三七通舒在大鼠脑缺血耐受和自体神经干细胞增殖中的作用

Effect of traditional Chinese medicine three seven three alcohol saponinon on brain ischemic tolerance and proliferation of endogenous neural stem cells in rat

目的 明确缺血预处理（IPC）及中药三七三醇皂苷（PTS）对大鼠脑梗死后7d脑海马区自体神经干细胞增殖、神经行为学评分,以及δ阿片受体（DOR）、Bax和Bcl-2 mRNA表达水平的影响. 方法 采用二次线栓法建立局灶性SD大鼠脑缺血耐受动物模型.80只SD雄性大鼠随机分为：假手术（Sham）组、缺血（MCAO）组、假手术＋缺血（Sham＋ MCAO）组、预缺血＋缺血（IP＋MCAO）组、三七三醇皂苷-缺血（PTS＋ MCAO）组.每组取10只行BrdU荧光标记细胞免疫组化染色,另6只行荧光定量PCR测定DOR、Bax、Bcl-2 mRNA水平.大鼠在处死前1d予腹腔注射BrdU.采用Zea-Longa评分方法对大鼠脑梗死后7d进行神经行为学评分,运用荧光免疫组织化学技术检测大鼠脑缺血侧海马区BrdU标记阳性细胞数量. 结果 SD大鼠脑梗死后7d,Zea-Longa神经行为学评分MCAO、Sham＋ MCAO两组与IP＋ MCAO、PTS＋ MCAO间均差异有统计学意义（P＜0.01）,而MCAO和Sham＋ MCAO组间、IP＋ MCAO和PTS＋ MCAO组间差异无统计学意义（P＞0.05）.SD大鼠脑梗死后7d,缺血侧海马区BrdU标记阳性细胞Sham、MCAO、Sham＋ MCAO3组与IP＋ MCAO、PTS＋ MCAO间均有差异（P＜0.01）;而MCAO和Sham＋MCAO组间、IP＋MCAO和PTS＋ MCAO组间差异无统计学意义（P＞0.05）.IP＋ MCAO组,DOR、Bcl-2 mRNA的表达水平明显高于MCAO、Sham＋ MCAO和PTS＋ MCAO组（P＜0.01）,而Bax mRNA的表达水平明显低于MCAO、Sham＋ MCAO和PTS＋ MCAO组（P＜0.01）.MCAO、Sham＋ MC AO和PTS＋ MCAO3组间差异无统计学意义（P＞0.05）. 结论 IPC可促进大鼠脑梗死后海马区成体神经干细胞增殖,并能改善其神经功能缺损症状;IPC可促使DOR、Bcl-2 mRNA水平的上调,并降低BaxmRNA的表达水平;PTS可改善大鼠脑梗死后的神经功能缺损症状,促使大鼠海马齿状回颗粒成体神经干细胞增殖,但对DOR、Bcl-2、Bax mRNA表达水平无影响.

Objective To investigate the effect of brain ischemic preconditioning （IP） combined with traditional Chinese medicine three seven three alcohol saponin （PTS） on proliferation of endogenous neural stem cells and the mRNA expressions of delta opioid receptor （DOR）,Bax,Bcl-2 in hippocampus at 7d post middle cerebral artery occlusion （MCAO）.Methods The focal-focal ischemic tolerance models were established with twice suture method.80 SD rats were included and randomly divided into 5 groups：sham group,MCAO group,sham＋ MCAO group,IP＋ MCAO group,PTS＋MCAO group （n=16 each）.We chose 10 SD rats from each group to evaluate their neurological status,and made BrdU fluorescent immunolabeling.In addition,we chose the other 6 SD rats to detect the expression levels of DOR,Bax and Bcl-2 mRNA in ischemic region in hippocampusby using RT-PCR.Animals were given one set of BrdU injections （on day 6,three times,4h apart,50mg/kg） to label the proliferating cells.The neurological status was assessed by using Zea Longa neurological deficit scores at 7 days following cerebral infarction.Results Zea longa neurologic deficit scores in MCAO group and sham＋ MCAO） group had significantly differences with IP＋ MCAO group and PTS＋ MCAO group respectively at 7d post MCAO（P＜0.01）.There was no significant differeuce in Zea-longa neurologic deficit scores between MCAO group versus sham＋ MCAO group,and IP＋ MCAO group versus PTS＋ MCAO group（P＞0.05）.The number of BrdU＋ ceils in hippocampus had significant differences between IP＋ MCAO and PTS＋ MCAO groups at 7d post MCAOand three groups of sham,MCAO and sham＋ MCAO respectively （P＜0.01）.There was no difference in the number of BrdU＋ cells between MCAO versus Sham ＋ MCAO groups and IP ＋ MCAO versus PTS＋MCAO groups（P＞0.05）.DOR and Bcl-2 mRNA expression levels were higher and Bax mRNA expression level was lower in IP＋ MCAO group than in MCAO,Sham＋ MCAO and PTS＋MCAO groups （P＜0.01）.There were no significant differences in DOR,Bcl-2 and Bax mRNA expressions among MCAO,Sham ＋ MCAO and PTS ＋ MCAO groups （P＞ 0.05）.Conclusions Acute cerebral infarction can induce the proliferation of endogenous neural stem cells in hippocampus in SD rats.IPC can facilitate the proliferation of endogenous neural stem cells in hippocampus afteracute cerebral infarction,improve the symptoms of neurologic dysfunction,increase DOR and Bcl 2 mRNA expressions,and reduce Bax mRNA expression in SD rats.PTS can facilitate the proliferation of endogenous neural stem cells in hippocampus after acute cerebral infarction in SD rats,and improve the symptoms of neurologic dysfunction,but it has no influence on the expressions of DOR,Bcl-2 and Bax mRNA.