Printer Friendly

Effect of total saponins of "panax notoginseng root" on aortic intimal hyperplasia and the expressions of cell cycle protein and extracellular matrix in rats.

ABSTRACT

Aim of the study: the effect of total saponins of "panax notoginseng root" on aortic intimal hyperplasia and the expressions of cell cycle protein and extracellular matrix in rats

Materials and methods: Sprague-Dawley rats were randomly divided into sham-operated, control, TSPN and atorvastatin group. Rat aorta intima in all groups were injured by insertion of domestic balloon catheter into the aortae except sham-operated rats. Drugs were administrated orally from the second day after vascular injury and continued for 14 days. The injured segments of aortae were collected on the sixteenth day after operation to observe the morphological changes of vascular structure and to examine the expressions of proliferating cell nuclear antigen(PCNA), cyclinDl, cyclinE, collagen I(Col-I), fibronect(FN), matrix metalloproteinase-9(MMP-9) and tissue inhibitor metalloproteinase-1 (TIMP-l). Results: TPNS significantly inhibited the vascular intimal hyperplasia. TPNS significantly lowered the expression of PCNA, cyclinE, cyclinD1, FN and MMP-9. TPNS had no significant impacts on the expression of Col-I and TIMP-1.

Conclusions: Our studies indicated that TSPN could inhibit vessel restenosis after vascular intimal injury, and its mechanisms may be related to the blockage of the excessive proliferation of VSMC, the reduction of ECM protein deposition in the endometrium, and the degradation of ECM protein.

[C] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Total saponins of "panax notoginseng root" Atorvastatin Vascular smooth muscle cell Proliferating cell nuclear antigen Cyclind [D.sub.1] Cycline Extracellular matrix Collagen I Fibronectin Matrix metalloproteinase-9 Tissue inhibitor metalloproteinase-1

1. Introduction

TSPN was the saponins effective component, which was extracted from the dried roots of Panax Notoginseng (Burk.) F.H.Chen. The content of TSPN reached high to 8-12% and was the chief pharmacological active component in the Panax Notoginseng. its main effective components included Ginsenoside [Rg.sub.1]([Rg.sub.1]). Ginsenoside [Rb.sub.1]([Rb.sub.1]), Notoginsenoside [R.sub.1]([R.sub.1]) and so on (Fig. 1). Recent studies suggested that, TSPN had the wider effects of angiocarpysuch as inhibiting thrombosis, expanding peripheral vessels, protecting ischemic myocardial cells, anti-ischemic brain damage, etc (Dong et al. 2003). The cardiovascular effects of TSPN were closely related to its calcium channel blockade action and free radical scavenging (Nah et al. 1995; Xu et al. 2003).

[FIGURE 1 OMITTED]

Restenosis incidence rate after percutaneous transluminal coronary angioplasty (PTCA) remains as high as 30-40% in coronary heart disease (Landau et al. 1994), which seriously affected the long-term curative effects of the therapy. The basic pathological process of restenosis after PTCA involves the tearing and denudation of coronary intima, platelet accumulation and adhesion to injury sites, the proliferation and migration of VSMC from media to intima, and synthesis of extracellular matrix (ECM). Therefore, the excessive proliferation of VSMC and synthesis increases of ECM were the key factors in restenosis occurred after PTCA, and inhibiting the proliferation of VSMC and promoting the clearance of ECM became important measures to attenuate the restenosis happened after PTCA.

As Xia, etc study showed that (Xia et al. 1995), TSPN could inhibit the proliferation of VSMC after carotid artery endothelial denudation in rabbits, which played the role in anti-vessel restenosis. It suggested that TSPN may have the great significance on inhibiting the proliferation of VSMC and vessel restenosis. But the exact mechanism of TSPN on inhibiting the proliferation of VSMC and vessel restenosis had not been still known up to now. This study aimed at investigating the mechanism of TSPN on inhibiting vascular intima hyperplasia by cell cyclin and ECM.

1. Materials and methods

1.1. Experimental materials

1.1.1. Experimental animals

Male adult Sprague-Dawley rats, weighed from 300 g to 350 g, were provided by Experimental Animal Center, Epidemic Prevention Station of Hunan Province, PRC Animals were allowed to drink and eat freely, caged in an environment of 18-20 [degrees]C and 65-70% relative humidity.

1.1.2. Balloon catheters

Balloon catheters were bought from Tianjin Zhongtuo Latex Technology Development Limited Company. Catheters were retrofited by the needle, No. 5 1/2. A domestic 2.0F balloon catheter was assembled with balloon and catheter in the same way as described before (Wu et al. 2008).

1.1.3. Drugs and reagents

The standard preparations such as Ginsenoside [Rg.sub.1] (content: 97.7%, batch number: 0703-200516), Ginsenoside [Rb.sub.1], (content: 98.0%, batch number: 0704-200517), and Notoginsenoside [R.sub.1], (content: 98.0%, batch number: 0745-200508) were bought from Biological Products Analysis Bureau of Ministry of Public Health of China. TSPN was purchased from Guangxi Wuzhou Pharmaceutical Cooperation Limited Company(batch number061119). Ator-vastatin (specifications: 10 mg/tablet) was purchased from Godecke GmbH (Germany) and packaged by Pfizer Pharmaceuticals Ltd.(USA) (batch number: 65837019).

Acetonitrile and methanol were of chromatographic pure grade, and silica gel beamethanol, xylene, brilliant green were of analytical pure grade, which were all provided by LiXin Biotechnology Co., LTD (Changsha, PRC). PCNA immunohistochemical staining kits, Col-I, FN, TIMP-1, MMP-9 and cyclinE polyclonal antibody, DAB Kits and general type of secondary antibody kits, positive control tissue sections were provided by Wuhan Boster Biological Technology Co., LTD (Wuhan, Hubei Province, PRC). Sp-9001 immunohistochemical staining kits, poly-L-lysine were provided by Beijing Zhongshan Golden Bridge Biotechnology Co., LTD (Beijing, PRC). SM[alpha]-actin polyclonal antibody were provided by Santa Cruz Biotechnology, Inc. (America), and cyclin[D.sub.1] monoclonal antibody were provided by Cell Signaling Technology, Inc. (America). Double distilled water was used in all experiments.

1.1.4. Apparatus

ODP-504E chromatographic column (4.6 mm x 250 mm, 5 [micro]m) was manufactured by Asahipak Company (Japan). High Performance Liquid Chromatograph (HPLC) named 2487 Dual Absor-bance Detector was manufactured by Breeze work station, Waters Company (USA). Type MV-265 uv detector was bought from Shimadzu (Japan). Type 20A electronic balance was bought from Precisa Company (Switzerland).

1.1.5. Drug certification and assay of effective contents

1.1.5.1. Drug certification. Chinese Herb, Panax Notoginseng, (Tian Chi or San Chi, Sanqi in Chinese), a perennial herbaceous plant, has been widely used in Asia for over centuries. TSPN was the active component of saponins extracted from the Panax Notoginseng. TSPN, the exact of Panax Notoginseng roots, was collected from Guangxi of China, and identified by drug identification room of Guangxi Wuzhou Pharmaceutical (Group) Limited Company. The original plant were preserved in drug specimen room of Guangxi Wuzhou Pharmaceutical (Group) Limited Company.

After testing, TPNS for the amorphous yellow powder, tasted bitter and slightly sweet, were solubled easily in water (with pH 6.1).

1.1.5.2. Effective contents of TSPN detected by HPLC. Chromatographic condition: chromatographic column: column C18,4.6 mm x 250 mm, 5 [micro]m, wave length: 203 nm, mobile phase: acetonitrile/water(33/67), flow rate: 1 mL [min.sup-1], temperature: 35 [degrees]C, inject volume 10 [micro]l. According to the theoretical plate number, calculation of notoginsenoside [R.sub.1] peak shall not be lower than 4000.

Preparation of control solution: 4.0 mg control article of Ginsenosides [Rg.sub.1], 4.0 mg control article of Ginsenosides [Rb.sub.1], 1.0 mg control article of Notoginsenoside [R.sub.1] weighed precisely was metered volume to 5 ml through methanol. Mixed control solution was made, containing Ginsenoside [Rg.sub.1] 0.8 mg * [l.sup.-1], Ginsenoside [Rb.sub.1] 0.8 * mg[l.sup.-1] and Notoginsenoside [R.sub.1] 0.2 mg * [l.sup.-1].

Preparation of the sample solution: 4.0 mg TSPN weighed precisely was metered volume to 5 ml through methanol.

The control solution and sample solution were assayed respectively under the chromatographic condition mentioned above and then the contents could be calculated. The content of each component was calculated by the common peak retention time (PRT) and peak area ratio(PAR) of the control solution and sample solution. The PRT of Ginsenoside [Rg.sub.1], Ginsenoside [Rb.sub.1] and Notoginsenoside [R.sub.1] respectly were 13.80 min, 20.24 min and 12.68 min. The main ingredients content of TSPN were tested by HPLC, and the characteristic chromatography peak of Notoginsenoside [R.sub.1], Ginsenoside [Rb.sub.1], and Ginsenoside [Rg.sub.1] could be seen clearly, with Ginsenosides [Rg.sub.1] 50.4%, Ginsenosides [Rb.sub.1] 30.9% and Notoginsenoside [R1.sub.1] 12.5% (Fig. 2). Total content of three above reached 93.8%, which were in line with the herbal medicine standars about TSPN promulgated by China's Health Ministry. All of above were in line with the "Chinese Pharmacopoeia" standards (Numbers: WS3-B-3829-98).

[FIGURE 2 OMITTED]

1.2. Animal model

The rat aorta injury model was made as described (Wu et al. 2008): the rats were anesthetized with 10% chloral hydrate and an incision in the middle of neck was made. The left common carotid artery (1-1.5 cm) was isolated and the artery distal end from heart was ligated. After occlusion of the proximal end of the left common carotid artery with an artery clamp, a v-shape incision was made between the ligation and clamp. A 2.0F balloon catheter was inserted from the V-shape incision and the clamp was released. The balloon catheter was then forwarded carefully through aortic arch down to abdominal aorta, with a depth of about 6-7 cm. About 0.2-0.4 ml physiological saline was injected into the catheter to fill the balloon inflated until resistance was felt when pulling the catheter back. Keeping the same resistance, the catheter was pulled back to the aortic arch and then push forward to its position in abdominal aorta where the balloon was filled and the procedure was repeated 6 times. The catheter was then rotated 180[degrees] to make the balloon turned to the opposite side in the artery lumen and the same steps described as above were made. After the catheter was withdrawn from the artery, the common carotid artery was ligated at the proximal end to stop bleeding. The muscle, subcutaneous tissue and skin were sutured separately. Penicillin (200,000 units) was injected intraperitone-ally at the end of the operation and in the following two days. The animals with a failed operation were excluded and 10 to 12 rats in each group were used for next experimental procedures.

Sprague-Dawley rats were randomly divided into sham-operated, control, TSPN (100 mg * [kg.sup-1]) and atorvastatin (10 mg * [kg.sup-1]) group. Left common carotid artery was dissociated, but the balloon catheter was not inserted in the sham-operated group. TSPN was made into the solution by distilled water (concentration: 10 mg [ml.sup.-1]) and atorvastatin was made into the suspension by distilled water (concentration: 1 mg [ml.sup.-1]) when used. Drugs or water (in sham-operated and control groups) were administrated by gavage from the second day after operation and one time a day for 7 days. The rats were then weighed and the amount of drugs given were adjusted according to the weights of rats with the dosage of per kilogram unchanged. The adjusted amount of drugs were administrated by gavage for other 7 days.

1.4. Vessel acquisition and indices determination

1.4.1. The method of vessel acquisition

All protocols for animal experiments were approved by Experimental Animal Management Committee of Hunan Province, PRC, and were in line with the requirements of animal ethics. The rats were bled after chloral hydrate anesthesia at the sixteenth day after operation. Three segments (1 cm) of injured thoracic aortae from aortic arch, the manhole of diaphragm and intermediate section were collected. The vessels were placed quickly into frozen tubes, and fixed with 4% paraformaldehyde at 4 [degrees]C for 8 hours. The vessel samples were then gradient dehydrated with ethanol and were embedded in paraffin in vertical orientation.

1.4.2. Determination of vascular morphological indices

3 paraffin embedded aorta segments of each rat aorta specimen were taken out and each segment was sliced discontinuously to get 8 to 10 sections of 5 [micro]m thick. Three sections were selected randomly from each segment of each rat aorta specimen at each time point for examination and photographing after masson staining under microscope. Determinated the morphological index of each vascular section, and calculated the average value as measurements.

The intramembranous area of the internal elastic tunica, intramembranous area of external elastic tunica and cross-section of lumen, the perimeters of internal elastic tunica and external elastic tunica and the lumen were measured with MIAS medical image analysis system. Other indices were calculated based on the above measurements as following: the area of mesolamella = intramembranous area of the external elastic tunica-intra-membranous area of the internal elastic tunica, the area of the intima = intramembranous area of the internal elastic tunica-the area of the lumen, the thickness of the mesolamella = (perimeter of the external elastic tunica-perimeter of the internal elastic tunica)/2[pi], the thickness of the intima = (the perimeter of the internal elastic tunica-the perimeter of the lumen)/2[pi], the hyperplasia ratio of the intimal area = the area of the intima/ (area of the intima+area of mesolamella) x 100%, the hyperplasia ratio of intimal thickness = thickness of the intima/(the thickness of the intima+the thickness of the mesolamella) x 100%.

1.4.3. Determination of expressions of PCNA, SM[alpha]-actin, cyclin[D.sub.1], cyclinE, Col-I, FN, MMP-9 and T1MP-1

Each vessel segment of rats was sliced discontinuously to get 8 to 10 sections. Three sections were selected randomly from each vessel segment. The expressions of PCNA, SM[alpha]-actin, cyclin[D.sub.1], cyclinE, Col-I, FN, MMP-9, and T1MP-1 in aortic cells were all detected by immunohistochemical staining as following.

Briefly, the sections were immersed in xylene, 95% alcohol and 80% alcohol for l0 min respectively and washed thrice with PBS (pH 7.4) after each immersion. After protein denature using microwave and non-specific biding blocking with normal goat serum for 20 min at RT, sections were incubated with primary antibodies (diluted as 1:200) against specific antigens overnight at 4[degrees]C. The sections were washed thrice with PBS and incubated with relevant secondary antibody for 20 minutes at 37 [degrees]C. The sections were again washed thrice with PBS and then incubated for 20 minutes with SABC. After further three time wash with PBS, the sections were incubated with 3,3-di-aminobenzidine (DAB) for 3-5 min, and the reaction was stopped the reaction by washing in PBS. At last 8-10 sections were counterstained with hematoxylin.

Positive tissue sections, provided by the Wuhan Boster Biological Technology Co., LTD, were used as positive controls and IgG was used instead of first antibodies as negative controls. The observable brown yellow particles appeared within the cells with microscope indicate positive expression of the protein molecules assessed. The immunohistochemical staining results were analyzed using image analysis system and a gray value was used to measure the expression levels of protein molecules assessed. The immunohistochemical staining results were analyzed using image analysis system and a gray value was used to measure the expression levels of protein molecules assessed. Three visual field of the vascular hyperplastic intima in each vessel slice were chosed to determinate, and took the average gray value of every vision of each section of as measurements. A lower gray value indicates lower expression of protein molecules stained. PCNA expression was taken as a measurement to evaluate the proliferation of VSMC, SM[alpha]-actin expression was used for identifying the cell types, the cyclin[D.sub.1] and cyclinE expression were used as markers of cell cycle status in vascular hyperplasia tissue, and the Col-I, FN, MMP-9 and TIMP-1 expression were used as evaluating ECM protein deposition in hyperplasia intima.

1.5. Statistical analysis

All data were analyzed using SPSS13.0 statistical package (SPSS incorporated, Chicago) and the values were expressed as mean [+ or -] SD. Log transition was made in the case that data were not complying with normal distribution and homogeneity of variance. One-way analysis of variance (ANOVA) was used for the data comparison in multiple groups and LSD test was used for the comparison between each group. Values of P<0.05 were considered statistically significant.

2. Results

2.1. Comparisons of vascular morphological indices in every group

Compared with the sham-operated group, the area and the thickness of intima, hyperplasia ratio of intimal area and thickness, were significantly higher in all other groups (P<0.01). All of above indices in TSPN group were were significantly lower than that in the control group (P<0.05), and so were than that in the atorvastatin group (P<0.01). But there were no differences between TSPN group and atorvastatin group (P>0.05) (Table 1).
Table 1
Measurable indices of vascular morphology from different groups
(x[+ or -]s)

groups               n   intima area ([mu][m.sup.2])

Sham-operated group  10     0.00 [+ or -] 0.00
Control group        10  4173.75 [+ or -] 1804.88 [DELTA][DELTA]
TSPN group           10  2910.45 [+ or -] 733.43 [DELTA][DELTA] *
Atorvastatin group   10  2369.25 [+ or -] 788.19 [DELTA][DELTA]**

groups               intima thickness ([mu].m)

Sham-operated group  0.00 [+ or -] 0.00
Control group        5.01 [+ or -] 2.11 [DELTA][DELTA]
TSPN group           3.50 [+ or -] 1.11 [DELTA][DELTA]*
Atorvastatin group   2.92 [+ or -] 1.07 [DELTA][DELTA] **

groups               hyperplasia ratio of intimal) area (%)

Sham-operated group   0.00 [+ or -] 0.00
Control group        27.00 [+ or -] 9.00 [DELTA][DELTA]
TSPN group           20.00 [+ or -] 5.00 [DELTA][DELTA]*
Atorvastatin group   17.00 [+ or -] 5.00 [DELTA][DELTA] **

groups               hyperplasia ratio of intimal thickness (%)

Sham-operated group   0.00 [+ or -] 0.00
Control group        29.00 [+ or -] 10.00 [DELTA][DELTA]
TSPN group           21.00 [+ or -] 5.00 [DELTA][DELTA]*
Atorvastatin group   18.00 [+ or -] 5.00 [DELTA][DELTA]**

[DELTA] P < 0.05 vs. Sham-operated group. [DELTA][DELTA] P < 0.01
vs. Sham-operated group, * P < 0.05 vs. Control group, **P < 0.01
vs. Control group.


2.2. Comparison of PCNA and SM[alpha]-actin expression among the groups in hyperplasial intima

No PCNA positive cells were observed in sham-operated group. A large number of positive cells were observed, and PCNA expression in the neotenic intima and mesolamella increased statistically in the control group. Compared with the sham-operated group, the expression of PCNA in the control group increased significantly (P<0.01). The expressions of PCNA in TSPN and atorvastatin groups were much higher than that of the sham-operated group (P<0.01-P<0.05), but lower than that of the control group (P<0.01). There were no differences between TSPN group and atorvastatin group (P>0.05). (Figs. 3a and 4a)

[FIGURE OMITTED]

SM[alpha]-actin, the specific biomarkers of VSMC, was used to identify the cell type in neotenic intima. The SM[alpha]-actin expression in the hyperplasial intima was detected by immunohistochemical staining. No SM[alpha]-actin positive cells were observed in intima of sham-operated group, but a large number of SM[alpha]-actin positive cells were found in the neointima of vessel with intimal hyperplasia, suggesting that VSMC was a major component in neotenic intima of injured vessel (Fig. 3b).

2.3. Comparison of cyclin[D.sub.1] expression among the groups in hyperplasial intima

No cyclin[D.sub.1]-positive cells could be observed in the sham-operated group. Lots of positive cells were observed, and the expression of cyclin[D.sub.1] inhanced significantly in the control group compared with the sham-operated group (P<0.01). The expressions of cyclin[D.sub.1] in the TSPN and atorvastatin group were markedly higher than that of sham-operated group (P<0.01-P<0.05), while the expressions of cyclin[D.sub.1] in TSPN and atorvastatin group were much lower than that of control group (P<0.01-P<0.05). But there were no differences between TSPN group and atorvastatin group (P>0.05) (Figs. 3c and 4a)

[FIGURE 4 OMITTED]

2.4. Comparison of cyclinE expression among the groups

There were no cyclinE-positive cells in the sham-operated group. A Lot of cyclinE-positive cells were observed in the control group. The differences of cyclinE expressions between the control group and the sham-operated group were significant statistically (P<0.01). The expression of cyclinE in TSPN group was much lower than that of control group (P<0.05). Although the expression of cyclinE in atorvastatin group was lower than that of control group, the differences between them were not significant statistically(P>0.05). (Figs. 3d and 4a)

2.5. Comparison of collagen I expression among the groups

Compared with the sham-operated group, the expression of collagen I in the control group increased significantly (P<0.01). Although the expression of collagen I in TSPN group was much higher than that of sham-operated group (P<0.01), the differences between the TSPN group and the control group were not significant statistically (P>0.05). The expression of collagen I in atorvastatin group was remarkedly lower than that of control group (P<0.01), and was significantly lower than that of TSPN group (P<0.05). (Figs. 3e and 4b)

2.6. Comparison of FN expression among the groups

The expression of FN in the control group was remarkedly higher than that of the sham-operated group (P<0.01). The expression of FN in the TSPN group was much higher than that of the sham-operated group (P<0.05), but much lower than that of the control group (P<0.05). The expression of FN in atorvastatin group was remarkedly lower than that of the control group (P<0.01). There were no differences between TSPN group and atorvastatin group (P>0.05) (Figs. 3f and 4b).

2.7. Comparison of MMP-9 expression among the groups

The differences in MMP-9 expressions between the sham-operated group and the control group were not significant statistically (P>0.05). The expressions of MMP-9 in the TSPN and atorvastatin group were much higher than those of the sham-operated and control group (P<0.05). The expression of MMP-9 in the atorvastatin group were remarkedly higher than that of TSPN group (P<0.05) (Figs. 3g and 4b).

2.8. Comparison of TIMP-1 expression among the groups

The expression of TIMP-1 in the control group was remarkedly higher than that of the sham-operated group (P<0.05). Although the expressions of TIMP-1 in TSPN and atorvastatin group were much higher than that of the sham-operated group (P<0.01-P<0.05), the differences among TSPN, atorvastatin and control group were not significant statistically (P>0.05). There were no differences between TSPN group and atorvastatin group (P>0.05) (Figs. 3h and 4b).

3. Discussion

In rat aorta injury model by the balloon catheter it was observed that the proliferation of VSMC was obvious after just a few hours of vessel injury and the neointima that covered the injured regions 2-3 weeks later after artery injury mostly came from the proliferating VSMC and deposited ECM protein (Karas et al. 1992). Therefore, inhibiting the over proliferation of VSMC and the over deposition of ECM, even inhibiting intima hyperplasia after injury would be the effective methods to preventing restenosis after PTCA.

The effect of TSPN and atorvastatin on vascular intimal hyperplasia were comparativeluy studied on the vessel restenosis model established by denuding the arterial endothelium with domestic-made balloon catheter in rats. The results showed that, the vessel restenosis was induced by vascular intimal hyperplasia on the sixteenth day after de-endothelialization. Atorvastatin decreased significantly the measurable indices of vascular morphology such as the area and the thickness of intima, the proliferous ratio of intimal area and thickness. It suggested that atorvastatin had the significant inhibition on vascular intimal hyperplasia. TPNS also inhibit vascular intimal hyperplasia, which showed that TPNS had the same suppressive effect.

PCNA is an important nucleoprotein in DNA synthesis, whose synthesis and expression are associated with cell proliferation. PCNA appears at the late stage of [G.sub.1] phase, reaches its peak at the end of S phase and declines at [G.sub.2] phase. There is no expression of PCNA at [G.sub.o] and M phase. PCNA is a specific marker of S phase in cell cycle(Suzuki et al. 2000), and a sensitive index to reflect cell proliferation.

In our study, it was found SM[alpha]-actin expressed strongly positive in the hyperplasia intima, further illustrated VSMC mainly occupied in the neointima, which suggested that the intimal hyperplasia causd by the proliferation of VSMC for the most. Therefore, detecting PCNA expression in hyperplasia intima mainly reflected the proliferation of VSMC, and the expression level was linearly related to the cell proliferation (Miniati et al. 2000). Our studies showed that vascular intima proliferated obviously and the PCNA expression increased, which suggested the proliferative activity of VSMC enhanced after de-endothelia-lization. Atorvastatin was shown to significantly decrease the PCNA expression in the neointima, suggesting that it had an inhibitory effect on VSMC proliferative activity. TSPN could lower the expression of PCNA in hyperplasia intima, which suggested it had an inhibitory effect on proliferation of VSMC. Inhibiting the proliferation of VSMC after vascular injury may be one of the important machanisms for TSPN and atorvastatin to guard against vascular intimal hyperplasia.

Cell proliferation depends on cell cycle progression orderly initiated by extracellular stimulating signals. As the final common pathway of cell proliferation, cell cycle is successively composed of [G.sub.1], S, [G.sub.2] and M phases. Cell cycle has two check-points to determine going on with proliferating or entering static status for cells, one of which is at the beginning of DNA synthesis and is called [G.sub.1]-S check-point, and the other is at the beginning of mitosis and is called [G.sub.2]-M check-point. [G.sub.1]-S check-point is the action site of growth factors and other extracellular stimulators and seemes more important. [G.sub.1]-S check-point is mainly regulated by a series of cyclins and cyclin-dependent kinase (CDK), which predominantly are cyclinD/[CDK.sub.4], [CDK.sub.6] and cyclinE/[CDK.sub.2]. CyclinD and cyclinE play important roles in the progression from [G.sub.0] to [G.sub.1] phase and from [G.sub.1] to S phase respectively in cell cycle and are the two key regulatory enzymes in cell proliferation (Cheng et al. 2003). Cyclin [D.sub.1] and cyclinE are decisive in the transformation of cells from differentiation state to dedifferentiated status and therefore play critical roles in promoting VSMC proliferation.

In this research it was clearly shown that hyperplastic intima was mainly composed of VSMC, and cyclin [D.sub.1] and cyclinE positive cells were mainly observed in the neointima after vascular intimal injury. It was found that the more remarkable the intima hyperplasia was, the higher the gray value of cyclin expression that reflected the abundance of cyclin positive cells. This correlation suggested that cyclin[D.sub.1] and cyclinE possibly played an important role in the formation of hyperplasia intima. There fore, cell circle of VSMC was quickly activated after vascular intimal injury and VSMC entered the proliferative status. The cyclin[D.sub.1]] expression was decreased significantly in atorvastatin group, suggesting that atorvastatin may exhibit its inhibitory activity on VSMC proliferation by blocking the cell circle progression from [G.sub.0] to [G.sub.0] phase. The fact that atorvastatin did not inhibit the expression of cyclinE in hyperplasia intima suggested that atorvastatin probably played its inhibitory role only in early stage of cell cycle in cell proliferation. TSPN could inhibit the expressions of both cyclin[D.sub.1] and cyclinE in the neointima. These results illustrated that TSPN may probably inhibit the first step and the course especially from [G.sub.1] into S stage of cell cycle, thus further inhibiting the proliferation of VSMC.

VSMC was transformed from differentiated state into dediffer entiated state after PTCA. VSMC proliferation and the deposition of newly synthesized ECM proteins by differentiated VSMC and other vascular cells all contributed to the formation of neointima. The synthesized ECM proteins included collagen, FN and LN, and collagen was one of the main ingredients, especially type 1 and III collagen. The cellular components accounted for about 11% and the remains were ECM in the neointima of restenosis model. A lot of collagen, FN and so on were deposited in hyperplasial intima, which benefited to the proliferation and migration of VSMC (Schwartz et al. 1992).

This study showed that the expressions of Col-I and FN in the hyperplasial intima enhanced obviously after balloon injury, and atorvastatin had inhibitory effects on potentiation about the expressions of collagenland FN. TSPN could also inhibit the expression of FN in the hyperplasial intima. All results suggested both atorvastatin and TSPN inhibited the proliferation and migration of VSMC through suppressing the expression and deposition of ECM after intimal injury, futher inhibited vascular intimal hyperplasia. The study also showed that TSPN could lowered the expression of FN, but atorvastatin lowed not only the expression of FN but also the expression of Col-I, which suggested the inhibitory effect of atorvastatin on the expression and deposition of ECM were stronger than that of TSPN.

In the degradation and synthesis of ECM, matrix metalloproteinase(MMPs) played an important role. MMPs have been found more than 20 at present, among which gelatinase including MMP-2, MMP-9 and so on played important roles in the synthesis and degradation of the collagen. Under normal circumstances, the metabolism of MMPs were inhibited by the endogenous tissue inhibitor of metalloproteinase(TIMPs), and TIMP-I, TIMP-2, TIMP-3, TIMP-4 have been found in all till now. TIMPs all could non-covalently link with MMPs of biological activity at the 1:1 molecular ratio in order to inhibit the effect of MMPs. Apart from restricting the migration of VSMC, the excessive expression of TIMPs could also inhibit the proliferation of VSMC and induce its apoptosis after PTCA (Baker et al. 1998).

Southgate, etc. confirmed the activity of [MMP.sub.2] and [MMP.sub.9] increased 3 days after pig coronary artery injury, and the two enzyme activity continued to increase to 21 days (Southgate et al. 1996). Collagen components reduced to 33 percent the first week after Iliac artery balloon injury in rat when MMPs inhibitor was applicated, and the neointimal formation were alleviated (Mason et al. 1999).

Our study showed that the expression of MMP-9 did not increase significantly 16 days after injury, but the expression of TIMP-1 enhanced significantly. These were different from the findings of Southgate, which may relate to different time point we chose, ft reflected that the neointimal had been formated 16 days after the injury. Though the expression of MMP-9 did not enhanced, TIMP-1 expressed reaction-enhancedly. The degradation and removal of ECM were weakened and the local deposition of ECM were promoted on the vascular injury point. Atorvastatin and TSPN promoted the expression of MMP-9, which suggested atorvastatin and TSPN may enhance the activity of MMP-9 in order to facilitate the clearance and degradation of ECM, then reduce the degree of neointimal formation. Atorvastatin and TSPN had been no significant impact on the expression of TIMP-1, which may show that the relationship were small between the two drugs for the inhibition of intimal hyperplasia and the change of TIMP-1.

Acknowledgment

This study was supported by a grant from the National Natural Science Foundation of China (No. 30572301).

References

Baker, A.H., Zaltsman, A.B., George, S.J., Newby, A.C., 1998. Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis. The Journal of Clinical Investigation 101 (6), 1478-1487.

Cheng, Y., Liu, P., Chen, H., Zeng, F., 2003. Antiproliferative effects of trapidil in vascular smooth muscle cells are associated by inhibition of MAPK and P34(cdc2) activity. Journal of Cardiovascular Pharmacology 35 (1), 1-6.

Dong, T.X., Cui, X.M., Song, Z.H., Zhao, K.J., Ji, Z.N., Lo, C.K., Tsim, K.W., 2003. Chemical assessment of roots of Panax notoginseng in China: regional and seasonal variations in its active constituents. Journal of Agricultural and Food Chemistry 51 (16), 4617-4623.

Karas, S.P., Gravanis, M.B., Santoian, E.C., Robinson, K.A., Anderberg, K.A., King, S.B., 1992. Coronary intimal proliferation after balloon injury and stenting in swine: an animal model of restenosis. Journal of the American College of Cardiology 20(2), 467-474.

Landau, C, Lange, R.A., Hillis, L.D., 1994. Percutaneous transluminal coronary angioplasty. The New England Journal of Medicine 330 (14), 981-993.

Mason, D.P., Kenagy, R.D., Hasenstab, D., Bowen-Pope, D.F., Seifert, R.A., Coats. S., Hawkins, S.M., Clowes, A.W., 1999. Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery. Circulation Research 85 (12), 1179-1185.

Miniati, D.N., Hoyt, E.G., Feeiey, B.T., Poston, R.S., Robbins, R.C., 2000. Ex vivo antisense oligonucleotides to proliferating cell nuclear antigen and [Cdc.sub.2] kinase inhibit graft coronary artery disease. Circulation 102 (19 (Suppl. 3)), III237-242.

Nah, S.Y., Park, H.J., McCleskey, E.W., 1995. A trace component of ginseng that inhibit [Ca.sup.2+] channels through a pertussis toxion-sensitive G protein. Proceedings of the National Academy of Sciences of the United States of America 92 (19), 8739-8743.

Schwartz, R.S., Holmes, D.R,, Topol Jr., E.J., 1992. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. Journal of the American College of Cardiology 20 (5), 1284-1293.

Southgate, K.M., Fisher. M., Banning, A.P., Thurston, V.J., Baker, A.H., Fabunmi, R.P., Groves, P.H., Davies, M., Newby, A.C., 1996. Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries. Circulation Research 79 (6), 1177-1187.

Suzuki, J., Isobe, M., Morishita, R., Nishikawa, T., Amano, J., Kaneda, Y., 2000. Prevention of cardiac allograft arteriosclerosis using antisense proliferating-cell nuclear antigen oligonucleotide. Transplantation 70 (2), 398-400.

Wu, L, Zhang, W., Deng, C.Q., 2008. Characteristics and feasibility of vessel restenosis models produced by denuding arterial endothelium with domestic-made balloon catheter in rats. Journal of Clinical Rehabilitative Tissue Engineering Research 12 (17), 3372-3375.

Xia, H., Li, G.S., Xu, J.L., 1995. Effects of total spaonins of panax notoginseng on c-myc gene expression and proliferation of rabbit carotid smooth muscle following endothelial removal. Chinese Journal of Interventional Cardiology 5 (1), 45-47.

Xu, Q.F., Fang, X.L., Chen, D.F., 2003. Pharmacokinetics and bioavailability of ginsenoside [Rb.sub.1] and [Rg.sub.1] from Panax notoginseng in rats. Journal of Ethnopharmacology 84 (2-3), 187-192.

Lu Wu (a), Wei Zhang (b), Ying-Hong Tang (c), Hua Li (d), Bei-Yang Chen (d), Guo-Min Zhang (d), Chang-QingDeng (b), *

(a) The Second Affiliated Traditional and Western Medicine Hospital of Hunan University of Traditional Chinese Medicine, Liuyang, Changsha, Hunan, China

(b) Pathophysiology Laboratory, Hunan University of Traditional Chinese Medicine, Changsha, Hunan, China

(c) Department of Pharmacology, Hunan University 0f Traditional Chinese Medicine, Changsha, Hunan, China

(d) Department of Pathology, Hunan University of Traditional Chinese Medicine, Changsha, Hunan, China

* Corresponding author. School of Integrated Chinese and Western Medicine, Hunan University of Traditional Chinese Medicine, Xiangzui Road, Hanpu, Yuelu

County, Changsha, China. Tel.: +867318458258; fax: +867318458257.

E-mail address: dchangq@yahoo.cn (C.-Q. Deng).

0944-7113/$-see front matter [C] 2009 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2009.07.021
COPYRIGHT 2010 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2010 Gale, Cengage Learning. All rights reserved.

 
Article Details
Printer friendly Cite/link Email Feedback
Author:Wu, Lu; Zhang, Wei; Tang, Ying-Hong; Li, Hua; Chen, Bei-Yang; Zhang, Guo-Min; Deng, Chang-Qing
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:1USA
Date:Mar 1, 2010
Words:5863
Previous Article:Preventive effect of crocin of Crocus sativus on hemodynamic, biochemical, histopathological and ultrastuctural alterations in isoproterenol-induced...
Next Article:Aged garlic extract delays the appearance of infra area in a cerebral ischemia model, an effect likely conditioned by the cellular antioxidant...
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters