流变性的角度,探讨其对脑微循环保护作用的机制。
方法
1.梓葛冻干粉针对大鼠局灶性脑缺血/再灌注损伤脑微循环障碍的改善作用
取雄性SD大鼠80只,采用随机数字法分为假手术组(10只)、缺血1h再灌注组(20只,造模成功16只)、缺血2h再灌注组(20只,造模成功14只)、缺血4h再灌注组(20只,造模成功8只)和尼莫地平组(缺血2h后再灌注,2.00mg·kg-1,10只,造模成功8只),大鼠再灌注时即刻给药(再灌注0h),尾静脉注射给药,每天1次(10mL·kg-1,体质量),连续给药14d,假手术组和模型组给同等体积的生理盐水。采用大鼠大脑左侧中动脉线栓法(MCAO)复制脑缺血/再灌注(I/R)模型,进行神经行为学评定,分别在缺血1h、2h和4h后再灌注,并于造模后1d、4d、7d、14d对大鼠进行改良神经功能缺损评分(mNSS)。
取雄性SD大鼠540只,线栓法复制I/R模型,随机分为假手术组(60只)、模型组(80只,造模成功61只)、葛根素注射液组(30.00mg·kg-1,80只,造模成功68只)、尼莫地平组(2.00mg·kg-1,80只,造模成功69只)、梓葛冻干粉针16.40mg·kg-1(80只,造模成功65只)、32.70mg·kg-1(80只,造模成功68只)和65.40mg·kg-1组(80只,造模成功70只),大鼠再灌注时即刻给药(再灌注0h),尾静脉注射给药,每天1次(10mL·kg-1,体质量),考察时间点分为缺血2h再灌注1d、4d、7d和14d,假手术组和模型组给同等体积的生理盐水。大鼠进行神经功能缺损评分,应用激光多普勒血流量仪测定软脑膜血流量的变化；HE染色光镜下观察大鼠皮层区病理形态学变化和损伤状况；TTC染色测定梗死体积；干湿法测定脑组织含水量；伊文氏蓝(EB)含量测血管的通透性；免疫组化染色法检测CD31表达。
取雄性SD大鼠960只,随机分为假手术组(96只)、模型组(144只,造模成功109只)、葛根素注射液组(30.00mg·kg-1,144只,造模成功120只)、尼莫地平组(2.00mg·kg-1,144只,造模成功123只)、梓葛冻干粉针16.40mg·kg-1(144只,造模成功117只)、32.70mg·kg-1(144只,造模成功122只)和65.40mg·kg-1(144只,造模成功126只)组,大鼠再灌注时即刻给药(再灌注0h),尾静脉注射给药,每天1次(10mL·kg-1,体质量),考察时间点分为缺血2h再灌注1d、4d、7d和14d,假手术组和模型组给同等体积的生理盐水。酶免疫试剂盒检测患侧脑皮层微血管中一氧化氮合成酶(NOS)、内皮素(ET-1)、6-酮-前列腺素F1α(6-Ke-to-PGF1α)和血栓素B2(TXB2)的含量；应用荧光定量PCR(qPCR)和免疫印迹(Western blot)检测脑皮层微血管的MMP-9、ZO-1、Occludin及Claudin-5mRNA和蛋白的表达变化。2.梓葛冻干粉针对高分子葡聚糖大鼠脑微循环障碍的改善作用
取雄性SD大鼠120只,随机分为正常对照组、模型组、尼莫地平(2.00mg-kg-1)、梓葛冻干粉针16.40ng·kg-1、32.70mg·kg-1和65.40mg·kg1组,每组20只,尾静脉注射给药,每天1次(10mL·kg-1,体质量),连续给药14d后开颅窗,并于末次给药30min后,大鼠(除正常对照组)再经股静脉给予10%高分子葡聚糖T-500(10nL·kg-1)复制大鼠急性脑微循环障碍模型,生物显微镜下观察大鼠造模前5min和造模后1、5、10、20、40、60min时间点软脑膜微动静脉直径,激光多普勒血流仪监测血流量,电阻法测红细胞压积,全自动血液流变仪测流变学指标。
3.梓葛冻干粉针对体外缺氧/复氧损伤大鼠脑微血管内皮细胞mmp-9蛋白表达的影响
采用氧糖剥夺的方法,建立体外培养大鼠脑微血管内皮细胞的缺氧复氧模型(模拟体内脑缺血再灌注损伤模型)。MTT法检测内皮细胞的增殖。内皮细胞缺糖缺氧2h再复氧24h损伤后,Western blot检测mmp-9蛋白表达。结果
1.梓葛冻干粉针对大鼠局灶性脑缺血/再灌注的脑微循环障碍的改善作用
(1)大鼠局灶性脑缺血/再灌注模型复制及神经行为学评定
脑缺血/再灌注损伤后大鼠神经功能缺损明显,其中损伤后1dmNSS评分1h、2h、4h组分别为5.68±0.75、9.23±1.05、11.53±1.88；14d梗死面积比率分别为0.1026±0.0214、0.2245±0.0228、0.3828±0.0318。缺血1h组主要的梗死部位为皮质,2h、4h组主要梗死部位为皮质及基底节区。确定缺血2h为最佳的造模时间进行后续实验。
(2)梓葛冻干粉针对脑缺血／再灌注大鼠脑微循环血流量、梗死体积及脑水肿的影响
梓葛冻干粉针(16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg-1)可在再灌注时使脑血流缓慢恢复。TTC染色结果显示,假手术组脑组织均呈深红色未见梗死灶；模型组可见大面积的梗死灶,多位于左侧皮质和基底节区；再灌注14d,与模型组相比,梓葛冻干粉针65.40mg·kg-1组梗死体积显著减少(P<0.05),提示梓葛冻干粉针可减轻大鼠脑缺血/再灌注的损伤。
与假手术组相比,模型组大鼠脑组织含水量和EB含量显著升高(P<0.01),显示造模成功。再灌注7d和14d,与模型组相比,梓葛冻干粉针(16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg1)可明显抑制大鼠的病变侧脑组织含水量和EB含量升高(P<0.05,P<0.01),提示其可改善脑缺血/再灌注损伤大鼠的血管通透性。
HE染色观察,脑缺血再灌注后大鼠大脑缺血侧细胞层次紊乱,细胞坏死,胞核萎缩成不规则状,水肿,疏松,炎性细胞浸润。而梓葛冻干粉针(16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg1)组在相应考察时间点可减少炎性细胞浸润,减小细胞周围间隙和水肿。
(3)梓葛冻干粉针对局灶性脑缺血/再灌注大鼠脑微血管密度的影响
免疫组化结果显示,假手术组CD31阳性细胞表达很少。与假手术组相比,模型组再灌注后1d缺血侧大鼠皮层区有少量CD31阳性细胞表达,到4d、7d有较高表达,7d以后阳性细胞表达增多缓慢或基本不增长(P<0.01)。与模型组阳性细胞表达相比,葛根素注射液组(30.00mg·kg-1)、尼莫地平组(2.00ng·kg-1)、梓葛冻干粉针16.40ng·kg-1、32.70mg·kg-1和65.40mg-kg-1组的梗死灶周边区阳性细胞表达,除再灌注后1d的无显著差异外,其余各个时间点的阳性细胞表达均显著增多(P<0.01),提示其可能促进了脑缺血/再灌注损伤大鼠血管新生。
(4)梓葛冻干粉针对脑缺血/再灌注大鼠的脑微循环中微血管舒缩功能的影响
与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中总NOS (TNOS)和诱生型NOS (iNOS)活力均显著升高(P<0.01),而原生型NOS (cNOS)活力显著降低(P<0.01),显示造模成功。与模型组相比,再灌注1d,葛根素注射液组(30.00mg·kg1)、尼莫地平组(2.00mg·kg-1)、梓葛冻干粉针16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg-1组均显著降低TNOS和iNOS活力(P<0.05,P<0.01)；再灌注4d、7d和14d,各给药组均能显著降低TNOS、iNOS活力(P<0.01),且也能显著升高cNOS活力(P<0.01),提示梓葛冻干粉针有改善脑缺血/再灌注损伤大鼠微血管舒缩功能的效果。
与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中ET-1含量均升高(P<0.01),且随着观察时间延长而逐渐增多,显示造模成功。再灌注1d,与模型组相比,梓葛冻干粉针32.70mg-kg-1组能显著降低大鼠缺血侧脑皮质层ET-1含量(P<0.05)；再灌注4、7和14d时,与模型组相比,梓葛冻干粉针各剂量组均能极显著降低大鼠缺血侧脑皮质层微血管中ET-1含量(P<0.01),提示其有减轻血管收缩和血管内皮细胞损伤的作用。
再灌注1、4、7和14d时,与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中TXB2含量升高和6-Ke-to-PGF1α含量降低(P<0.01),显示造模成功。而与模型组相比,梓葛冻干粉针16.40mg·kg-1、32.70ng·kg-1和65.40mg·kg-1组能显著抑制大鼠缺血侧脑皮质层TXB2含量升高和6-Ke-to-PGF1α含量降低(P<0.05,P<0.01),且能降低TXB2/6-Ke-to-PGF1比值(P<0.01),提示其有改善脑缺血／再灌注损伤大鼠微血管舒缩功能的效果。
(5)梓葛冻干粉针对脑缺血/再灌注大鼠的脑微循环中微血管通透性的影响
荧光定量PCR结果显示,假手术组皮层脑组织中有少量MMP-9mRNA表达,脑缺血再灌注后,各时间点(1、4、7和14d),与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中MMP-9mRNA表达均显著升高(P<0.01),显示造模成功。与模型组相比,梓葛冻干粉针16.40mg·kg-1、32.70mg、kg-1和65.40mg·kg-1组均能显著降低大鼠缺血侧脑皮质层微血管中MMP-9mRNA表达水平(P<0.05,P<0.01)；与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中Claudin-5mRNA、Occludin mRNA、ZO-1mRNA表达均显著降低,而与模型组相比,梓葛冻干粉针各剂量组在相应时间点能显著增加大鼠缺血侧脑皮质层微血管中Claudin-5mRNA、Occludin mRNA、ZO-1mRNA表达(P<0.05,P<0.01)。
Western blot检测蛋白结果与其基因水平结果基本一致。在再灌注1、4、7和14d,与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中MMP-9蛋白表达均显著升高(P<0.01),显示造模成功。与模型组相比,各给药组均能显著降低大鼠缺血侧脑皮质层微血管中MMP-9蛋白表达(P<0.01)；与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中Claudin-5、Occludin、ZO-1蛋白水平均显著降低,而与模型组相比,梓葛冻干粉针各剂量组在相应时间点能显著升高大鼠缺血侧脑皮质层微血管中Claudin-5、Occludin、ZO-1蛋白表达水平(P<0.05,P<0.01),提示其有改善脑缺血/再灌注损伤大鼠微血管通透性的效果。2.梓葛冻干粉针对高分子葡聚糖大鼠急性脑微循环障碍的改善作用
与正常组相比,在考察相对应的时间点,模型组的大鼠软脑膜微动脉,微静脉内径显著缩小,且血流量呈显著下降(P<0.01),显示造模成功；与模型组相比,梓葛冻干粉针16.40mg·kg-1,32.70mg·kg-1和65.40mg-kg-1组中大鼠软脑膜的微动脉、微静脉内径恢复显著(P<0.01),且能使血流量在10min以后基本恢复至造模前的血流量。与正常组相比,模型组的全血粘度、血浆粘度、红细胞压积和红细胞聚集指数均显著升高,显示造模成功；与模型组相比,梓葛冻干粉针65.40mg·kg-1组能显著降低血液流变学的各项指标(P<0.01)；梓葛冻干粉针16.40mg·kg-1口32.70mg·kg-1组能显著降低除红细胞压积以外的其它项血液流变学的指标(P<0.05,P<0.01),提示梓葛冻干粉针具有对大鼠急性脑微循环障碍的改善作用。
3.梓葛冻干粉针对体外缺氧/复氧损伤大鼠脑微血管内皮细胞mmp-9蛋白表达的影响
MTT法结果显示,梓葛冻干粉针作用于脑微血管内皮细胞的ICso值为860μg·mL-1。缺氧/复氧损伤下,与正常对照组相比,复氧24h后,模型组的BMECs细胞中MMP-9蛋白表达显著升高(P<0.01),显示造模成功。与模型组相比,梓葛冻干粉针24.50μg·ML-1、49.00μg·mL1和98.00μg·mL-1可抑制BMECs缺氧／复氧损伤下MMP-9蛋白升高,提示其对血管内皮细胞完整性具有保护作用。结论
1.采用Longa线栓法制备脑缺血模型最佳的缺血/再灌注时间为缺血2h,模型稳定,可控性和可重复性好。梓葛冻干粉针对脑缺血/再灌注损伤大鼠,能使脑血流在再灌注时缓慢恢复,减小脑梗死体积,减小脑组织含水量和EB含量,可以减轻脑水肿和血脑屏障的破坏程度；梓葛冻干粉针能促进缺血侧大鼠皮层区CD31的表达,表明可促进脑缺血/再灌注损伤大鼠的血管新生；梓葛冻干粉针改善脑微循环可能与调节微血管舒缩功能,抑制iNOS的活化,减少TNOS和ET-1的生成,抑制大鼠缺血侧脑皮质层TXB2和6-Ke-to-PGF1。表达有关；同时,梓葛冻干粉针可抑制MMP-9mRNA及蛋白表达和促进Claudin-5、Occludin、ZO-1mRNA及蛋白表达,保护微血管完整性,降低微血管通透性,改善脑微循环。
2.梓葛冻干粉针对急性脑微循环障碍大鼠,可扩张脑微动静脉管径,逆转脑血流量下降的趋势和血液粘稠,提示梓葛冻干粉针具有对急性脑微循环障碍的改善作用。
3.梓葛冻干粉针能抑制BMECs缺氧／复氧损伤下MMP-9蛋白表达,提示其对血管内皮细胞完整性的保护作用机制可能与降低MMP-9蛋白表达有关。
Background
Our research team researched a pharmaceutical preparation with the anti-ischemic effect of Zige lyophilized powder that contained catalpol and puerarin and had a patent. Zige lyophilized powder for injection had the effects of the neural functional recovery in permanent cerebral ischemia of rats, reducing cerebral infarction area, angiogenesis, improving blood stasis and blood brain barrier (BBB) permeability, and so on. But we had not research the effects of Zige lyophilized powder for injection in the celebral microcirculation.
Objective
It aims to research possible mechanism of Zige lyophilized powder for injection against cerebral ischemia-reperfusion injury in the celebral microcirculation through the pathological causes of contraction, permeability and blood rheology of microvessel in experiments of animal and cell.
Methods
1. The improving effects of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in the disturbance of microcirculation
80male sprague-dawley rats were randomly divided into sham group (10), ischemia1h group (16/20), ischemia2h group (16/20), ischemia4h group (16/20), nimodipine injection (ischemia1h and reperfusion,2.00mg·kg-1,8/10), and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution. It made a cerebral ischemia-reperfusion (I/R) model using the line in the left middle cerebral artery occlusion (MCAO) of rats and test neuro-behavioral evaluation by a method of modified neurological severity scores (mNSS) in ischemia for1h,2h,4h and reperfusion for1d,4d,7d and14d.
540male sprague-dawley rats were randomly divided into sham group (60), model group (61/80), puerarin injection group(30.00mg·kg-1,68/80), nimodipine injection (2.00mg·kg-1,69/80), Zige lyophilized powder groups(16.40mg·kg-1,65/80;32.70mg·kg-1,68/80;65.40mg·kg-1,70/80) in ischemia for2h and reperfusion for1d,4d,7d and14d, and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution. Blood flow volume of pia mater in rats was observed by laser doppler flowmetry. Pathomorphism changes and damaged conditions in rat cortex were observed by HE staining. The volume of the cerebral infarction was estimated by2%TTC staining. The water content was calculated by dry and wet weight method. The disruption of micro vessel permeability was detected by the EB content. The expression of CD31was detected by immunohistochemical staining method.
960male sprague-dawley rats were randomly divided into sham group (96), model group (109/144), puerarin injection group(30.00mg·kg-1,120/144), nimodipine injection (2.00mg·kg-1,123/144), Zige lyophilized powder groups(16.40mg·kg-1,117/144;32.70mg·kg-1,122/144;65.40mg·kg-1,126/144) in ischemia for2h and reperfusion for1d,4d,7d and14d, and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution.Nitric oxide synthase (NOS), endothelin-1(ET-1),6-ketone-prostaglandin F1α (6-Ke-to-PG-F1α) and thromboxane B2(TXB2) content of the brain microvessel in the lateral ischemic cerebral cortex was detected by enzyme immunoassay kits. The expression of MMP-9、ZO-1、Occludin and Claudin-5protein and mRNA of the brain microvessel in the lateral ischemic cerebral cortex was detected by fluorescence quantitative PCR (qPCR) and Western blot (WB).
2. The protective effect of Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats by intravenous injection of high molecule dextran
120male rats were divided into normal control group, model group, nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) with20for each goup, and made window craniotomy after the administration of drugs for14days (tail vein injection,1time per day), the model of rats with the disturbance of cerebral microcirculation was estabished by intravenous injection of high molecule dextran (10%T-500,10mL·kg-1) after the last administrat-ion of drug for30min. The effects of micro-vein diameter for5min in normal rats and1,5,10,20,40,60min in making model of rats were observed through the inverted microscope. Blood flow volume on brain meninx was observed through laser doppler flowmetry. HCT was measured by electric resistance method, hemorheological indexes were observed by auto-hemorheological instrument.
3. The protein expression levels of mmp-9to Zige lyophilized powder for injection on brain microvascular endothelial cells after hypoxia-reoxygenation injury were detected in vitro
It made a hypoxia-reoxygenation injury model on cultured BMECs of rat (simulated cerebral ischemia-reperfusion injury model) in vitro by oxygen-glucose deprivation. The proliferation of endothelial cells was detected by MTT method. Endothelial cells were cultured by oxygen-glucose deprivation for2h and reoxygenation for24h, it determined to detect the protein expression levels of mmp-9by WB method.
Results
1. The improving effects of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in the celebral microcirculation
(1) The focal cerebral ischemia-reperfusion model of rats and neuro-behavioral test after I/R rats have significantly neurological deficit,1d after I/R, the mNSS score of1h,2h and4h groups were5.68±0.75,9.23±1.05,11.53±1.881, respectively. Percentages of the infarction area of three groups in14d were0.1026±0.0214,0.2245±0.0228,0.3828±0.0318, respectively. The main infarction area of1h group was cortex, and the main infarction area of2h and4h groups was cortex and basal ganglia. The best time of making the ischemia-reperfusion model is2h to ischemia for subsequent experiments.
(2) The effect of Zige lyophilized powder for injection against the model rats of blood flow volume, infarction volume and brain edema in cerebral microcirculation.
In this experiment, Zige lyophilized powder(16.40,32.70and65.40mg-kg-1) showed a trend of slow recovery during reperfusion. In TTC staining, sham group appeared in deep red and had not the brain infarcts, model group had a large area of infarcts and located in the left side of the cortex and basal ganglia. Compared with model group, Zige lyophilized powder groups (65.40mg-kg-1) decreased significantly infarction volume in14d(P<0.05).
Compared with sham group, the water content and EB content increased significantly (P＜0.05, P<0.01) in model group,and it made a successful model. Compared with model group in reperfusion for7d and14d, Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit significantly the water content and EB content in lesion side of the rat brain tissue (P<0.05,P<0.01), these showed that Zige lyophilized powder protected against cerebral ischemic injury by improving microvessel permeability.
In HE staining, cells of brain ischemia side in all groups after cerebral ischemia-reperfusion appeared necrosis, shrinking nucleus into irregular shape, edema, loosen and infiltration by inflammatory cells. Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit infiltration by inflammatory cells, and reduce intercellular space and edema in the corresponding time.
(3) The effect of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in microvascular density of the celebral microcirculation.
In immunohistochemical experiment, a few of CD31cells expressed in sham group. It showed that the expression of CD31after ischemia-reperfusion in lateral cortex of rats had a few in reperfusion for1d, and had higher in reperfusion for4d and7d, and increased slowly or no growth in reperfusion after7d. Compared with model group in reperfusion for1d,4d,7d and14d, the expression of CD31of puerarin injection group (30.00mg·kg-1), Nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had higher significantly in the corresponding time except reperfusion for1d (P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by promoting angiogenesis.
(4) The effect of Zige lyophilized powder against the microvascular contraction function after cerebral ischemia-reperfusion injury of rats in the microcirculation
In this experiment, compared with sham group, it showed that TNOS and iNOS enzyme activity had increased and cNOS enzyme activity had decreased significantly after ischemia-reperfusion in lateral cortex of rats (P<0.01). Compared with model group in reperfusion for1d, puerarin injection group (30.00mg·kg-1), Nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had reduce significantly TNOS and iNOS enzyme activity (P<0.05, P<0.01). Compared with model group in reperfusion for4,7and14d, all drug groups had reduce significantly TNOS and iNOS enzyme activity and increased significantly cNOS enzyme activity. These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving the microvascular contraction function in the celebral microcirculation.
Compared with sham group, it showed that the level of ET-1after ischemia-reperfusion in lateral cortex of rats had a rising trend and increased gradually with prolonged observation. In reperfusion for1d, compared with model group, Zige lyophilized powder group (32.70mg·kg-1) could reduce significantly the level of ET-1 (P<0.05). In reperfusion for4,7and14d, compared with model group, Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had reduce significantly the level of ET-1in micro vessel of the lateral ischemic cerebral cortex (P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving the microvascular contraction function and endothelial cell damage.
In reperfusion for1,4,7and14d, compared with sham group, it showed that the level of TXB2had increased and6-Ke-to-PGF1α had decreased significantly in model group. Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit significantly the level of TXB2and6-Ke-to-PGF1α(P<0.05,P<0.01)and the ratio of TXB2/6-Ke-to-PGF1α(P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving microvascular contraction function in microcirculation.
(5) The effect of Zige lyophilized powder for injection against the microvascular permeability after cerebral ischemia-reperfusion injury of rats in the celebral microcirculation
In real-time quantitative PCR, the cortex of sham group had a few of mmp-9mRNA levels. After focal cerebral ischemia-reperfusion, comparing with sham group in reperfusion for1,4,7and14d, it showed that mmp-9mRNA levels in lateral cortex of rats had increased significantly in model group. Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could reduce significantly mmp-9mRNA levels. Comparing with sham group, it showed that claudin-5mRNA、occludin mRNA、zo-1mRNA levels in lateral cortex of rats had reduced significantly in model group. Three groups of Zige lyophilized powder could increase significantly claudin-5mRNA、occludin mRNA、zo-1mRNA levels respectively (P<0.05,P<0.01).
In the experiment of western blot, a change trend of protein expression and genetic level is basically identical. Compared with sham group in reperfusion for1,4,7and14d, it showed that mmp-9protein expression in lateral cortex of rats had increase significantly in model group and all drug groups could reduce mmp-9protein expression significantly. Comparing with sham group, it showed that the protein expressions of claudin-5、occludin、zo-1in lateral cortex of rats had reduced significantly in model group. Three groups of Zige lyophilized powder could increase significantly claudin-5、occludi、zo-1levels in the corresponding time, respectively (P <0.05,P<0.01).These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving microvascular permeability.
2. The protective effect of Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats by intravenous injection of high molecule dextran
Compared with normal control group in the corresponding time, it showed that pia mater microvenule diameter and microartery diameter in rats had shrink significantly and blood flow volume had decrease significantly in model group. Compared with model group, Zige lyophilized powder for injection(16.40,32.70and65.40mg·kg-1) had significantly expended micro-vein diameter (P<0.01),and returned normal trend of blood flow volume after10min of making model. Compared with normal control group, it showed that blood viscosity, erythrocyte agglutination index, plasma viscosity and hematocrit in rats had increase significantly in model group. Compared with model group, Zige lyophilized powder (65.40mg·kg-1)could significantly reduce blood rheology indicators (P<0.01),and Zige lyophilized powder for injection(16.40,32.70mg·kg-1)had could significantly reduce other blood rheology indicators except hematocrit (P<0.05,P<0.01).
3. The protein expression levels of mmp-9to Zige lyophilized powder on brain microvascular endothelial cells (BMECs) after hypoxia-reoxygenation injury were detected in vitro
The IC50value is860μg·ml-1by MTT method in cultured BMECs by Zige lyophilized powder for injection. Compared with normal control group in the reoxygenation time for24h, it showed that the level of mmp-9protein had increase significantly in model group. Zige lyophilized powder groups (24.50、49.00and98.00μg·ml-1)had inhibit significantly the increased levels of mmp-9protein in a hypoxia-reoxygenation injury.
Conclusions
1. The best time of making I/R model by Longa method is2h to ischemia, and it is stability, controllability and repeatability. Zige lyophilized powder for injection showed a trend of slow recovery during reperfusion, and could decrease infarction volume, and inhibit significantly the water content and EB content in lesion side of the rat brain tissue. These showed the fact that it could reduce the damage rate of the blood brain barrier and brain edema. Zige lyophilized powder for injection can promote CD31expression in lateral cortex of rats, it would be promote angiogenesis after ischemica-reperfusion injury, and improve the cerebral microcirculation, which is beneficial to the recovery of damaged nerve tissue. The possible mechanisms of Zige lyophilized powder for injection in the improving celebral microcirculation would adjust the microvascular contraction function, and inhibit the enzyme activity of iNOS, and reduce the generated TNOS and ET-1content, and inhibit the level of TXB2and6-Ke-to-PGF1a. Zige lyophilized powder for injection can protect the microvascular integrity, and reduce microvascular permeability, and improve cerebral microcirculation through it can inhibit the increasing trend of mmp-9mRNA and protein expression and the decreasing trend of claudin-5, occludin, zo-1mRNA and protein level.
2. Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats can change trend of pia mater microartery and microvenule diameter, and improve celebral blood flow and blood viscosity, these showed that it would impove the effect of the acute disturbance of cerebral microcirculation.
3. Zige lyophilized powder for injection had inhibited the increased levels of mmp-9protein in a hypoxia-reoxygenation injury on cultured BMECs. It showed that the possible mechanism of Zige lyophilized powder for injection on protecting the cell integrity would be associated with the decreasing mmp-9protein expression.
“梓葛冻干粉针”为本实验室自主创制的抗脑缺血卒中的中药单体复方新药,由梓醇和葛根素组成,已经申请技术发明专利。课题组前期已经进行了该制剂对大鼠永久性脑缺血后神经功能恢复,对脑梗死面积、脑血管新生和改善气虚血瘀证等方面的研究。但该制剂对脑部微循环是否有影响,尚不确定,有待研究。目的
在整体动物与离体细胞实验中,观察梓葛冻干粉针对脑缺血再灌注损伤的保护作用效果,并从调节微血管的舒缩功能、通透性和血液流变性的角度,探讨其对脑微循环保护作用的机制。
方法
1.梓葛冻干粉针对大鼠局灶性脑缺血/再灌注损伤脑微循环障碍的改善作用
取雄性SD大鼠80只,采用随机数字法分为假手术组(10只)、缺血1h再灌注组(20只,造模成功16只)、缺血2h再灌注组(20只,造模成功14只)、缺血4h再灌注组(20只,造模成功8只)和尼莫地平组(缺血2h后再灌注,2.00mg·kg-1,10只,造模成功8只),大鼠再灌注时即刻给药(再灌注0h),尾静脉注射给药,每天1次(10mL·kg-1,体质量),连续给药14d,假手术组和模型组给同等体积的生理盐水。采用大鼠大脑左侧中动脉线栓法(MCAO)复制脑缺血/再灌注(I/R)模型,进行神经行为学评定,分别在缺血1h、2h和4h后再灌注,并于造模后1d、4d、7d、14d对大鼠进行改良神经功能缺损评分(mNSS)。
取雄性SD大鼠540只,线栓法复制I/R模型,随机分为假手术组(60只)、模型组(80只,造模成功61只)、葛根素注射液组(30.00mg·kg-1,80只,造模成功68只)、尼莫地平组(2.00mg·kg-1,80只,造模成功69只)、梓葛冻干粉针16.40mg·kg-1(80只,造模成功65只)、32.70mg·kg-1(80只,造模成功68只)和65.40mg·kg-1组(80只,造模成功70只),大鼠再灌注时即刻给药(再灌注0h),尾静脉注射给药,每天1次(10mL·kg-1,体质量),考察时间点分为缺血2h再灌注1d、4d、7d和14d,假手术组和模型组给同等体积的生理盐水。大鼠进行神经功能缺损评分,应用激光多普勒血流量仪测定软脑膜血流量的变化；HE染色光镜下观察大鼠皮层区病理形态学变化和损伤状况；TTC染色测定梗死体积；干湿法测定脑组织含水量；伊文氏蓝(EB)含量测血管的通透性；免疫组化染色法检测CD31表达。
取雄性SD大鼠960只,随机分为假手术组(96只)、模型组(144只,造模成功109只)、葛根素注射液组(30.00mg·kg-1,144只,造模成功120只)、尼莫地平组(2.00mg·kg-1,144只,造模成功123只)、梓葛冻干粉针16.40mg·kg-1(144只,造模成功117只)、32.70mg·kg-1(144只,造模成功122只)和65.40mg·kg-1(144只,造模成功126只)组,大鼠再灌注时即刻给药(再灌注0h),尾静脉注射给药,每天1次(10mL·kg-1,体质量),考察时间点分为缺血2h再灌注1d、4d、7d和14d,假手术组和模型组给同等体积的生理盐水。酶免疫试剂盒检测患侧脑皮层微血管中一氧化氮合成酶(NOS)、内皮素(ET-1)、6-酮-前列腺素F1α(6-Ke-to-PGF1α)和血栓素B2(TXB2)的含量；应用荧光定量PCR(qPCR)和免疫印迹(Western blot)检测脑皮层微血管的MMP-9、ZO-1、Occludin及Claudin-5mRNA和蛋白的表达变化。2.梓葛冻干粉针对高分子葡聚糖大鼠脑微循环障碍的改善作用
取雄性SD大鼠120只,随机分为正常对照组、模型组、尼莫地平(2.00mg-kg-1)、梓葛冻干粉针16.40ng·kg-1、32.70mg·kg-1和65.40mg·kg1组,每组20只,尾静脉注射给药,每天1次(10mL·kg-1,体质量),连续给药14d后开颅窗,并于末次给药30min后,大鼠(除正常对照组)再经股静脉给予10%高分子葡聚糖T-500(10nL·kg-1)复制大鼠急性脑微循环障碍模型,生物显微镜下观察大鼠造模前5min和造模后1、5、10、20、40、60min时间点软脑膜微动静脉直径,激光多普勒血流仪监测血流量,电阻法测红细胞压积,全自动血液流变仪测流变学指标。
3.梓葛冻干粉针对体外缺氧/复氧损伤大鼠脑微血管内皮细胞mmp-9蛋白表达的影响
采用氧糖剥夺的方法,建立体外培养大鼠脑微血管内皮细胞的缺氧复氧模型(模拟体内脑缺血再灌注损伤模型)。MTT法检测内皮细胞的增殖。内皮细胞缺糖缺氧2h再复氧24h损伤后,Western blot检测mmp-9蛋白表达。结果
1.梓葛冻干粉针对大鼠局灶性脑缺血/再灌注的脑微循环障碍的改善作用
(1)大鼠局灶性脑缺血/再灌注模型复制及神经行为学评定
脑缺血/再灌注损伤后大鼠神经功能缺损明显,其中损伤后1dmNSS评分1h、2h、4h组分别为5.68±0.75、9.23±1.05、11.53±1.88；14d梗死面积比率分别为0.1026±0.0214、0.2245±0.0228、0.3828±0.0318。缺血1h组主要的梗死部位为皮质,2h、4h组主要梗死部位为皮质及基底节区。确定缺血2h为最佳的造模时间进行后续实验。
(2)梓葛冻干粉针对脑缺血／再灌注大鼠脑微循环血流量、梗死体积及脑水肿的影响
梓葛冻干粉针(16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg-1)可在再灌注时使脑血流缓慢恢复。TTC染色结果显示,假手术组脑组织均呈深红色未见梗死灶；模型组可见大面积的梗死灶,多位于左侧皮质和基底节区；再灌注14d,与模型组相比,梓葛冻干粉针65.40mg·kg-1组梗死体积显著减少(P<0.05),提示梓葛冻干粉针可减轻大鼠脑缺血/再灌注的损伤。
与假手术组相比,模型组大鼠脑组织含水量和EB含量显著升高(P<0.01),显示造模成功。再灌注7d和14d,与模型组相比,梓葛冻干粉针(16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg1)可明显抑制大鼠的病变侧脑组织含水量和EB含量升高(P<0.05,P<0.01),提示其可改善脑缺血/再灌注损伤大鼠的血管通透性。
HE染色观察,脑缺血再灌注后大鼠大脑缺血侧细胞层次紊乱,细胞坏死,胞核萎缩成不规则状,水肿,疏松,炎性细胞浸润。而梓葛冻干粉针(16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg1)组在相应考察时间点可减少炎性细胞浸润,减小细胞周围间隙和水肿。
(3)梓葛冻干粉针对局灶性脑缺血/再灌注大鼠脑微血管密度的影响
免疫组化结果显示,假手术组CD31阳性细胞表达很少。与假手术组相比,模型组再灌注后1d缺血侧大鼠皮层区有少量CD31阳性细胞表达,到4d、7d有较高表达,7d以后阳性细胞表达增多缓慢或基本不增长(P<0.01)。与模型组阳性细胞表达相比,葛根素注射液组(30.00mg·kg-1)、尼莫地平组(2.00ng·kg-1)、梓葛冻干粉针16.40ng·kg-1、32.70mg·kg-1和65.40mg-kg-1组的梗死灶周边区阳性细胞表达,除再灌注后1d的无显著差异外,其余各个时间点的阳性细胞表达均显著增多(P<0.01),提示其可能促进了脑缺血/再灌注损伤大鼠血管新生。
(4)梓葛冻干粉针对脑缺血/再灌注大鼠的脑微循环中微血管舒缩功能的影响
与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中总NOS (TNOS)和诱生型NOS (iNOS)活力均显著升高(P<0.01),而原生型NOS (cNOS)活力显著降低(P<0.01),显示造模成功。与模型组相比,再灌注1d,葛根素注射液组(30.00mg·kg1)、尼莫地平组(2.00mg·kg-1)、梓葛冻干粉针16.40mg·kg-1、32.70mg·kg-1和65.40mg·kg-1组均显著降低TNOS和iNOS活力(P<0.05,P<0.01)；再灌注4d、7d和14d,各给药组均能显著降低TNOS、iNOS活力(P<0.01),且也能显著升高cNOS活力(P<0.01),提示梓葛冻干粉针有改善脑缺血/再灌注损伤大鼠微血管舒缩功能的效果。
与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中ET-1含量均升高(P<0.01),且随着观察时间延长而逐渐增多,显示造模成功。再灌注1d,与模型组相比,梓葛冻干粉针32.70mg-kg-1组能显著降低大鼠缺血侧脑皮质层ET-1含量(P<0.05)；再灌注4、7和14d时,与模型组相比,梓葛冻干粉针各剂量组均能极显著降低大鼠缺血侧脑皮质层微血管中ET-1含量(P<0.01),提示其有减轻血管收缩和血管内皮细胞损伤的作用。
再灌注1、4、7和14d时,与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中TXB2含量升高和6-Ke-to-PGF1α含量降低(P<0.01),显示造模成功。而与模型组相比,梓葛冻干粉针16.40mg·kg-1、32.70ng·kg-1和65.40mg·kg-1组能显著抑制大鼠缺血侧脑皮质层TXB2含量升高和6-Ke-to-PGF1α含量降低(P<0.05,P<0.01),且能降低TXB2/6-Ke-to-PGF1比值(P<0.01),提示其有改善脑缺血／再灌注损伤大鼠微血管舒缩功能的效果。
(5)梓葛冻干粉针对脑缺血/再灌注大鼠的脑微循环中微血管通透性的影响
荧光定量PCR结果显示,假手术组皮层脑组织中有少量MMP-9mRNA表达,脑缺血再灌注后,各时间点(1、4、7和14d),与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中MMP-9mRNA表达均显著升高(P<0.01),显示造模成功。与模型组相比,梓葛冻干粉针16.40mg·kg-1、32.70mg、kg-1和65.40mg·kg-1组均能显著降低大鼠缺血侧脑皮质层微血管中MMP-9mRNA表达水平(P<0.05,P<0.01)；与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中Claudin-5mRNA、Occludin mRNA、ZO-1mRNA表达均显著降低,而与模型组相比,梓葛冻干粉针各剂量组在相应时间点能显著增加大鼠缺血侧脑皮质层微血管中Claudin-5mRNA、Occludin mRNA、ZO-1mRNA表达(P<0.05,P<0.01)。
Western blot检测蛋白结果与其基因水平结果基本一致。在再灌注1、4、7和14d,与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中MMP-9蛋白表达均显著升高(P<0.01),显示造模成功。与模型组相比,各给药组均能显著降低大鼠缺血侧脑皮质层微血管中MMP-9蛋白表达(P<0.01)；与假手术组相比,模型组的大鼠缺血侧脑皮质层微血管中Claudin-5、Occludin、ZO-1蛋白水平均显著降低,而与模型组相比,梓葛冻干粉针各剂量组在相应时间点能显著升高大鼠缺血侧脑皮质层微血管中Claudin-5、Occludin、ZO-1蛋白表达水平(P<0.05,P<0.01),提示其有改善脑缺血/再灌注损伤大鼠微血管通透性的效果。2.梓葛冻干粉针对高分子葡聚糖大鼠急性脑微循环障碍的改善作用
与正常组相比,在考察相对应的时间点,模型组的大鼠软脑膜微动脉,微静脉内径显著缩小,且血流量呈显著下降(P<0.01),显示造模成功；与模型组相比,梓葛冻干粉针16.40mg·kg-1,32.70mg·kg-1和65.40mg-kg-1组中大鼠软脑膜的微动脉、微静脉内径恢复显著(P<0.01),且能使血流量在10min以后基本恢复至造模前的血流量。与正常组相比,模型组的全血粘度、血浆粘度、红细胞压积和红细胞聚集指数均显著升高,显示造模成功；与模型组相比,梓葛冻干粉针65.40mg·kg-1组能显著降低血液流变学的各项指标(P<0.01)；梓葛冻干粉针16.40mg·kg-1口32.70mg·kg-1组能显著降低除红细胞压积以外的其它项血液流变学的指标(P<0.05,P<0.01),提示梓葛冻干粉针具有对大鼠急性脑微循环障碍的改善作用。
3.梓葛冻干粉针对体外缺氧/复氧损伤大鼠脑微血管内皮细胞mmp-9蛋白表达的影响
MTT法结果显示,梓葛冻干粉针作用于脑微血管内皮细胞的ICso值为860μg·mL-1。缺氧/复氧损伤下,与正常对照组相比,复氧24h后,模型组的BMECs细胞中MMP-9蛋白表达显著升高(P<0.01),显示造模成功。与模型组相比,梓葛冻干粉针24.50μg·ML-1、49.00μg·mL1和98.00μg·mL-1可抑制BMECs缺氧／复氧损伤下MMP-9蛋白升高,提示其对血管内皮细胞完整性具有保护作用。结论
1.采用Longa线栓法制备脑缺血模型最佳的缺血/再灌注时间为缺血2h,模型稳定,可控性和可重复性好。梓葛冻干粉针对脑缺血/再灌注损伤大鼠,能使脑血流在再灌注时缓慢恢复,减小脑梗死体积,减小脑组织含水量和EB含量,可以减轻脑水肿和血脑屏障的破坏程度；梓葛冻干粉针能促进缺血侧大鼠皮层区CD31的表达,表明可促进脑缺血/再灌注损伤大鼠的血管新生；梓葛冻干粉针改善脑微循环可能与调节微血管舒缩功能,抑制iNOS的活化,减少TNOS和ET-1的生成,抑制大鼠缺血侧脑皮质层TXB2和6-Ke-to-PGF1。表达有关；同时,梓葛冻干粉针可抑制MMP-9mRNA及蛋白表达和促进Claudin-5、Occludin、ZO-1mRNA及蛋白表达,保护微血管完整性,降低微血管通透性,改善脑微循环。
2.梓葛冻干粉针对急性脑微循环障碍大鼠,可扩张脑微动静脉管径,逆转脑血流量下降的趋势和血液粘稠,提示梓葛冻干粉针具有对急性脑微循环障碍的改善作用。
3.梓葛冻干粉针能抑制BMECs缺氧／复氧损伤下MMP-9蛋白表达,提示其对血管内皮细胞完整性的保护作用机制可能与降低MMP-9蛋白表达有关。
Background
Our research team researched a pharmaceutical preparation with the anti-ischemic effect of Zige lyophilized powder that contained catalpol and puerarin and had a patent. Zige lyophilized powder for injection had the effects of the neural functional recovery in permanent cerebral ischemia of rats, reducing cerebral infarction area, angiogenesis, improving blood stasis and blood brain barrier (BBB) permeability, and so on. But we had not research the effects of Zige lyophilized powder for injection in the celebral microcirculation.
Objective
It aims to research possible mechanism of Zige lyophilized powder for injection against cerebral ischemia-reperfusion injury in the celebral microcirculation through the pathological causes of contraction, permeability and blood rheology of microvessel in experiments of animal and cell.
Methods
1. The improving effects of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in the disturbance of microcirculation
80male sprague-dawley rats were randomly divided into sham group (10), ischemia1h group (16/20), ischemia2h group (16/20), ischemia4h group (16/20), nimodipine injection (ischemia1h and reperfusion,2.00mg·kg-1,8/10), and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution. It made a cerebral ischemia-reperfusion (I/R) model using the line in the left middle cerebral artery occlusion (MCAO) of rats and test neuro-behavioral evaluation by a method of modified neurological severity scores (mNSS) in ischemia for1h,2h,4h and reperfusion for1d,4d,7d and14d.
540male sprague-dawley rats were randomly divided into sham group (60), model group (61/80), puerarin injection group(30.00mg·kg-1,68/80), nimodipine injection (2.00mg·kg-1,69/80), Zige lyophilized powder groups(16.40mg·kg-1,65/80;32.70mg·kg-1,68/80;65.40mg·kg-1,70/80) in ischemia for2h and reperfusion for1d,4d,7d and14d, and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution. Blood flow volume of pia mater in rats was observed by laser doppler flowmetry. Pathomorphism changes and damaged conditions in rat cortex were observed by HE staining. The volume of the cerebral infarction was estimated by2%TTC staining. The water content was calculated by dry and wet weight method. The disruption of micro vessel permeability was detected by the EB content. The expression of CD31was detected by immunohistochemical staining method.
960male sprague-dawley rats were randomly divided into sham group (96), model group (109/144), puerarin injection group(30.00mg·kg-1,120/144), nimodipine injection (2.00mg·kg-1,123/144), Zige lyophilized powder groups(16.40mg·kg-1,117/144;32.70mg·kg-1,122/144;65.40mg·kg-1,126/144) in ischemia for2h and reperfusion for1d,4d,7d and14d, and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution.Nitric oxide synthase (NOS), endothelin-1(ET-1),6-ketone-prostaglandin F1α (6-Ke-to-PG-F1α) and thromboxane B2(TXB2) content of the brain microvessel in the lateral ischemic cerebral cortex was detected by enzyme immunoassay kits. The expression of MMP-9、ZO-1、Occludin and Claudin-5protein and mRNA of the brain microvessel in the lateral ischemic cerebral cortex was detected by fluorescence quantitative PCR (qPCR) and Western blot (WB).
2. The protective effect of Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats by intravenous injection of high molecule dextran
120male rats were divided into normal control group, model group, nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) with20for each goup, and made window craniotomy after the administration of drugs for14days (tail vein injection,1time per day), the model of rats with the disturbance of cerebral microcirculation was estabished by intravenous injection of high molecule dextran (10%T-500,10mL·kg-1) after the last administrat-ion of drug for30min. The effects of micro-vein diameter for5min in normal rats and1,5,10,20,40,60min in making model of rats were observed through the inverted microscope. Blood flow volume on brain meninx was observed through laser doppler flowmetry. HCT was measured by electric resistance method, hemorheological indexes were observed by auto-hemorheological instrument.
3. The protein expression levels of mmp-9to Zige lyophilized powder for injection on brain microvascular endothelial cells after hypoxia-reoxygenation injury were detected in vitro
It made a hypoxia-reoxygenation injury model on cultured BMECs of rat (simulated cerebral ischemia-reperfusion injury model) in vitro by oxygen-glucose deprivation. The proliferation of endothelial cells was detected by MTT method. Endothelial cells were cultured by oxygen-glucose deprivation for2h and reoxygenation for24h, it determined to detect the protein expression levels of mmp-9by WB method.
Results
1. The improving effects of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in the celebral microcirculation
(1) The focal cerebral ischemia-reperfusion model of rats and neuro-behavioral test after I/R rats have significantly neurological deficit,1d after I/R, the mNSS score of1h,2h and4h groups were5.68±0.75,9.23±1.05,11.53±1.881, respectively. Percentages of the infarction area of three groups in14d were0.1026±0.0214,0.2245±0.0228,0.3828±0.0318, respectively. The main infarction area of1h group was cortex, and the main infarction area of2h and4h groups was cortex and basal ganglia. The best time of making the ischemia-reperfusion model is2h to ischemia for subsequent experiments.
(2) The effect of Zige lyophilized powder for injection against the model rats of blood flow volume, infarction volume and brain edema in cerebral microcirculation.
In this experiment, Zige lyophilized powder(16.40,32.70and65.40mg-kg-1) showed a trend of slow recovery during reperfusion. In TTC staining, sham group appeared in deep red and had not the brain infarcts, model group had a large area of infarcts and located in the left side of the cortex and basal ganglia. Compared with model group, Zige lyophilized powder groups (65.40mg-kg-1) decreased significantly infarction volume in14d(P<0.05).
Compared with sham group, the water content and EB content increased significantly (P＜0.05, P<0.01) in model group,and it made a successful model. Compared with model group in reperfusion for7d and14d, Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit significantly the water content and EB content in lesion side of the rat brain tissue (P<0.05,P<0.01), these showed that Zige lyophilized powder protected against cerebral ischemic injury by improving microvessel permeability.
In HE staining, cells of brain ischemia side in all groups after cerebral ischemia-reperfusion appeared necrosis, shrinking nucleus into irregular shape, edema, loosen and infiltration by inflammatory cells. Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit infiltration by inflammatory cells, and reduce intercellular space and edema in the corresponding time.
(3) The effect of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in microvascular density of the celebral microcirculation.
In immunohistochemical experiment, a few of CD31cells expressed in sham group. It showed that the expression of CD31after ischemia-reperfusion in lateral cortex of rats had a few in reperfusion for1d, and had higher in reperfusion for4d and7d, and increased slowly or no growth in reperfusion after7d. Compared with model group in reperfusion for1d,4d,7d and14d, the expression of CD31of puerarin injection group (30.00mg·kg-1), Nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had higher significantly in the corresponding time except reperfusion for1d (P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by promoting angiogenesis.
(4) The effect of Zige lyophilized powder against the microvascular contraction function after cerebral ischemia-reperfusion injury of rats in the microcirculation
In this experiment, compared with sham group, it showed that TNOS and iNOS enzyme activity had increased and cNOS enzyme activity had decreased significantly after ischemia-reperfusion in lateral cortex of rats (P<0.01). Compared with model group in reperfusion for1d, puerarin injection group (30.00mg·kg-1), Nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had reduce significantly TNOS and iNOS enzyme activity (P<0.05, P<0.01). Compared with model group in reperfusion for4,7and14d, all drug groups had reduce significantly TNOS and iNOS enzyme activity and increased significantly cNOS enzyme activity. These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving the microvascular contraction function in the celebral microcirculation.
Compared with sham group, it showed that the level of ET-1after ischemia-reperfusion in lateral cortex of rats had a rising trend and increased gradually with prolonged observation. In reperfusion for1d, compared with model group, Zige lyophilized powder group (32.70mg·kg-1) could reduce significantly the level of ET-1 (P<0.05). In reperfusion for4,7and14d, compared with model group, Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had reduce significantly the level of ET-1in micro vessel of the lateral ischemic cerebral cortex (P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving the microvascular contraction function and endothelial cell damage.
In reperfusion for1,4,7and14d, compared with sham group, it showed that the level of TXB2had increased and6-Ke-to-PGF1α had decreased significantly in model group. Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit significantly the level of TXB2and6-Ke-to-PGF1α(P<0.05,P<0.01)and the ratio of TXB2/6-Ke-to-PGF1α(P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving microvascular contraction function in microcirculation.
(5) The effect of Zige lyophilized powder for injection against the microvascular permeability after cerebral ischemia-reperfusion injury of rats in the celebral microcirculation
In real-time quantitative PCR, the cortex of sham group had a few of mmp-9mRNA levels. After focal cerebral ischemia-reperfusion, comparing with sham group in reperfusion for1,4,7and14d, it showed that mmp-9mRNA levels in lateral cortex of rats had increased significantly in model group. Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could reduce significantly mmp-9mRNA levels. Comparing with sham group, it showed that claudin-5mRNA、occludin mRNA、zo-1mRNA levels in lateral cortex of rats had reduced significantly in model group. Three groups of Zige lyophilized powder could increase significantly claudin-5mRNA、occludin mRNA、zo-1mRNA levels respectively (P<0.05,P<0.01).
In the experiment of western blot, a change trend of protein expression and genetic level is basically identical. Compared with sham group in reperfusion for1,4,7and14d, it showed that mmp-9protein expression in lateral cortex of rats had increase significantly in model group and all drug groups could reduce mmp-9protein expression significantly. Comparing with sham group, it showed that the protein expressions of claudin-5、occludin、zo-1in lateral cortex of rats had reduced significantly in model group. Three groups of Zige lyophilized powder could increase significantly claudin-5、occludi、zo-1levels in the corresponding time, respectively (P <0.05,P<0.01).These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving microvascular permeability.
2. The protective effect of Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats by intravenous injection of high molecule dextran
Compared with normal control group in the corresponding time, it showed that pia mater microvenule diameter and microartery diameter in rats had shrink significantly and blood flow volume had decrease significantly in model group. Compared with model group, Zige lyophilized powder for injection(16.40,32.70and65.40mg·kg-1) had significantly expended micro-vein diameter (P<0.01),and returned normal trend of blood flow volume after10min of making model. Compared with normal control group, it showed that blood viscosity, erythrocyte agglutination index, plasma viscosity and hematocrit in rats had increase significantly in model group. Compared with model group, Zige lyophilized powder (65.40mg·kg-1)could significantly reduce blood rheology indicators (P<0.01),and Zige lyophilized powder for injection(16.40,32.70mg·kg-1)had could significantly reduce other blood rheology indicators except hematocrit (P<0.05,P<0.01).
3. The protein expression levels of mmp-9to Zige lyophilized powder on brain microvascular endothelial cells (BMECs) after hypoxia-reoxygenation injury were detected in vitro
The IC50value is860μg·ml-1by MTT method in cultured BMECs by Zige lyophilized powder for injection. Compared with normal control group in the reoxygenation time for24h, it showed that the level of mmp-9protein had increase significantly in model group. Zige lyophilized powder groups (24.50、49.00and98.00μg·ml-1)had inhibit significantly the increased levels of mmp-9protein in a hypoxia-reoxygenation injury.
Conclusions
1. The best time of making I/R model by Longa method is2h to ischemia, and it is stability, controllability and repeatability. Zige lyophilized powder for injection showed a trend of slow recovery during reperfusion, and could decrease infarction volume, and inhibit significantly the water content and EB content in lesion side of the rat brain tissue. These showed the fact that it could reduce the damage rate of the blood brain barrier and brain edema. Zige lyophilized powder for injection can promote CD31expression in lateral cortex of rats, it would be promote angiogenesis after ischemica-reperfusion injury, and improve the cerebral microcirculation, which is beneficial to the recovery of damaged nerve tissue. The possible mechanisms of Zige lyophilized powder for injection in the improving celebral microcirculation would adjust the microvascular contraction function, and inhibit the enzyme activity of iNOS, and reduce the generated TNOS and ET-1content, and inhibit the level of TXB2and6-Ke-to-PGF1a. Zige lyophilized powder for injection can protect the microvascular integrity, and reduce microvascular permeability, and improve cerebral microcirculation through it can inhibit the increasing trend of mmp-9mRNA and protein expression and the decreasing trend of claudin-5, occludin, zo-1mRNA and protein level.
2. Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats can change trend of pia mater microartery and microvenule diameter, and improve celebral blood flow and blood viscosity, these showed that it would impove the effect of the acute disturbance of cerebral microcirculation.
3. Zige lyophilized powder for injection had inhibited the increased levels of mmp-9protein in a hypoxia-reoxygenation injury on cultured BMECs. It showed that the possible mechanism of Zige lyophilized powder for injection on protecting the cell integrity would be associated with the decreasing mmp-9protein expression.
引文
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