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金妙文 周仲瑛 方泰惠 张培祥 王志英 吴勉华
（南京中医药大学，江苏南京，210029）
摘要 目的：探讨养心通脉口服液治疗充血性心力衰竭的作用机理。方法：1)具有益阴助阳、活血通脉功
效的养心通脉口服液理论依据。2)按Fletchor（1981）法，复制心衰大鼠的模型，动物随机分为正常组、
假手术组、心衰组（模型组）、卡托普利组、养心通脉小剂量组、大剂量组、随后三组进行等容量灌胃给
药，每日1 次；正常组、假手术组和模型组，给予等容量生理盐水，每日1 次；连续给药4 周。结果：养
心通脉大剂量组与卡托普利组均能增加心衰大鼠左室压峰值（LVSP）、左心室内压变化最大速率
（+dp/dtmax）。养心通脉大剂量组作用更强。养心通脉大剂量组与卡托普利组均能增加左室重量和LV/BW，
且以大剂量组为显著。养心通脉小剂量组和卡托普利组均能抑制右室肥厚，且小剂量组同时能降低肺脏指
数。临床研究指示本品有强心、利尿作用。结论：养心通脉口服液通过增强心肌收缩力的同时，可以在一
定程度上改善心肌舒张、改善心泵、增加肾血流量、减轻心肌损伤、降低心脏前负荷、改善心脏的功能，
使心衰达到缓解和控制。
关键词 益阴助阳、活血通脉法；养心通脉口服液；大鼠心衰模型
Effect Evaluation and Mechanism of Yangxin Tongmai Liquid in Treating Congestive Heart Failure
Jin Miaowen,Zhou Zhongying,Fang Taihui,Zhang Peixiang,
Wang Zhiying,Wu Mianhua
(Nanjing University of Chinese Medicine,Nanjing Jiangsu,210029)
ABSTRACT Objective:To investigate the effect and mechanism of Yangxin Tongmai liquid in treating
congestive heart failure. Methods: 1.To find out theoretic proofs of Yangxin Tongmai liquid which has
functions of tonifying Yin and Yang and promoting coronary circulation. 2.In experiment research,congestive
heart failure rat model was established by Fletchor's method (1981).All rats were randomly divided into six
groups:control group,sham group,congestive heart failure group(model group),captopril group,small dose of
Yangxin Tongmai liquid group,large dose of Yangxin Tongmai liquid group. Subsequently, the former three
groups were orally administrated with equal volume of saline,while the later with corresponding drugs,once a day
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·249·
实 验 研 究
Experimental study
for four weeks. Results:Both large dose of Yangxin Tongmai liquid group and captopril group had an influence
on chronic heart failure rats in raising LVSP,left ventricle weight,LV/BW and utmost ascending rate of left
ventricle inner pressure(+dp/dtmax),the large dose group was more obviously.Both small dose of Yangxin
Tongmai liquid group and captopril group had an obvious influence in restraining right ventricle hypertrophy, the
small dose group could also decrease the lung finger.The clinical research showed that Yangxin Tongmai liquid
has a good function of strengthening the heart and diuresis. Conclusions: Yangxin Tongmai liquid can reinforce
myocardium contractility,improve myocardium energy metabolism,reinforce blood pumping function,increase
renal blood flow,relieve the injury of myocardium,decrease heart preload and improve cardiac function, therefore
relieve or control congestive heart failure.
Key words tonifying Yin and Yang and promoting coronary circulation; Yangxin Tongmai liquid; congestive
heart failure rat model
充血性心力衰竭是指在静脉回流正常的情况下，由于原发的心脏损害引起心排血量减少，不能满足组
织代谢需要的—种综合征。临床上以肺循环和(或)体循环瘀血以及组织血液灌注不足为主要特征。是临床
常见的危重症。目前随着心血管疾病发病率上升，充血性心力衰竭发病率亦有增加趋势，且为心血管疾病
患者丧失劳动力和死亡的重要原因之一。现代医学虽然应用强心、利尿、扩张血管药物等治疗本病，但死
亡率仍较高，并有一定副作用，临床应用具有益阴助阳、活血通脉功效的养心通脉口服液治疗充血性心力
衰竭取得明显疗效的基础上，对其作用机理进行了探讨，总结如下。
1 益阴助阳、活血通脉法立论依据
根据充血性心力衰竭的临床表现，一般隶属于中医学喘息、水肿、心悸、怔忡、心痹、心水诸病证范
畴。由于各种心系原发病，迁延日久，耗气伤津，损阴戕阳，加之外感六淫，内伤情志，体劳过度，药物
失宜以及妊娠等耗损气血津液，致使心之阴阳虚衰，心脉瘀滞，脏腑功能失调而发病。
充血性心力衰竭多有一个积渐至突变的过程，始则多因心气虚弱，气不运血；心阴亏耗，阴虚血涩，
表现气阴两虚，心营不畅。进而气虚阳衰或阴损及阳，心阳亏虚，势必导致气血运行障碍，心脉瘀阻不畅。
因此，阴阳两虚，心脉瘀滞是其病理基础，且以心阳(气)亏虚，心脏鼓动减弱，营运无力为其病理变化的
主要方面。
由于心气(阳)虚衰，心失营运，而致血脉瘀滞，瘀血内停。阳虚阴盛则饮邪内生，阳虚血瘀则水邪潴
留，本虚标实，因果交错为病，促使疾病演变发展。主病之脏在心，损及他脏，或他脏及心，表现心失营
(血)养，肺不主气，脾失健运，肾气虚衰，肝血虚滞，驯致五脏精气虚衰，多脏同病，功能失调，但其病
变重点在于心、肾，因心为火脏，为五脏六腑之大主；肾主水，为真阴真阳之所寄，心肾两脏，一阴一阳，
络脉相连，在维系人体阴阳平衡，协调脏腑生理功能方面起着极为重要的作用，“心主身之血脉”，是营运
气血的动力器官，肾阳上温心阳而益心气，肾阴上济心阴而制亢火，既提供心脏鼓动射血的物质基础，又
调节心主血脉的功能活动。各种病因一旦导致心气(阳)虚衰，则鼓动无力，心不主血，心脉瘀滞，而致心
悸、怔忡、紫绀；肾气虚衰，元阳不振，则心气(阳)更虚，或水不济火，心气浮越，或阳不制水，水邪内
停，或肾不纳气，气不归根，而致心肾交病，出现喘脱，故心衰重症，每见心肾阳衰或阴竭阳亡的危候。
此外，由于心主血脉，肺主气，百脉朝会于肺，病则互为影响，肺心同病，肺失肃降，而致喘咳痰多；
心主血，气为血帅，气行则血行，脾为气血生化之源，化源亏乏，气血不足，则心失所养，而致心脾两虚，
脾运不健，为肿为胀：心主血而肝藏血，心脉瘀滞，营运不畅，则肝郁血瘀，疏泄失司形成癥积。
综上所述，主病之脏在心，与肺肝脾互相影响，病理性质为本虚标实，气血阴阳亏虚为本，瘀血水饮
为标。气血水三者相互作用，瘀从气虚来，水自阳虚生，血不利为水，而瘀血、水饮又可阻遏心之气阳，
长此以往，正虚邪实，因果循环，使病情反复迁延。可知“阴阳两虚，心脉瘀滞”是充血性心力衰竭的基本
病机病证，从而为应用益阴助阳，活血通脉治法提供了理论依据，据此组方用药研制成养心通脉口服液。
2 实验研究
2.1 材料
SD 大鼠90 只，体重220～270 g，雌雄各半，由南京中医药大学动物实验中心提供。养心通脉口服液
含生药2.7 g/g，由南京中医药大学中医药研究院提供，文中剂量按生药量计算。其余药物为临床常用的卡
托普利、肝素钠、氯化钠、盐酸利多卡因等注射液。
2.2 实验方法
2.2.1 充血性心衰大鼠模型的复制 按Fletcher(1981)法，腹腔注射乌拉坦(0.8 g/kg)麻醉，仰卧固定。
经舌下静脉注射利多卡因(10 mg/kg)，行喉管插管术，于第4～5 左肋间开胸，接通人工呼吸机，迅速将心
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·25 0·
实 验 研 究
Experimental study
脏挤出，于肺动脉圆锥和左心耳之间，距主动脉根部约2 mm 处结扎左冠状动脉(冠脉)主干，然后将心脏
复位，挤出胸腔内空气，迅速闭胸，恢复自主呼吸后关闭呼吸机。
2.2.2 实验分组 动物随机分为正常组、假手术组、心衰组(模型组)、卡托普利组(0.016 g/kg)、养心通
脉小剂量组(YX1，12 g/kg)和大剂量组(YX2，36 g/kg)。卡托普利和养心通脉口服液按等容量灌胃给药，每
日1 次；正常组、假手术组和模型组给予等容量生理盐水，每日1 次；连续给药4 周。
2.2.3 充血性心衰大鼠血流动力学指标检测方法 制模后连续给药4 周，眼眶静脉取血4 mL 用于放射
免疫测定，用质量分数为25%的乌拉坦(0.8 g/kg)腹腔麻醉。舌下静注体积分数为1%的肝素生理盐水0.1
mL/100 g 体内抗凝。分离右侧颈总动脉，将充有0.1%肝素生理盐水的导管插入左室腔，连接压力换能器，
测量动脉收缩压(SAP)、动脉舒张压(DAP)、平均动脉压(MAP)，经载波放大器记录左室内压(LVP)；将LVP
信号输入直流放大器放大10 倍后测取左心室舒张末期压力(LVEDP)；将LVP 信号输入压力处理器(时间常
数0.5 ms，定标667 kPa·s-1·10 mm-1)，分别描记左室压变化速率曲线(dp/dt 曲线)和左室压的对数变化速率
曲线〔(dp/dt)· p-1 曲线〕。由血压搏动信号触发AT601G 并直接读出心率(HR)。上述观察指标均同步记录于
RM 6000 型8 导生理记录仪上(快速走纸50 mm/s)。在LVP 曲线上，由零点至曲线顶点的数值为左室压峰
值(LVSP)。左心室内压变化最大速率(±dp/dtmax)为在dp/dt 曲线上，零线至峰顶的数值。由dp/dt 曲线上升
支的起点至对应上升支峰顶的间隔时间为t dp/dtmax(左心室开始收缩至左心室内压上升速率峰值时间)。在
(dp/dt)·p-1 曲线上，曲线从零点至正向峰顶的数值为左心室肌收缩成分的实测最大缩短速率(Vpm)。
2.2.4 充血性心衰大鼠心肺重量的测定 在测定心功能后，立即打开腹腔经十二指肠给药，30min 后,
取肠系膜动脉，制备肠系膜上动脉灌流模型。然后开胸取出心脏、肺脏,并用生理盐水洗净，称量全心重和
肺重。切去心脏房室沟以上部分，沿室间隔切开左、右心室分别称重。
2.2.5 统计学方法 数据以均数±标准差(x±s)表示，各组间的比较采用方差分析和两样本均数比较的
t 检验。方差不齐时，通过变量变换或非参数统计方法进行分析。
2.3 结果
2.3.1 对充血性心衰大鼠血流动力学的影响 由表7 可知，模型组与正常组比较，各项指标均存在较
为显著的差异，表明心衰模型组大鼠左心泵血功能显著下降，收缩功能明显减弱，循环系统有明显充血现
象，佐证制模较为成功。与模型组比较，养心通脉大剂量组与卡托普利组均能增加心衰大鼠的LVSP、
+dp/dtmax，显著降低LVEDP，且养心通脉大剂量组与卡托普利组相比无明显差异。养心通脉大剂量组与
卡托普利组均能使t dp/dtmax 下降，似养心通脉大剂量组作用更强。养心通脉小剂量组大鼠的LVSP 明显
提高、t dp/dtmax 显著下降。同时卡托普利组和养心通脉大、小剂量组大鼠的SAP 均有明显升高，似以养
心通脉大剂量组作用最强。
2.3.2 对充血性心衰大鼠心、肺的影响 由表8 可知，模型组与正常组比较，各项指标均有明显差异。
与模型组比较，养心通脉大剂量组与卡托普利组均能增加左室重量和LV/BW，且以大剂量组为显著。养心
通脉小剂量组和卡托普利组均能抑制右室肥厚，且小剂量组同时能降低肺脏指数。
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·251·
实 验 研 究
Experimental study
3 讨 论
3.1 充血性心衰病机特点
审证求因，充血性心衰的发病多为各种原发病直接或间接损伤“心主血”的功能，导致心气不足，心阳
不振，进而气血阴阳亏虚，血脉瘀阻，以致心血不畅，肺失肃降，脾运不健，肾虚失纳，肝失疏泄等多脏
同病，导致瘀阻水停，痰饮内聚，此即《灵枢·刺节真邪篇》所指“手少阴气绝,则脉不通，脉不通则血不流”
的气虚致瘀理论。由于气(阳)虚血滞，脏腑气化功能障碍，水液输布排泄失常，必致体内水湿痰饮潴留，
但尤以血瘀为其主要病理因素，如《金匮要略·水气篇》之“血不利则为水”，而致本虚与标实互为因果，故
阴阳两虚、心脉瘀滞为其基本病机特点。
3.2 疗效分析
以往临床研究表明，养心通脉口服液由附子、人参、玉竹、葶苈子、泽兰、石菖蒲等组成，治疗组总
有效率明显优于西药对照组，并有明显强心利尿作用，随之主要症状体征明显改善。目前西药治疗以洋地
黄、利尿剂、血管扩张剂为主，洋地黄的治疗量与中毒量很接近，安全系数小；而利尿剂易造成电解质紊
乱。本品毒副作用小、安全系数大，有良好的应用开发价值。
3.3 实验研究表明养心通脉口服液具有强心、利尿作用
实验结果表明，用养心通脉口服液治疗后，大鼠+dp/dtmax、LVSP 升高，LVEDP 降低，说明养心通脉
口服液在增强心肌收缩力的同时，可以在一定程度上改善心肌舒张功能。其强心作用主要与人参、附子等
所具有的正性变力作用有关。以往研究表明，人参无论是临床观察还是动物实验都显示出明显的强心作用，
它通过抑制心肌细胞膜Na+K+ATP 酶活性，增加细胞内Ca2+浓度，促进儿茶酚胺释放及提高心肌
cAMP/cGMP 比值等机制，从而使心肌收缩增强。附子的有效成分去甲乌药碱与异丙基肾上腺素作用相似，
有兴奋β 受体作用，对心肌细胞的激动作用能被β 受体阻滞剂减弱和完全对抗。临床研究表明本品能增加
尿量，提示其有利尿作用。本品还具有升压作用，能使SAP 和MAP 升高，以SAP 升高幅度更大。DAP
似乎也呈升高趋势，但无统计学意义。以往实验研究表明，本品具有活血化瘀，改善微循环，调节血管舒
缩等作用，对大鼠离体心肌缺血/再灌注损伤亦有保护作用，提高过氧化物歧化酶，清除自由基，能使戊巴
比妥钠诱发家猫心衰的TXA2 降低，升高6-keto-PGF1a，降低T/K 比值，提示具有调节TXA2-PGF1a 的作用。
综上所述，养心通脉口服液通过增强心肌收缩力，降低肾素-血管紧张素-醛固酮系统、交感系统活性及
血浆内皮，扩张血管，改善心肌能量代谢，改善心泵功能，增加肾血流量，抗氧化、调节TXA2-PGF1a 系统
功能等作用，达到减轻心肌损伤，降低心脏前负荷，改善心功能，使心衰患者的症状体征明显改善或消失。
Effect of the serum contained Radix Astragali and Hirudo and Hirudin and
the compoundings on the grow th cycle and apoptosis of rats Glomerular
Mesangial Cells
Ren Xianzhi1 Jiang Shuming2 Zhai Wensheng3
( 1 Mobile station for the post-doctors of Nanjing University of TCM, Nanjing, 210029; 2 China pharmaceutical University, Nanjing, 210009; 3 No.1 affiliated
hospital of Henan college of TCM, Zhengzhou, 450000)
Abstract Objective: The study was aimed to investigate the effect of the serum contained Radix Astragali and
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·25 2·
实 验 研 究
Experimental study
Hirudo and Hirudin and the compounding of these two herbs respectively on the growth cycle and apoptosis of
rats Glomerular Mesangial Cells and to observe the morphology of apoptosis. [Method] The growth cycle and
apoptosis of GMCs was measured by flow cytometer. Meanwhile morphous of apoptosis was visualized by
Wright's staining and observed by microscope. [Result] The serum contained Radix Astragali and Hirudo and
Hirudin and the compoundings respectively can keep most MC in the stage of G0/G1, and it had a significant
deviation compared to the control (P< 0.01) . It also increased the rate of MC apoptosis, and it had a significant
deviation compared to the control (P<0.01) . [Conclusion] The serum contained Radix Astragali and Hirudo and
Hirudin and the compounding respectively can prevent GMCs entering the stage of to inhibit its growth and it also
can increase the rate of MC apoptosis.
Keywords Rat; Glomerulum Mesangial Cells; growth cycle ; apoptosis ; morphological ; Radix Astragali;
Hirudo; Hirudin
Growing glomerulum nephritis is children’s common kidney disease, with the progress of pathological change,
GMCs hyperplasia, the ground substance (ECM) outside cells Increase. The experiment aims to take
representative medicine of silt as target, inqire the influlence of the medicine on the GMCs apotosis and growth
cycle , observe the function intensity of the two medicine and offer reference for clinical and dialectical
administration, inqire into how the mechanism develop in the growing glomerulus nephritis.
1 Material
1.1 Animal: Animal: SD rats, 2 - 3 months old, male, weight 150- 200g. Offered by centre of experimental
animal of nanjing university of TCM(SPF grade) . Divided into 11 groups, 6 every groups at random.
1.2 Cells: Membrane cell one of rats‘ department, offered (HBZY-1) by the biology department of Wuhan
university.
1.3 medicines: Radix Astragali of Inner Mongol (Astragalus mongholicusBge.) , the leech (Japan cures leeches,
hirudo nipponia Whitman. talc powder fries,) ,Offered by nanjing university of TCM. The hirudin (injected
recombinate leeches plainly, Hirudin) 10mg each, lot number (20001001) ,Than living≥ 2000AIU, purity≥ 99% ,
mingkang biological engineering company,Guangzhou.
1.4 Reagents: MEM culture medium: Gibco products, lot number: 41500-067. EDTA: Sigma products, lot
number: 40-4. Trypsin: Gibco products, lot number: 27250-018. Cycle TEST PLUS DNA reagent box: The
products of U.S.A. BECTON DICKINSON Company, lot number: 340242. Auspicious dye liquor: Sichuan
Province Mike Science Technologies company, lot number: 0603012. Lipopolysaccharide (LPS) : 10mg each,
sigma products, lot number: DH183-1, offered by the biomedical engineering company of three leaves of
Zhengzhou .
2 Method
2.1 the prepration of the serum contained medicine
Fry traditional Chinese medicine Radix Astragali water and concentrate to including crude medicine 1.5g/ml,
grinds the leech to powder with the high-speed pulverizer, mix into the mixing and hanging liquid of 0.3g/ml.
With 2 times (Radix Astragali 12g/d, leech 2.4g/d) of the rat's equivalent dosage of clinical adult's administration
quantity ,Irritate, take medicinal solution, 2 times a day, 5 day continuously, 2h after the last dosage, fetch blood
by eyeball. calculates the equivalent hirudin dosage of the rat according to 0.5mg/kg, with 15 times of
dosage, gives medicine by the end intravenous injection, fetch blood after 20 minutes. 0.22¦Ìm aseptic filter
filters after deactivating the complement, packages, kept in the low-temperature refrigerator .
2.2 measure of the nonpoisonous boundary line of GMCs cells of the handling factor
Fetch serum of normal rat,serum contained high density of Radix Astragli and hirudo, dilute the serum by the
MEM cell maintaining liquid not including serum from 1:1,1:2 to 1:16, each dilute degree set 4 replying holes,
set the normal cells as contrast. The biggst dilute degree not leading to cell retreat is the nonpoisonous boundary,
through observation, the serum of normal rat and the serum when dilute as 1:2 not influence the growth of GMCs
cell clearly.
2.3 Divide into groups in experiment
After melting in step, GMCs are divided at random: Control group ( Add LPS (1.8mg/ml) 10ul + mouse serum
without medicine 360ul +MEM1430ul) , prescription group ( Add LPS (1.8mg/ml) 10ul + Radix Astragali
serum 360ul +MEM1430ul ) , the hirudo group(Add LPS (1.8mg/ml) 10ul + leech serum 360ul + MEM1430ul) ,
the hirudin group (Add LPS (1.8mg/ml) 10ul + hirudin serum 360ul + MEM1430ul) , the Radix Astragali
group (Add LPS (1.8mg/ml) 10ul + Radix Astragali serum 360ul + MEM1430ul) five groups.
2.4 Testing method of cell cycle and apotosis
2.4.1 cell preparation Fetch a well growing cell to digest, blow and beat cell to make monocyte hang liquid,
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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Experimental study
transfer cell density to 1* 105 / ml and inoculate into 15ml cell bottle, inoculate 1.8ml in each bottle, every
group set up 4 replying bottles. 37℃5% CO2 training case train, when cells cover with 80% of bottle wall, suck
and abandon the serum on the top,cultivate the cells with MEM liquid including 0.5% FCS fou 24h to make
them in step. Divided into every group described above at random, join the corresponding treatment factor,
continue cultivating for 48h.
2.4. 2 cells collecting After cultivating for 48h, digest cells routinely, wait for cells to turn into round and
suck pancreas enzyme, stop digesting. 2 PBS wash, join 3mlPBS ,blow and treat the cells and move into a tube,
1000 tuns each minute,fall go PBS, aad about 0.5mlPBS,then blow and beat to monocyte hanging liquid. The
liquid is packed into the test tube of 17* 100mm to hang cells, centrifuge for 5 minutes at room temperature 300*
g. Suck and abandon the serum on the top, aad 1ml and buffer the liquid and hang cells again, Repeated the last
step . regulate cell density into 1.0* 106 / ml.
2.4.3 cell dying centrifuge for 5 minutes at room temperature 400* g ,suck and abandon the serum at the top;
add 250ul A liquid (contain trypsin, four hydrogen chlorine precise amine stain-remover, used for digesting the
cell membrane and cell skeleton) into each tube ,Mix lightly and lightly, don't shake, put gently and quietly for 10
minutes at room tempreture. add 250ul B liquid (contain trypsin inhibitor, RNA enzyme, citrate and buffer liquid,
four hydrogen chlorine precise amine, used for inhibiting the function of trypsin and digesting RNA) into each
tube ,Mix lightly and lightly, don't shake, put gently and quietly for 10 minutes at room tempreture. add 250ul C
liquid (contain PI dye liquor, four hydrogen chlorine precise amine, citrate and buffer the liquid) into each
tube,Mix lightly and lightly, don't shake, put photophobically and quietly for 10 minutes in the low temperature
(2- 8 ℃).
2.4.4 measuring cell cycle by flowing type cell appearances filter and enter into sample tube with 50um nylon
membance, operate the computer test. Use BD CellQuest1.0 software to analysis.
2.5 cells apotosis and morphology observation
2.5.1 blow and beat the well growing cell to make monocyte hanging liquid, adjust cell density to 2.5* 104 / ml,
inoculate with 24 holes in advance in the cells with small glass slice cultivating board, every group has 4 replying
holes. 37℃5% CO2 case cultivate, when cells sticks to the wall, suck and abandon the top , cultivate with
MEM liquid including 0.75% for 24h to make the cell in step. Divided into every group described above at
random, join the corresponding treatment factor, continue cultivatinging 48h.
2.5.2 auspicious dye After cultivating for 48h, take the small glass slice in the holes sightly, after PBS washing,
0.4% of the formaldehyde is fixed for 5- 10 minutes at room temperature, air dry; Add the auspicious dye liquor to
dye for 1- 2 minutes; In addition auspicious to buffer liquid ( Auspicious the dye liquor 10ml adds PBS20ml)
1- 2 drops are dyed for 4- 10 minutes; Distilled water is washed, air dry, the neutral gum is sealed slice.
2.5.3 cells observation observe the changes of cell shape and nucleus under the
microscope, count positive cells, calculate the rate of apotosis cells by two pathologic personnel who are
familiar with cell apotosis.
2.6 statistics analysis
The experiment result is showed by X ±s 。Employ SPSS 10. 0 statistical analysis software to deal with
experimental data, every group materials spend single factor analysis of variance relatively, the second Liang are
compared and examined with Student-Newman-Keuls.
3 Result
3.1 comparative of Radix Astragali, hirudo and compoundings that influence MC cycle of growing and apotosis.
Hirudo, Radix Astragali, prescription filling by hirudo and Radix Astragali, can make a large number of MC in
G0/G1 stage, prevent the cell from entering S stage to achieve the purpose of inhibiting MC outgrowth.
The result is shown in Table 1.
Table 1 comparative of Radix Astragali, hirudo and compoundings that influence MC cycle of growing and
apotosis. （%， X ±S）
Group n G0/G1 Apoptosis
Control group 4 50.78± 4.09 1.63± 0.76
Radix Astragali
group 4 74.49± 2.34* 6.19±2.45
hirudo group 4 85.58± 8.92* 11.86± 3.16*
Hirudin group 4 75.64±5.34* 1.90± 1.49
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
Radix Astragali
andhirudo group 4 89.12± 2.57* 16.20± 6.12*
Note: *P<0.01 compared with control group, P> 0.05; Compared with Radix Astragali group P< 0.05, P<
0.01; Compared with hirudo group P< 0.05
We can find in Table 1, the cell proportion of every group in G0/G1 compared with control group obviously
rises (P<0.01) ,And hirudo group, prescription group are stronger than the Radix Astragali group (P parts <0.05
and 0.01) ,hirudo group does not have prominent difference (P> 0.05) to compare with prescription group. It
proves the function of traditional Chinese medicine hirudo inhibiting MC outgrowth is stronger than Radix
Astragali, if use in combination with Radix Astragali and act on stronger, it is not so obvious as hirudo group
that the hirudo usually organizes function.
From the result of influence on MC apotosis,we can find hirudo, hirudo and Radix Astragali fill a prescription, can
improve the rate of rats‘ MC apotosis, there are extremely obvious differences compared with control group (P<
0.01); The Radix Astragali group does not have obvious difference compared with control group (P>
0.05),Maybe it relates to the greater virationg in the group. The hirudin group does not have obvious difference
(P> 0.05). Among the groups,hirudo group, prescription group are obviously superior to the Radix Astragali
group (P<0.01) , prescription group is superior to hirudo group (P<0.05) . It proves that the function traditional
Chinese medicine hirudo can promote MC apotosis of rat is superior to Radix Astragali when stimulated by LPS,
if use in combination with Radix Astragali act on stronger. (See Fig. 1- 4)
Control group of Fig. 1 Radix Astragali group of Fig.2
Hirudo group of Fig. 3 Hirudo compound group of Radix Astragali of Fig. 4
3.2 cells apotosis and morphology observation
Observe the changes of cell shape and nucleus under the microscope, whether the cell volume appears to
shrink , cell quality becomes dense, dyeing acidly more, and can form small apotosis body; Whether nuclear
interior chromatin concentrates and forms the chromatin lump to assemble on the edge in the nucleus, form the
varied distribution shape, or take the form of hemisphere, or take the form of crescent, or taking the form of sand
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
dune form, horse's hoof, boat form,etc..
The number of apotosis cells in control group, Radix Astragali group,hirudo group,hirudin group and
prescription group were finally: 2.54±0.86, 5.26±2.76, 9.2±4.72, 2.84±1.26 and 15.63±5.71 (%) , among them,
MC apotosis of the leech, prescription group are more prominent, compared with control group, there is a
prominent difference (P<0.05) ,It is basically identical to the result measured with the flowing type cell
appearance, prove traditional Chinese medicine can really lead to MC apotosis. It appears shape change of cell
apotosis in every group, in the control group,apotosis cell is not typical,in small quantity and distribute loosely
(Fig. 5, Fig. 6) ;In Leech group, prescription group apotosis cell is typical , in large quantity and distribute
tightly.(Fig. 7,Fig.
.
Control group of fig.5 Radix Astragali group*200 of fig.6
Hirudo group*200 of fig.7 prescription group of fig.8
4 Discussion
The membrane hyperplasia glomerulus nephritis of natural disposition (MsPGN) accounts for 40%- 50% of child's
nephropathy, it is child's common kidney disease. The membrane cells (MC) hyperplasia is common pathology
change of many glomerulus disease, is outstanding pathological characteristic of MsPGN. The excessive
hyperplasia of kidney membrane cell, causes ECM to accumulate in the glomerulus, leads to the fact that kidney
goes on fiber and hardens., it is the important mechanism of development in glomerulus sclerosis, it is also the
important links [1 ] that renal function damages. However, there is not effective therapeutic method to prevent the
MC hyperplasia, ground substance accumulate and glomerulus sclerosis at present.
The clinical manifestations of growing glomerulus nephritis are edema, weak, pain in the back and so on. we can
see blood in the urine, proteinuria, high lipoprotein blood disease and low protein blood disease to check in the
laboratory, it indicates to " Edema " , " hematuria " , " empty consumptive disease " in Chinese medicine Category.
Its root belongs to deficiency while its branch belongs to excess in traditional Chinese medicine. The disease
relies mainly on the deficiency of lung, spleen, renal Qi ,while blood stasis,wind,water and dampness,
dampness-heat,turbid and dampness are the most common. With the thoroughly realize on the disease, " blood
stasis " plays a more and more important role in this disease, we can say this disease to a certain extent from"
stasis " to " deficiency" .This is a great breakthrough known to the tradition. The pathology characteristic that
immune compoundings deposited, cell hyperplasia, glomerulus fiber, sclerosis all accord with Chinese medicine
" blood stasis " .Blood stasis is a nephropathy change that run through the diseases all the way ,from watching
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·25 6·
实 验 研 究
Experimental study
dialectical microly, the basic nephropathy change is blood stasis.
Using traditional Chinese medicine to treat kidney disease has better curative effect , the representative medicine
is reinforcing Qi and removing blood stasis , this medicine can prevent or slow down renal fiber on a certain
level. Radix Astragali, hirudo are the most frequently using medicine in reinforcing Qi and removing blood stasis
medicine, so we use Radix Astragali and hirudo as representatives to carry on the reearch, use molecular biology
and serum pharmacology method to probe into the influence on MC hyperplasia , apotosis, cell cycle of growing
and produce TGF – B, IL-6, IL-13, collagenic(Col- And the fibrous connection albumen (FN),compare the
function intensity between reinforcing Qi and removing blood stasis medicine ,provide reference for theory and
clinical administrations dialecticals. We have already verified in the previous work that Radix Astragali, hirudo
and prescription can reduce the content [2 ] of the principal ingredients Col- ⅳof ground substance and FN outside
cells effectively.
The research on rat MC cycle influence indicates that hirudo, Radix Astragali, hirudo Radix Astragali fill a
prescription, can make a large number of MC in G0/G1 stage, prevent cells from entering S stage to achieve a
purpose of inhibiting MC outgrowth. The cell proportion that every group in G0/G1 compared with control group
obviously rises, and hirudo group and prescription group have the most obvious function.
The research on MC apotosis influence indicates that hirudo , Radix Astragali, hirudo Radix Astragali fill a
prescription, can improve rat ‘s rate of MC apotosis ,there are extremely obvious differences compared with
control group and hirudo group, prescription group, and present the typical peak of apotosis. It proves that
traditional Chinese medicine hirudo has a stronger act on promoting cell apotosis when stimulate by LPS than
Radix Astragali, if uses in combination with Radix Astragali and acts stronger. After observing MC morphology
apotosis, we find that after using traditional Chinese medicine ,every group have morphology apotosis change, the
nucleus creases, dyes, cracked densely, nuclear interior chromatin concentrates and gathers the edge in the nucleus,
or take the form of hemisphere, or take the form of crescent, form the varied distribution shape.
Through the research on inhibiting MC hyperplasia and promoting apotosis, the hirudo group, prescription group
have more prominent function, and present the typical peak of apotosis.It proves that the function of hirudo
promoting MC apotosis and inhibiting hyperplasia is superior to Radix Astragali., if use in combination with
Radix Astragali and act stronger. We can infer, in the prescription group of Radix Astragali and hirudo , in
order to strengthen the function of inhibiting MC hyperplasia and promoting apotosis, hirudo should be the main
medicine . It proves blood stasis is an important factor in the development of growing glomerulus nephritis .
Activating blood circulation and removing blood stasis are the basic curing methods in children‘s kidney disease.
it is indispensable to reinforce Qi and strengthen the body resistance, maybe it is more important than that.
Activating blood circulation should run through nephropathy treatment all the way.
References:
[1] Floege J, Johnson RJ, Gordon K, et al. Increased synthesis of extracelluar matrix in mesangial prolifertive mephritis[J]. Kidney Int, 1991, 40:477-488.
[2] Let will now, deep and vast to pass, Zhai WenSheng. Radix Astragali, leech and different proportions fill a prescription and suck serum of medicine to the
fibrous connection albumen and collagenic influence of the type outside the membrane cells of loud mouse's department. Chinese traditional Chinese medicine
magazine, 2006, 21(11) : 690-691.
[3] Ren Xianzhi, Wang Shouchuan, Zhai Wensheng.Influence of the contained-herb serum of Radix Astragali and Hirudo in different propor tion of
compounding on the Fibrillar connection Protein and Collagen type IV of Glomerular Mesangial Cells in vitro Rats. China Journal of Traditional Chinese
Medicine and Pharmacy, 2006, 21(11) : 690-691.
（南京中医药大学，江苏南京，210029）
摘要 目的：探讨养心通脉口服液治疗充血性心力衰竭的作用机理。方法：1)具有益阴助阳、活血通脉功
效的养心通脉口服液理论依据。2)按Fletchor（1981）法，复制心衰大鼠的模型，动物随机分为正常组、
假手术组、心衰组（模型组）、卡托普利组、养心通脉小剂量组、大剂量组、随后三组进行等容量灌胃给
药，每日1 次；正常组、假手术组和模型组，给予等容量生理盐水，每日1 次；连续给药4 周。结果：养
心通脉大剂量组与卡托普利组均能增加心衰大鼠左室压峰值（LVSP）、左心室内压变化最大速率
（+dp/dtmax）。养心通脉大剂量组作用更强。养心通脉大剂量组与卡托普利组均能增加左室重量和LV/BW，
且以大剂量组为显著。养心通脉小剂量组和卡托普利组均能抑制右室肥厚，且小剂量组同时能降低肺脏指
数。临床研究指示本品有强心、利尿作用。结论：养心通脉口服液通过增强心肌收缩力的同时，可以在一
定程度上改善心肌舒张、改善心泵、增加肾血流量、减轻心肌损伤、降低心脏前负荷、改善心脏的功能，
使心衰达到缓解和控制。
关键词 益阴助阳、活血通脉法；养心通脉口服液；大鼠心衰模型
Effect Evaluation and Mechanism of Yangxin Tongmai Liquid in Treating Congestive Heart Failure
Jin Miaowen,Zhou Zhongying,Fang Taihui,Zhang Peixiang,
Wang Zhiying,Wu Mianhua
(Nanjing University of Chinese Medicine,Nanjing Jiangsu,210029)
ABSTRACT Objective:To investigate the effect and mechanism of Yangxin Tongmai liquid in treating
congestive heart failure. Methods: 1.To find out theoretic proofs of Yangxin Tongmai liquid which has
functions of tonifying Yin and Yang and promoting coronary circulation. 2.In experiment research,congestive
heart failure rat model was established by Fletchor's method (1981).All rats were randomly divided into six
groups:control group,sham group,congestive heart failure group(model group),captopril group,small dose of
Yangxin Tongmai liquid group,large dose of Yangxin Tongmai liquid group. Subsequently, the former three
groups were orally administrated with equal volume of saline,while the later with corresponding drugs,once a day
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
for four weeks. Results:Both large dose of Yangxin Tongmai liquid group and captopril group had an influence
on chronic heart failure rats in raising LVSP,left ventricle weight,LV/BW and utmost ascending rate of left
ventricle inner pressure(+dp/dtmax),the large dose group was more obviously.Both small dose of Yangxin
Tongmai liquid group and captopril group had an obvious influence in restraining right ventricle hypertrophy, the
small dose group could also decrease the lung finger.The clinical research showed that Yangxin Tongmai liquid
has a good function of strengthening the heart and diuresis. Conclusions: Yangxin Tongmai liquid can reinforce
myocardium contractility,improve myocardium energy metabolism,reinforce blood pumping function,increase
renal blood flow,relieve the injury of myocardium,decrease heart preload and improve cardiac function, therefore
relieve or control congestive heart failure.
Key words tonifying Yin and Yang and promoting coronary circulation; Yangxin Tongmai liquid; congestive
heart failure rat model
充血性心力衰竭是指在静脉回流正常的情况下，由于原发的心脏损害引起心排血量减少，不能满足组
织代谢需要的—种综合征。临床上以肺循环和(或)体循环瘀血以及组织血液灌注不足为主要特征。是临床
常见的危重症。目前随着心血管疾病发病率上升，充血性心力衰竭发病率亦有增加趋势，且为心血管疾病
患者丧失劳动力和死亡的重要原因之一。现代医学虽然应用强心、利尿、扩张血管药物等治疗本病，但死
亡率仍较高，并有一定副作用，临床应用具有益阴助阳、活血通脉功效的养心通脉口服液治疗充血性心力
衰竭取得明显疗效的基础上，对其作用机理进行了探讨，总结如下。
1 益阴助阳、活血通脉法立论依据
根据充血性心力衰竭的临床表现，一般隶属于中医学喘息、水肿、心悸、怔忡、心痹、心水诸病证范
畴。由于各种心系原发病，迁延日久，耗气伤津，损阴戕阳，加之外感六淫，内伤情志，体劳过度，药物
失宜以及妊娠等耗损气血津液，致使心之阴阳虚衰，心脉瘀滞，脏腑功能失调而发病。
充血性心力衰竭多有一个积渐至突变的过程，始则多因心气虚弱，气不运血；心阴亏耗，阴虚血涩，
表现气阴两虚，心营不畅。进而气虚阳衰或阴损及阳，心阳亏虚，势必导致气血运行障碍，心脉瘀阻不畅。
因此，阴阳两虚，心脉瘀滞是其病理基础，且以心阳(气)亏虚，心脏鼓动减弱，营运无力为其病理变化的
主要方面。
由于心气(阳)虚衰，心失营运，而致血脉瘀滞，瘀血内停。阳虚阴盛则饮邪内生，阳虚血瘀则水邪潴
留，本虚标实，因果交错为病，促使疾病演变发展。主病之脏在心，损及他脏，或他脏及心，表现心失营
(血)养，肺不主气，脾失健运，肾气虚衰，肝血虚滞，驯致五脏精气虚衰，多脏同病，功能失调，但其病
变重点在于心、肾，因心为火脏，为五脏六腑之大主；肾主水，为真阴真阳之所寄，心肾两脏，一阴一阳，
络脉相连，在维系人体阴阳平衡，协调脏腑生理功能方面起着极为重要的作用，“心主身之血脉”，是营运
气血的动力器官，肾阳上温心阳而益心气，肾阴上济心阴而制亢火，既提供心脏鼓动射血的物质基础，又
调节心主血脉的功能活动。各种病因一旦导致心气(阳)虚衰，则鼓动无力，心不主血，心脉瘀滞，而致心
悸、怔忡、紫绀；肾气虚衰，元阳不振，则心气(阳)更虚，或水不济火，心气浮越，或阳不制水，水邪内
停，或肾不纳气，气不归根，而致心肾交病，出现喘脱，故心衰重症，每见心肾阳衰或阴竭阳亡的危候。
此外，由于心主血脉，肺主气，百脉朝会于肺，病则互为影响，肺心同病，肺失肃降，而致喘咳痰多；
心主血，气为血帅，气行则血行，脾为气血生化之源，化源亏乏，气血不足，则心失所养，而致心脾两虚，
脾运不健，为肿为胀：心主血而肝藏血，心脉瘀滞，营运不畅，则肝郁血瘀，疏泄失司形成癥积。
综上所述，主病之脏在心，与肺肝脾互相影响，病理性质为本虚标实，气血阴阳亏虚为本，瘀血水饮
为标。气血水三者相互作用，瘀从气虚来，水自阳虚生，血不利为水，而瘀血、水饮又可阻遏心之气阳，
长此以往，正虚邪实，因果循环，使病情反复迁延。可知“阴阳两虚，心脉瘀滞”是充血性心力衰竭的基本
病机病证，从而为应用益阴助阳，活血通脉治法提供了理论依据，据此组方用药研制成养心通脉口服液。
2 实验研究
2.1 材料
SD 大鼠90 只，体重220～270 g，雌雄各半，由南京中医药大学动物实验中心提供。养心通脉口服液
含生药2.7 g/g，由南京中医药大学中医药研究院提供，文中剂量按生药量计算。其余药物为临床常用的卡
托普利、肝素钠、氯化钠、盐酸利多卡因等注射液。
2.2 实验方法
2.2.1 充血性心衰大鼠模型的复制 按Fletcher(1981)法，腹腔注射乌拉坦(0.8 g/kg)麻醉，仰卧固定。
经舌下静脉注射利多卡因(10 mg/kg)，行喉管插管术，于第4～5 左肋间开胸，接通人工呼吸机，迅速将心
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·25 0·
实 验 研 究
Experimental study
脏挤出，于肺动脉圆锥和左心耳之间，距主动脉根部约2 mm 处结扎左冠状动脉(冠脉)主干，然后将心脏
复位，挤出胸腔内空气，迅速闭胸，恢复自主呼吸后关闭呼吸机。
2.2.2 实验分组 动物随机分为正常组、假手术组、心衰组(模型组)、卡托普利组(0.016 g/kg)、养心通
脉小剂量组(YX1，12 g/kg)和大剂量组(YX2，36 g/kg)。卡托普利和养心通脉口服液按等容量灌胃给药，每
日1 次；正常组、假手术组和模型组给予等容量生理盐水，每日1 次；连续给药4 周。
2.2.3 充血性心衰大鼠血流动力学指标检测方法 制模后连续给药4 周，眼眶静脉取血4 mL 用于放射
免疫测定，用质量分数为25%的乌拉坦(0.8 g/kg)腹腔麻醉。舌下静注体积分数为1%的肝素生理盐水0.1
mL/100 g 体内抗凝。分离右侧颈总动脉，将充有0.1%肝素生理盐水的导管插入左室腔，连接压力换能器，
测量动脉收缩压(SAP)、动脉舒张压(DAP)、平均动脉压(MAP)，经载波放大器记录左室内压(LVP)；将LVP
信号输入直流放大器放大10 倍后测取左心室舒张末期压力(LVEDP)；将LVP 信号输入压力处理器(时间常
数0.5 ms，定标667 kPa·s-1·10 mm-1)，分别描记左室压变化速率曲线(dp/dt 曲线)和左室压的对数变化速率
曲线〔(dp/dt)· p-1 曲线〕。由血压搏动信号触发AT601G 并直接读出心率(HR)。上述观察指标均同步记录于
RM 6000 型8 导生理记录仪上(快速走纸50 mm/s)。在LVP 曲线上，由零点至曲线顶点的数值为左室压峰
值(LVSP)。左心室内压变化最大速率(±dp/dtmax)为在dp/dt 曲线上，零线至峰顶的数值。由dp/dt 曲线上升
支的起点至对应上升支峰顶的间隔时间为t dp/dtmax(左心室开始收缩至左心室内压上升速率峰值时间)。在
(dp/dt)·p-1 曲线上，曲线从零点至正向峰顶的数值为左心室肌收缩成分的实测最大缩短速率(Vpm)。
2.2.4 充血性心衰大鼠心肺重量的测定 在测定心功能后，立即打开腹腔经十二指肠给药，30min 后,
取肠系膜动脉，制备肠系膜上动脉灌流模型。然后开胸取出心脏、肺脏,并用生理盐水洗净，称量全心重和
肺重。切去心脏房室沟以上部分，沿室间隔切开左、右心室分别称重。
2.2.5 统计学方法 数据以均数±标准差(x±s)表示，各组间的比较采用方差分析和两样本均数比较的
t 检验。方差不齐时，通过变量变换或非参数统计方法进行分析。
2.3 结果
2.3.1 对充血性心衰大鼠血流动力学的影响 由表7 可知，模型组与正常组比较，各项指标均存在较
为显著的差异，表明心衰模型组大鼠左心泵血功能显著下降，收缩功能明显减弱，循环系统有明显充血现
象，佐证制模较为成功。与模型组比较，养心通脉大剂量组与卡托普利组均能增加心衰大鼠的LVSP、
+dp/dtmax，显著降低LVEDP，且养心通脉大剂量组与卡托普利组相比无明显差异。养心通脉大剂量组与
卡托普利组均能使t dp/dtmax 下降，似养心通脉大剂量组作用更强。养心通脉小剂量组大鼠的LVSP 明显
提高、t dp/dtmax 显著下降。同时卡托普利组和养心通脉大、小剂量组大鼠的SAP 均有明显升高，似以养
心通脉大剂量组作用最强。
2.3.2 对充血性心衰大鼠心、肺的影响 由表8 可知，模型组与正常组比较，各项指标均有明显差异。
与模型组比较，养心通脉大剂量组与卡托普利组均能增加左室重量和LV/BW，且以大剂量组为显著。养心
通脉小剂量组和卡托普利组均能抑制右室肥厚，且小剂量组同时能降低肺脏指数。
第九届世界中医药大会
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实 验 研 究
Experimental study
3 讨 论
3.1 充血性心衰病机特点
审证求因，充血性心衰的发病多为各种原发病直接或间接损伤“心主血”的功能，导致心气不足，心阳
不振，进而气血阴阳亏虚，血脉瘀阻，以致心血不畅，肺失肃降，脾运不健，肾虚失纳，肝失疏泄等多脏
同病，导致瘀阻水停，痰饮内聚，此即《灵枢·刺节真邪篇》所指“手少阴气绝,则脉不通，脉不通则血不流”
的气虚致瘀理论。由于气(阳)虚血滞，脏腑气化功能障碍，水液输布排泄失常，必致体内水湿痰饮潴留，
但尤以血瘀为其主要病理因素，如《金匮要略·水气篇》之“血不利则为水”，而致本虚与标实互为因果，故
阴阳两虚、心脉瘀滞为其基本病机特点。
3.2 疗效分析
以往临床研究表明，养心通脉口服液由附子、人参、玉竹、葶苈子、泽兰、石菖蒲等组成，治疗组总
有效率明显优于西药对照组，并有明显强心利尿作用，随之主要症状体征明显改善。目前西药治疗以洋地
黄、利尿剂、血管扩张剂为主，洋地黄的治疗量与中毒量很接近，安全系数小；而利尿剂易造成电解质紊
乱。本品毒副作用小、安全系数大，有良好的应用开发价值。
3.3 实验研究表明养心通脉口服液具有强心、利尿作用
实验结果表明，用养心通脉口服液治疗后，大鼠+dp/dtmax、LVSP 升高，LVEDP 降低，说明养心通脉
口服液在增强心肌收缩力的同时，可以在一定程度上改善心肌舒张功能。其强心作用主要与人参、附子等
所具有的正性变力作用有关。以往研究表明，人参无论是临床观察还是动物实验都显示出明显的强心作用，
它通过抑制心肌细胞膜Na+K+ATP 酶活性，增加细胞内Ca2+浓度，促进儿茶酚胺释放及提高心肌
cAMP/cGMP 比值等机制，从而使心肌收缩增强。附子的有效成分去甲乌药碱与异丙基肾上腺素作用相似，
有兴奋β 受体作用，对心肌细胞的激动作用能被β 受体阻滞剂减弱和完全对抗。临床研究表明本品能增加
尿量，提示其有利尿作用。本品还具有升压作用，能使SAP 和MAP 升高，以SAP 升高幅度更大。DAP
似乎也呈升高趋势，但无统计学意义。以往实验研究表明，本品具有活血化瘀，改善微循环，调节血管舒
缩等作用，对大鼠离体心肌缺血/再灌注损伤亦有保护作用，提高过氧化物歧化酶，清除自由基，能使戊巴
比妥钠诱发家猫心衰的TXA2 降低，升高6-keto-PGF1a，降低T/K 比值，提示具有调节TXA2-PGF1a 的作用。
综上所述，养心通脉口服液通过增强心肌收缩力，降低肾素-血管紧张素-醛固酮系统、交感系统活性及
血浆内皮，扩张血管，改善心肌能量代谢，改善心泵功能，增加肾血流量，抗氧化、调节TXA2-PGF1a 系统
功能等作用，达到减轻心肌损伤，降低心脏前负荷，改善心功能，使心衰患者的症状体征明显改善或消失。
Effect of the serum contained Radix Astragali and Hirudo and Hirudin and
the compoundings on the grow th cycle and apoptosis of rats Glomerular
Mesangial Cells
Ren Xianzhi1 Jiang Shuming2 Zhai Wensheng3
( 1 Mobile station for the post-doctors of Nanjing University of TCM, Nanjing, 210029; 2 China pharmaceutical University, Nanjing, 210009; 3 No.1 affiliated
hospital of Henan college of TCM, Zhengzhou, 450000)
Abstract Objective: The study was aimed to investigate the effect of the serum contained Radix Astragali and
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
Hirudo and Hirudin and the compounding of these two herbs respectively on the growth cycle and apoptosis of
rats Glomerular Mesangial Cells and to observe the morphology of apoptosis. [Method] The growth cycle and
apoptosis of GMCs was measured by flow cytometer. Meanwhile morphous of apoptosis was visualized by
Wright's staining and observed by microscope. [Result] The serum contained Radix Astragali and Hirudo and
Hirudin and the compoundings respectively can keep most MC in the stage of G0/G1, and it had a significant
deviation compared to the control (P< 0.01) . It also increased the rate of MC apoptosis, and it had a significant
deviation compared to the control (P<0.01) . [Conclusion] The serum contained Radix Astragali and Hirudo and
Hirudin and the compounding respectively can prevent GMCs entering the stage of to inhibit its growth and it also
can increase the rate of MC apoptosis.
Keywords Rat; Glomerulum Mesangial Cells; growth cycle ; apoptosis ; morphological ; Radix Astragali;
Hirudo; Hirudin
Growing glomerulum nephritis is children’s common kidney disease, with the progress of pathological change,
GMCs hyperplasia, the ground substance (ECM) outside cells Increase. The experiment aims to take
representative medicine of silt as target, inqire the influlence of the medicine on the GMCs apotosis and growth
cycle , observe the function intensity of the two medicine and offer reference for clinical and dialectical
administration, inqire into how the mechanism develop in the growing glomerulus nephritis.
1 Material
1.1 Animal: Animal: SD rats, 2 - 3 months old, male, weight 150- 200g. Offered by centre of experimental
animal of nanjing university of TCM(SPF grade) . Divided into 11 groups, 6 every groups at random.
1.2 Cells: Membrane cell one of rats‘ department, offered (HBZY-1) by the biology department of Wuhan
university.
1.3 medicines: Radix Astragali of Inner Mongol (Astragalus mongholicusBge.) , the leech (Japan cures leeches,
hirudo nipponia Whitman. talc powder fries,) ,Offered by nanjing university of TCM. The hirudin (injected
recombinate leeches plainly, Hirudin) 10mg each, lot number (20001001) ,Than living≥ 2000AIU, purity≥ 99% ,
mingkang biological engineering company,Guangzhou.
1.4 Reagents: MEM culture medium: Gibco products, lot number: 41500-067. EDTA: Sigma products, lot
number: 40-4. Trypsin: Gibco products, lot number: 27250-018. Cycle TEST PLUS DNA reagent box: The
products of U.S.A. BECTON DICKINSON Company, lot number: 340242. Auspicious dye liquor: Sichuan
Province Mike Science Technologies company, lot number: 0603012. Lipopolysaccharide (LPS) : 10mg each,
sigma products, lot number: DH183-1, offered by the biomedical engineering company of three leaves of
Zhengzhou .
2 Method
2.1 the prepration of the serum contained medicine
Fry traditional Chinese medicine Radix Astragali water and concentrate to including crude medicine 1.5g/ml,
grinds the leech to powder with the high-speed pulverizer, mix into the mixing and hanging liquid of 0.3g/ml.
With 2 times (Radix Astragali 12g/d, leech 2.4g/d) of the rat's equivalent dosage of clinical adult's administration
quantity ,Irritate, take medicinal solution, 2 times a day, 5 day continuously, 2h after the last dosage, fetch blood
by eyeball. calculates the equivalent hirudin dosage of the rat according to 0.5mg/kg, with 15 times of
dosage, gives medicine by the end intravenous injection, fetch blood after 20 minutes. 0.22¦Ìm aseptic filter
filters after deactivating the complement, packages, kept in the low-temperature refrigerator .
2.2 measure of the nonpoisonous boundary line of GMCs cells of the handling factor
Fetch serum of normal rat,serum contained high density of Radix Astragli and hirudo, dilute the serum by the
MEM cell maintaining liquid not including serum from 1:1,1:2 to 1:16, each dilute degree set 4 replying holes,
set the normal cells as contrast. The biggst dilute degree not leading to cell retreat is the nonpoisonous boundary,
through observation, the serum of normal rat and the serum when dilute as 1:2 not influence the growth of GMCs
cell clearly.
2.3 Divide into groups in experiment
After melting in step, GMCs are divided at random: Control group ( Add LPS (1.8mg/ml) 10ul + mouse serum
without medicine 360ul +MEM1430ul) , prescription group ( Add LPS (1.8mg/ml) 10ul + Radix Astragali
serum 360ul +MEM1430ul ) , the hirudo group(Add LPS (1.8mg/ml) 10ul + leech serum 360ul + MEM1430ul) ,
the hirudin group (Add LPS (1.8mg/ml) 10ul + hirudin serum 360ul + MEM1430ul) , the Radix Astragali
group (Add LPS (1.8mg/ml) 10ul + Radix Astragali serum 360ul + MEM1430ul) five groups.
2.4 Testing method of cell cycle and apotosis
2.4.1 cell preparation Fetch a well growing cell to digest, blow and beat cell to make monocyte hang liquid,
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
transfer cell density to 1* 105 / ml and inoculate into 15ml cell bottle, inoculate 1.8ml in each bottle, every
group set up 4 replying bottles. 37℃5% CO2 training case train, when cells cover with 80% of bottle wall, suck
and abandon the serum on the top,cultivate the cells with MEM liquid including 0.5% FCS fou 24h to make
them in step. Divided into every group described above at random, join the corresponding treatment factor,
continue cultivating for 48h.
2.4. 2 cells collecting After cultivating for 48h, digest cells routinely, wait for cells to turn into round and
suck pancreas enzyme, stop digesting. 2 PBS wash, join 3mlPBS ,blow and treat the cells and move into a tube,
1000 tuns each minute,fall go PBS, aad about 0.5mlPBS,then blow and beat to monocyte hanging liquid. The
liquid is packed into the test tube of 17* 100mm to hang cells, centrifuge for 5 minutes at room temperature 300*
g. Suck and abandon the serum on the top, aad 1ml and buffer the liquid and hang cells again, Repeated the last
step . regulate cell density into 1.0* 106 / ml.
2.4.3 cell dying centrifuge for 5 minutes at room temperature 400* g ,suck and abandon the serum at the top;
add 250ul A liquid (contain trypsin, four hydrogen chlorine precise amine stain-remover, used for digesting the
cell membrane and cell skeleton) into each tube ,Mix lightly and lightly, don't shake, put gently and quietly for 10
minutes at room tempreture. add 250ul B liquid (contain trypsin inhibitor, RNA enzyme, citrate and buffer liquid,
four hydrogen chlorine precise amine, used for inhibiting the function of trypsin and digesting RNA) into each
tube ,Mix lightly and lightly, don't shake, put gently and quietly for 10 minutes at room tempreture. add 250ul C
liquid (contain PI dye liquor, four hydrogen chlorine precise amine, citrate and buffer the liquid) into each
tube,Mix lightly and lightly, don't shake, put photophobically and quietly for 10 minutes in the low temperature
(2- 8 ℃).
2.4.4 measuring cell cycle by flowing type cell appearances filter and enter into sample tube with 50um nylon
membance, operate the computer test. Use BD CellQuest1.0 software to analysis.
2.5 cells apotosis and morphology observation
2.5.1 blow and beat the well growing cell to make monocyte hanging liquid, adjust cell density to 2.5* 104 / ml,
inoculate with 24 holes in advance in the cells with small glass slice cultivating board, every group has 4 replying
holes. 37℃5% CO2 case cultivate, when cells sticks to the wall, suck and abandon the top , cultivate with
MEM liquid including 0.75% for 24h to make the cell in step. Divided into every group described above at
random, join the corresponding treatment factor, continue cultivatinging 48h.
2.5.2 auspicious dye After cultivating for 48h, take the small glass slice in the holes sightly, after PBS washing,
0.4% of the formaldehyde is fixed for 5- 10 minutes at room temperature, air dry; Add the auspicious dye liquor to
dye for 1- 2 minutes; In addition auspicious to buffer liquid ( Auspicious the dye liquor 10ml adds PBS20ml)
1- 2 drops are dyed for 4- 10 minutes; Distilled water is washed, air dry, the neutral gum is sealed slice.
2.5.3 cells observation observe the changes of cell shape and nucleus under the
microscope, count positive cells, calculate the rate of apotosis cells by two pathologic personnel who are
familiar with cell apotosis.
2.6 statistics analysis
The experiment result is showed by X ±s 。Employ SPSS 10. 0 statistical analysis software to deal with
experimental data, every group materials spend single factor analysis of variance relatively, the second Liang are
compared and examined with Student-Newman-Keuls.
3 Result
3.1 comparative of Radix Astragali, hirudo and compoundings that influence MC cycle of growing and apotosis.
Hirudo, Radix Astragali, prescription filling by hirudo and Radix Astragali, can make a large number of MC in
G0/G1 stage, prevent the cell from entering S stage to achieve the purpose of inhibiting MC outgrowth.
The result is shown in Table 1.
Table 1 comparative of Radix Astragali, hirudo and compoundings that influence MC cycle of growing and
apotosis. （%， X ±S）
Group n G0/G1 Apoptosis
Control group 4 50.78± 4.09 1.63± 0.76
Radix Astragali
group 4 74.49± 2.34* 6.19±2.45
hirudo group 4 85.58± 8.92* 11.86± 3.16*
Hirudin group 4 75.64±5.34* 1.90± 1.49
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
Radix Astragali
andhirudo group 4 89.12± 2.57* 16.20± 6.12*
Note: *P<0.01 compared with control group, P> 0.05; Compared with Radix Astragali group P< 0.05, P<
0.01; Compared with hirudo group P< 0.05
We can find in Table 1, the cell proportion of every group in G0/G1 compared with control group obviously
rises (P<0.01) ,And hirudo group, prescription group are stronger than the Radix Astragali group (P parts <0.05
and 0.01) ,hirudo group does not have prominent difference (P> 0.05) to compare with prescription group. It
proves the function of traditional Chinese medicine hirudo inhibiting MC outgrowth is stronger than Radix
Astragali, if use in combination with Radix Astragali and act on stronger, it is not so obvious as hirudo group
that the hirudo usually organizes function.
From the result of influence on MC apotosis,we can find hirudo, hirudo and Radix Astragali fill a prescription, can
improve the rate of rats‘ MC apotosis, there are extremely obvious differences compared with control group (P<
0.01); The Radix Astragali group does not have obvious difference compared with control group (P>
0.05),Maybe it relates to the greater virationg in the group. The hirudin group does not have obvious difference
(P> 0.05). Among the groups,hirudo group, prescription group are obviously superior to the Radix Astragali
group (P<0.01) , prescription group is superior to hirudo group (P<0.05) . It proves that the function traditional
Chinese medicine hirudo can promote MC apotosis of rat is superior to Radix Astragali when stimulated by LPS,
if use in combination with Radix Astragali act on stronger. (See Fig. 1- 4)
Control group of Fig. 1 Radix Astragali group of Fig.2
Hirudo group of Fig. 3 Hirudo compound group of Radix Astragali of Fig. 4
3.2 cells apotosis and morphology observation
Observe the changes of cell shape and nucleus under the microscope, whether the cell volume appears to
shrink , cell quality becomes dense, dyeing acidly more, and can form small apotosis body; Whether nuclear
interior chromatin concentrates and forms the chromatin lump to assemble on the edge in the nucleus, form the
varied distribution shape, or take the form of hemisphere, or take the form of crescent, or taking the form of sand
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
dune form, horse's hoof, boat form,etc..
The number of apotosis cells in control group, Radix Astragali group,hirudo group,hirudin group and
prescription group were finally: 2.54±0.86, 5.26±2.76, 9.2±4.72, 2.84±1.26 and 15.63±5.71 (%) , among them,
MC apotosis of the leech, prescription group are more prominent, compared with control group, there is a
prominent difference (P<0.05) ,It is basically identical to the result measured with the flowing type cell
appearance, prove traditional Chinese medicine can really lead to MC apotosis. It appears shape change of cell
apotosis in every group, in the control group,apotosis cell is not typical,in small quantity and distribute loosely
(Fig. 5, Fig. 6) ;In Leech group, prescription group apotosis cell is typical , in large quantity and distribute
tightly.(Fig. 7,Fig.
.Control group of fig.5 Radix Astragali group*200 of fig.6
Hirudo group*200 of fig.7 prescription group of fig.8
4 Discussion
The membrane hyperplasia glomerulus nephritis of natural disposition (MsPGN) accounts for 40%- 50% of child's
nephropathy, it is child's common kidney disease. The membrane cells (MC) hyperplasia is common pathology
change of many glomerulus disease, is outstanding pathological characteristic of MsPGN. The excessive
hyperplasia of kidney membrane cell, causes ECM to accumulate in the glomerulus, leads to the fact that kidney
goes on fiber and hardens., it is the important mechanism of development in glomerulus sclerosis, it is also the
important links [1 ] that renal function damages. However, there is not effective therapeutic method to prevent the
MC hyperplasia, ground substance accumulate and glomerulus sclerosis at present.
The clinical manifestations of growing glomerulus nephritis are edema, weak, pain in the back and so on. we can
see blood in the urine, proteinuria, high lipoprotein blood disease and low protein blood disease to check in the
laboratory, it indicates to " Edema " , " hematuria " , " empty consumptive disease " in Chinese medicine Category.
Its root belongs to deficiency while its branch belongs to excess in traditional Chinese medicine. The disease
relies mainly on the deficiency of lung, spleen, renal Qi ,while blood stasis,wind,water and dampness,
dampness-heat,turbid and dampness are the most common. With the thoroughly realize on the disease, " blood
stasis " plays a more and more important role in this disease, we can say this disease to a certain extent from"
stasis " to " deficiency" .This is a great breakthrough known to the tradition. The pathology characteristic that
immune compoundings deposited, cell hyperplasia, glomerulus fiber, sclerosis all accord with Chinese medicine
" blood stasis " .Blood stasis is a nephropathy change that run through the diseases all the way ,from watching
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
·25 6·
实 验 研 究
Experimental study
dialectical microly, the basic nephropathy change is blood stasis.
Using traditional Chinese medicine to treat kidney disease has better curative effect , the representative medicine
is reinforcing Qi and removing blood stasis , this medicine can prevent or slow down renal fiber on a certain
level. Radix Astragali, hirudo are the most frequently using medicine in reinforcing Qi and removing blood stasis
medicine, so we use Radix Astragali and hirudo as representatives to carry on the reearch, use molecular biology
and serum pharmacology method to probe into the influence on MC hyperplasia , apotosis, cell cycle of growing
and produce TGF – B, IL-6, IL-13, collagenic(Col- And the fibrous connection albumen (FN),compare the
function intensity between reinforcing Qi and removing blood stasis medicine ,provide reference for theory and
clinical administrations dialecticals. We have already verified in the previous work that Radix Astragali, hirudo
and prescription can reduce the content [2 ] of the principal ingredients Col- ⅳof ground substance and FN outside
cells effectively.
The research on rat MC cycle influence indicates that hirudo, Radix Astragali, hirudo Radix Astragali fill a
prescription, can make a large number of MC in G0/G1 stage, prevent cells from entering S stage to achieve a
purpose of inhibiting MC outgrowth. The cell proportion that every group in G0/G1 compared with control group
obviously rises, and hirudo group and prescription group have the most obvious function.
The research on MC apotosis influence indicates that hirudo , Radix Astragali, hirudo Radix Astragali fill a
prescription, can improve rat ‘s rate of MC apotosis ,there are extremely obvious differences compared with
control group and hirudo group, prescription group, and present the typical peak of apotosis. It proves that
traditional Chinese medicine hirudo has a stronger act on promoting cell apotosis when stimulate by LPS than
Radix Astragali, if uses in combination with Radix Astragali and acts stronger. After observing MC morphology
apotosis, we find that after using traditional Chinese medicine ,every group have morphology apotosis change, the
nucleus creases, dyes, cracked densely, nuclear interior chromatin concentrates and gathers the edge in the nucleus,
or take the form of hemisphere, or take the form of crescent, form the varied distribution shape.
Through the research on inhibiting MC hyperplasia and promoting apotosis, the hirudo group, prescription group
have more prominent function, and present the typical peak of apotosis.It proves that the function of hirudo
promoting MC apotosis and inhibiting hyperplasia is superior to Radix Astragali., if use in combination with
Radix Astragali and act stronger. We can infer, in the prescription group of Radix Astragali and hirudo , in
order to strengthen the function of inhibiting MC hyperplasia and promoting apotosis, hirudo should be the main
medicine . It proves blood stasis is an important factor in the development of growing glomerulus nephritis .
Activating blood circulation and removing blood stasis are the basic curing methods in children‘s kidney disease.
it is indispensable to reinforce Qi and strengthen the body resistance, maybe it is more important than that.
Activating blood circulation should run through nephropathy treatment all the way.
References:
[1] Floege J, Johnson RJ, Gordon K, et al. Increased synthesis of extracelluar matrix in mesangial prolifertive mephritis[J]. Kidney Int, 1991, 40:477-488.
[2] Let will now, deep and vast to pass, Zhai WenSheng. Radix Astragali, leech and different proportions fill a prescription and suck serum of medicine to the
fibrous connection albumen and collagenic influence of the type outside the membrane cells of loud mouse's department. Chinese traditional Chinese medicine
magazine, 2006, 21(11) : 690-691.
[3] Ren Xianzhi, Wang Shouchuan, Zhai Wensheng.Influence of the contained-herb serum of Radix Astragali and Hirudo in different propor tion of
compounding on the Fibrillar connection Protein and Collagen type IV of Glomerular Mesangial Cells in vitro Rats. China Journal of Traditional Chinese
Medicine and Pharmacy, 2006, 21(11) : 690-691.
