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Original Experimental Research
Journal of Chinese Integrative Medicine: Volume 3   January, 2005   Number 1

DOI: 10.3736/jcim20050112
Effects of Yangyin Shengjin Decoction on hemorheological parameters and coagulation factors in model rabbits with syndrome of excessive heat consuming body fluid and blood stasis
1. MA Hong (College of Basic Medical Sciences, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu Province 210029, China E-mail: mhhome603@sina.com)
2. YANG Jin (College of Basic Medical Sciences, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu Province 210029, China E-mail: jyang@njutcm.edu.cn)
3. NAN Shu-Ling (College of Basic Medical Sciences, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu Province 210029, China )
4. GONG Jie-Ning (College of Basic Medical Sciences, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu Province 210029, China )
5. LIU Xue-Feng (College of Basic Medical Sciences, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu Province 210029, China )

Objective: To investigate the effects of Yangyin Shengjin Decoction (YYSJD) on hemorheological parameters and coagulation factors in model rabbits with syndrome of excessive heat consuming body fluid and blood stasis.

Methods: Rabbit model with syndrome of excessive heat consuming body fluid and blood stasis was produced. The effects of YYSJD on the blood viscosity, erythrocyte sedimentation rate (ESR), hematocrit, platelet aggregation rate, prothrombin time (PT), thrombin time (TT), kaolin partial thromboplastin time (KPTT), fibrinogen (Fg), thromboxane B2 (TXB2), and 6-keto-prostaglandin F (6-keto-PGF) in the model rabbits were observed.

Results: YYSJD decreased the whole blood viscosity and hematocrit, inhibited the platelet aggregation, prolonged PT, TT and KPTT, and reduced the content of Fg. It also regulated the balance between TXB2 and 6-keto-PGF.

Conclusion: YYSJD can promote the blood circulation, adjust the blood agglutinating function, and decrease the formation of thrombus. This is one of the pharmacological mechanisms of the therapeutic method of "nourishing yin to promote blood circulation" in the theory of traditional Chinese medicine for seasonal febrile diseases.

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J Chin Integr Med, 2005, 3(1): 39-42

Correspondence to: Prof. YANG Jin. E-mail: jyang@njutcm.edu.cn 

基金项目: 江苏省自然科学基金资助项目(No.BK99139)

作者简介: 马红(1957-),女,博士,副教授.E-mail: mhhome603@sina.com

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     外感温病热壅血滞,因热炽而致瘀,或津枯血凝,因水涸而致瘀,使瘀热证成为温病病程中常见且重要的证型。因而治疗中,在运用清热凉血、活血散瘀方药的同时,养阴生津亦是不可忽视的一个方面。本研究观察了养阴生津方对瘀热证模型家兔血液流变性和凝血因子的影响。

 
   

1 材料与方法
1.1 实验材料
1.1.1 药物及试剂
 养阴生津方(Yangyin Shengjin Decoction, YYSJD),由麦冬、芦根、玄参、生地黄组成,以2∶2∶1∶1的比例按常规制成含生药1 g/ml的水煎液,4℃冰箱保存备用。精制大肠杆菌内毒素(LPS, Escherichia coli serotype O127∶B8),美国Sigma公司实验用产品(批号:28H4042),用前以生理盐水在无菌条件下配制成0.5 μg/ml的溶液。呋塞米注射液,20 mg/支,南通制药厂产品(批号:010628)。地塞米松磷酸钠注射液,5 mg/支,江苏省江阴制药厂产品(批号:010518)。5’-腺苷二磷酸二钠盐(adenosine-5’-diphosphate disodium salt,ADP),上海伯奥生物科技公司产品(批号:990527)。柠檬酸三钠,江苏宜兴第二化学试剂厂产品(批号:991005),用时配成3.8%的溶液。消炎痛-EDTA、血栓素B2(thromboxane B2, TXB2)和6-酮-前列腺素F(6-keto-prostaglandin F1α,6-keto-PGF1α)放免药盒,解放军总医院科技开发中心放免研究所产品。纤维蛋白原(fibrinogen,Fg)试剂盒,上海复旦张江生物医药有限公司产品。凝血酶原时间(prothrombin time,PT)、凝血酶时间(thrombin time, TT)和部分凝血活酶时间(kaolin partial thromboplastin time, KPTT)测定试剂盒,复旦大学附属华山医院技协生物试剂公司产品。
1.1.2 动物 新西兰兔,雌雄各半,体质量2.0~2.8 kg,由南京中医药大学实验动物中心提供。
1.1.3 仪器 锥板式血黏度测定仪、血小板聚集凝血因子分析仪,北京世帝科学仪器公司产品。半自动生化分析仪,意大利英诺威生物技术有限公司产品。低温冷冻高速离心机,德国Hermle公司产品。低速自动平衡离心机,北京医用离心机厂产品。
1.2 实验方法
1.2.1 造模方法 新西兰兔18只,随机分为3组,即模型组、养阴生津方组和正常对照组,每组6只。模型组和养阴生津方组以地塞米松2.5 mg·kg-1·d-1后肢肌肉注射,正常对照组注射等量生理盐水,连续7 d。于实验第8天,前两组家兔分别自耳缘静脉注射呋塞米注射液,剂量25 mg/kg,1 h后同法等剂量重复注射1次;2 h后按0.3 μg/kg将大肠杆菌内毒素自耳缘静脉缓缓注入。正常对照组注射等量生理盐水。3 h后全部实验动物由颈动脉采血,分离血浆,进行指标测定。测试TXB2及6-keto-PGF所用血浆以消炎痛-EDTA抗凝,低温离心机4℃ 3 500 r/min离心15 min;富血小板血浆以3.8%柠檬酸三钠溶液抗凝,800 r/min离心10 min;余用血浆以3.8%柠檬酸三钠溶液抗凝,3 000 r/min离心10 min。
1.2.2 给药方法 养阴生津方组于攻毒前1 d下午灌胃给药1次,给药量3.36 g/kg。在攻毒当日攻毒前3 h和攻毒即刻再分别灌胃1次,给药量同前。正常对照组及模型组分别给予等量温开水。
1.3 观察指标及方法 血液黏度(全血黏度和血浆黏度)采用锥板式黏度仪测定;血沉、红细胞压积用温氏管法测定[1];血小板聚集率采用血小板聚集凝血因子分析仪测定;PT、TT、KPTT、Fg、TXB2和6-keto-PGF分别按所用试剂盒方法测定。
1.4 统计学方法 采用t检验。

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2 结 果
2.1 对全血黏度和血浆黏度的影响
 养阴生津方在低切变率、中切变率及高切变率处均能显著降低全血黏度,同时还能显著降低血浆黏度。见表1。

表1 各组全血黏度和血浆黏度比较
Tab 1 Comparison of whole blood viscosity and plasma viscosity among 3 groups
                                                                                                                               (`x±s, mPa·s)

Group
n
Whole blood viscosity
Plasma viscosity
200/s
60/s
5/s
1/s
Normal control group
6
2.96±0.36
3.57±0.37
7.92±0.99
18.94±3.96
1.43±0.07
Model group
6
3.40±0.24*
4.11±0.21*
9.66±1.15*
24.91±4.77*
1.62±0.09**
YYSJD-treated group
6
2.87±0.13 △△
3.42±0.18 △△
7.06±0.33 △△
16.33±0.84 △△
1.51±0.06
*P<0.05, **P<0.01, vs normal control group; P<0.05, △△P<0.01 vs model group

2.2 对血沉和红细胞压积的影响 养阴生津方能显著降低模型家兔红细胞压积,但对其血沉无明显影响。见表2

表2 各组血沉和红细胞压积比较
Tab 2 Comparison of ESR and hematocrit among 3 groups
                                                                           (`x±s)

 Group

n

ESR (mm/h)

Hematocrit (%)

Normal control group

6

0.48±0.43

33.58±2.73

Model group

6

10.17±14.83

42.67±3.56**

YYSJD-treated group

6

2.58±1.74

31.50±1.05△△

**P<0.01, vs normal control group; △△P<0.01, vs model group

2.3 对ADP诱导血小板聚集的影响 养阴生津方能显著降低血小板聚集率,并能显著延长其聚集时间。见表3。

表3 各组ADP诱导血小板聚集的比较
Tab 3 Comparison of ADP-induced platelet aggregation rate among 3 groups
                                                                                                                           (`x±s)

Group
n
Platelet aggregation rate (%)
Platelet aggregation time (s)
1 min
Maximum
5 min
Normal control group
6
10.48±5.66
15.75±9.02
6.23±3.56
180.00±85.02
Model group
6
25.72±10.75*
32.13±14.66*
15.50±7.72*
85.00±17.96*
YYSJD-treated group
6
7.25±4.12 △△
9.33±4.81 △△
3.23±1.02 △△
144.33±59.36
*P<0.05, vs normal control group; P<0.05, △△P<0.01, vs model group

2.4 对血浆PT、TT、KPTT、Fg的影响 养阴生津方能显著延长模型家兔PT、TT、KPTT时间,显著降低其血浆Fg含量。见表4。

表4 各组血浆PT、TT、KPTT、Fg比较
Tab 4 Comparison of PT, TT, KPTT and Fg in plasma among 3 groups
                                                                                                                              (`x±s)

  Group

n

PT (s)

TT (s)

KPTT (s)

Fg (g/L)

Normal control group

6

21.83±0.98

102.17±5.78

99.67±9.20

3.27±0.55

Model group

6

18.67±1.03**

89.83±9.87*

81.33±10.29**

5.20±0.65**

YYSJD-treated group

6

20.50±1.38

105.00±4.94△△

103.17±5.74△△

3.27±0.49△△

*P<0.05, **P<0.01, vs normal control group;P<0.05, △△P<0.01, vs model group

2.5 对血浆TXB2、6-keto-PGF的影响 养阴生津方能显著降低血浆TXB2含量,显著升高6-keto-PGF含量,并使两者比值下降。见表5。

表5 各组血浆TXB2、6-keto-PGF比较
Tab 5 Comparison of TXB2 and 6-keto-PGF among 3 groups
                                                                                                                     (`x±s)

  Group

n

TXB2 (ng/L)

6-keto-PGF (ng/L)

TXB2/6-keto-PGF

Normal control group

6

168.75±52.97

939.41±189.48

0.18

Model group

6

254.65±62.39*

382.12±171.50**

0.64

YYSJD-treated group

6

141.89±26.19△△

831.62±285.30△△

0.17

*P<0.05, **P<0.01, vs normal control group; △△P<0.01, vs model group 

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3 讨 论
     瘀热证是温病病程中的常见证型,它是多因素、多环节病理共同作用的结果。近年来临床和实验研究发现,阴津损伤与温病瘀热证形成密切相关,热盛津伤、血行涩滞是温病瘀热证的重要病理基础之一,在温病治疗中合理使用养阴生津方药,有助于提高临床疗效。为探讨养阴生津方药治疗瘀热证的作用机制,我们在南京中医药大学温病教研室以往研究的基础上[2],采用大肠杆菌内毒素、呋塞米和地塞米松复合造模,制作家兔热盛津伤血瘀证动物模型,并观察了养阴生津方对该模型家兔血液流变性和凝血因子的影响。
     建立疾病动物模型,应使该模型尽可能与人类疾病的病理改变相类似。温病瘀热证形成的重要病理之一是邪热亢盛致阴津亏耗而血行瘀滞,故模型动物必须同时具备热毒、阴伤、血瘀的病理特点。由于温病学中的“温邪”实际包含了多种致病微生物,而大肠杆菌内毒素又是效应很强的致热原,且有容易控制、使用安全等优点,所以本实验选用大肠杆菌内毒素作为致病因素进行造模。考虑到使用内毒素后受试动物有血液凝固性下降和血黏度降低的情况,以及模型动物必须具有阴伤存在的特点,我们采用攻毒前先大剂量肌肉注射皮质激素地塞米松1周的方法,使动物处于阴虚和高凝、高黏状态。并且为使模型动物的阴津损伤更为明显,在攻毒前还给予实验动物大剂量呋塞米注射2次,使本模型尽可能地体现热盛伤津血瘀证候的特点。造模后模型家兔各项指标检测显示:全血黏度和血浆黏度上升,红细胞压积增高,血小板聚集率增高和聚集时间缩短,Fg含量升高,TXB2上升,6-keto-PGF下降,二者比值升高,PT、TT、KPTT缩短,血液呈高黏、高凝状态。
     血液黏度作为血液流动时所表现出的内摩擦,是决定血液阻力的因素之一,而血液黏度本身是由血浆和血细胞的流变特性决定的。影响血液黏度的主要因素有红细胞压积、红细胞变形性、红细胞聚集、血浆黏度以及血小板聚集等。红细胞压积是影响血液黏度的主要因素,黏度随红细胞压积呈指数增长。红细胞在流场中发生变形和定向是影响高切变率时血液黏度的重要因素之一,而低切变率时血液黏度主要受红细胞聚集的影响。血浆黏度对全血黏度影响很大,血浆中链状蛋白质分子的链越长、分子量越大对血浆黏度的影响越大,因此纤维蛋白原对血浆黏度的影响最大[3]。血小板具有黏附、聚集和释放等主要功能,在止血、凝血和血栓形成方面起着重要的作用,许多血栓性疾病均具有血小板聚集、释放功能亢进。而ADP诱导血小板聚集的一个重要条件是必需有纤维蛋白原的存在。血栓素A2(thromboxane A2,TXA2)主要是由血小板微粒体合成并释放的一种具有强烈促进血管收缩和血小板聚集的生物活性物质。其生物半衰期约30 min,而迅速代谢为无活性的TXB2。血管壁内皮细胞合成和释放的另一种抗血小板聚集和舒张的生物活性物质是前列环素(prostaglandin I2, PGI2),生物半衰期约为3 min,迅速代谢生成6-keto-PGF。血浆或组织中TXA2和PGI2平衡失调是造成血小板聚集、血管痉挛收缩或血栓形成的原因之一。由于TXA2和PGI2的不稳定,目前难以直接测定,故国内外均以测定TXB2和6-keto-PGF1α作为判断其浓度的指标。凝血活酶能使血浆中的凝血酶原变成凝血酶,从而促进纤维蛋白原变为纤维蛋白而使血浆凝固,PT、TT是检测外源性凝血系统较敏感的筛选试验;KPTT测定血浆中凝血第一阶段内源性凝血因子(除第Ⅶ和Ⅻ因子外)是否缺乏的灵敏过筛试验[4]。在对血瘀证模型家兔应用养阴生津方治疗后,家兔全血黏度和血浆黏度均有明显降低,其中全血黏度在200/s、60/s处接近正常组,与正常组比较无显著性差异,红细胞压积显著下降,Fg含量显著减低,血浆TXB2下降,6-keto-PGF上升,两者比值降低,血浆PT、TT、KPTT显著延长。因受设备条件的限制,本次实验未能进行红细胞变形和红细胞聚集实验,但从理论上讲,由于已观察到养阴生津方对高切变率时全血黏度改善作用最为明显,故推测该方可能对红细胞变形的影响较大。实验结果表明,养阴生津方滋阴生津、增水行血之效,与其能降低血液黏度和红细胞压积,抑制血小板聚集,降低Fg含量,对抗PT、TT、KPTT缩短,协调TXB2、6-keto-PGF间平衡,以改善血液流变性,调节凝血功能,抑制血栓形成有关。养阴生津法“滋而能通”具有科学的药理基础。

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References
1. 翁维良, 廖福龙, 吴云鹏, 等. 血液流变学研究方法及其应用[M]. 第1版. 北京: 科学出版社, 1989. 64.
2. 王秋. 家兔“温病阴虚热盛证”动物模型的实验研究[J]. 辽宁中医杂志, 1995, 22(9) : 427-429.
  
3. 陈文杰. 血液流变学[M]. 第1版. 天津: 天津科学技术出版社, 1987. 43.
4. 李家增, 王鸿利, 韩忠朝. 血液实验学[M]. 第1版. 上海: 上海科学技术出版社, 1997. 343.
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