• 247 •Chin J Integr Med 2010 Jun;16(3):247-251

T h e l e s i o n o f t h r o m b o s i s i n c a r d i o - /cerebrovascular diseases is based on atherosclerosis, and many clinical and lab researches have confirmed that thrombosis due to atherosclerosis is closely related to inflammation(1-3). The etiology and pathogenesis of platelet activation and thrombosis in the process of cardiac and cerebral thrombotic diseases are considered as "blood stasis" in Chinese medicine (CM). A series of therapeutic principles and methods of promoting blood circulation and removing blood stasis was formed under the guidance of this theory, which showed a positive therapeutic effect on the prevention and treatment of cardio-/cerebrovascular diseases. However, the pathological changes such

as tissue necrosis and inflammation can not be considered only as CM blood stasis(4). Based on the combination of the basic theory of blood stasis and pathogenic toxins of CM, experimental carotid artery thrombosis rats model were used in this study. The pathophysiological changes were reflected

EXPERIMENTAL RESEARCH

Effect of Chinese Herbal Medicine for Activating Blood Circulation and Detoxifying on Expression of Inflammatory

Reaction and Tissue Damage Related Factors in Experimental Carotid Artery Thrombosis Rats

XUE Mei (薛 梅), ZHANG Lu (张 璐), YANG Lin (杨 琳), JIANG Yue-rong (蒋跃绒),GUO Chun-yu (郭春雨), YIN Hui-jun (殷惠军), and CHEN Ke-ji (陈可冀)

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2010Supported by National Basic Research Program of China (No. 2006CB504803)Center of Cardiovascular Diseases, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing (100091), ChinaCorrespondence to: Prof. YIN Hui-jun, Tel: 86-10-62874093, E-mail: huijunyin@yahoo.com.cnDOI: 10.1007/s11655-010-0247-4

ABSTRACTABSTRACT ObjectiveObjective: To observe the pharmaceutical effect of Chinese drugs for activating blood circulation : To observe the pharmaceutical effect of Chinese drugs for activating blood circulation

(Xiongshao Capsule, XSC, 芎芍胶囊) and for activating blood circulation and detoxification (Xiongshao Capsule (Xiongshao Capsule, XSC, 芎芍胶囊) and for activating blood circulation and detoxification (Xiongshao Capsule

and Huanglian Capsule, XSHLC, 黄连胶囊) in terms of the indices of thrombosis, inflammatory reaction and and Huanglian Capsule, XSHLC, 黄连胶囊) in terms of the indices of thrombosis, inflammatory reaction and

tissue damage related factors in experimental carotid artery thrombosis rats. tissue damage related factors in experimental carotid artery thrombosis rats. MethodsMethods: Fifty Wistar rats were : Fifty Wistar rats were

randomly divided into the sham operation group, the model group, the Simvastatin group (SG), the activating randomly divided into the sham operation group, the model group, the Simvastatin group (SG), the activating

blood circulation (ABC) group, and the activating blood circulation and detoxifying (ABCD) group, with 10 blood circulation (ABC) group, and the activating blood circulation and detoxifying (ABCD) group, with 10

rats in each group. Simvastatin (1.8 mg/kg), XSC (0.135 g/kg) and XSHLC (0.135 g/kg) were administered to rats in each group. Simvastatin (1.8 mg/kg), XSC (0.135 g/kg) and XSHLC (0.135 g/kg) were administered to

Simvastatin, ABC and ABCD group by gastrogavage, and an equal volume of normal saline was given to the Simvastatin, ABC and ABCD group by gastrogavage, and an equal volume of normal saline was given to the

sham operation group and the model group. After 2 weeks of successive medication, the rats in the model sham operation group and the model group. After 2 weeks of successive medication, the rats in the model

and all drug therapy groups were made into experimental carotid artery thrombosis model. The serum levels and all drug therapy groups were made into experimental carotid artery thrombosis model. The serum levels

of matrix metalloproteinases (MMP-9), tissue inhibitors to metalloproteinase (TIMP-1), granule membrane of matrix metalloproteinases (MMP-9), tissue inhibitors to metalloproteinase (TIMP-1), granule membrane

protein-140 (GMP-140), tissue-type plasminogen activator (t-PA), high-sensitivity C-reactive protein (hs-CRP) protein-140 (GMP-140), tissue-type plasminogen activator (t-PA), high-sensitivity C-reactive protein (hs-CRP)

and interleukin-6 (IL-6) were detected with enzyme-linked immunoassay 24 h later. and interleukin-6 (IL-6) were detected with enzyme-linked immunoassay 24 h later. ResultsResults: Compared with the : Compared with the

model group, the levels of serum GMP-140, hs-CRP, IL-6 and MMP-9 were significantly decreased, and the model group, the levels of serum GMP-140, hs-CRP, IL-6 and MMP-9 were significantly decreased, and the

level of t-PA was significantly increased in the ABC and ABCD group (level of t-PA was significantly increased in the ABC and ABCD group (P<0.05), while the level of serum hs-CRP <0.05), while the level of serum hs-CRP

in ABCD group decreased significantly compared with that in the ABC group (in ABCD group decreased significantly compared with that in the ABC group (P<0.05). <0.05). ConclusionsConclusions: Chinese : Chinese

drugs both for activating blood circulation and for activating blood circulation and detoxifying have good effects drugs both for activating blood circulation and for activating blood circulation and detoxifying have good effects

on regulating indices of thrombosis, inflammatory reaction and tissue damage in experimental carotid artery on regulating indices of thrombosis, inflammatory reaction and tissue damage in experimental carotid artery

thrombosis rats. The effect of activating blood circulation and detoxifying drugs on regulating the level of serum thrombosis rats. The effect of activating blood circulation and detoxifying drugs on regulating the level of serum

hs-CRP is superior to that of activating blood circulation drug alone. hs-CRP is superior to that of activating blood circulation drug alone.

KEYWORDSKEYWORDS activating blood circulation, activating blood circulation and detoxifying, thrombosis, tissue activating blood circulation, activating blood circulation and detoxifying, thrombosis, tissue

damage, inflammatory reaction damage, inflammatory reaction

• 248 • Chin J Integr Med 2010 Jun;16(3):247-251

in thrombosis, inflammatory reaction and tissue damage in three aspects, and the difference of the therapeutic effects of Chinese drugs for activating blood circulation to that for activating blood circulation and detoxifying were compared as well. We then tried to determine the micro-pathological basis of the relationship between blood stasis and the pathogenic toxin, and provide an objective experimental basis for the CM treatment of cardiac and cerebral thrombotic diseases.

METHODS

AnimalsFifty SPF Wistar rats, half male and half female,

weighing 190±10 g, were provided by Experimental Institute of Chinese Academy of Medical Sciences [Certificate No. SCXK (Beijing) 2005-0013].

Main Reagents and InstrumentsEnzyme-linked immunosorbent assay (ELISA)

kit, matrix metalloproteinases (MMP-9, batch number: 20080413), tissue inhibitors to metalloproteinase (TIMP-1, batch number: 20080617), granule membrane protein-140 (GMP-140, batch number: 20080621), tissue-type plasminogen activator (t-PA, batch number: 080523), high-sensitivity C-reactive protein (hs-CRP, batch number: 20080617), interleukin-6 (IL-6, batch number: 20080524), were provided by R & D Systems Inc, USA and were sub-packaged by Shanghai Boatman Biotech Inc., which were detected by micro-plate reader (Thermo MK3, USA).

MedicationSimvastatin (Hangzhou MSD Pharmaceutical

Co., Ltd., batch number: 07432), 40 mg each tablet, was made into powder in mortar and dissolved into suspension with double-distilled water. Xiongshao Capsule (芎芍胶囊, XSC, Dalian Institute of Chemical Physics, batch number: 070929), 0.25 g each capsule, contained paeoniflorin (more than or equal to 28 mg each capsule), ferulate (more than or equal to 3.5 mg each capsule) and total phenolic acid (more than or equal to 34 mg each capsule). Huanglian Capsule (黄连胶囊, HLC, Hubei Xianglian Pharmaceutical Co., Ltd.,), each capsule contained 0.25 g Coptis chinensis Franch.

Grouping and Methods of Model EstablishmentFifty Wistar rats were randomly divided into

the sham operation group, the model group, the

simvastatin group, the activating blood circulation (ABC) group and the activating blood circulation and detoxifying (ABCD) group, with 10 in each group. The dose was determined by equivalent conversion method according to the animals' body surface area ratio(5). Simvastatin (1.8 mg/kg), XSC (0.135 g/kg) and Xiongshao Capsule and Huanglian Capsule (XSHLC, 0.135g /kg) were administered to the simvastatin group, ABC group and ABCD group by gastrogavage, respectively, once every 24 h for two weeks, and an equal volume of normal saline was given to the sham operation group and the model group. One hour after the last administration, the anesthetized rats (20% urethane solution 0.6 mL/100g, intraperitoneal injection) were set on the operating table in supine position with head fixed, stretched limbs lashed and neck fully exposed.

The right side of the neck was shaved, with local skin sterilized with 75% alcohol and incised. According to Kurz's method(6), the right common carotid artery was separated about 2 cm, a small piece of plastic film was put (4 cm×1.8 cm) under it to protect surrounding tissues, and then filter paper (1 cm×1 cm) soaked in 20 μL FeCl3 (70%) was used, which was placed on the vessel to induce the injury. Filter paper soaked in an equal volume of normal saline was used in the sham operation group. Thirty mintues later the filter paper was removed, local tissue was flushed with normal saline and sutured and the rats were put back to the feeding room. After 24 h, the blood samples were collected from the abdominal aorta of rats and kept in a red biochemical procoagulant tube at room temperature for 30 min. The serum was separated by low-speed centrifugation (2 000 r/min for 15 min) and then was stored at -20 ℃ for use. Two rats in each group were randomly selected, and about 1.5 cm of the right common carotid artery was cut down and fixed with 10% formaldehyde for the follow-up biopsy.

Items of Observation and MethodsThe serum levels of MMP-9, TIMP-1, GMP-140,

t-PA, hs-CRP and IL-6 were detected with enzyme-linked immunosorbent assay. The thrombosis from the common carotid artery was observed by HE staining with an optical microscope (Olympus BX51, Japan).

Statistical Analysis Al l s ta t is t ica l analyses were per formed

with SPSS 11.5, and P＜0.05 was considered as

• 249 •Chin J Integr Med 2010 Jun;16(3):247-251

statistically significant. The analysis of variance (ANOVA) were applied in the intergroup comparison of measurement data, and the χ2 test was used in the comparison of enumeration data.

RESULTS

Pathological Changes of Common Carotid Artery

The histophysiological structure of the sham operation group was clear and complete, arranged in neat rows. The structures of the tunica media and tunica externa were normal, and the tunica intima was smooth and complete without foreign bodies. In the other groups, the vessel wall was thin and a cord-like mixed thrombus was formed, which had a homogeneous red staining region (mainly composed of platelets and fibrin) and a small amount of plasma with some cracks. There were also large stained areas (mainly composed by cracked red blood cells and white blood cells) in the vessel. The tunica intima was damaged, covered with hemosiderin and cracked macrophages, and the basic structure of the tunica media and tunica externa maintained integrity while the elastic layer was thinner. (Figure 1).

Comparison of Thrombosis-related Indicators among Groups

Compared with the sham operation group, the serum level of GMP-140 was significantly increased and the serum level of t-PA was signif icantly decreased in the model group (P<0.01, P<0.05). The serum level of GMP-140 was significantly decreased

and the serum level of t-PA was significantly increased in the simvastatin, ABC and ABCD groups compared with those in the model group (P<0.05, Table 1).

Comparison of Inflammation-related Indicators among Groups

Compared with the sham operation group, the serum levels of hs-CRP and IL-6 were significantly increased in the model group (P<0.01). The serum levels of hs-CRP and t-PA were signif icantly decreased in the simvastatin group, ABC group and ABCD group compared with those in the model group (P<0.05, P<0.01).The level of serum hs-CRP in ABCD decreased significantly compared with that in ABC (P<0.05, Table 2).

Table 2. Comparison of the Serum Levels ofhs-CRP and IL-6 among Groups ( ±s)

Group n hs-CRP (ng/mL) IL-6 (pg/mL)

Sham operation 10 6.29±2.64 234.07±99.22

Model 10 13.55±3.56 433.55±224.34

Simvastatin 10 8.96±4.28 246.23±147.07

ABC 10 10.14±3.07 277.67±152.56

ABCD 10 7.17±2.50 △ 246.40±161.76

Notes: P<0.05, P<0.01, compared with the model group; △P<0.05, compared with the ABC group

Table 1. Comparison of the Serum Levels of GMP-140 and t-PA among Groups (pg/mL, ±s)

Group n GMP-140 t-PA

Sham operation 10 657.18±247.75 1089.14±299.19

Model 10 1319.37±403.94 689.90±292.63

Simvastatin 10 946.95±404.67 1095.38±451.44

ABC 10 956.64±366.72 1055.29±377.24

ABCD 10 967.77±432.22 1130.23±505.08

Notes: P<0.05, P<0.01, compared with the model group

Figure 1. Pathological Changes of Common Carotid Artery in Each Group (HE staining, ×200)Notes: A: sham operation group; B: model group; C: simvastatin group; D: ABC group; E: ABCD group

Compar ison of T issue Damage-re la ted Indicators among Groups

Compared with the sham operation group, the serum level of MMP-9 was significantly increased in the model group (P< 0.01). The serum levels of MMP-9 were significantly decreased in simvastatin group, ABC group and ABCD group compared with the model group (P< 0.05). There was no significant difference in the serum leves of TIMP-1 among the different groups (P> 0.05, Table 3).

DISCUSSION

Blood stasis syndrome and its treatment by promoting blood circulation is one of the most active fields in CM research, which has made remarkable progress on the pathogenic character is t ics ,

A B DC E

• 250 • Chin J Integr Med 2010 Jun;16(3):247-251

determination of standards, scoring and pathologic changes. This has significantly improved the clinical diagnosis and treatment efficacy towards cardiac and cerebral thrombotic diseases. Although there were many discourses about the status of the pathogenic toxin in the pathogenesis of cardio-/cerebrovascular diseases, no common understanding with regards to its pathogenic characteristics, whole- and micro-pathological changes, and syndrome differentiation and disease identification has been up to standard. Especially, there has been inadequate understanding of whether the toxin or the combination and transformation of the toxin and blood stasis of CM are involved in the pathogenesis of cardio-/cerebrovascular diseases. This limited the in-depth study and improvement of clinical efficacy of CM in the treatment and prevention of cardio-/cerebrovascular diseases.

Modern research has confirmed that modern physiochemistry-related indicators of cardio-/cerebrovascular thrombosis diseases considered to be CM blood stasis syndrome are involved in platelet activation, adhesion, congregation and thrombosis, etc. However, some scholars have made modern biological indices and connotation on the toxin and the therapeutic method of detoxifying. The combined use of Chinese herbal medicine for detoxifying and activating blood circulation (Polygonum Cuspidatum Sieb. et Zucc., XSC) could reduce the expression of nuclear factor-κB and MMP-9 in the aorta of ApoE(-/) mice，and the effect of the combination is superior to that of use of either of them alone(7), which could also reduce the serum hs-CRP level(8). Active ingredients from Chinese drugs for activating blood circulation and detoxifying, including Panax notoginseng saponins, Rhizoma Coptidis, Polygonum Cuspidatum Sieb. et Zucc could lower the blood lipid levels in ApoE knockout mice, and a significant difference was still seen between Polygonum Cuspidatum Sieb. et Zucc and the others. However, a significant decrease in the

level of tumor necrosis factor-α was seen only in the rhubarb treated group(9). But whether toxin and blood stasis are the reciprocal causations involved in the disease process, or which modern pathological and biochemical indicators may be involved, or whether the therapeutic method of activating blood circulation and detoxifying is superior to that of activating blood circulation, or which indices are sensitive indicators, remain unanswered and there are still no systematic researches about these.

Atherosclerosis (AS) is a common basis for thrombosis in cardio-/cerebrovascular diseases. According to CM theory, the pathogenetic factors involved in the formation of AS such as platelet aggregation, thrombosis, and vascular endothelial proliferation have an intrinsic relationship with blood stasis, and Chinese drugs for activating blood circulation have shown better efficacy in clinics in the prevention and treatment of AS and its related diseases(10). Meanwhile, the pathological changes such as inflammatory cell infiltration and increased levels of inflammation markers in the AS process have similar presentations with toxin and blood stasis. According to the CM theory of blood stasis and pathogenic toxins, we made the experimental carotid artery thrombosis rat model using Kurz's method, tried to imitate the pathological process of disease due to blood stasis combined with understanding of the etiology and pathogenesis of blood stasis and the pathogenic toxin of CM, and compared the therapeutic effects of Chinese drugs for activating blood circulation to that for activating blood circulation and detoxifying. The results showed that the serum levels of GMP-140, hs-CRP, IL-6, and MMP-9 increased and the serum level of t-PA decreased in the model group compared with those in the sham operation group.

XSC is, based on experience, an effective prescription for the treatment of coronary heart disease in our hospital, whose composition is based on the classic prescription of Xuefu Zhuyu Decoction (血府逐瘀汤) and its modern pharmacological study. The capsule is composed of the effective part of Chuanxiong total phenols and red peony glycoside. HLC is a heat-clearing and detoxifying herb, so we used XSC as the therapeutic drug of ABC and used XSC and HLC as the therapeutic drugs of ABCD. Compared with the model group, the levels of serum

Table 3. Comparison of the Serum Levels of MMP-9 and TIMP-1 among Groups (pg/mL, ±s)

Group n MMP-9 TIMP-1

Sham operation 10 228.12±74.51 272.83±55.11

Model 10 466.05±173.15 284.25±78.27

Simvastatin 10 341.17±123.71 305.94±75.20

ABC 10 336.84±88.34 328.79±107.78

ABCD 10 334.47±110.16 298.07±101.75

Notes: P<0.05, P<0.01, compared with the model group

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GMP-140, hs-CRP, IL-6 and MMP-9 in ABC group and ABCD group were significantly reduced (P<0.05), while the level serum of hs-CRP in ABCD group decreased more significantly compared with that in ABC group (P<0.05).

GMP-140, a member of the selectin family, can be only expressed on the surface of the platelet after degranulation, which is the specific marker for late-stage platelet activation and one of the micro-cosmic indicators for blood stasis syndrome(11). t-PA is mainly secreted by vascular endothelial cells, which can reflect the state of fibrinolysis and coagulation. AS is a chronic inflammation of the endarterium, and CRP is closely related with cardiovascular diseases such as acute coronary syndrome, while hs-CRP may be a predictor for the risk of cardiovascular disease. IL-6 has extensive biological activities, and plays many biological roles in inflammatory reaction, anti-infection and injury(12). In our study, the results showed that platelets and the coagulation system could be activated after FeCl3 injured the tunica intima, increased the expression of GMP-140 and formed the obstructive thrombus in the injured local tissue. Thus, the content of t-PA in the blood decreased and the thrombosis model of "blood stasis" was formed. The increase of serum hs-CRP and IL-6 levels compared with that in the sham operation group just confirmed an inflammatory response.

XSC used alone or combined with HLC can reduce the serum levels of GMP-140, hs-CRP, IL-6 and MMP-9 and increase the serum level of t-PA, so as to inhibit platelet activation and relieve inf lammation. XSC combined with HLC could decrease the serum level of hs-CRP than either used alone, which showed that hs-CRP might be one of the sensitive indicators for evaluation of the therapeutic effect of ABCD drugs. The relative balance between MMP and TIMP plays an important role in many physiological and pathological conditions, such as infection, autoimmune reactions and hypoxia/ischemia etc(13). In this study, the serum level of MMP-9 could be decreased in the simvastatin, ABC and ABCD groups compared with that in the model group, but the level of serum TIMP-1 showed no significant difference among the groups, which may be related to blood samples drawing time after modeling, and should be explored in our future study.

REFERENCESPaoletti R, Gotto AM Jr, Hajjar DP. Inflammation in

atherosclerosis and implications for therapy. Circulation

2004; 109(23 SuppI 1): Ⅲ 20-26.

Zouridakis E, Avanzas P, Arroyo-Espliguero R, Fredericks

S, Kaski JC. Markers of inflammation and rapid coronary

artery disease progression in patients with stable angina

pectoris. Circulation 2004;110:1747-1753.

Nijm J, Wikby A, Tompa A, Olsson AG, Jonasson L.

Circulating levels of proinflammatory cytokines and

neutrophil-platelet aggregates in patients with coronary

artery disease. Am J Cardiol 2005; 95: 452-456.

Shi DZ, Xu H, Yin HJ, Zhang JC, Chen KJ. Combination

and t ransformat ion o f tox in and b lood s tas is in

etiopathogenesis of thrombotic cerebro-/cardiovascular

diseases. J Chin Integr Med (Chin) 2008;6:1105-1108.

Chen Q, ed. Methodology of research on pharmacology

of traditional Chinese medicine. Beijing: People's Medical

Publishing House; 1996:1103-1105.

Kurz KD, Main BW, Sandusky GE. Rat model of arterial

thrombosis induced by ferric chloride. Thromb Res 1990;

60: 269-280.

Zhang JC, Chen KJ, Zheng GJ, Zhang WG, Shi DZ, Yin

HJ, et al. Regulatory effect of Chinese herbal compound for

detoxifying and activating blood circulation on expression

of NF-κB and MMP-9 in aorta of apolipoprotein E gene

knocked-out mice. Chin J Integr Tradit West Med (Chin)

2007;27:40-44.

Zhang JC, Chen KJ, Liu JG, Zhang WG, Shi DZ, Liu LT, et

al. Effect of assorted use of Chinese drugs for detoxifying

and activating blood circulation on serum high sensitive

C-reactive protein in apolipoprotein E gene knock-out mice.

Chin J Integr Tradit West Med (Chin) 2008;28: 330-333.

Zhou MX, Xu H, Chen KJ, Pan L, Wen C, Liu JG. Effects

of some active ingredients of Chinese drugs for activating

blood circulation and detoxicating on blood lipids and

atherosclerotic plaque inflammatory reaction in ApoE-gene

knockout mice. Chin J Integr Tradit West Med (Chin) 2008;

28:126-130.

Shi DZ, Ma XC, Gao XA. Research overview of the

prevention and treatment of atherosclerosis by prescriptions

for activating blood circulation. J Tradit Chin Med (Chin)

1995;36:433-435.

Chen KJ, Xue M, Yin HJ. The relationship between platelet

activation and coronary heart disease and blood-stasis

syndrome. J Capit Med Univ (Chin) 2008;29:266-269.

Ma XJ, Yin HJ, Chen KJ. Research progress of correlation

between blood-stasis syndrome and inflammation. Chin J

Integr Tradit West Med (Chin) 2007;27:669-672.

Candelario-Jalil E, Yang Y, Rosenberg GA. Diverse

roles of matrix metalloproteinases and tissue inhibitors

of metalloproteinases in neuroinflammation and cerebral

ischemia. Neuroscience 2009;158:983-994.

(Received December 8, 2009)

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