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ffects of Panax notoginseng saponins on proliferation and apoptosis of vascularmooth muscle cells
i Xua,b, Jun-tian Liua,∗, Na Liuc, Pei-pei Lua, Xiao-ming Panga
Department of Pharmacology, Xi′an Jiaotong University School of Medicine, Xi′an 710061, ChinaDepartment of Pharmacy, the Affiliated Hospital of Xi′an Medical College, Xi′an 710061, ChinaDepartment of Pharmacy, the Second Affiliated Hospital of Xi′an Jiaotong University School of Medicine, Xi′an 710004, China
r t i c l e i n f o
rticle history:eceived 13 October 2010eceived in revised form 10 May 2011ccepted 11 May 2011vailable online 17 May 2011
Aim of the study: Atherosclerosis is a common cardiovascular disease, and linked with the developmentof many cardiovascular complications, such as myocardial ischemia and stroke. Although pathogene-sis of atherosclerosis is not completely elucidated, increasing evidence has demonstrated that abnormalproliferation of vascular smooth muscle cells (VSMCs) plays an important role in formation of atheroscle-rosis. Previous studies showed that saponins from Panax notoginseng (PNS) possess anti-atheroscleroticproperties. However, the mechanism of PNS against atherosclerosis is not well understood. Therefore,the present study observed the effects of PNS on proliferation and apoptosis of VSMCs.Materials and methods: Rat VSMCs were cultured, and platelet-derived growth factor (PDGF) was used tostimulate cell proliferation. The viability of VSMCs was assessed with the MTT method. VSMCs apoptosiswas detected by flow cytometry. Expressions of apoptosis related protein p53, Bax, caspase-3 and Bcl-2were determined using Western blot.Results: Pretreatment of the cells with PNS (200, 400, 800 �g/mL) significantly inhibited proliferation ofPDGF-stimulated VSMCs, and induced apoptosis of the proliferated VSMCs in a concentration-dependent
way. Western blot analysis showed that PNS upregulated expressions of pro-apoptotic protein p53, Baxand caspase-3, downregulated expression of anti-apoptotic protein Bcl-2, and enlarged Bax/Bcl-2 ratioin the proliferated VSMCs induced by PDGF.Conclusions: This study demonstrates that PNS both inhibits VSMCs proliferation and induces VSMCsapoptosis through upregulating p53, Bax, caspase-3 expressions and downregulating Bcl-2 expression,which constitute the pharmacological basis of its anti-atherosclerotic action.
. Introduction
Atherosclerosis is one of the common cardiovascular diseases,hich may result in a host of complications like myocardial
schemia, acute coronary syndromes and stroke. Although itsathogenesis has not been completely elucidated, it seems thattherosclerosis results from a complex interplay of multiple fac-ors, including endothelial dysfunction, inflammation, proliferationf vascular smooth muscle cells (VSMCs) and matrix alterationMercer et al., 2007). Among these, abnormal proliferation of
SMCs is a critical factor, which is linked to other cellular pro-esses such as inflammation, apoptosis, matrix alterations, andontributes to the pathogenesis of atherosclerosis-related events
∗ Corresponding author at: Postbox 58, Xi′an Jiaotong University School ofedicine, 76 West Yanta Road, Xi′an 710061, China. Tel.: +86 29 82655188;
like restenosis after percutaneous transluminal angioplasty, in-stent restenosis, transplant vasculopathy and vein bypass graftfailure (Dzau et al., 2002; Zakar and Ken, 2003). Therefore, inhi-bition of the abnormal VSMCs proliferation has been a focus for thetreatment of cardiovascular diseases.
Panax notoginseng (Buck.) F. H. Chen (Araliaceae) is a tradi-tional Chinese herb, which has been used for the treatment ofinflammation, body pains, trauma, internal and external bleed-ing due to injury and circulatory disorders for hundreds of yearsin China. Panax notoginseng saponins (PNS) are the bioactivecomponents extracted from Panax notoginseng. Previous studiesshowed that PNS exerts multiple beneficial effects on cardiovas-cular system, such as improving myocardial relaxation function(Chan et al., 2002a), lowering blood lipids (Cicero et al., 2003),anti-inflammation in atherosclerotic lesion of the aorta (Jia et al.,
2008), protecting arterial endothelium from injury (Chen et al.,2004), blocking Ca2+ influx into VSMCs (Guan et al., 2006), andestrogen-like activity (Chan et al., 2002b). On the basis of the above-mentioned bioactivities, PNS has been recently applied in the
Fig. 1. Effect of PNS on the viability of normal VSMCs. The cells were incubated
L. Xu et al. / Journal of Ethnop
revention and treatment of ischemic cardiocerebrovascular dis-rders including atherosclerosis. Considering the ability of Panaxotoginseng to inhibit proliferation of VSMCs (Wang and Hu, 2006),e observed the effects of PNS on proliferation and apoptosis ofSMCs so as to provide a pharmacological basis for treatment of
he proliferative cardiovascular disorders in the present study.
. Materials and methods
.1. Reagents
PNS was provided by Panax Notoginseng Research Institute ofenshan Prefecture (Yunnan, China), and dissolved in Dulbecco’sodified eagles medium (DMEM). DMEM, fetal bovine serum (FBS),
enicillin, streptomycin were purchased from Gibco (Gaithers-urg, MD, USA). Antibodies against p53, Bax, Bcl-2, caspase-3ere obtained from Cell Signaling (Beverly, MA, USA). Fluores-
ein isothiocyanate-conjugated annexin V (FITC annexin V) andropidium iodide (PI), HEPES, trypsin, antibody against actin ofSMCs, and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumromide (MTT) were from Sigma Chemical (St. Louis, MO, USA).latelet-derived growth factor (PDGF, PDGF-BB) was produced bynD (Minneapolis, MN, USA). ECL kit and BCA protein assay kit wererdered from Pierce (Rockford, IL, USA).
.2. Cell culture
VSMCs were isolated from the thoracic aorta of Sprague–DawleySD) rats as described previously (Griendling et al., 1991), and main-ained in DMEM supplemented with 10% FBS, 100 U/mL penicillinnd 100 �g/mL streptomycin at 37 ◦C in a humidified atmospheref 95% air and 5% CO2. The cells were passaged every 4–7 days.inally, identity of VSMCs was confirmed by the morphologi-al examination, and showed 99% purity as estimated with themmunocytochemical staining for �-actin. The cells at passages 3–6
ere used for the experiments. When the cells were grown to con-uence, the medium was changed to the serum-free medium forn additional 24 h incubation before the experiments.
.3. Assessment of cell viability
Cell viability was assayed by the MTT method. Briefly, VSMCsere seeded in 96-well plates at 104 cells/well. After overnight
ncubation, the cells were starved in the serum-free medium for4 h. Then, the cells were treated with 200, 400, 800 �g/mL PNSr DMEM for control. After 24 h, 20 �L of MTT was added into theedium, and the cells were continuously incubated for a further
h. Next, the medium was removed, and the cells were lysed in50 �L of DMSO with shaking for 10 min. Finally, the absorbance at90 nm was measured with ELISA reader (Thermo, USA). Cell via-ility was expressed as percentage of control. Each group containedix parallel wells, and the experiments were repeated three times.
In another experiment, the cells in drug-treated groups werereincubated with 200, 400, 800 �g/mL PNS for 12, 24 and 48 h,nd the cells in control and PDGF groups were preincubated withMEM instead. Then, the cells were stimulated for 24 h with PDGFt the final concentration of 50 ng/mL except control. Finally, theiability of VSMCs was assayed by the MTT method.
.4. Detection of cellular apoptosis
Apoptosis of VSMCs was detected by flow cytometry. In brief, the
ells were seeded in 6-well plates at a density of 1 × 106 cells/well.fter pretreatment with the same concentrations of PNS or DMEM,
he cells were stimulated with PDGF for 24 h. Then, the cellsere harvested by trypsinization, and washed twice with cold
for 24 h with the different concentrations of PNS or DMEM. Then, cell viability wasassayed by the MTT method. The data represent mean ± S.D. of three experiments.**P < 0.01 vs. control.
PBS (0.15 M, pH 7.2). Next, the cells were centrifuged at 3000 rpmfor 5 min, and resuspended in the binding buffer at a density of1.0 × l06 cells/mL. The cells were incubated with 5 �L of annexinV-FITC and 5 �L of PI for 15 min at room temperature in the dark.The cellular apoptosis were analyzed by FACS (Becton Dickinson)using Cell Quest Research Software (Becton Dickinson).
2.5. Western blot analysis
After the same treatment and stimulation as above, the cellswere harvested by scraping, and lysed with 250 �L of ice-coldlysis buffer containing protease inhibitor cocktail. Protein wasquantified by BCA protein assay kit. Equal amount of proteinextract (40 �g) was loaded, separated by 12% SDS-PAGE, and blot-ted onto nitrocellulose membrane. Then, the membranes wereincubated with primary antibodies (anti-p53, anti-Bcl-2, anti-Bax,anti-caspase-3, anti-�-actin) overnight at 4 ◦C. After washing threetimes, the membranes were incubated with horseradish peroxidase(HRP)-conjugated secondary antibody for 1 h at room temperature.Signals were detected using the enhanced chemiluminescence.Band intensity was quantified by densitometry of immunoblotsusing Quantity One Software.
2.6. Statistical analysis
All values were shown as mean ± S.D. Statistical significancebetween groups was assessed by using one-way ANOVA, followedby modified LSD test (SPSS-software). A value of P < 0.05 was con-sidered to be statistically significant.
3. Results
3.1. Effect of PNS on normal VSMCs viability
As shown in Fig. 1, the viability of VSMCs was slightly decreasedwith the increase of PNS concentrations. Compared with control,400 and 800 �g/mL PNS showed an inhibitory effect (P < 0.01). Thissuggests that higher concentrations of PNS cause a decrease inviable cell number.
3.2. Effect of PNS on PDGF-stimulated proliferation of VSMCs
As seen from Fig. 2, PDGF at concentration used in the exper-iment significantly increased the viability of VSMCs (P < 0.01 vs.
228 L. Xu et al. / Journal of Ethnopharmacology 137 (2011) 226– 230
Fig. 2. Effect of PNS on the viability of PDGF-stimulated VSMCs. The cells were pre-treated with the different concentrations of PNS or DMEM for 12, 24 and 48 h, andsubsequently stimulated for 24 h with 50 ng/mL PDGF except control. Then, cellve
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Fig. 4. Effects of PNS on p53 expression in the proliferated VSMCs induced by PDGF.The cells were pretreated for 24 h with the different concentrations of PNS or DMEM,and subsequently stimulated for 24 h with 50 ng/mL PDGF except control. Then,protein expression of p53 was determined by Western blot. The data represent
iability was assayed by the MTT method. The data represent mean ± S.D. of threexperiments. **P < 0.01 vs. PDGF alone.
ontrol), revealing that PDGF stimulates proliferation of VSMCs.ut, pretreatment of the cells with PNS markedly reduced the celliability in time- and concentration-dependent manners (P < 0.01s. PDGF alone) and EC50 was 676, 297, 252 �g/mL for 12, 24,8 h pretreatment, demonstrating that PNS is able to inhibit PDGF-timulated proliferation of VSMCs.
.3. Effect of PNS on apoptosis of the proliferated VSMCs inducedy PDGF
Fig. 3 showed that apoptosis ratio of VSMCs was not significantlyhanged in comparison to control after the cells were exposed toDGF for 24 h. However, pretreatment of the cells with 200, 400nd 800 �g/mL PNS increased the ratio of apoptotic cells to 20.2%,3.4%, and 34.5% respectively from 9.8% in the PDGF-treated groupP < 0.05 or 0.01 vs. PDGF alone), confirming that PNS is capable of
romoting apoptosis of the proliferated VSMCs.
ig. 3. Effect of PNS on apoptosis of the proliferated VSMCs induced by PDGF. Theells were pretreated for 24 h with the different concentrations of PNS or DMEM,nd subsequently stimulated for 24 h with 50 ng/mL PDGF except control. Then,ell apoptosis was assayed by annexin V-FITC and PI staining, and analyzed withow cytometry. The results were expressed as apoptosis ratio. The data representean ± S.D. of three experiments. *P < 0.05 and **P < 0.01 vs. PDGF alone.
mean ± S.D. of three experiments. *P < 0.05 and **P < 0.01 vs. PDGF alone.
3.4. Effects of PNS on expressions of p53, Bax and caspase-3 in theproliferated VSMCs induced by PDGF
To investigate the signal pathway involved in PNS-induced apo-ptosis of VSMCs, expressions of pro-apoptotic protein p53, Bax andcaspase-3 in the proliferated VSMCs were assessed with Westernblot. The results in Figs. 4–6 displayed that protein expressionsof p53, Bax, and caspase-3 in PDGF-treated VSMCs were similarto control. But, preincubation of the cells with PNS significantlyupregulated protein expressions of p53, Bax, and caspase-3 in theproliferated VSMCs in a concentration-dependent manner (P < 0.05or 0.01 vs. PDGF alone).
Fig. 5. Effects of PNS on Bax expression in the proliferated VSMCs induced by PDGF.The cells were pretreated for 24 h with the different concentrations of PNS or DMEM,and subsequently stimulated for 24 h with 50 ng/mL PDGF except control. Then,protein expression of Bax was determined by Western blot. The data representmean ± S.D. of three experiments. **P < 0.01 vs. PDGF alone.
L. Xu et al. / Journal of Ethnopharmacology 137 (2011) 226– 230 229
Fig. 6. Effects of PNS on caspase-3 expression in the proliferated VSMCs induced byPDGF. The cells were pretreated for 24 h with the different concentrations of PNS orDTr
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Fig. 8. Effects of PNS on Bax/Bcl-2 ratio in the proliferated VSMCs induced by PDGF.The cells were pretreated for 24 h with the different concentrations of PNS or DMEM,and subsequently stimulated for 24 h with 50 ng/mL PDGF except control. Then, pro-
MEM, and subsequently stimulated for 24 h with 50 ng/mL PDGF except control.hen, protein expression of caspase-3 was determined by Western blot. The dataepresent mean ± S.D. of three experiments. **P < 0.01 vs. PDGF alone.
.5. Effects of PNS on Bcl-2 expression in the proliferated VSMCsnduced by PDGF
To further probe the mechanism of PNS-induced VSMCs apo-tosis, expression of anti-apoptotic protein Bcl-2 in the proliferatedSMCs was also detected. As indicated in Figs. 7 and 8, stimulationf the cells with PDGF did not lead to a significant change of Bcl-2rotein expression in the proliferated VSMCs and Bax/Bcl-2 ratioompared with control. However, PNS obviously downregulatedcl-2 protein expression in the proliferated VSMCs, and enlargedax/Bcl-2 ratio (P < 0.01 vs. PDGF alone).
. Discussion
Atherosclerosis is a proliferative vascular disease, and its devel-pment is regulated by a large number of growth factors, cytokines
ig. 7. Effects of PNS on Bcl-2 expression in the proliferated VSMCs induced by PDGF.he cells were pretreated for 24 h with the different concentrations of PNS or DMEM,nd subsequently stimulated for 24 h with 50 ng/mL PDGF except control. Then,rotein expression of Bcl-2 was determined by Western blot. The data representean ± S.D. of three experiments. **P < 0.01 vs. PDGF alone.
tein expressions of Bax and Bcl-2 were determined by Western blot, and Bax/Bcl-2ratio was calculated. The data represent mean ± S.D. of three experiments. **P < 0.01vs. PDGF alone.
and vasoactive substances. In response to multiple stimuli, VSMCsin the arterial media are activated, and subsequently migrate toand proliferate in the intima, which seem to be early steps inthe formation of atherosclerosis (Rodriguez-Campos et al., 2001).Until recently, VSMCs have been viewed as directly responsible forgenerating the atherosclerotic plaque via proliferation and syn-thesis of matrix proteins in the intima. So, inhibition of VSMCsproliferation is an important therapeutic strategy for atheroscle-rosis. In this study, we found that PNS significantly inhibitedproliferation of PDGF-stimulated VSMCs, which contributes to itsanti-atherosclerotic action.
Excessive accumulation of VSMCs in intima and media ofatherosclerotic lesion involves both the abnormal proliferationand the reduced apoptosis of the cells. Alteration of the balancebetween proliferation and apoptosis of VSMCs is thought to playan important role in atherosclerosis formation and subsequent car-diovascular complications (Hsieh et al., 2000; Clarke and Bennett,2006). Therefore, modulation of the balance between proliferationand apoptosis of VSMCs has been proposed as an effective thera-peutic method for prevention and treatment of vascular diseasesincluding atherosclerosis (Meiners et al., 2002). The result in thepresent study indicated that PNS was able to promote apoptosis ofthe proliferated VSMCs, which may relieve the pathological prolif-eration of VSMCs observed in atherosclerosis.
In order to probe the mechanism of PNS-induced apoptosisof VSMCs, we further examined expressions of apoptosis relatedproteins, including p53, Bcl-2, Bax and caspase-3. In response tocellular stress or DNA damage, p53 binds to specific sequencesin DNA to initiate transcription of genes that induces apoptosis(Kim et al., 2003). Caspase activation also involves p53 (Hauptet al., 2003; Yuan et al., 2008). In vitro studies furnish further evi-dence that p53 regulates apoptosis in cultured VSMCs (Mercer et al.,2007) and then, prevents inappropriate growth of VSMCs (Mnjoyanet al., 2003). Previous studies also indicated that p53 deficiencymay lead to enhanced atherosclerosis (van Vlijmen et al., 2001). Inthis study, PNS obviously increased p53 protein expression, imply-ing that PNS induces apoptosis of the proliferated VSMCs via p53pathway.
Bcl-2 plays a central regulatory role to decide the fate of cells via
the interaction between pro- and anti-apoptotic members, suchas Bax and Bcl-2 (Cory and Adams, 2002). The balance of pro-apoptotic Bax and anti-apoptotic Bcl-2 is known to be importantin determining cells death or survival. The Bax/Bcl-2 ratio in cells
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30 L. Xu et al. / Journal of Ethnop
cts to regulate apoptosis (Huang et al., 2003). In addition, Baxlso is a target of p53 (Borner, 2003). Our results showed thatretreatment of VSMCs with PNS increased Bax expression andecreased Bcl-2 expression to result in enlargement of Bax/Bcl-2atio. Increased Bax expression seems to result from p53 accumu-ation. So, regulation by PNS of the balance between Bax and Bcl-2
ay be associated with its pro-apoptotic role in PDGF-stimulatedSMCs.
Upregulation of Bax by activating p53 helps to initiate a cas-ade of events that ultimately lead to cell death through theechanism likely involving caspase activation (Zhu et al., 2006).
aspases, a family of cysteine proteases, is the final execution-rs of apoptosis. Caspase-3 is a key factor in the completion ofpoptosis (Zhu et al., 2006). Upon triggering the apoptosis signalransduction pathway, caspases are activated and contribute tohe degradation of cellular proteins and disassembly of the cellular
atrix (Borner, 2003). In the present experiment, we demonstratedhat PNS enhanced caspase-3 expression in PDGF-stimulatedSMCs, suggesting that caspase-3 is also involved in PNS-inducedpoptosis.
Even though the primary pro-apoptotic mechanism of PNS isrovided in the present experiment, the actual molecular mecha-ism is not well defined. More questions need to be answered, suchs whether PNS affects extrinsic apoptosis pathway, and how PNSnteracts with the apoptotic signal transduction pathway.
. Conclusion
This study demonstrates that PNS has the ability both to inhibitroliferation of VSMCs, and to induce apoptosis of VSMCs. Thero-apoptotic mechanism of PNS involves upregulation of P53,ax, caspase-3 expressions, and downregulation of Bcl-2 expres-ion. These results provide important information needed to betternderstand the effects and mechanisms of PNS as a potentialherapeutic strategy for cardiovascular disorders involving VSMCroliferation and atherogenesis. However, the precise pharmaco-
ogical mechanisms of PNS’s anti-proliferative properties are stillnclear and require further study.
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