实验研究
醒脑开窍针刺法对不同时间局灶性脑缺血/再灌
注损伤时活体脑片海马神经元[Ca 2+
]i 的影响
马玉侠1
,王舒2
,倪光夏2
,石学敏
23
(1.山东中医药大学博士后流动站,山东济南250014;2.天津中医学院第一附属医院,天津300193)
摘 要:目的:研究醒脑开窍针刺法对局灶性脑缺血/再灌注大鼠脑细胞内Ca 2+
的调节作用。方法:利
用激光共聚焦扫描显微镜观察海马C A1区活体脑片中锥体细胞内Ca 2+
的分布及动态变化。结果:脑缺血1h 时海马C A1区神经元的[Ca
2+
]i 明显升高,再灌注后,神经元内[Ca
2+
]i 随再灌注时间延长而进一
步升高,到再灌注6h ,达到最高峰,此后虽有下降,但仍高于缺血前水平,再灌注24h 时又有第二次钙积聚高峰。针刺对脑缺血/再灌注大鼠各时程的[Ca
2+
]i 均有降低作用,但对其下调程度,醒脑开窍针刺组
明显优于传统针刺组。结论:醒脑开窍针刺法可迅速调节缺血/再灌注后海马C A1区神经元细胞内的Ca 2+
含量,抑制胞内钙超载,拮抗脑缺血Π再灌注后继发神经元的损伤。
关键词:醒脑开窍;针刺;激光共聚焦扫描显微镜;局灶性脑缺血/再灌注;海马;钙离子中图分类号:R245-0 文献标识码:A 文章编号:1002-2392(2007)05-0009-03收稿日期:2007-05-28 修回日期:2007-07-28
基金项目:国家自然基金课题面上项目(N o :30572414);天津市卫生局中
医、中西医结合科研基金重点课题(N o :03040)。
作者简介:马玉侠(1976-),博士后,医学博士,主要从事针灸治疗脑血
管病的机制研究。
3通讯作者:石学敏,中国工程院院士,教授,T el :022-********。
脑血管病是现今人类死亡率最高的三大疾病之一,而缺血性脑血管病的发病率约占脑血管病的75～80%,且近年来不断上升,严重危害了人类健康。脑缺
血-再灌注损伤是一个十分复杂的病理生理过程[1]
,其中心环节是各种原因引起的细胞内钙超载,缺血后
神经元和神经胶质细胞内过度Ca 2+
积聚是细胞死亡
的最后共同通路[2]
。因此本实验选择线栓法制作大脑中动脉阻塞的局灶性脑缺血/再灌注大鼠模型,利用激光共聚焦扫描显微镜技术,观察海马C A1区活体脑片
上针刺对缺血/再灌注后细胞内的Ca 2+
含量影响,进一步探讨醒脑开窍针刺法对缺血/再灌注损伤的保护作用机制。1 材料和方法1.1 材料
1.1.1 试剂 Fluo -3ΠAM 购于美国M olecular Probes 公司,多聚赖氨酸(P oly -L -lysine hydrobromide )、H o 2
chest33258、肝素(heparin s odium salt )购于美国Sigma 公
司,Pluronic F -127购于美国Biotium 公司。
1.1.2 动物 健康雄性S D 成年大鼠(Ⅱ级),体重200～250g ,由北京大学医学部实验动物中心提供。1.1.3 实验仪器 Leica T CS -NT 型激光共聚焦扫描显微镜(德国Leica 公司),1Π10000电子分析天平(瑞士Mettler AEI00公司)。
1.2 方法1.
2.1 分组 将动物随机分为6组,每组5只:正常组,假手术组,缺血/再灌注模型组,醒脑开窍针刺组,传统针刺组,非穴针刺组。后四组分设缺血1h 及缺血1h 后再灌注1h 、3h 、6h 、12h 、24h 组,共计6个观察时
间点。1.2.2 大鼠大脑中动脉阻塞(MC AO )模型制作 将大鼠仰卧固定于鼠板,在20min 内制作MC AO 再灌注或
假手术模型。采用Longa 等[3-4]
线栓法制作左侧MC AO 大鼠模型,钓丝经10%P oly -L -lysine hydrobro 2mide (多聚赖氨酸)溶液处理,1h 后抽出尼龙钓丝,使缺血区血流再灌。假手术组除钓丝不插入颈内动脉外,其余处理同缺血组。1.2.3 针刺方法 根据中国针灸学会实验针灸研究会制定的“动物针灸穴位图谱”,醒脑开窍针刺组选石学敏教授“醒脑开窍”主穴人中、内关(双侧);非穴针刺组予针刺双侧腋中线下3mm 各一非穴点及尾骨尖旁
?
9?中医药学报2007年第35卷第5期
开3mm 一非穴点;传统针刺组选患侧曲池、足三里穴。三组均采用电针刺激10分钟,电针强度3mA ,频率2H z 。缺血1h 组在造模成功后即刻针刺1次;缺血1h 后再灌注1h 、3h 、6h 、12h 组在造模成功后即刻和再灌注后即刻各针刺1次;缺血1h 后再灌注12h 、24h 组在造模成功后即刻和再灌注后即刻各针刺1次,此外,每6h 针刺1次。1.2.4 大鼠海马C A1区活体脑片制备 在不同的时间观察点,将大鼠迅速断头取脑(均在3min 内),浸入4℃ACSF (人工脑脊液)中(ACSF 组成:NaCl 126、K Cl 5,G lucose 10、NaH 2PO 41.25、NaHC O 326、MgS O 41.5、CaCl 22,pH 为7.4),插管通入95%O 2和5%C O 2混合气体5min 。取出脑组织去掉小脑,沿两侧大脑半球连接处将大脑半球切开,剥离出病变侧海马,将海马放置于软组织切片机样品台湿润的滤纸上,连续切3～5张,制
备400μm 活体海马脑片。
1.2.5 大鼠脑片细胞内Ca 2+
荧光探针的负载 将脑
片迅速放到含有10μm ol ΠL Fluo -3ΠAM 、1ug ΠL H o 2
ches t33258及5μL pluronicF -127(12.5%重量比)的ACSF 中,插管通入95%O 2、5%C O 2混合气体,室温避
光孵育30min ,然后用不含荧光探针的ACSF 液清洗3次,待上机测定。
1.2.6 海马脑片细胞内Ca 2+
荧光强度的测定 将负载后的脑片放入C LS M 样品槽中,在40倍油镜下观察脑片,选取海马C A1区的锥体细胞作为观察对象,以激发波长488nm ,发射波长520nm ,电子放大为2,紫外
激发波长361nm ,发射波长420～470nm ,利用C LS M 的
无损伤光切功能,对活体的海马进行不同层面光切,随机寻找数个视野,每个样品应采集细胞数100个以上,
观察海马不同层面锥体细胞内的Ca 2+
相对荧光强度。每只动物检测两张脑片。
用激光扫描共聚焦显微镜图像处理系统(T CS SP2confocal s oftware )进行图像分析与数据处理,以测得的
荧光绝对强度FI 代表Ca 2+
相对强度。1.3 统计学方法
使用SPSS 11.5统计软件进行统计学处理,检测结果以均数±标准差( x ±s )表示;多组间差异的显著性采用单因素方差分析(F 检验),用Student -Newman -K euls 法进行组间的两两比较。2 结果2.1 脑缺血Π再灌注时不同时段大鼠海马C A1区神经
元[Ca 2+
]i 的变化(见图1)
大鼠脑缺血1h 时,脑组织海马C A1区神经细胞[Ca 2+
]i 开始升高;大鼠脑缺血1h 再灌注1h 后,脑组
织神经元细胞[Ca 2+
]i 继续升高,脑缺血再灌注3h 时
[Ca
2+
]i 仍持续升高,到再灌注6h 时达到高峰,再灌注
12h 后脑组织海马C A1区神经元细胞[Ca
2+
]i 较再灌
注6h 时有所下降,但仍然高于再灌注3h 组;再灌注
24h 后C A1区神经元细胞[Ca 2+
]i 比12h 又有所增高,但是仍然较6h 低(均P <0.05)
。
图1 Changes of Ca 2+
relative fluorescent intensity in brain slices with different time of CIR.( x ±s ,n =5)
2.2 醒脑开窍针刺法对缺血/再灌注后不同时间点大
鼠海马神经细胞内Ca 2+
含量的影响
醒脑开窍针刺组及传统针刺组对脑缺血/再灌注
大鼠各时程的[Ca 2+
]i 均有降低作用,与模型组比较,
差异具有显著性意义(P <0.05),传统针刺组与醒脑
开窍针刺组比较,在缺血1h 时,差异无显著性意义,但在再灌注损伤的各时程,差异具有显著性意义,非穴位
针刺组与模型组同一时间点比较,[Ca 2+
]i 无显著变化(P >0.05),见表1。
?01?AC MP 1Aug ,2007,V ol 135,N o 15
表1 醒脑开窍针刺法对脑缺血Π再灌注后海马C A1区脑片[Ca2+]i含量的影响( x±s,n=5)
缺血1h再灌注1h再灌注3h再灌注6h再灌注12h再灌注24h
正常组16.61±3.3916.61±3.3916.61±3.3916.61±3.3916.61±3.3916.61±3.39
模型组21.67±4.23327.43±3.043331.66±4.543346.61±6.033338.41±4.473340.48±5.2733
非穴针刺组22.01±3.86326.52±3.963330.99±4.043344.37±5.623337.64±4.373341.08±4.2933
传统针刺组18.32±3.51323.01±2.983#25.44±4.013#39.41±3.6633#31.56±4.3433#33.75±5.1233#
醒脑开窍针刺组17.03±4.12#18.68±3.72#△20.03±4.26##△25.02±5.103##△△22.68±3.9133##△△24.45±4.8533##△△
注:与正常组相比,3P<0.05,33P<0.01;与同时间段模型组相比#P<0.05,##P<0.01;与同时段传统针刺组比较,△P<0.05,△△P<0.01。3 讨论
活体定位定量观察细胞内Ca2+含量对于深入揭
示在体脑缺血Π再灌注损伤机制有重大意义,由于脑片
具有定位的特征,直接在脑片上观察钙离子的成像的
意义是不言而喻的。亲脂性Fluo-3ΠAM易于透过细
胞膜,激发光的波长恰好在可见光区范围内,适于激光
共聚焦扫描显微镜,使得此种标记方法可能成为观察
钙离子变化的理想手段之一[5]。本研究正是运用了
LCS M无损伤断层光切扫描的技术特性和荧光探针激
发/发射波长不同的特点,联合应用细胞核荧光探针
H ochest33258和Fluo-3ΠAM,能够较清晰的定位定量
的测定脑片上的单细胞内的Ca2+含量。
脑缺血时氧和葡萄糖供应不足,引起一系列病理
生化改变,最后导致神经元损伤或死亡。其产生机制
并不十分清楚,目前认为至少涉及以下几个不同的方
面:能量耗竭、细胞内钙超负荷、兴奋性氨基酸(E AA)
的释放增加、酸中毒、毒性氧自由基产生、花生四烯酸
产生等。其中胞内钙超载是神经细胞损伤和死亡的主
要原因。钙不仅在急性神经元损伤(缺血5h内发生的
神经元损伤)、成熟神经元损伤(缺血5～24h期间发生
的神经元损伤),而且在迟发性神经元死亡(缺血后
24h发生的再灌注损伤)中都起着非常重要的作用[6],
缺血损伤随时间的延长而加重[7-9]。本研究结果显
示,与正常大鼠相比,脑缺血1h时海马C A1区神经元
的[Ca2+]
i 开始升高,再灌注后,神经元内[Ca2+]
i
浓度
随再灌注时间延长而进一步升高,到再灌注6h,神经元内的游离钙浓度达到最高峰,此后[Ca2+]i虽有下降,但仍高于缺血前水平,再灌注24h时又有第二次钙积聚高峰。与文献报道的缺血Π再灌注后的[Ca2+]i变化规律[9-11]相一致。脑缺血再灌注后脑损伤的发生和发展与神经细胞[Ca2+]
i
的变化有密切关系,神经细胞胞内钙超载随着再灌注时间的延长而逐渐加重,由此所引发的神经细胞的损害也在逐渐加重。但是[Ca2+]i升高的时间与神经细胞形态学上损伤的出现是否同步及它们之间的关系,还需要进一步研究。
并且本研究发现给以醒脑开窍针刺治疗的大鼠,在缺血及再灌注初期就显示出了良好的抑制钙超载的作用,但是随着再灌注损伤的加重,尽管醒脑开窍针刺组的胞内钙高于正常组和假手术组,但是远低于模型组,而施以传统针刺治疗法的对照组尽管也对钙超载有抑制作用,但这种作用远低于醒脑开窍治疗组。非穴针刺组对于缺血再灌注后的钙超载没有治疗作用。因此,对脑缺血Π再灌注施以醒脑开窍针刺法的早期干预治疗效果明显优于其他针刺疗法,这种作用将有利于挽救半暗带区组织,减轻钙超载带来的损伤,从而达到对脑组织的保护作用。
“窍闭神匿、神不导气”是中风病的根本病理基础和关键环节,并且本病的病变部位在脑,反应于脏腑经络,从辨证论治的角度来切断这一关键环节才是治疗中风病的关键。虽然缺血性脑血管病的发生是由于脑组织缺血缺氧而出现局部细胞受损,它的病变部位在脑,曲池、足三里作为手阳明大肠经、足阳明胃经的合穴,能够补益并通畅阳明经的气血运行。因此,在脑梗塞之抢救和治疗过程特别是急性期治疗过程中,针刺曲池、足三里的效应对脑供血的影响是间接的,不足以使气达病所治疗脑腑病变。醒脑开窍针刺法主穴人中为督脉与手、足阳明经之会穴,督脉起于胞中,上行入脑达巅,故泻人中可调督脉、开窍启闭以醒脑安神;内关为八脉交会穴,通于阴维,属手厥阴心包经之络穴,有宁心安神、疏通气血之功。二穴效应皆直接作用于脑,开窍醒神通络,达到对脑组织的保护作用。本实验既证实了针刺腧穴与非穴之间对缺血Π再灌注后的钙超载具有显著的干预治疗差异,具有腧穴特异性;也证实了醒脑开窍针刺法对脑缺血Π再灌注的钙超载的抑制作用明显优于传统针刺组,更适合于脑缺血Π再灌注早期的干预治疗。
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中医药学报2007年第35卷第5期
ABSTRACTS FROM ORIGINAL ARTICL ES
E ffects of“XNKQ”acupuncture on[C a2+]i after different lengths of cerebral ischemia reperfusion(CIR)in rat hippocampus
MA Yu-xia1,WANG Shu2,NI Guang-xia2,Shi Xue-min2 Abstract:Objective:T o study the“X NK Q”acupuncture’s intervention in fluence on hippocam pus C A1neuron[Ca2+]i during CIR.Methods: Using con focal laser scanning microscope(C LS M)to observe the distri2 bution and dynamic changes of the intracellular free calcium in hipp2 ocam pus C A1neuron.R esults:The neuron[Ca2+]i of hippocam pus C A1regions in the rat began to increase after ischemia1h,and sus2 tained to elevate after reper fusion,at6h of ischemia reper fusion time course the[Ca2+]i elevated at the highest level.Then the[Ca2+]i be2 came to decrease,however is still higher than that before CIR.While at 24h the[Ca2+]i elevated at the second peak of calcium accumulation, but is lower than6h time course.Both“X NK Q”1acupuncture and tra2 ditional acupuncture could decrease the ischemic accumulation of calci2 um of any time course during CIR.But the inhibitory effect to Ca2+ overloading of“X NK Q”acupuncture is obviously better than that of tra2 ditional acupuncture.Conclusion:The“X NK Q”acupuncture could reg2 ulate the content of intracellular calcium of the region of ischemia and reper fusion,inhibited Ca2+overloading,and maintained the calcium homeostasis,s o as to protect neurons from injury of brain ischemia reper fusion.
Author’s address:1.P ostD octoral S tation of Shandong University of T C M,Jinan250014;2.First H ospital A ffiliated to T ianjin C ollege of T C M,T ian-jin300193
K ey w ords:“X NK Q”(activating brain and opening orifices)acupunc2 ture;con focal laser scanning microcope;cerebral ischemia reper fusion; hippocam pus;calcium
(Original article on page9) Study on inhibitory effects of extract of Cirsium setosum(Willd.) MB on grow th of four kinds of hum an carcinom a cells
LI Yu,WANG Zhen-fei,JIA Rui-zhen
Abstract:Objective:T o investigate the inhibitory effects of the extract of Cirsium setosum(Willd.)M B on human carcinoma cells.Methods: M orphological change observation and live cell accounting were used to investigate the inhibitory effects of Cirsium setosum(Willd.)M B on human chronic myloid leukemia K562cell,hepatoma HepG2cell,cervi2 cal carcinoma Hela cell and gastric carcinoma BG C823cell.R esults: G rowth of the four kinds of cells was significantly inhibited by the ex2 tract of Cirsium setosum(Willd.)M B,highest inhibit rate was88.27% and cell m orphological changes included shrinkage,roundness,floating and crumbling.Conclusion:Cirsium setosum(Willd.)M B has reliable carcinoma-inhibiting effects and the effects con form to the qi and blood theory,the Meridian-reaching theory and the liver-kidney hom ology theory of T raditional Chinese Medicine,which shows once again the abundance of basic theories of T raditional Chinese Medicine which can als o be exhibited in vitro and at cell level and have im portant guidance meaning to T raditional Chinese Medicine research by laboratory meth2 ods.
Author’s address:K ey Laboratory of Mammal Reproductive Biology and Biotechnology,M inistry of Education,Inner M ong olia University, Huhhot010021,China
K ey w ords:Cirsium setosum(Willd.)M B;carcinoma cell;growth;in2 hibitory effects
(Original article on page12)Experimental study of Dunhu ang Xiaobi Dingtong Tincture’s ef2 fects on TNF-a,I L-1and I L-6in the rat models of collagen-induced arthritis
ZENG Zhao-yang1,G UO Ying-qiang1,Y ANG Guo-dong1,SONG Peng-cheng1,MI Zhong-xiang2,LI Yu-wei1,QIU Tong1,SONG Gui-jie1
Abstract:Objective:T o explore the active mechanism of Dunhuang X i2 aobi Dingtong T incture treating RA and the effects on the contents of T NF-a,I L-1and I L-6in joint liquid of the rat m odel of collagen-induced arthritis(CI A).Methods:The rat m odels of CI A were made. The contents of T NF-a,I L-1and I L-6were measured with the method of E LIS A.R esults:Dunhuang X iaobi Dingtong T incture could significantly deceased the contents of T NF-a,I L-1and I L-6in joint liquid of the rats of CI A.Conclusion:Dunhuang X iaobi Dingtong T inc2 ture have certain inhibitory effects,and can alleviate in flammation of ar2 ticular synovium by inhibiting the contents of T NF-a,I L-1and I L-6 in joint liquid of rats,which may be one of the mechanism of the thera2 peutic effect on rheumatoid arthritis.
Author’s address:1.The H ospital A ffiliated to G ansu C ollege of T C M, Lanzhou730020,China;2.The H ospital to G ansu Province of T C M, Lanzhou730000,China
K ey w ords:Dunhuang X iaobi Dingtong T incture;collagen-induced ar2 thritis;T NF-a;I L-1;I L-6
(Original article on page17)
Determination of hesperidin in Zhisou T ablet with HP LC
LI Fang1,ZH ANG Qing-bo1,ZH ANG Xiao-chen2
Abstract:Objective:T o establish HP LC method to determine the hes2 peridin in Zhis ou T ablet.Methods:The HP LC method for determination of Hesperidin was Aglient S B-18(4.6×150mm),mixture Acetonitrile -0.3%phosphoric acid(20:80)as m obile phase,detection wave2 length at283nm.R esults:This method showed a g ood linear relation2 ship.The mean recovery was98.7%(n=6).Conclusion:This method is accurate and g ood recovery.
Author’s address:1.Heilongjiang Provincial Institute for Drug C on2 trol,Harbin150001;2.Mudanjiang LingT ai Pharmaceutical H olding C O.LT D,Mudanjiang157011
K ey w ords:HP LC;Zhis ou T ablet;hesperidin
(Original article on page37) The report of Xiaohu ang Decoction combined with Potenlin in
treating for80cases with acute icteric hep atitis
XIE Ming1,SHI Changsheng2
Abstract:Objective:T o observe the effect of self-drafting X iaohuang decotion combined with protenlin.Methods:By randomized controlled trail,161patients were divided into mamanagement team and cotrolled team,and all related indexes,clinical therapeutic effect,and hospital days were observed pretherapy and post-treatment.R esult:The general therapeutic effect,clinical effect,recovery of Hepatic function,and hos2 pital days of mamanagement team are better than those of cotrolled team.(P<0.05).Conclusion:X iaohuang Decoction combined with P otenlin in treating for patients with acute icteric hepatitis can get obvi2 ous therapeutic efficacy.
Author’s address:1.N o255H ospital of P LA,T angshan063000;2. Hebei Province C orps H ospital of CPAPF,Shijiazhuang050082
K ey w ords:X iaohuang Decoction;P otenlin;acute icteric hepatitis;clini2 cal observation
(Original article on page53)