Background: Ginseng is the most popular herb used for treatment of ischemic heart diseases in Chinesecommunity; ginsenosides are considered to be the major active ingredients. However, whether ginseno-sides can enhance the coronary artery flow of ischemic heart and, if so, by what mechanisms they do this,remains unclear.Methods: Isolated rat hearts with ischemia/reperfusion injury in Langendorff system were employed forexamining the effect of total ginsenosides (TGS) on coronary perfusion flow (CPF). In addition, humanaortic endothelial cells (HAECs) were used for mechanistic study. Levels of various vasodilative molecules,intracellular calcium concentration ([Ca2+]i), and expressions and activation of proteins involving regu-lation of nitric oxide (NO) signaling pathways in heart tissues and HAECs were determined.Results: TGS dose-dependently and significantly increased CPF and improved systolic and diastolic func-tion of the ischemia/reperfused rat heart, while inhibitors of NO synthase (NOS), soluble guanylate cyclase(sGC), heme oxygenase (HO), cyclooxygenase (COX), and potassium channel abolished the vasodilationeffect of TGS. Positive control verapamil was effective only in increasing CPF. TGS elevated levels of NO and
6-keto-prostaglandin F1�, a stable hydrolytic product of prostacyclin I2 (PGI2), in both coronary effluentsand supernatants of HAECs culturing medium, and augmented [Ca2+]i in HAECs. TGS significantly up-regulated expression of phosphoinositide 3-kinase (PI3K) and phosphorylations of Akt and endothelialNOS (eNOS) as well.Conclusions: TGS significantly increased CPF of ischemia/reperfused rat hearts through elevation of NOproduction via activation of PI3K/Akt-eNOS signaling. In addition, PGI2, EDHF and CO pathways also
asod
partially participated in v
ntroduction
Ischemic heart disease (IHD), a leading cause of human deathn industrialized countries, is caused by reduced blood supply toeart due to coronary pathology such as arthrosclerosis in whichhe blood vessel becomes narrowing (Ferdinandy et al., 2007). Anmportant treatment strategy for IHD is therefore the inductionf dilation in coronary artery, which can restore the blood supplyreperfusion) to the ischemic area of heart and minimize the dam-ge caused by ischemic attack. Vasodilation is mainly induced byhree endothelium-derived factors, i.e. nitric oxide (NO), prosta-
yclin I2 (PGI2) and endothelium-derived hyperpolarizing factorEDHF) (Vanhoutte, 1998). In addition, recent researches have alsoevealed the participation of carbon monoxide (CO) and heme oxy-enase (HO) in regulation of vasodilation and protection of heart
in IHD (Nishikawa et al., 2004; Zipes, 2006; Sun et al., 2008). So, toelucidate underlying action mechanisms of many vasodilative orcardioprotective agents these endogenous vasodilators are oftenemployed.
Ginseng is the most popular and important herbal product usedby Chinese community in daily health care or disease treatment forat least 2000 years. In Western countries, it has been employed asa complementary and alternative medicine or herbal supplementfor several decades. Traditionally and clinically, Chinese medicinedoctors usually employ ginseng to prevent or treat cardiovasculardiseases, such as angina pectoris (myocardial ischemia) and heartfailure. Clinical and laboratory evidences have demonstrated thatthe cardioprotective effect of ginseng against ischemia/reperfusion(I/R) injury of heart is closely in association with vasodilation and
promotion of NO release (Chen et al., 1984; Chen, 1996; Gillis, 1997;Maffei Facino et al., 1999; Li et al., 2001). However, these results onvasodilation induced by ginseng were mainly obtained from exper-iments using aortic rings in vitro. Whether ginseng can dilate thecoronary artery of heart remains unclear. In addition, underlying
echanisms of the vasodilative effect of ginseng remain elusive.n this report, we firstly investigated the direct dilative effect ofotal ginsenosides (TGS), the major bioactive constituents of gin-eng (Attele et al., 1999; Furukawa et al., 2006), to the coronaryrtery in Langendorff rat heart preparation and then explored theotential roles of vasodilators NO, HO, CO, PGI2 and EDHF in vasodi-
ation induced by TGS in the rat hearts or human aortic endotheliumells (HAECs).
ethods and materials
nimals
Male Sprague–Dawley (SD) rats weighing 200–220 g were pur-hased from the Laboratory Animal Services Center of the Chineseniversity of Hong Kong (Hong Kong, China). Animals were housed
our per cage with food and water provided ad libitum andcclimated in the laboratory for at least one week prior to thexperiment. All procedures involving animals and their care werepproved by the Committee on Use of Human & Animal Subjectsn Teaching and Research of the Hong Kong Baptist University andhe Department of Health of the Hong Kong Special Administrationegion.
reparation of total ginsenosides (TGS)
Ginseng, the roots of Panax ginseng, was purchased from aholesale market in Tonghua County of Jilin Province, China. TGSas prepared by refluxing with 70% ethanol followed by column
hromatographic separation with D101 resin (The Chemical Plantf Nan Kai University, China). A total of 17.5 g TGS with good waterolubility was obtained from 1 kg ginseng. To control the qual-ty consistency of TGS, chemical fingerprints (Fig. 1) of TGS werestablished on a Phenomenex ODS column using high performanceiquid chromatography at 203 nm. Twelve ginsenosides, i.e. Rg1, Re,0, malonyl Rb1 (mRb1), malonyl Rc (mRc), malonyl Rb2 (mRb2),alonyl Rd (mRd), Rf, Rb1, Rc, Rb2 and Rd were identified from
GS through comparison of the retention times with authenticompounds. Contents of 12 ginsenosides were 77.56, 71.86, 70.8,4.37, 13.46, 5.82, 14.04, 24.38, 140.19, 93.2, 61.52, and 54.86 mg/g,espectively, accounting for over 66% of TGS.
rug treatment protocols in Langendorff rat heart preparation
Langendorff perfusion system was established according to theethod described by Petzelbauer et al. (2005) with modifications.
n brief, the rat hearts were isolated and retrogradely perfused withrebs–Henseleit (K–H) buffer equilibrated with 95% O2 and 5% CO2
pH 7.4) in Langendorff perfusion system (ADInstruments, Aus-ralia) at a constant coronary perfusion pressure of 65 ± 1 mm Hg at7 ◦C for 20–30 min (stabilization), then perfused at 20 ± 1 mm Hgor 40 min to produce a global low flow ischemia, and finally reper-used at 65 ± 1 mm Hg for 10 min. Left ventricular pressure (LVP)nd coronary perfusion flow (CPF) were recorded by Chart 5.0 soft-are (ADInstruments, Australia). Heart rate, dp/dtmin and dp/dtmax
ere calculated off-line. The hearts were randomly allocated toifferent groups for drug treatment according to the following pro-ocols:
Protocol 1: The hearts were randomly perfused with K–H buffer,GS in K–H buffer at 12.5, 25, or 50 mg/l or verapamil (posi-
ive control) at 0.5 mg/l for 60 min, starting from the last 10 minf stabilization until the end of reperfusion. Coronary effluentsere collected from each heart for a period of 40 s before drug
dministration or after 10 min reperfusion for determination of NOynthase (NOS).
17 (2010) 1006–1015 1007
Protocol 2: The hearts were randomly perfused with K–H bufferor K–H buffer conditioned with some inhibitors during the first20 min of stabilization. These inhibitors include: 100 �M N(G)-nitro-l-arginine methyl ester (l-NAME, an NOS inhibitor) (Pablaand Curtis, 2007), 1.5 �M protoporphyrin IX zinc (ZnPPIX, an HOinhibitor) (Sun et al., 2008), or 10 �M 1-H-[1,2,4] oxadiaxolo [4,3-a]quinolalin-1-one [ODQ, a soluble guanylate cyclase (sGC) inhibitor](Bolognesi et al., 2007), or 25 �M indomethacin [a cyclooxygenase(COX) inhibitor to show the effect of PGI2] (Grbovic and Jovanovic,1997), or 25 mM tetraethyl ammonium chloride (TEA, a potassiumchannel inhibitor to show the effect of EDHF) (Karamsetty et al.,1998). Starting from the last 10 min of stabilization until the endof reperfusion, the hearts were perfused with K–H buffer or TGS(50 mg/l in K–H buffer. For some hearts, the coronary effluentswere collected for a period of 40 s at 10 min after reperfusion fordetermination of PGI2.
At the end of reperfusion in both protocols, the hearts werecollected and the left ventricles were quickly separated, weighed,frozen in liquid nitrogen, and stored at −80 ◦C for bioassays.
Drug treatment protocols in human aortic endothelium cells
Human aortic endothelium cells (HAECs) were obtained asproliferating cultures at the third passage from Cascade Biolog-ics (USA) and cultured in Medium 200 supplement with LSGS(low serum growth supplement, Invitrogen, USA) with a finalconcentration of 2% fetal bovine serum, 1 g/ml hydrocortisone,10 ng/ml human epidermal growth factor, 3 ng/ml basic fibrob-last growth factor and 10 g/ml heparin. The cells were maintainedin a 37 ◦C humidified atmosphere of 95% air/5% CO2 as describedby Welter et al. (2003). The cells passaged for additional fourto six times were used for experimental protocols described asbelow.
Protocol 1: To examine the effect of TGS on HAECs undernormoxic condition, the cells were cultured in 96-well plates(104/well) in the absence or presence of TGS for 48 or 72 h.
Protocol 2: To examine the effect of TGS on HAECs underhypoxic/reoxygenated condition, the cells were cultured in 96-wellplates (104/well) or 100 mm dishes (8 × 105) under normoxic con-dition (95% air/5% CO2) overnight; then the medium was replacedwith fresh medium with or without TGS. After 10 min incuba-tion, the cells were placed under a hypoxic condition producedin a custom-made sealed chamber (850 ml, 16.5 × 10.5 × 6.5 cm3)by flushing with a mixture of 5% CO2–95% N2 for 5 min at a rateof 0.5 l/min to bring the concentration of O2 from 20.1 down to2.1%, showing a hypoxic condition (Bordoni et al., 2002). Afterexposure to the hypoxic condition for 40 min, the cells were reoxy-genated under normoxia for 10 min. At the indicated time points,the medium and cells were collected for bioassays and West-ern blot analysis. Cytotoxicity of TGS on HAECs was examinedby 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay (Gerlier and Thomasset, 1986).
NOS, NO and PGI2 assay
Level of NOS in coronary perfusion effluents was determinedusing an NOS assay kit (Nanjing Jiancheng Bioengineering Insti-tute, Nanjing, China) with a spectrophotometer (Bio-RAD, USA)at 530 nm according to manufacturer’s instructions with modifi-cations as described by Ma et al. (1997). Level of NO in coronaryperfusion effluents and culturing medium of HAECs was measured
using a nitrate/nitrite colorimetric assay kit (Cayman ChemicalCompany, USA) at 540 nm according to manufacturer’s instruc-tions. In addition, level of 6-keto-prostaglandin F1�, a stablehydrolytic product PGI2 that thus services as an indicator of PGI2,in the coronary perfusion effluents and culturing medium of HAECs
1008 X.Q. Yi et al. / Phytomedicine 17 (2010) 1006–1015
F ristic pm d mar
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ig. 1. HPLC fingerprint of total ginsenosides (TGS) derived from ginseng. Charactealonyl Rb2 (mRb2), malonyl Rd (mRd), Rf, Rb1, Rc, Rb2 and Rd were identified an
as measured using an EIA kit (Cayman Chemical Company, USA)t 410 nm according to the manufacturer’s instructions.
easurement of intracellular calcium level in HAECs
To determine the role of calcium in vasodilation induced byGS, intracellular level of calcium ([Ca2+]i) in HAECs was deter-ined by flow cytometry using Fluo-3/AM and Fura-RedTM/AM
ccording to the methods described by Novak and Recht (Novaknd Rabinovitch, 1994; Recht et al., 2004; Lin et al., 2007). In brief,ml of HAECs (105/ml) were cultured in 35 mm dishes for two days,nd then incubated for 35 min at 37 ◦C in fresh medium contain-ng 5 �M Fluo-3/AM, 9 �M Fura-red/AM and 0.04% Pluronic F-127Molecular Probes, USA). The cells were washed once with PBS (pH.4) and then incubated with fresh medium in the absence or pres-nce of TGS under normoxic or hypoxic/reoxygenated conditions.he cells were then washed with PBS once and treated with 0.25%rypsin/EDTA for about 3 min at 37 ◦C. Medium 200 was added totop the reaction. The cells were washed with cold PBS twice ande-suspended to 4 × 105 cells/ml. Analyses were performed on theACS Calibur Flow Cytometer using CellQuest software (BD Bio-ciences, San Diego, USA). Fluo-3 and Fura Red were excited at88 nm with Fluo-3 and Fura Red emission detected at 515–535nd 665–685 nm, respectively. The results were expressed in dotlots.
estern blot analysis
Expressions of the total and/or phosphorylated proteins relatedo vasodilation and cell survival signaling were determined using
estern blot analysis according to the methods described byarton et al. (2001), Uruno et al. (2005), and Sun et al. (2008).
n brief, equal amounts of proteins (approximately 100 �g foreart tissue or 30 �g for HAECs) were separated by 10% SDS-olyacrylamide gel electrophoresis (SDS-PAGE), and then theroteins were electro-transferred onto nitrocellulose membranes.he nitrocellulose membranes were blocked by 5% dried milkn Tris-buffered saline (TBS, 20 mM Tris, 500 mM NaCl, pH 7.5)or 60 min and then incubated with primary antibodies againstndothelial NOS (eNOS), phosphorylated eNOS (p-eNOS), inducible
OS (iNOS), HO-1, HO-2, phosphoinositide 3-kinase (PI3K), p-PI3K,kt, or p-Akt, glucocorticoid receptor (GR), p-GR, or glyceralde-yde 3-phosphate dehydrogenase (GAPDH, as loading control) (BDransduction Laboratories, Assay Designs, Cell Signaling Technol-gy, Santa Cruz Biotechnology, or Upstate, USA) overnight at 4 ◦C.
eaks of TGS, i.e. ginsenosides Rg1, Re, R0, malonyl Rb1 (mRb1), malonyl Rc (mRc),ked at the corresponding peaks in the fingerprint.
The membranes were washed six times with TBS-Tween (TBST,20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20), and thenincubated with horseradish peroxidase conjugated secondary anti-bodies for 60 min. The membranes were washed six times andthe immunoreactive proteins were detected by enhanced chemi-luminescence (ECL) using hyperfilm and ECL reagent (AmershamInternational Plc., Buckinghamshire, UK). The relative optical den-sity of bands was quantified by densitometric scanning of theWestern blots with Quantity One software (version 4.4.1).
Statistical analysis
Data are expressed as mean ± SEM. Statistical analysis was per-formed with one-way ANOVA followed by post hoc test with leastsignificant difference (LSD) method or a Student’s t-test as appro-priate. All analyses were performed with SPSS 15.0 (SPSS Inc.,Chicago, USA). Values were considered as significantly differencewhen P < 0.05.
Results
TGS dose-dependently increased CPF of the isolated rat hearts
The isolated rat hearts in Langendorff system were perfused for10 min under perfusion pressure of 65 mm Hg to establish a base-line of CPF, and then the perfusion was conducted with low flow at20 mm Hg for 40 min to induce global ischemia in the heart tissues.After that, the hearts were further perfused under the pressure of65 mm Hg for 10 min to induce I/R injury. Taken the basal CPF as100%, the CPF in non-treated rat hearts was found about 35% dropafter 10 min perfusion (Fig. 2A and B). In contrast, CPF in TGS orverapamil group was significantly elevated to 115–170% and thehighest elevation was found in 50 mg/l TGS group (Fig. 2A andB). This indicates that TGS can significantly and dose-dependentlydilate the coronary artery and thus increase CPF of the perfusedhearts under normal condition.
During 40-min period of ischemia, CPF in all groups was droppeddown to about 20% of the basal value. However, during the reper-fusion period, CPF in all groups was elevated to some degreesalthough it never recovered to the level before ischemia (Fig. 2A
and B). Interestingly, TGS significantly enhanced the recovery ofCPF in a dose-dependent manner. After 10 min of reperfusion, CPFin non-treated rat hearts was recovered up to only 65% of basalvalue. In contrast, CPF in TGS-treated rat hearts was recovered to100, 130, and 170% of the basal value in the group treated with TGS
X.Q. Yi et al. / Phytomedicine 17 (2010) 1006–1015 1009
Fig. 2. Influence of TGS on coronary perfusion flow (CPF) and cardiac functions of the isolated rat hearts in Langendorff system. (A) Effect and dose-dependence of TGS onCPF, (B) typical CPF diagrams treated with or without TGS and (C–F) effect of TGS and verapamil on LVP, LVEDP, dp/dtmax and dp/dtmin after 10 min reperfusion. Data areexpressed as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control rat hearts.
1010 X.Q. Yi et al. / Phytomedicine 17 (2010) 1006–1015
F uents*
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ig. 3. Effect of TGS on the levels of NO (A), NOS (B) and PGI2 (C) in coronary artery effl*P < 0.01 and ***P < 0.001 compared with the control rat hearts.
t 12.5, 25, and 50 mg/ml, respectively. This elevation of CPF recov-ry in 25 and 50 mg/ml TGS groups was more potent than that ofhe positive control drug, verapamil. These results suggest that, inhe perfused rat hearts after I/R insult, TGS can still significantlynd dose-dependently dilate the coronary artery and thus increasehe CPF.
GS improved systolic and diastolic function of the ischemic ratearts
TGS and verapamil showed different influences on systolic andiastolic function of the ischemic rat hearts although both could
ncrease CPF of the hearts under I/R injury condition. As seen inig. 2C–E, treatment with 50 mg/l of TGS significantly elevated LVPnd dp/dtmax, and decreased LVEDP as compared to the control
earts. In contrast, the positive drug verapamil demonstrated anpposite effect, significant decreases in LVP, dp/dtmax and dp/dtminut marked increase in LVEDP (Fig. 2C–F).
ig. 4. Influence of vasodilation inhibitors on the vasodilative effect of TGS. Iso-ated rat hearts were pretreated with l-NAME (NOS inhibitor, 100 �M), ZnPPIXHO inhibitor, 1.5 �M), ODQ (sGC inhibitor, 10 �M), indomethacin (COX inhibitor,5 �M) or TEA (K+ channel inhibitor, 25 mM) for 20 min before TGS treatment. Datare expressed as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with theontrol hearts. #P < 0.05 and ##P < 0.01 compared with the TGS-treated rat hearts.
of the isolated rat hearts in Langendorff system. Data are expressed as mean ± SEM.
TGS increased levels of NO, NOS and PGI2 in the perfusion effluentsof isolated rat hearts
NO and PGI2 are two major mediators involved in regulation ofcoronary artery dilation. So, we determined levels of NO, NOS and6-keto-prostaglandin F1� (a stable hydrolytic product of PGI2) inthe perfusion effluents of isolated rat hearts in Langendorff systemso as to see whether NO and PGI2 are involved in coronary arterydilation induced by TGS. As shown in Fig. 3A and B, levels of NO andNOS at 10 min of reperfusion in the perfusion effluents of rat heartstreated with 50 mg/l TGS were significantly higher than that of thecontrol rat hearts. In addition, there was a trend that TGS elevatedthe level of PGI2 in the perfusion effluents (Fig. 3C).
Vasodilative effect of TGS was blocked by inhibitors of NO, PGI2,EDHF, sGC and HO
In addition to NO and PGI2, vasodilators of EDHF, sGC and HOare also involved in regulation of coronary artery dilation. To under-stand whether these vasodilators are involved in the vasodilativeaction of TGS in rat hearts, we used inhibitors of NO, PGI2, EDHF,sGC and HO to perfuse hearts before TGS (50 mg/l) treatment andexamined if inhibition of these mediators could block the vasodila-tive effect of TGS. As shown in Fig. 4, all except PGI2 – namely, NO(l-NAME), sGC (ODQ), EDHF (TEA, the inhibitor of potassium chan-nel serving as a down-stream molecule of EDHF) and HO (ZnPPIX)inhibitors – significantly diminished the effect of TGS, showing assuppressed elevation of CPF induced by TGS treatment after 5 and10 min basal perfusion and 5 and 10 min reperfusion. And, PGI2inhibitor indomethacin also diminished the vasodilative effect ofTGS but to a much less extent compared to the extents inducedby other four inhibitors. These results suggest that the vasodilativeeffect of TGS is mainly related to the production of NO, sGC, EDHFand HO in rat heart tissues.
TGS elevated NO and PGI2 levels in HAECs
In order to elucidate the underlying molecular mecha-nisms by which TGS induces vasodilation, in vitro cell culturesusing HAECs were performed. Cytotoxicity of TGS was firstly
examined, the results showed that 3.13–100 mg/ml TGS undernormoxic condition for 72 h (Fig. 5A) and 50 �g/ml TGS underhypoxic/reoxygenated condition for 60 min (Fig. 5B) did not showsignificant cytotoxicity to HAECs. Therefore, 50 �g/ml TGS was usedfor subsequent experiments on HAECs. Under normoxic condi-
X.Q. Yi et al. / Phytomedicine 17 (2010) 1006–1015 1011
Fig. 5. Cytotoxicity test and effect of TGS on the productions of NO and 6-keto-prostaglandin-F1� in human aortic endothelium cells (HAECs) under normoxic orhypoxic/reoxygenated condition. (A) Cell viability of HAECs influenced by TGS under normoxia, (B) cell viability of HAECs influenced by TGS at 50 mg/ml underh m oft lturet
ticP6wm
ypoxia/reoxygenation, (C) dose-dependence of TGS on NO level in culture mediureatment and (D–E) effect of TGS on NO and 6-keto-prostaglandin-F1� levels in cuo the control cells at the same time points. Data are expressed as mean ± SEM.
ion, TGS treatment for 72 h dose-dependently and significantlyncreased NO level of HAECs (Fig. 5C). Under hypoxic/reoxygenated
ondition, TGS caused a time-dependent increase in both NO andGI2 levels (Fig. 5D and E). The increase in NO level induced by0 min treatment of TGS under hypoxic/reoxygenated conditionas obviously higher than that by 72 h treatment of TGS under nor-oxic condition (102% vs. 51%, Fig. 5C and D). Elevation of NO level
HAECs under normoxia. *P < 0.05 and **P < 0.01 compared to the cells without TGSmedium of HAECs under hypoxia/reoxygenation. *P < 0.05 and **P < 0.01 compared
induced by TGS under a hypoxic/reoxygenized condition was grad-ually potentiated from 10 to 60 min and peaked at 60 min (Fig. 5D),
implying that effect of TGS on promotion of NO production inHAECs is stronger in injured cells than in normal cells. TGS-inducedPGI2 level quickly increased after 10 min treatment and then grad-ually declined thereafter under hypoxic/reoxygenated condition(Fig. 5E).
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GS increased intracellular concentration of calcium in HAECs
NO production in endothelial cells has been proven to be con-rolled by intracellular calcium, we therefore determined [Ca2+]in HAECs under normoxic and hypoxic/reoxygenated conditions inhe presence or absence of TGS by flow cytometry using Fluo-3/AMnd Fura-RedTM/AM staining. Fig. 6A shows that under normoxicondition, [Ca2+]i of HAECs treated with TGS at 50 �g/ml for 30nd 60 min were increased by 39 and 33%, respectively. In con-rast, [Ca2+]i was increased by 43% under hypoxic/reoxygenatedondition by 60 min treatment of TGS at concentration of 50 �g/mlFig. 6B), implying that TGS may have stronger influence on [Ca2+]if HAECs under hypoxic/reoxygenated condition than under nor-oxic condition.
GS up-regulated PI3K and p-Akt and p-eNOS expressions
Accumulated evidences have shown that NO signaling playsivotal role in induction of vasodilatation of the heart arter-
es (Vanhoutte, 1998). In the current study, we found that TGSncreased CPF and elevated NO level while inhibitors of NOS, HO,nd sGC suppressed TGS’s effect. We therefore examined the effectf TGS on expression and/or phosphorylation of proteins in NOignaling pathway in HAECs, i.e. GR, PI3K/Akt, eNOS, iNOS and HO.
As shown in Fig. 7, TGS could significantly up-regulate expres-ion of PI3K, p-Akt and p-eNOS compared to the control HAECsnder hypoxic/reoxygenated condition (P < 0.05). However, TGCreatment did not cause significant change in expression of p-PI3K,kt and eNOS as well as other regulator proteins in NO signalingathway, i.e. GR, p-GR, iNOS, HO-1, and HO-2. Similar results werebtained from the isolated rat hearts in Langendorff system (dataot shown). These suggest that TGS-induced NO production andubsequent vasodilation of coronary arteries of rat hearts may pre-ominantly result from up-regulation of PI3K, p-Akt and p-eNOSroteins evoked by TGS treatment.
iscussion
In previous studies, ginsenosides have been proven as the majorioactive components of ginseng, which can prevent heart tis-ues from injury caused by hyperbaric oxygen in rats (Maffeiacino et al., 1999) and reduce deterioration of cardiac contrac-ion and incidence of arrhythmias (Scott et al., 2001). Promotionf NO release from cardiomyocytes and thus produced vasodila-ion are considered as one of the major molecular mechanismsf ginsenosides in cardioprotection (Persson et al., 2006). In addi-ion, activation of calcium-activated potassium channel in vascularmooth muscle cells (Li et al., 2001) and inhibition of influx ofhe excessive calcium to cardiomyocytes (Bai et al., 2004) arenvolved in cardioprotection of ginsenosides. These reports mayartly explain the beneficiary effect of ginseng to heart. However,here is not direct evidence showing the vasodilative effect of gin-enosides on coronary artery. In the current report, we for therst time report the direct vasodilative effect of TGS on coronaryrtery.
In Langendorff preparation, the hearts were perfused underormal pressure (65 ± 1 mm Hg) or low pressure (20 ± 1 mm Hg)onditions to mimic the normal and ischemic situations in clinic.nder these conditions, TGS significantly and dose-dependently
ncreased coronary perfusion flow rate, indicating the dilation of
oronary artery. This suggests that TGS could be beneficial for bothealthy people and patients with ischemic heart condition. Impor-antly, the pattern of CPF recovery in hearts perfused with highose of TGS (25 and 50 mg/ml) is different from that of non-treatedontrol hearts or verapamil treated hearts. The recovery of CPF in
17 (2010) 1006–1015
control hearts was not maintained in 10 min reperfusion period,showing by temporal recovery to about 80% of basal value after5 min reperfusion and then dropping again thereafter (to about70% of basal value after 10 min reperfusion). This implies that thereperfusion could have caused endothelium dysfunction due tonon-timely reperfusion. In contrast, the recovery in TGS-treatedhearts was maintained in 10 min reperfusion period, implying thatTGS treatment have partially reserved the endothelium functionof coronary artery from which an important protection to heartagainst I/R injury is produced. This suggests that TGS treatmentnot only induce vasodilation but also provide protection to hearts.This could also partially explain the improved systolic and diastolicfunction of the ischemic rat hearts treated with TGS, showing aselevation of LVP and dp/dtmax and reduction of LVEDP.
In contrast, positive control drug verapamil showed a deterio-ration in CPF recovery and systolic/diastolic function of the hearts.Verapamil is a calcium channel blocker with vasodilative actionand negative inotropic effect on the heart (Urquhart et al., 1984),which reduce oxygen consumption of the heart but may not ben-efit the systolic and diastolic functions of ischemic hearts (Kolaret al., 1990). Our results imply that TGS and verapamil possess dis-tinct pharmacological properties and advantages for ischemic heartconditions, although both of them were effective in elevating CPFof isolated rat hearts. These results suggest that the clinical benefitsof these two drugs may vary as well.
With respect to molecular mechanisms of vasodilation in hearts,it has been proven that calcium-NO-cGMP, PGI2-cAMP, and EDHF-Ca2+-dependent K+ channels are the major pathways to relax bloodvessels, and that they work through decreasing [Ca2+]i in vascularsmooth muscle cells (Vanhoutte, 1998). Production of NO has par-ticularly benefits to hearts with I/R injury (Bolli, 2000). Our resultsdemonstrated that TGS can effectively elevate NO and NOS levelsin isolated rat hearts and induce NO production and calcium influxin HAECs. On the other hand, NO inhibitor, l-NAME, abolished theeffect of TGS. These data strongly support that NO is involved inTGS-induced vasodilation.
Increasing evidence shows that rapid activation of the GRthrough non-genomic pathway which leads to sequential activa-tions of PI3K, Akt, eNOS, NO production and vasodilation representsan important cardiovascular protective effect of corticosteroidtherapy (Hafezi-Moghadam et al., 2002; Klouche, 2006; Murphyand Steenbergen, 2007). Our current experiments demonstratedthat TGS significantly up-regulated expression of PI3K and phos-phorylation of Akt and eNOS, showing a similar regulation ascorticosteroid does and thus implying that TGS’s protective effectsis produced by augmenting NO production via activation of PI3K,Akt and eNOS. Reports from others on the activation of non-genomic pathways of GR by ginsenosides Rb1, Rg1, and Re supportour speculation (Furukawa et al., 2006; Leung et al., 2006; Nakayaet al., 2007; Yu et al., 2007).
In addition to NO signaling, PGI2 is another endothelium-derived vasodilator and may be involved in the vasodilativeaction of TGS (Pennacchio et al., 1999; Wu et al., 2005). Inthe current study, indomethacin, a COX inhibitor (the up-streammodulator of PGI2) partially abolished the vasodilative effect ofTGS. Furthermore, TGS treatment increased 6-keto-prostaglandinF1� production both in isolated rat hearts and HAECs underischemia/reperfusion conditions. This evidence clearly supports theinvolvement of PGI2 in TGS-induced vasodilation.
EDHF induced coronary artery dilation is related to potassiumchannel (Medhora et al., 2001). Previous report showed that gin-
senosides could activate KCa channels, resulting in relaxation ofisolated aortic rings (Li et al., 2001). Studies also showed that gin-senoside Re induced KCa channels activation in vascular smoothmuscle cells may attribute to NO signaling enhancement throughPI3K/Akt activation.(Nakaya et al., 2007) In our experiments, we
X.Q. Yi et al. / Phytomedicine 17 (2010) 1006–1015 1013
Fig. 6. Effect of TGS on intracellular level of calcium ([Ca2+]i) in HAECs under normoxia and hypoxia/reoxygenation. (A) Time-dependence of TGS on [Ca2+]i in HAECs undernormoxia. [Ca2+]i level was measured by flow cytometry and expressed as dot plots. Area ratio of the fourth quarter (Q4) represents relative [Ca2+]i level and (B) effect of TGSon [Ca2+]i in HAECs under hypoxia/reoxygenation. Data are expressed as mean ± SEM of three independent experiments. *P < 0.05 compared with the control group.
Fig. 7. Effect of TGS on activation of NO and HO signaling pathways. The expression of proteins was normalized to GADPH. Typical blots were shown on top the expressionbar chart of each protein. Data are expressed as mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.05 compared with the control group.
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ound that potassium channel inhibitor TEA partially abolishedGS-induced CPF elevation in isolated rat hearts under both basalnd I/R injury conditions, implying involvement of EDHF and potas-ium channel in TGS’s vasodilative effect.
CO has been found in endothelium and smooth muscle cellsf blood vessels and is important in protection of heart tissuesia vasodilatation. It is degraded from heme by HO that exists inascular endothelium (Nishikawa et al., 2004). After diffused intoascular smooth muscle cells, CO dilates blood vessels by activat-ng sGC, increasing cGMP level and decreasing [Ca2+]i (Zipes, 2006).O can also elevate activity of eNOS which subsequently stimulatesO synthesis (Sun et al., 2008). Our results showed that ZnPPIX andDQ (HO and sGC inhibitors, respectively) partially abolished theffect of TGS in the isolated rat hearts, demonstrating that CO, HOnd sGC participate in the vasodilative and cardioprotective effectf TGS. However, Western blot analysis showed no significant influ-nces on HO-1 and HO-2 expressions by TGS treatment in HAECs.his may be due to the difference between the two model systems.
Taken together, our researches indicates that TGS treatmentan activate PI3K, which in turn phosphorylates Akt and eNOS inequence, and then the activated eNOS catalyzes l-arginine andesults in an increase of NO level in endothelial cells of coronaryrtery. The NO level is further increased due to elevation of [Ca2+]induced by TGS and finally causes vasodilation of coronary artery.ther than NO signaling, we for the first time demonstrate thatGI2, EDHF and CO pathways partially participate in vasodilationnduced by TGS in rat hearts. However, the exact roles and mecha-isms of PGI2, EDHF and CO in this effect need further investigation.
onclusion
In conclusion, we for the first time demonstrated that TGS canlevate coronary perfusion flow of isolated rat hearts in Langen-orff system under either basal perfusion or I/R injury condition,consequence of coronary artery dilation, which also protected
eart tissues from I/R injury. This effect of TGS is dominantly medi-ted by activation of PI3K/Akt-eNOS signaling and NO production.n addition, PGI2, EDHF and CO in some degrees participate in thisffect. The practical implications of this study are that TGS may ben-fit healthy people in preventing ischemia and people who alreadyave an ischemic heart condition.
isclosure statement
The authors declare that no author has conflict of interest.
cknowledgements
This research was funded by the Innovative Technology Fundf Hong Kong (GHP/054/05) and the Faculty Research Grants ofong Kong Baptist University (FRG/06-07/II-32, FRG/07-08/II-71).he authors wish to thank Dr. Martha Dahlen for her English editingo this article and Dr. Shao Zhen Hou and Dr. Bao Zeng to provideechnical supports.
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