1、脑出血后血管新生及中医药的作用机制研究背 景1）流行病学资料流行病学资料ICH13%SAH13%Lacunar19%Other 3%Unknown32%Ischemic 71%Hemorrhagic 26%Cardioembolic14%Thromboembolic6%NINCDS Stroke Data Bank:Foulkes,Stroke,1988；Yang QD,et al.Cerebrovasc Dis 2004，17:303313 脑出血占脑卒中的32.6%长沙的社区资料则达55.4%2)前期基础前期基础新药开发：新药开发：脑溢安颗粒（脑溢安颗粒（ICHICH急性期）急性期）脑溢安
2、脑溢安2 2号（号（ICHICH恢复期）恢复期）天龙熄风颗粒（缺血性中风急性期）天龙熄风颗粒（缺血性中风急性期）国家自然科学基金项目“脑溢安对出血性中风大鼠脑缺血损伤的保护作用研究”（39670907）国家自然基金青年项目“出血性中风大鼠神经胶质反应及中药干 预的研究”（39800189）iNOS in astrocyteHO-1 in Microglia 国家自然科学基金项目“从神经干细胞分化角度探讨脑溢安对脑出血的脑保护机制”（编号：30171192）脑出血对微血管损伤脑出血对微血管损伤 原发于血肿的机械性破坏 继发于炎症介导的损伤研究的思路研究的思路微血管对脑的重要性微血管对脑的重要性
3、脑是血流高度依赖性器官有利于维持脑内微环境的稳定 血脑屏障的组成 物质交换:血氧的输运,废物的清除 外周干预手段到达损伤部位的基本途径：药物，细胞移植、各种修饰载体微血管系统重建是脑组织修复、神经元抗损伤的必须过程Luo EH,et al.Nat Rev Neurosci,2003,4:399-415.Wei L,et al.Pathophysiology,2005,12:47-62.Change of mirovessels around and in the hematoma.No hemorrhage was found in sham control group(A).In ICH g
4、roup,there was a hematoma in the right basal ganglion(inlet of panel B);no vessels were observed around and in the clot before 4days(B),but many enlarged and thin-walled mirovessels(arrow)appeared close to the clot at 7days(C)and extended into it from 14days(D)then almost spread all over the clot at
5、 about 21days(E).Dash lines marked the border of hematoma,and solid lines marked the border of the area where few vessels invaded in the hematoma.H&E stain was used.Scale bars,100m,1mm for the inlets.血肿区有血管新生血肿区有血管新生研究内容研究内容 研究脑出血后血管新生的调控机制Thrombin-inducedangiogenesisfollowingICHAngiogenesisviaVEGFR
6、2afterICH 从微血管系统重建的角度，以益气活血经典方补阳还五汤和电针足三里为干预手段，探讨中医治疗脑出血的机制结结 果果1.Thrombin-induced angiogenesis following ICHProliferated cerebral endothelial cells after ICH.A and B:Many BrdU-positive nuclei(arrows showing brown spots)in vWF-positive dilated vessels(blue)around the hematoma.C:Labeled nuclei in ves
7、sels increased until Day 14 after ICH.However,hirudin reduced significantly the BrdU-positive nuclei in cerebral ECs(*p 0.05,*p 0.01;N/mm2,mean SD,n=5);DAB(brown)enhanced with ammonium nickel sulfate(blue),scale bar=100 m(A)and 25 m(B).d=days.Immunohistochemistry for detection of HIF-1,VEGF,Ang-1,an
8、d Ang-2 after ICH.No HIF-1(A)or Ang-2(J)immunoreactivity was found,and there were few slim microvessels of VEGF(D)or Ang-1(G)immunoreactivity scattered in the brains of sham-operated animals.However,numerous HIF-1(B),VEGF-(E),Ang-1(H),and Ang-2(K)positive microvessels of the enlarged profile were de
9、tected in the perihematomal tissue after ICH.Quantitative analysis showed that HIF-1(C)and Ang-2(L)immunoreactivity declined at about 7 days following a striking upregulation at 3 days,while VEGF(F)and Ang-1(I)immunoreactivity increased notably as long as 2 weeks after ICH.In hirudin-treated brains,
10、the immunoreactivity of HIF-1(C),VEGF(F),Ang-1(I),and Ang-2(L)decreased significantly(*p 0.05,*p 0.01;values presented as mean SD,n=5).DAB,scale bar=100 m.MOD=mean optical density.Quantitative analysis of HIF-1,VEGF,Ang-1,and Ang-2 mRNA after ICH.Real-time RT-PCR demonstrated that the upregulation o
11、f VEGF(B)and Ang-1(C)persisted until 14 days after ICH,while Ang-2(D)mRNA peaked at 3 days.However,the upregulation of VEGF(B),Ang-1(C),and Ang-2(D)mRNA levels was inhibited by hirudin.No change was detected in HIF-1 mRNA(A)in any group.*p 0.05;*p 0.01.The values are presented as mean SD(n=5).Gray b
12、ars show levels after ICH;black bars indicate levels after ICH+hirudin.n fold=change in gene expression relative to the sham control sample.Proliferated cerebral endothelial cells after thrombin treatment.BrdU-positive nuclei(A;arrows showing brown spots)could be found in vWF-positive vessels(blue)u
13、ntil 7 days after thrombin injection(B;*p 0.05,*p 0.01,N/mm2;values presented as mean SD,n=5);DAB(brown)enhanced with ammonium nickel sulfate(blue),scale bar=25 m(A).2.从微血管系统重建的角度，以益气活血经从微血管系统重建的角度，以益气活血经典方补阳还五汤和电针足三里为干预手段，典方补阳还五汤和电针足三里为干预手段，探讨中医治疗脑出血的机制探讨中医治疗脑出血的机制1)Buyang huanwu decoction promotes
14、 angiogenesis via VEGFR2 activation after ICHBYHWD upregulates angiogenesis via VEGFR2 activation following ICH.Double-labeling revealed that BrdU+nuclei(brown,arrow)were shown in vWF+vessels(blue)in either hemisphere of the sham-operated animals(H),ICH group(I),SU5416-treated(J),BYHWD-treated(K)and
15、 BYHWD+SU5416-treated(L)groups.The number of BrdU+/vWF+(M)nuclei were compared between BYHWD-treated and non-treated groups.BYHWD upregulates VEGFR2 activation and angiogenesis via the PI3K/Akt signaling pathway.A representative immunoblot showed that the effects of ICH,SU5416 and BYHWD on the prote
16、in levels of pVEGFR2,VEGFR2,PI3K,pAkt and Akt at the ipsilateral basal ganglion(N and Q).The ratios of pVEGFR2/VEGFR2(O),pVEGFR2/-actin,VEGFR2/-actin(P),PI3K/-actin(R)and the ratios of pAkt/Akt(S)were compared in ICH and BYHWD with or without SU5416(meanSD,n=5,*P 0.05,*P 0.01).Bar=50 m.Neurological
17、evaluation scores.The forelimb asymmetric use rate(AUR)of the SNE group gradually decreased as the observation time progressed,and all AUR values were greater than those in the SHAM group at the corresponding time points.In addition,the AUR values of the SEZ group were significantly less than those
18、of the SNE group.However,EA at a nonacupoint had little effect on the neurological recovery after ICH.P0.01 vs.the SHAM group;P0.05,*P0.01 vs.the SNE group.P0.05 vs.the former time point in the SNE group(meanSD,n=5 for each time point).2)2)针刺足三里能促进脑出血后的血管新生针刺足三里能促进脑出血后的血管新生针刺足三里能促进脑血管内皮针刺足三里能促进脑血管内皮
19、细胞增殖细胞增殖.脑出血后，脑出血后，血肿周围有一些BrdU+/vWF+细胞核（A）。在电针足三里能显著增加BrdU+/vWF+细胞核，而电针非穴位处无效（B）。*P0.05,*P0.01.Scalebar50m。HIF-1在脑组织表达在脑组织表达.假手术假手术动物脑内的动物脑内的HIF-1信号很少信号很少(A).脑出血后,在血肿周围的扩张的微血管上可看到HIF-1阳性信号(B).双标技术显示：HIF-1（红色）表达在vWF（绿色）阳性血管内皮细胞上(C-E).Scalebar,100mforpanelsA-B,50mforpanelsC-E.电针足三里对电针足三里对HIF-1mRNA和蛋白和
20、蛋白表达的影响。表达的影响。脑出血后，HIF-1mRNA的表达在各时间点和各组间没有差异(A)，但HIF-1蛋白水平在3天达峰值，而电针足三里能显著提高该蛋白水平，而而电针非穴位处无效(B,C).*P0.05,*P0.01.电针足三里对电针足三里对VEGF mRNA和蛋和蛋白表达的影响。白表达的影响。VEGFmRNA在假手术组未见明显表达，脑出血后3天VEGFmRNA表达即明显增强，一直持续到14天，针刺组VEGFmRNA表达水平在各时间点均明显高于脑出血组，而假针刺组与脑出血组比较差异无统计学意义。*P0.01Quantitative analysis of Ang-1 and Ang-2
21、mRNA after ICH.After ICH induction,notable upregulation of Ang-1(A)and Ang-2(B)mRNAs could be detected at 3 days,and the upregulation of Ang-1 persisted until 14 days,while the expression of Ang-2 mRNA decreased after 7 days.Post-hoc analysis indicated striking increases in the expression of Ang-1 m
22、RNA from 3 days to 14 days and Ang-2 mRNA at 3 days in the SEZ group as compared with the SNE group.However,EA at a nonacupoint had little effect on the expression of Ang-1 and Ang-2 at the mRNA level.*P0.05 vs.the SNE group.P0.01 vs.the former time point in the SNE group(meanSD,n=5 for each time point).小结 凝血酶是脑出血的血管新生的启动因素之一 益气活血法可通过改变VEGFR2的活化，调节血管新生 电针足三里能显著促进脑出血后血管新生，此作用可能与其上调脑血管内皮上HIF-1、VEGF和Angs的表达有关
