Genistein attenuates low temperature induced pulmonary hypertension in broilerchicks by modulating endothelial function
Ying Yang a, Mingyu Gao b, Zhenlong Wu a,⁎, Yuming Guo a,⁎a State key Lab of Animal Nutrition, College of Animal Science and Technology, China Agricultural University (CAU), Beijing, 100193, PR Chinab Department of Basic Veterinary Medicine, College of Veterinary Medicine, CAU, Beijing, PR China
Pulmonary arterial hypertension is characterized by high pulmonary blood pressure, vascular remodeling andright ventricular hypertrophy. In the present study, we investigated whether genistein would prevent thedevelopment of low temperature-induced pulmonary hypertension in broilers. Hemodynamic parameters,vascular remodeling, the expression of endothelial nitric oxide and endothelin-1 content in lung tissue wereevaluated. The results demonstrated that genistein significantly reduced pulmonary arterial hypertension andsuppressed pulmonary arterial vascular remodeling without affecting broilers' performance. The beneficialeffects appeared to be mediated by restoring endothelial function especially endothelial nitric oxide andendothelin-1, two critical vasoactive molecules that associated with the development of hypertension.Genistein supplementation might be a potential therapeutic strategy for the treatment of pulmonaryhypertension.
Pulmonary arterial hypertension is a disease of small pulmonaryarteries characterized by high pulmonary blood pressure, vascularremodeling and right ventricular hypertrophy (Runo and Loyd, 2003;Humbert et al., 2004). The pathogenesis of pulmonary arterialhypertension involves a complex and multi-factorial process inwhich endothelium-derived vasoactive molecules, such as nitricoxide (NO), prostacyclin, endothelin-1 (ET-1), serotonin, and throm-boxane. These molecules have been increasingly recognized as criticalfactors (Siow et al., 2007) and potential therapeutic targets intreatment of pulmonary arterial hypertension (Budhiraja et al.,2004; Siow et al., 2007). Recent studies showed that impaired NOsignaling plays an important role in maintaining the balance betweenendothelial mediators with opposing action on pulmonary vascula-ture during the development of hypertension (Demoncheaux et al.,2005). Restoration of endothelial NO synthase activity prevents orreverses this process in experimental pulmonary arterial hyperten-sion in rats (Kanno et al., 2001; Abe et al., 2004).
Epidemiological studies and clinical data suggest that estrogenshave cardiovascular protective effects by various mechanisms (Lissinand Cooke, 2000). However, an increased incidence of side effectslimits their therapeutic potential (Armitage et al., 2003). Given thedemonstrated risks of conventional estrogen therapy, the phytoestro-gens, including genistein and daidzein, are currently receiving more
attention because of their potential health benefits in preventingchronic diseases such as cardiovascular disease, obesity and osteopo-rosis (Setchell and Lydeking-Olsen, 2003; Altavilla et al., 2004; Parket al., 2005). In vitro and in vivo studies showed that genistein canlower blood pressure and alleviate oxidative stress in human subjectsand experimental hypertensive rats (Hodgson et al., 1999; Rivas et al.,2002; Homma et al., 2006) which can be partly explained by therestoration of NO-mediated vasorelaxation (Mishra et al., 2000;Karamsetty et al., 2001; Walker et al., 2001; Squadrito et al., 2002). Allthese data suggest that genistein may be able to suppress pulmonaryarterial hypertension by modulating NO-mediated signaling pathway.
In addition, pulmonary arterial hypertension is one of the mostfrequent signs in broiler's ascites syndrome which is still one of theleading causes of death in poultry industry. However, the underlyingmechanism is not fully known as yet. Therefore we hypothesize thatgenistein might prevent low temperature induced pulmonary hyper-tension throughmodulating endothelial function in broilers and explorethe potential mechanisms that contribute to these protective effects.To this aim, broilers were bred under normal or low ambient tem-perature in the presence or absence of genistein. The hemodynamicparameters, vascular remodeling and the expression of endothelialNO synthase and endothelin-1 in lung tissue were examined.
2. Materials and methods
2.1. Animals
One day-old commercial male Arbor Acre broiler chicks weremaintained in an environmental chamber with continuous lighting
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at a temperature of 28–30 °C with free access to water and a com-mercial broiler starter diet (23% crude protein, metabolizableenergy=13.4 MJ/kg from 1 to 14 days of age) and then growerdiets (20% crude protein, metabolizable energy=13.4 MJ/kg from 15to 42 days of age). From 15 days of age, birds were randomly assignedinto each of six groups (10 replicate pens per group, 10 chicks perpen), non-supplemented basal diet, basal diet supplemented with 20or 50 mg/kg of genistein under normal temperature (28–30 °C), orbasal diet supplemented with 0, 20 or 50 mg/kg of genistein underlow temperature (15–18 °C). The concentrations of genistein selectedin this study were based on our preliminary experiments (data notshow). The basic content of genistein in the grower diet is 11.3 μg/kg.Birds for time interval sampling were randomly selected from eachgroup on days 21, 35 and 42. All animals were handled in accordancewith the Guide for the Care and Use of Laboratory Animals publishedby the National Institute of Health. All experiments and protocolswere approved by Committee of Animal Care and Use of ChinaAgricultural University.
2.2. Diagnosis of pulmonary hypertension syndrome
All birds that died during the experimental periodwere necropsiedto identify pulmonary hypertension syndrome related mortality bycharacteristic symptoms, such as the presence of ascites fluid in theabdominal cavity, right ventricular dilation, hydropericardium, andvascular congestion, as have been previously reported (Druyan et al.,2007).
2.3. Performance, pulmonary arterial pressure, right ventricularhypertrophy, heart rate and saturation of hemoglobin with oxygen inarterial blood
Feed consumption and bodyweight gainwere recorded on days 21and 42, respectively. The mean body weight gain and the ratio of feedto weight gain were calculated for each treatment.
At 21, 35 and 42 days of age, heart rate, pulmonary arterial systolicand diastolic pressure (mm Hg) of broilers were measured using aright cardiac catheter as previously described (Yang et al., 2005).Briefly, birds were restrained in a dorsal position on the operating-table and locally anesthetized with 5% procaine chloride in the middleof the right side of the neck. A polyethylene plastic catheter wasinserted into the jugular vein after the jugular vein was separated. Thecatheter was pushed forward slowly to the right ventricle to measureheart rate and then pulmonary artery for pulmonary arterial pressuredetermination. Pressure signals were sent to the host computer ofRM-6000 type Polygraph (Nihon Kohden Ltd., Japan) throughpressure sensors.
After the in vivo measurements were completed, saturation ofhemoglobinwith oxygen in arterial blood and the right ventricle to totalventricle weight ratio were measured respectively (Huchzermeyer,1988).
2.4. Determination of plasma genistein levels in broiler
Plasma genistein levels in broilers were measured by using a high-performance liquid chromatography (HPLC) system equipped withUV detection as previously described (Squadrito et al., 2002).
2.5. Histological examination
Segments with a thickness of 0.5 cm adjacent to the bronchi wereremoved from the lungs and fixed with 10% formaldehyde solution formore than 24 h and dehydrated in an ascending gradient of ethanol.After becoming transparent in dimethylbenzene, the lung tissueswere embedded in paraffin and routinely processed into sections of5 μm in depth followed by Weigert–van Gieson staining for elastin
(Herget, 1991). Small pulmonary arterioles with an external diameterin the range of 20 to 50, 50 to 100 and 100 to 200 μm were studiedusing an automatic image analyzer (BH2, Olympus, Japan) with theadvanced software (Motic 3.0). Twelve average regions of crosssection were chosen. The adventitia and the lumen diameters weremeasured respectively, following which the relative medial thickness(%) was recorded and analyzed. The relative medial thickness ofpulmonary arterioles with different cut angles and conditions ofeither contractile or relaxation was computed from the abovemeasurements according to the previous methods (Barth et al.,1993; Tan et al., 2005).
2.6. Measurement of lung eNOS activity
Pulmonary vascular eNOS activity was determined by measuringthe calcium-dependent conversion of [3H]L-arginine to [3H]L-citrullineas previously reported (Fadel et al., 2000). Briefly, the lungs werequickly frozen in liquid nitrogen immediately after removal. Tissuewas homogenized on ice in lysis buffer containing 1 μM leupeptin,1 μM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. Homog-enates were incubated at 37 °C for 30 min in 50 mM Tris/HCl buffer(pH 7.4) in the presence of 1 mM NADPH, 10 μM L-valine and amixture of unlabelled and 10 uM L-[3H]arginine (1 μCi/ml). Theradioactivity was measured in the supernatant by a liquid scintillationcounter. eNOS activity was expressed as picomoles of [3H] L-citrullineproduced per milligram protein in 30 min.
2.7. Western blot analysis of endothelial NO synthase expression
The expression of NO synthase in broiler lungs was analyzed bywestern blot as previously reported (Tan et al., 2007). Briefly, lungtissues were homogenized in lysis buffer containing 1 mM NaF, 1 mMprotease inhibitor cocktail and centrifuged for 15 min at 12,000 g toremove cellular debris. The protein concentration was determinedusing a Bio-Rad protein assay kit. Equal amounts of protein wereseparated on SDS-page gels and transferred to PVDF membranes(Millipore). The membranes were blocked in a 5% skimmed milksolution at room temperature for 1 h, and then incubated with dilutedanti-eNOS antibody.
2.8. Measurement of lung ET-1 and cGMP
Broiler lung tissueswere separated and immediately frozen in liquidnitrogen. ET-1 was measured in lung homogenate using a radioimmu-noassaykit asdescribedpreviously (Naruse et al., 1989). LungcGMPwasdetermined as previously described (Takashima et al., 2006) using theBiotrad enzyme immunoassay system from Amersham BiosciencesCorp (Buckinghamshire, UK) and resultswere expressed as femtomolesof cGMP per milligram of dry weight.
2.9. Statistical analysis
Comparisons between groups were performed using two-wayANOVA followed by the Duncan test. Differences were consideredstatistically significant at the level of Pb0.05 and data are presentedas means±S.E.M. The statistical analysis was performed with thesoftware SPSS 11.0 for Windows (SPSS, Chicago, IL).
3. Results
3.1. Plasma genistein levels in broilers
The levels of genistein in plasma were determined and shown inTable 1. The plasma genistein is directly correlated with the genisteinsupplement in broiler diets. There is no difference between theplasma concentrations of genistein in broilers raised under low
Table 1Plasma genistein levels in broilers.
Treatment Temperature Normal temperature Low temperature
Genistein (mg/kg diets) 0 20 50 0 20 50
Serum 21 day 1.3±0.32 5.0±1.42 12.8±2.53 1.2±0.44 5.3±1.20 11.5±1.84GEN 35 day 1.7±030 6.1±0.93 16.1±1.22 1.8±0.62 6.4±0.91 17.2±3.44(ng/ml) 42 day 1.9±0.41 8.8±1.41 19.5±2.07 2.1±0.31 8.62±1.74 18.9±2.29
Data for plasma genistein levels were shown as means±S.E.M. from ten birds. GEN = genistein.
244 Y. Yang et al. / European Journal of Pharmacology 649 (2010) 242–248
temperature as compared with corresponding control raised undernormal temperature (Table 1).
3.2. Growth rate and ascites incidence
To explore whether the genistein supplementation has any effecton the performance of broilers, body weight gain and the ratio of feedto weight gain were determined. Both body weight gain and theratio of feed to weight gain in 0, 20, 50 mg/kg diet groups underlow temperature were kept at a similar level as compared with that ofcorresponding control during the first growth stage (0 to 21 days)(Table 2). However, these two parameters under low temperaturewere significantly lower than those in control when comparing themas a whole experimental period (0 to 42 days) suggesting that thechicks under low temperature had less weight gain and slowergrowth rates during the second growth period. Moreover, high dose ofgenistein (50 mg/kg) significantly improved body weight gain anddecreased feed to weight gain (0 to 42 days) compared with non-treatment group under low temperature, indicating a beneficial effectof genistein on the broilers' performance.
The incidence of hydropericardium and mortality associatedwith ascites were confirmed using previous method (Druyan et al.,2007). As shown in Table 3, low temperature increased the mortalityand incidence of hydropericardium in non-treatment group as com-paredwith that of control under normal temperature (from 1/100 and3/100 respectively under normal temperature to 27/100 and 21/100respectively under low temperature) during the whole experimentalperiods (0–42 days). However, genistein supplementation markedlydecreases the mortality associated with ascites from 27/100 to 6/100and 2/100 for 20, 50 mg/kg diet respectively. The incidence ofhydropericardium was also decreased from 21/100 to 7/100 and 4/100 for 20, 50 mg/kg diet respectively, suggesting a preventive effectof genistein supplementation on the incidence of hydropericardiumand related mortality in low temperature-induced pulmonaryhypertension in broilers, especially at a higher concentration of50 mg/kg diets.
3.3. Pulmonary arterial pressure and heart rate
As shown in Table 4, low temperature significantly increasedmeanpulmonary arterial pressure of broilers on 21, 35 and 42 days of age
Table 2Performance data for live broiler body weight gain and the ratio of feed to weight gain.
Treatment Temperature Normal temperature
Genistein (mg/kg diets) 0 20
BWG1(kg) 0–21 days 0.77±0.043 0.77±0.0640–42 days 2.05±0.064 ac 2.06±0.085a
F/G2 0–21 days 1.51±0.079 1.51±0.0440–42 days 1.90±0.035ab 1.90±0.068ab
1BWG = body weight gain; 2F/G = the ratio of feed to weight gain. Data were shown assignificantly (Pb0.05).
compared with that of control in non-treatment groups (Pb0.05 forthree time points). The higher dose genistein treatment (50 mg/kg)kept pulmonary arterial pressure at the similar level compared withthat of control under normal temperature.
Low temperature significantly increased heart rate of broilers onday 21 and decreased it on day 42 compared with corresponding timepoints of control in non-treatment groups (Pb0.05 for the two timepoints). Genistein supplementation under normal temperature didnot affect heart rate at the three time points (Table 4).
3.4. The ratio of right ventricle to total ventricle weight and arterialhemoglobin oxygen saturation
The right: total ventricle weight ratio in broilers under lowtemperature conditionwithout genistein supplementation on days 35and 42 were significantly increased compared with correspondingnon-treatment controls under normal temperature (Pb0.05),(Table 4). The supplementation of genistein at 50 mg/kg diet keptthe ratio of right ventricle to total ventricle weight at the similar levelcompared with that of control under normal temperature at the threetime points.
Low temperature significantly decreased the arterial hemoglobinoxygen saturation at the three time points compared with those ofcontrol (Table 4). Genistein supplementation, especially at highconcentration (50 mg/kg diets), abrogated the decrease of the arterialhemoglobin oxygen saturation, and kept it at similar levels to those ofcontrol.
3.5. Pulmonary artery remodeling
As shown in Table 5, the relative medial thicknesses of pulmonaryartery in the ranges of 20≤Φb50, 50≤Φb100 or 100≤Φb200 μmare significantly greater in the non-treatment group under lowtemperature than those under normal temperature on days 35 and 42,respectively (Pb0.05). Genistein supplementation suppressed lowtemperature induced vascular remodeling and maintained thethickness at similar levels of those in control under normal tem-perature. Fig. 1 showed the morphological change of pulmonaryarteriole structure with pulmonary arteriole external diameterranging from 20 to 50 μm under normal temperature, low temper-ature or low temperature treated with genistein (50 mg/kg diet).
Low temperature
50 0 20 50
0.79±0.034 0.81±0.034 0.80±0.034 0.80±0.0272.10±0.045a 1.82±0.079b 1.86±0.10b 2.03±0.076 ac
means±S.E.M. from ten birds. a–c Means in a row with no common superscript differ
Table 3Ascites relatedmortality and the incidence of hydropericardium in broilers treated withor without genistein.
Treatment Temperature Normal temperature Low temperature
Genistein(mg/kg diets)
0 20 50 0 20 50
Mortalitya of ascites 0–21 days 0 0 0 0 0 00–42 days 1 0 0 27 6 2
Incidencea ofhydropericardium
0–21 days 0 0 0 5 2 10–42 days 3 2 1 21 7 4
a Absolute numbers, initial number of chickens per group is 100.
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3.6. Effects of genistein on pulmonary vascular NOS activity and itsprotein expression
As shown in Fig. 2, low temperature decreased eNOS activity ascompared with normal temperature control, this is in agreement withprevious studies indicating that eNOS is associated with hypertensionin various experimental systems (Chou et al., 1998; Safar et al., 2001).Genistein treatment (both low and high doses) significantly increasedeNOS activity in pulmonary vascular tissue in broilers under lowtemperature. Genistein also increased eNOS activity in normaltemperature groups; however, there was no significant difference inboth dose treatments (Fig. 2A). Consistent with the eNOS activity,western blot analysis showed a markedly down-regulated eNOS levelin low temperature induced hypertensive broilers which were largelyrestored by genistein treatment (Fig. 2B).
Table 4Mean pulmonary arterial pressure (mPAP), heart rate (HR), the ratio of the of right ventriclarterial blood (SaO2) of broilers in the different groups and investigated intervals, respectiv
Treatment Temperature Normal
Genistein (mg/kg diets) 0 20
mPAP (mmHg) 21 day 17.3±2.82a 17.0±2.77a
35 day 26.6±2.10a 25.9±2.10a
42 day 30.2±3.31a 30.7±3.31a
HR (beat/min) 21 day 433±21.9ab 430±17.9a
35 day 411±18.3 410±12.342 day 398±20.8a 397±22.8a
RV:TV (%) 21 day 0.20±0.033 0.19±0.09335 day 0.23±0.035a 0.21±0.015a
42 day 0.25±0.037a 0.24±0.087a
SaO2 (%) 21 day 92.1±1.99a 91.7±2.96a
35 day 88.2±3.27a 90.2±6.27a
42 day 89.0±2.06a 88.0±7.06a
a–cMeans in a row with no common superscript differ significantly (Pb0.05).
Table 5Relative medial thickness of pulmonary arteriole with external diameters ranging from 20 tomeans±S.E.M. from ten birds.
RMT = Relative medial thickness. a–cMeans in a row with no common superscript differ sig
3.7. Lung ET-1 peptide levels and cGMP levels
In consistent with our previously study, ET-1 level was markedlyincreased in low temperature control as compared with the normaltemperature control, suggesting that alteration of ET-1 is involved inthe development of pulmonary hypertension. Genistein administra-tion (higher and lower doses) significantly decreased the lung ET-1peptide levels in broilers under low temperature compared withthat of low temperature control (Fig. 3). Neither dose of genisteinaffects the ET-1 peptide level in the normal temperature counterparts.The cGMP levels in lung tissue were markedly lower comparedwith normal temperature control. However, genistein treatmentsignificant increased cGMP levels under low temperature (Fig. 4)which is in consistent with the eNOS activity and protein levels asdemonstrated in Fig. 2. Genistein did not affect lung cGMP levels innormal temperature groups.
4. Discussion
We have previously shown that Ca2+ channel antagonist or ET-1receptor antagonist can attenuate PH with ascites in broilers.However, the side effects on the growth and the performance ofbroilers limited their application (Yang et al., 2005; Yang et al., 2007).This encouraged us to identify small molecules that could be ofpotential therapeutic interest for the treatment of pulmonary arterialhypertension, without negative effects.
In the present study, we provided evidence that low ambienttemperature leads to a reduced eNOS and an increased endothelin-1
e to the total ventricle weight (RV:TV, %) and saturation of hemoglobin with oxygen inely. Data are expressed as means±S.E.M. from ten birds.
Low
50 0 20 50
16.8±2.53a 23.6±4.29b 18.3±2.20a 17.2±1.84a
25.1±3.22a 35.1±3.82b 31.4±4.21c 26.2±4.44a
30.1±3.07a 40.4±4.11b 36.2±3.34c 30.6±3.79a
428±19.4a 447±22.3b 442±20.8ab 429±21.3a
409±16.5 399±16.9 410±23.2 408±20.0395±18.2a 370±24.3b 380±22.6ab 397±12.7 a
Fig. 2. Genistein supplementation restored lung vascular tissue eNOS activity in lowtemperature induced hypertensive broilers. Broilers were supplemented with genistein(GEN, 0, 20 or 50 mg/kg diet) for 28 days (at 42 days old) under normal or low temperature,eNOS activity (A) and eNOS protein levels (B) were determined. The denitometricquantification is shown at the bottom. Data are shown as means±S.E.M., n=6. *Pb0.05compared with low temperature control.
A
C
B
Fig. 1. Genistein suppress vascular remodeling in low temperature induced hypertensivebroilers. The morphological change of pulmonary arteriole structure with pulmonaryarteriole external diameter ranging from20 to 50 μm(Weight and VanGieson×100) undernormal temperature (A), low temperature (B) or low temperature treated with genistein(50 mg/kg diets) for 28 days (at 42 days old). Data presentedherewere from representativeexperiment. Similar data were obtained from 2 more repeat experiments (n=4).
246 Y. Yang et al. / European Journal of Pharmacology 649 (2010) 242–248
levels in lung vascular arteries and induced pulmonary hypertensionas characterized by an increased blood pressure (Table 4), vascularremodeling in broilers (Table 5 and Fig. 1) and an increased mortalityincidence (Table 3), suggesting that impaired endothelial functionis associated with the development of hypertension in our animalmodel. Genistein supplementation significantly reduced low temper-ature induced pulmonary arterial pressure (Table 4), inhibitionof pulmonary arterial vascular remodeling (Fig. 1) and decrease theincidence of mortality in broilers by improving endothelial functioninvolving enhanced eNOS activity (Fig. 2) and lung cGMP levels(Fig. 4) and decreased ET-1 content (Fig. 3) without decreasingbroilers' performance.
Genistein is one of the main biologically active isoflavones in soy-derived products with various health benefits associated with chronicdiseases. It has been reported that populations that consume a diethigh in phytoestrogens have lower risks of hypertension or pulmo-nary hypertension implicated in cardiovascular disease (Lichtenstein,
1998; Mishra et al., 2000; Ambra et al., 2006; Homma et al., 2006; Choet al., 2007; Si and Liu, 2008), however, the underlying mechanism isnot fully understood. Moreover, the effects of genistein on broiler'svasculature function and its possible anti-hypertension mechanismremains unknown, even though pulmonary hypertension is com-monly observed in broilers and causes huge economic loss in poultryindustry. We hypothesized that genistein might reduce low temper-ature induced pulmonary hypertension by modulating vasoactivemediators in hypertensive broilers.
To test this hypothesis, we started with low temperature inducedpulmonary model in broilers which had been reported to display thecharacteristics of pulmonary hypertension and the results are con-sistent with the previous study from other labs and ours (Olkowskiand Classen, 1998;Wideman and Tackett, 2000; Tan et al., 2005; Yanget al., 2007). Supplementation of genistein markedly inhibited lowtemperature induced pulmonary hypertension and arrested thevascular remodeling (Table 4 and Fig. 1).
0
2000
4000
6000
0 20 50 0 20 50GEN:
ET
-1 (
pg/g
tiss
ue) *
*
Normal Low
Fig. 3. Genistein supplementation (GEN, 20 or 50 mg/kg diet) reduced lung ET-1peptide level in low temperature-induced hypertensive broilers. Broilers weresupplemented with genistein (GEN, 0, 20 or 50 mg/kg diet) for 28 days (at 42 daysold) under normal or low temperature, Lung ET-1 peptide levels were determined asdescribed in materials and methods. Values are means±S.E.M, n=6. *Pb0.05compared with low temperature control.
247Y. Yang et al. / European Journal of Pharmacology 649 (2010) 242–248
The imbalance of vasoactivemediators, such as NO and endothelin-1 (ET-1), has been recognized as a critical factor that contributesto endothelial dysfunction which consequently result in pulmonaryarterial hypertension. To investigate whether these factors areinvolved and responsible for the beneficial effect of genistein againstlow temperature induced pulmonary hypertension, we determinedthe eNOS activity, a critical vasodilator, in lung tissue of hypertensivebroilers and found that eNOS activity and expression were signifi-cantly reduced in low temperature induced hypertensive chicks,which were markedly restored by genistein, indicating improvedendothelial function, suggesting that the beneficial effect is related, atleast partly, to themodulation of eNOS activity. This observation, alongwith the increased lung cGMP levels, indicated that eNOS-NO-cGMPaxis was activated upon gensitein treatment. This result is consistentwith the previous studies showing that genistein up-regulates eNOSexpression in hypertensive rats (Chou et al., 1998; Squadrito et al.,2000; Safar et al., 2001; Si and Liu, 2008).
It has been reported that ET-1, a potent vasoconstrictor and pro-mitogenic peptide, plays an important in the initiation and evolutionof pulmonary hypertension in human and rats (Giaid et al., 1993; Celikand Karabiyikoglu, 1998). Our recent study also showed that ET-1 iselevated in low temperature induced pulmonary hypertension inbroilers which can be reversed by endothelin receptor antagonistBQ123 (Yang et al., 2005). In the present study, genistein treatment
0
50
100
150
0 20 50 0 20 50GEN:
**
cGM
P(f
mol
es/m
g w
eigh
t)
Normal Low
Fig. 4. Genistein increase lung cGMP levels in low temperature-induced hypertensivebroilers. Broilers were supplemented with genistein (GEN, 0, 20 or 50 mg/kg diet)for 28 days (at 42 daysold)undernormal or low temperature, lung tissue cGMP levelsweredetermined using the method described. Values were expressed as means±S.E.M, n=5.*Pb0.05 compared with low temperature control.
produced a significant decrease of lung ET-1 level in hypertensivebroilers as compared to the low temperature control even thoughthis effect is less than that of endothelin receptor antagonist (datanot shown). This finding is in agreement with a recent study in post-menopausal women (Squadrito et al., 2002), showing that improvedvascular function of genistein is associated with an increased ratioof nitric oxide to endothelin-1. However, in monocrotaline-inducedpulmonary hypertensive rats, genistein treatment improves thedown-regulation of expression of lung eNOS, consistent with ourdata, but did not affect the endotehlin-1 levels in the lungs (Hommaet al., 2006). This discrepancy may be due to the several importantdifferences between studies, for example, species, methods used toinduce hypertension, treatment periods and doses.
NO and ET-1 are endothelium-derived vasoactive factors thatinteract with one another (Lavallee et al., 2001). NO causes vaso-dilation and inhibits smooth muscle cell proliferation (Loscalzo,1995). ET-1 causes potent vasoconstriction of the systemic andcornonary vasculature through binding to endothelin receptors,increase monocyte adhesion and promote vascular smooth musclecell proliferation, opposing the effects of NO (Mathew et al., 1996).Studies in porcine aorta indicated that NO inhibits the production ofET-1 by a cyclic GMP-dependent pathway (Boulanger and Luscher,1990). On the other hand, endothelin-1 receptor B present inendothelial cells mediates the production of NO (de Nucci et al.,1988), thus constituting a feed-back regulatory loop to maintain anormal vascular tone. In the present study, genistein treatmentactivated eNOS-NO-cGMP signaling pathway (Figs. 2 and 4) anddecreased levels of ET-1 (Fig. 3) in lung tissue. However, in vitrostudy using endothelial cells or smooth muscle cells in combina-tion with eNOS or cGMP inhibitors as shown in previous study(Boulanger and Luscher, 1990), is required to test whether thedeceased ET-1 is regulated by genistein or genistein activated eNOS-NO-cGMP.
The mechanism by which genistein increases the expression ofeNOS and decreases ET-1 expression is unclear. There are at least twopossibilities that might contribute to the activation of eNOS. The firstone is that genistein, like estrogen, activates eNOS activity throughclassical actions of estrogen via transcriptional activation of estrogen-responsive genes involving estrogen receptors. However, it appearsnot the case in our system, because estrogen receptor antagonist ICI182, 780 did not block genistein induced eNOS activity (data notshown). Similar results were also observed in human endothelialcells (Rathel et al., 2005). Secondly, it might mediated by inhibitingtyrosine kinase (Minchenko and Caro, 2000). However, it should benoted that this inhibitory property requires a much higher concen-tration of genistein (100 μM) (Makela et al., 1999) which is quitehigher than the plasma level of genistein in our study (Table 1), thusexcluding the possibility that genistein activated eNOS through theinhibition of tyrosine kinase.
Moreover, genistein has also been suggested to inhibit hydrogenperoxide production and increase the activity of antioxidant enzymes,such as catalase, manganese superoxide dismutase, glutathioneperoxidase and glutathione reductase leading to a reduced oxidativestress and improved endothelial function and reduced blood pressurein vivo (Wei et al., 1995; Mahn et al., 2005). More study is requiredto test whether genistein is the main active component that isresponsible for the activation of antioxidant genes, because in theirstudy, soy protein rich diet was used which contains multiplephytoestrogens, including genistein and daidzein, instead of singlemolecule was used.
In summary, this is the first study providing evidence thatsupplementation of genistein significantly attenuated low tempera-ture induced pulmonary hypertension and inhibited vascular remo-deling in broilers without affecting the performance index. Thebeneficial effect of genistein appeares to be mediated by restoringendothelial function, especially eNOS and ET-1, two critical vasoactive
248 Y. Yang et al. / European Journal of Pharmacology 649 (2010) 242–248
mediators associated with the development of hypertension. There-fore, genistein might have therapeutic effects for the treatment ofpulmonary hypertension.
Acknowledgements
This work was supported by the Yangtz River Scholar andInnovation Research Team Development Program (Project No.IRT0945) and by the grants from National Natural Science Foundationof China (No. 30700576) and State Key laboratory of Animal Nutrition(Project No. 2004DA125184-0807).
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