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The promising effect of barberry (Zereshk) extract against experimental pulmonary microvascular remodeling and hypertension: A comparison with sildenafil ab
ab
Naser Mahdavi , Siyavash Joukar , Hamid Najafipour
bc
d
& Majid Asadi-Shekaari
a
Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran, b
Department of Physiology and Pharmacology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, c
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Cardiovascular Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran, and d
Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran Published online: 02 Jun 2015.
To cite this article: Naser Mahdavi, Siyavash Joukar, Hamid Najafipour & Majid Asadi-Shekaari (2015): The promising effect of barberry (Zereshk) extract against experimental pulmonary microvascular remodeling and hypertension: A comparison with sildenafil, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1050676
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1050676
ORIGINAL ARTICLE
The promising effect of barberry (Zereshk) extract against experimental pulmonary microvascular remodeling and hypertension: A comparison with sildenafil Naser Mahdavi1,2, Siyavash Joukar1,2, Hamid Najafipour2,3, and Majid Asadi-Shekaari4 Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran, 2Department of Physiology and Pharmacology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, 3Cardiovascular Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran, and 4Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
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Abstract
Keywords
Context: Despite the beneficial effects of barberry (Berberis integerrima Berberidaceae) on decreasing systemic hypertension, its influence has not been investigated on pulmonary hypertension. Objective: The objective of this study is to examine the effect of barberry fruit, on monocrotaline-induced pulmonary hypertension. Materials and methods: Nine groups were arranged as follows: the control group, the monocrotaline (M) group, the barberry groups with doses of 50, 100, and 200 (mg/kg), the M plus barberry groups, and the M plus sildenafil group. Two weeks after a single injection of monocrotaline (60 mg/kg, s.c.), barberry water extracts or sildenafil (30 mg/kg/d) were gavaged daily for 2 weeks. At the end of the 4th week, hemodynamic, biochemical, and histopathological parameters were assessed. Results: In comparison with the M group, barberry (200 mg/kg) or sildenafil significantly reduced the right ventricular systolic pressure (RVSP) (22.95 ± 1.78 mm Hg and 30.71 ± 1.64 mm Hg, versus 41.28 ± 1.5 mm Hg), right ventricular hypertrophy (RVH) (0.39 ± 0.03 and 0.42 ± 0.02, versus 0.57 ± 0.02), and the medial wall thickness (MWT) (4.56 ± 0.15 mm and 5.97 ± 0.19 mm, versus 7.02 ± 0.43 mm). Barberry or sildenafil had no significant effect on the plasma level of endothelin-1, glutathione peroxidase, and the malondialdehide of lung. Conclusion: 200 mg/kg of barberry has an improving effect on the monocrotaline-induced pulmonary hypertension. This effect was stronger than that of the sildenafil’s and may have been mediated through mechanisms other than the modulation of the endothelin-1 or redox system.
Berberine, endothelin-1, monocrotaline, pulmonary arterial hypertension
Introduction In clinics, pulmonary arterial hypertension (PAH) is described as a mean pulmonary artery pressure 25 mm Hg at rest or 30 mm Hg with exercise and with pulmonary capillary wedge pressure (15 mm Hg) (Barst et al., 2004). The survival time for the untreated idiopathic PAH (formerly included in primary pulmonary hypertension) was reported to be 3 years (Archer et al., 2010). PAH can be associated with various other diseases including HIV infection, connective tissue diseases, portal hypertension, schistosomiasis, chronic hemolytic anemia, and congenital heart disease, and the prognosis could depend on the underlying diseases (Chan & Loscalzo, 2008). The increased vasoconstriction of the pulmonary arteries in PAH Correspondence: Siyavash Joukar, Physiology Research Center, Institute of Neuropharmacology and Department of Physiology and Pharmacology, School of Medicine, Kerman University of Medical Sciences, PO Box 7616914115, Kerman, Iran. Tel/Fax: +98 341 3220081. E-mail: [email protected], [email protected]
History Received 6 November 2014 Accepted 8 May 2015 Published online 29 May 2015
has been attributed to the reduced expression/activity of the voltage-gated potassium channels and also dysfunction of the endothelium that lead to an imbalance in the production and release of both vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) and vasoconstrictors such as endothelin-1 (ET-1) and serotonin (5-HT) (Humbert et al., 2004). Many of these regulators of vascular tone also have played roles in controlling cell proliferation and apoptosis and can contribute to the remodeling of the pulmonary vasculature. The structural changes of pulmonary vessels in PAH can include the medial thickening due to the smooth muscle cell hyperplasia and hypertrophy (Yildiz, 2009), formation of a neointima composed of smooth muscle cells or myofibroblasts embedded in a mucopolysaccharide matrix and the plexiform lesions due to endothelial and smooth muscle cell proliferation (Widerman & Hamal, 2011). Currently, there are three main classes of drugs used for the treatment of this disease: prostanoids, endothelin-1 receptor antagonists, and phosphodiesterase-type 5 (PDE5) inhibitors (Rhodes et al., 2009).
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Although, to some extent, these drugs can decelerate the PAH development and attenuate its clinical symptoms, they cannot cure the disease completely (Rhodes et al., 2009). Hence, finding of new agents for treating of PAH is necessary. There are two famous barberry species, Berberis integerrima Berberidaceae and Berberis vulgaris Berberidaceae (Zereshk), in Iran. Berberis integerrima is a thorny shrub with fragile branches to a height of 1–3 m. Its fruits are 7– 10 mm long and 3–4 mm in diameter, with a mild sour taste (Ardestani et al., 2013; Joukar & Mahdavi, 2014). The major chemical compounds of barberry fruits include coumarin, ascorbic acid, berberine, caffeic acid, b-carotene, flavonoids, malic acid, palmatine, carbohydrates, tannin, and ursolic acid (Imanshahidi & Hosseinzadeh, 2008). Overall, the amounts of ash, fiber, protein, reducing sugars, total sugar, and pH are higher in B. integerrima whereas the moisture content, brix, and color indices are higher in B. vulgaris. The amounts of P, Zn, Fe, Na, and K in B. vulgaris are higher than in B. integerrima and the amounts of Mn, Mg, and Cu, Ca are higher in fruits of B. integerrima. In addition, the total phenolic and total anthocyanin contents of fresh fruits of B. integerrima are higher than in the fresh fruits of B. vulgaris (Ardestani et al., 2013). The various parts of Zereshk plant including its root, bark, leaf, and fruit have long been used in the West and East folk medicine (Imanshahidi & Hosseinzadeh, 2008). In traditional medicine, barberry has been used for the treatment of various heart diseases such as hypertension and arrhythmia (DerMaderosian, 2001; Fatehi et al., 2005). Several pharmacological studies have indicated the cardiovascular effects of barberry and berberine, its famous alkaloid constituent, such as preventing ischemia-induced ventricular tachyarrhythmia, improving cardiac contractility and lowering peripheral vascular resistance, and blood pressure (Chun et al., 1979; Marin-Neto et al., 1988). A study demonstrated that the administration of the barberry extract, the fruit of B. vulgari (0.05–1 mg/100 g body weight), significantly decreased the systemic arterial blood pressure and the heart rate dose dependently in rats (Fatehi-Hassanabad et al., 2005). However, so far the influence of this edible and medicinal fruit has not been investigated on pulmonary hypertension. The present study was conducted to examine the effects of the water extract of barberry, a popular seasoning especially used in the eastern societies, on the monocrotaline-induced pulmonary arterial hypertension, and to compare its effect with sildenafil.
Materials and methods Sildenafil sulfide (Kharazmi Pharmaceutical CO, Tehran, Iran) and sodium thiopental (Biocheme, Kundl, Austria) were dissolved in physiological saline and monocrotaline (MCT) (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) plus saline. Barberry (Berberis integerrima fruit) was collected from Baft area of Kerman, Iran, and it was identified by the Department of Botany, Bahonar University of Kerman. A voucher specimen (KF-1194) of the plant was deposited in the Herbarium Center of the Pharmacy Faculty of Kerman University of Medical Sciences. The glutathione peroxidase (GPX) assay kit was from Randox, Northern
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Ireland, UK, and the endothelin-1 (ET-1) kit was obtained from USCN Life Science Inc., Wuhan, China. Animal groups The study procedures were approved and performed based on the national guidelines for conducting animal studies (Ethic committee permission No. 90/315-Kerman University of Medical Sciences, Kerman, Iran). Seventy-two male Wistar rats (purchased from the Physiology Research Center, Kerman, Iran) weighing between 250 and 300 g were allowed free access to standard laboratory rat chow and tap water and were housed under controlled humidity and room temperature and a 12 h light/dark cycle. Animals were divided to nine groups as follows: the CTL (control) group which received a single injection of 0.5 ml saline plus DMSO (i.p.) on the first day of study as the vehicle of monocrotaline. The M (monocrotaline) group which received equal volume of single dose of MCT (60 mg/kg, i.p.) on the first day of study. The B50, B100, and B200 groups which received saline plus DMSO (i.p.) on the first day of study. Then, at the beginning of the third week, they were treated with barberry extracts by the gavage method with doses of 50, 100, and 200 mg/kg/d, for the next 2 weeks. The MB50, MB100, and MB200 groups received monocrotaline on the first day and then were treated with barberry extracts similar to the B50, B100, and B200 groups. The Msil group was treated with sildenafil (30 mg/kg/d, orally) for 2 weeks at the beginning of the third week after monocrotaline injection. Survival At the end of the experiment, the rats which received monocrotline had significant mortality, but no rat died in the MB200 group. Therefore, we designed our study so that 7–8 rats could stay alive at the treating period (4th week). Three out of the 11 rats of the M group (n ¼ 8, surviving), two out of nine in the MB50 group (n ¼ 7, surviving), one out of nine in the MB100 group (n ¼ 8, surviving), one out of eight in the MSil group (n ¼ 7, surviving) died during the treatment period. Induction of pulmonary arterial hypertension The pulmonary arterial hypertension was induced by a single intraperitoneal injection of 60 mg/kg monocrotaline on the first day of the study (Sauvageau et al., 2009). At the end of the fourth week, the animals showed obvious pulmonary hypertension. Barberry extracts preparation Each 10 g of dried barberry was boiled with 100 ml water for 5 min. The mixture was then filtered, and the liquid evaporated under vacuum at 45–50 C. The material was then dried by exposure to hot air (40 C) for 48 h (FatehiHassanabad et al., 2005). Then, barberry extracts were kept in glass vials at 20 C prior to use. Hemodynamic measurements Four weeks after monocrotaline or vehicle injection, on the 29th day, the animals were anesthetized with an
Anti-PAH effect of barberry
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intra-peritoneal injection of 50 mg/kg thiopental sodium. A polyethylene cannula containing saline-heparin solution (15 l/mL) was inserted into the isolated right carotid artery and then was connected to a pressure transducer to measure the arterial blood pressure (Joukar et al., 2010). The mean arterial pressure (MAP) was calculated by ‘‘MAP ¼ Pd + (PsPd)/3 formula’’, in which Pd is the diastolic arterial pressure and Ps is the systolic arterial pressure. The right ventricle was gently cannulated with a filled catheter (saline–heparin solution, 15 l/mL) connected to a pressure transducer of a PowerLab system (Power Lab Systems, Springs, CO) to record its systolic pressure as an equivalent parameter of the pulmonary systolic pressure.
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Biochemical parameters In the evening of the 28th day, under ether light anesthesia, 2 ml of blood sample taken by retro-orbital puncture, then centrifuged to later produce a serum which was frozen and kept at 80 C for measurement of ET-1. At the end of the experimental recording, a piece of the lung tissue was cut and frozen in liquid nitrogen and then was stored at 80 C for the measurement of the ET-1, GPX, and malondialdehide (MDA) levels of the lung tissue. The ET-1 content was measured by using an ET-1 immunoassay kit by a sandwich enzyme immunoassay technique (Zambelli et al., 2011). All reagents, samples, and standards were prepared according to the manufacturer protocol. The reaction product was measured by reading the absorbance at 450 nm. The GPX level was measured by using RANDOX kit (Randox, Northern Ireland, UK) via the ultra violet (UV) method. Briefly, 0.05 ml sample supernatant was mixed with 1 ml diluting agent. After incubating for 5 min, 1 ml Drobkin’s reagent was added. Samples were thoroughly mixed and, after 20 min, they were measured by reading the absorbance at 340 nm. The MDA level was measured using TBARS assessment (Joukar et al., 2012). Histopathological assessment
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Data analysis Statistical analysis was performed by statistical software SPSS 20 (SPSS Inc., Chicago, IL). All values were expressed as means ± SEM. Comparisons were performed among different groups by one-way ANOVA and post hoc Tukey’s test. p Value less than 0.05 were considered statistically significant.
Results Hemodynamic experiments Mean systemic arterial pressure (MAP) did not show any significant difference among the animal groups (Figure 1). The right ventricular systolic pressure (RVSP) significantly increased in the M group in comparison with the CTL, B50, B100, and B200 groups (p50.001). In the presence of monocrotaline, 2-week consumption of the barberry extracts with doses of 50, 100, and 200 mg/kg significantly reduced the RVSP. This effect was prominent in the MB100 and MB200 groups (p50.001 versus the M group) compared with the MB50 group (p50.01 versus the M group). The administration of sildenafil also reduced the RVSP (p50.01 versus the M group), although this beneficial effect was insignificantly weaker than the effect of higher doses of barberry (Figure 2). RVH index RVH index significantly increased in the M group compared with the control groups (CTL, B50, B100, and B200). The animal groups that received monocrotaline plus sildenafil (Msil group) or barberry extract with dosages of 100 and 200 mg/kg (MB100 and MB200) showed a reduction in the RVH index whenever compared with monocrotaline alone (p50.05 for Msil and MB100 versus the M group and p50.01 for MB200 versus the M group) (Figure 3). Histopathological assessment Because of the stronger beneficial effect of 200 mg/kg of the barberry extract on the improvement of the
At the end of the experiment, lungs were washed by saline infusion and then infused with 40 mL of 4% paraformaldehyde solution. Thereafter, lungs were removed and fixed in 10% buffered formalin, and after tissue processing, they were embedded in paraffin. Three micron-thick sections were prepared and stained with hematoxylin and eosin and were examined under light microscopy. The medial thickness and vessel diameter of 60 pulmonary arterioles, with a size of 25–100 mm, in each lung section were measured randomly in blind manner (Cowan et al., 2000; Mathew et al., 1997). Right ventricular hypertrophy index After hemodynamic measurements, the hearts of the animals were also excised and cleaned by saline. The right ventricular hypertrophy (RVH) index was calculated using the following formula: ‘‘RVH ¼ 2 (RVFWT)/(LVFWT + IST)’’, where RVFWT is the right ventricular free wall thickness, LVFWT is the left ventricular free wall thickness and IST is the interventricular septum thickness (Nishida et al., 2004).
Figure 1. Mean systemic arterial pressure (MSAP) in different experimental groups at the end of the 4th week. Values are mean ± SEM. n ¼ 7–8. The differences in data were not significant. CTL, control group; B50, 50 mg/kg barberry extract; B100, 100 mg/kg barberry extract; B200, 200 mg/kg barberry extract; M, monocrotaline group; MB50, monocrotaline + 50 mg/kg barberry extract; MB100, monocrotaline + 100 mg/kg barberry extract; MB200, monocrotaline + 200 mg/kg barberry extract; Msil, monocrotaline + 30 mg/kg sildenafil.
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Figure 2. Right ventricular systolic pressure (RVSP) in different experimental groups (mm Hg) in all groups at the end of the 4th week. Values are mean ± SEM. n ¼ 7–8. ***p50.001 compared with CTL, B50, B100, and B200 groups. #p50.01 compared with M and MB200 groups. zp50.001 compared with the M group. p50.001 compared with the M group. *p50.01 compared with the M group. RVSP increased in the monocrotaline group compared with other groups and barberry extracts (50, 100, and 200 mg/kg) dose dependently reduced this parameter. CTL, control group; B50, 50 mg/kg barberry extract; B100, 100 mg/kg barberry extract; B200, 200 mg/kg barberry extract; M, monocrotaline group; MB50, monocrotaline + 50 mg/kg barberry extract; MB100, monocrotaline + 100 mg/kg barberry extract; MB200, monocrotaline + 200 mg/kg barberry extract; Msil, monocrotaline + 30 mg/kg sildenafil.
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Figure 4. Medial wall thickness of pulmonary arterioles (mm). Data are presented as mean ± SEM. n ¼ 7–8, ***p50.001 compared with CTL and B200 groups. *p50.05 compared with the M group. zp50.001 compared with Msil and M groups. Medial wall thickness of pulmonary arteriols significantly decreased in MB200 compared with monocrotaline and Msil groups. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
Plasma and lung tissue ET-1 level The administration of monocrotaline was associated with the increase of serum ET-1 in the M group (p50.001 versus the CTL group). The consumption of 200 mg/kg barberry extract and the sildenafil could not attenuate the effect of the monocrotaline on the serum ET-1 level (Figure 6). The lung tissue ET-1 was lowered in the M group compared with the other groups, but it was not statistically significant. The usage of sildenafil or barberry extract plus monocrotaline insignificantly elevated the lung ET-1 to values of the CTL and B200 levels, respectively (Figure 7). Lung tissue GPX and MDA Figure 3. Right ventricular hypertrophy index. Data are presented as mean ± SEM. n ¼ 7–8. *p50.05 compared with CTL and B100 groups. #p50.01 compared with the B200 group. yp50.01 compared with the M group. p50.05 compared with the M group. RVH increased in the monocrotaline group. Two weeks consumption of barberry extracts attenuated this parameter especially in dose of 200 mg/kg. CTL, control group; B50, 50 mg/kg barberry extract; B100, 100 mg/kg barberry extract; B200, 200 mg/kg barberry extract; M, monocrotaline group; MB50, monocrotaline + 50 mg/kg barberry extract; MB100, monocrotaline + 100 mg/kg barberry extract; MB200, monocrotaline + 200 mg/kg barberry extract; Msil, monocrotaline + 30 mg/kg sildenafil.
hemodynamic parameters and the RVH index, histopathological and biochemical indices were assessed only in the CTL, B200, M, MB200, and Msil groups. MWT significantly increased in the M group compared with the CTL and B200 groups. Two weeks usage of 200 mg/kg barberry extract obviously decreased the growing effect of monocrotaline on the thickness of the arterioles wall (p50.001 compared with the M group). Sildenafil also attenuated the monocrotaline induced arteriole remodeling (p50.05 versus the M group); however, this valuable effect was weaker than in the barberry extract (p50.05) (Figures 4 and 5).
The animals with monocrotaline-induced pulmonary hypertension showed a mild but non-significant reduction in GPX (Figure 8) and a mild raise in MDA content of the lung tissue (Figure 9). Barberry extract consumption recovered these effects whereas sildenafil could not improve the elevated level of MDA (Figure 9).
Discussion This study was conducted to determine the therapeutic effect of aqueous extract of barberry on monocrotaline-induced pulmonary hypertension and to compare it with the effect of sildenafil – a known drug for PAH treatment. MCT injection had no significant effect on MAP in this study. This result is in accordance with the findings of previous studies (Mei et al., 2011; Nishida et al., 2004). In addition, different groups which received barberry extract alone or plus monocrotaline and also sildenafil plus monocrotaline group did not show significant changes in systemic arterial pressure. However, higher doses of barberry (100 and 200 mg/kg) to some extent reduced the systemic blood pressure which was statistically insignificant. A study reported that barberry aqueous extract significantly reduces the systemic blood pressure in normotensive and hypertensive
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Figure 5. Arterioles of lung tissue of different animals group. (A) CTL group, (B) B200 group, (C) M group, (D) MB200 group, and (E) Msil group. Arterial wall thickness in monocrotaline group increased compared with other groups and two weeks consumption of barberry extract (200 mg/kg) attenuated this remodeling and its effect was stronger than 2 weeks consumption of sildenafil.
rats (Fatehi-Hassanabad et al., 2005). It showed that barberry caused the enhancement of K+ current outflux and this could be accounted for the reduction of systemic blood pressure through peripheral resistance vessels relaxation. The discrepancy between our findings with the results of Fatehi et al. (2005) and Fatehi-Hassanabad et al. (2005) could be due to the difference in the administration method of the barberry extract. We gavaged the extract for 2 weeks and a day after the last administration, pressure recording was done. Fatehi et al. (2005) used hypertensive animals and injected the barberry extract intravenously and immediately measured the systemic blood pressure. The other possible reason may come from this fact that barberry extract like antihypertensive drugs have stronger effects on lowering the systemic blood pressure in hypertensive conditions than in normotensive conditions.
In agreement with previous studies (Mei et al., 2011; Nishida et al., 2004), we observed a significant increase in RVSP, as an equivalent parameter of pulmonary arterial pressure, and the RVH in the M group. In addition, monocrotaline increased the medial wall thickness of the pulmonary arterioles, which is consistent with previous studies (Mei et al., 2011; Nishida et al., 2004; Todd et al., 1985). Barberry extract significantly decreased the monocrotaline-induced pulmonary arterial hypertension, the RVH, and also the medial wall thickness of pulmonary arterioles. These beneficial effects were comparable with sildenafil and even it was more prominent in the case of barberry with the dose of 200 mg/kg. Barberry contains phenolic compounds such as caffeic and chlorogenic acids, which strengthen the K+ current outflux
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Figure 6. Serum Et-1 levels in experimental groups at the end of the 4th week. Data are presented as mean ± SEM. n ¼ 6–7. zp50.001 compared with the CTL group. *p50.05 versus the B200 group, #p50.05 compared with the B200 group. p50.01 compared with the CTL group. Barberry extract (200 mg/kg) and sildenafil (30 mg/kg) could not alter serum ET-1 level in the monocrotaline group. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
Figure 7. Lung ET-1 levels at the end of the 4th week in all groups. Values are mean ± SEM. Differences were not statistically significant. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
and consequently increase the vasorelaxation (Fatehi et al., 2005; Fatehi-Hassanabad et al., 2005). The lowering effects of barberry on the pulmonary arterial hypertension and also the RVH through the reduction of the right ventricular afterload may be attributable to its phenolic compounds. On the contrary, Ivanovska and Philipov (1996) showed that the alkaloid constituents with an isoquinolinic nucleus such as berberine, berbamine, and palmatine are important compounds of the barberry root extract. An in vitro study reported that berberine inhibits the rat’s vascular smooth muscle cell migration and its proliferation. In addition, in vivo findings suggest that berberine improves the neointima formation after balloon injury (Lee et al., 2006). Therefore, a part of the preventive effect of barberry on the pulmonary arteriolar remodeling and PAH may result from the effects of its berberine components. Biochemical measurements showed the significant increase of serum ET-1, the non-significant reduction of ET-1 and GPX and also the non-significant elevation of MDA levels of the lung tissue in the monocrotaline group.
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Figure 8. Glutation peroxidase (GPX) activity of lung tissue in different groups at the end of the 4th week. n ¼ 6. Difference in data was not statistically significant. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
Figure 9. Malondialdehide (MDA) content of lung tissue at the end of the 4th week in all groups. n ¼ 6. Difference in data was not statistically significant. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
In agreement with our findings, others demonstrated that monocrotaline increases the plasma ET-1 (Jasmin et al., 2003; Miyauchi et al., 1993), and it has been observed the elevated levels of plasma ET-1 in patients with PAH (Frasch et al., 1999). In addition, following monocrotaline-induced PAH, reduction has been observed in the level of the lung tissue ET-1 (Miyauchi et al., 1993). Increase in the plasma level of ET-1 may originate from other organs such as the kidney and the heart (Miyauchi et al., 1993). There is some evidence in favor to significant downregulation of the ETB receptor in the pulmonary resistance arteries of PAH rats (Sauvageau et al., 2009). Therefore, the other possible reason to explain high serum ET-1 in the M group is the attenuation of ET-1 clearance by ETB receptors. Barberry and sildenafil did not show any significant influences on the monocrotalineinduced elevated serum ET-l levels. It is in accordance with the previous study that sildenafil did not alter the elevated ET1 followed by pulmonary hypertension in the rat (Dai et al., 2006). As a result, the therapeutic effect of barberry or
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sildenafil on monocrotaline-induced PAH could not be mediated through the modulation of ET-1. As shown in Figures 8 and 9, barberry to some extent insignificantly recovered the harmful effect of monocrotaline on the GPX and MDA level of the lung tissues of the PAH rats. Previous studies confirmed the antioxidant effects of barberry (Tomosaka et al., 2008; Zovko et al., 2010). Therefore, a small part of the beneficial effects of barberry in the present study may result from its effect on redox balance.
Conclusion
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Treatment with aqueous extract of barberry particularly in the dose of 200 mg/kg attenuated the pulmonary arterial hypertension and pulmonary vascular remodeling in monocrotalineinduced PAH in rat and these effects were stronger than sildenafil. The results suggest that barberry involves the mechanisms other than the modulation of endothelin and redox imbalance in the prevention of PAH development.
Acknowledgements The data presented in this article are from a Master’s thesis (Naser Mahdavi) performed in the Department of Physiology of School of Medicine and Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran.
Declaration of interest The authors report that they have no conflicts of interest. The authors are thankful to the Vice Chancellor of Research, Kerman University of Medical Sciences, for financial support.
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Frasch HF, Marshall C, Marshall BE. (1999). Endothelin-1 is elevated in monocrotaline pulmonary hypertension. Am J Physiol 1276: L304–10. Humbert M, Morrell NW, Archer SL, et al. (2004). Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 43:13S–24. Imanshahidi M, Hosseinzadeh H. (2008). Pharmacological and therapeutic effects of Berberis vulgaris and its active constituent, Berberine. Phytother Res 22:999–1012. Ivanovska N, Philipov S. (1996). Study of the anti-inflammatory action of Berberis vulgaris root extract, alkaloid fractions and pure alkaloids. Int J Immunopharmacol 18:553–61. Jasmin JF, Cernacek P, Dupuis J. (2003). Activation of the right ventricular endothelin (ET) system in the monocrotaline model of pulmonary hypertension: Response to chronic ETA receptor blockade. Clin Sci 105:647–53. Joukar S, Mahdavi N. (2014). Alterations of blood pressure and ECG following two-week consumption of Berberis integerrima fruit extract. ISRN 2014:209683. Joukar S, Najafipour H, Malekpour-Afshar R, et al. (2010). The effect of passive opium smoking on cardiovascular indices of rabbits with normal and ischemic hearts. Open Cardiovasc Med J 4:1–6. Joukar S, Shahouzehi B, Najafipour H, et al. (2012). Ameliorative effect of black tea on nicotine induced cardiovascular pathogenesis in rat. EXCLI J 11:309–17. Lee S, Lim HJ, Park HY, et al. (2006). Berberine inhibits rat vascular smooth muscle cell proliferation and migration in vitro and improves neointima formation after balloon injury in vivo berberine improves neointima formation in a rat model. Atherosclerosis 186: 29–37. Marin-Neto JA, Maciel BC, Secches AL, Gallo L. (1988). Cardiovascular effects of berberine in patients with severe congestive heart failure. Clin Cardiol 11:253–60. Mathew R, Gloster ES, Sundararajan T, et al. (1997). Role of inhibition of nitric oxide production in monocrotaline induced pulmonary hypertension. J Appl Physiol 82:1493–8. Mei Y, Jin H, Tian W, et al. (2011). Urantide alleviates monocrotaline induced pulmonary arterial hypertension in Wistar rats. Pulm Pharmacol Ther 24:386–93. Miyauchi T, Yorikane R, Sakai S, et al. (1993). Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rats with monocrotaline-induced pulmonary hypertension. Circ Res 73:887–97. Nishida M, Okada Y, Akiyoshi K, et al. (2004). Role of endothelin ETB receptor in the pathogenesis of monocrotaline induced pulmonary hypertension in rats. Eur J Pharmacol 496:159–65. Rhodes CH, Davidson A, Wharton J, Wilkins MR. (2009). Therapeutic targets in pulmonary arterial hypertension. Pharmacol Ther 121: 69–88. Sauvageau S, Thorin E, Villeneuve L, Dupuis J. (2009). Change in pharmacological effect of endothelin receptor antagonists in rats with pulmonary hypertension: Role of ETB-receptor expression levels. Pulm Pharmacol Ther 22:311–17. Todd L, Mullen M, Olley PM, Rabinovitch M. (1985). Pulmonary toxicity of monocrotaline differs at critical periods of lung development. Pediatr Res 19:731–7. Tomosaka H, Chin YW, Salim AA, et al. (2008). Antioxidant and cytoprotective compounds from Berberis vulgaris (barberry). Phytother Res 22:979–81. Widerman RF, Hamal KR. (2011). Idiopathic pulmonary arterial hypertension: An avian model for plexogenic arteriopathy and serotonergic vasoconstriction. J Pharmacol Toxicol Methods 63: 283–95. Yildiz P. (2009). Molecular mechanisms of pulmonary hypertension. Clin Chim Acta 403:9–16. Zovko Koncic´ M, Kremer D, Karlovic´ K, Kosalec I. (2010). Evaluation of antioxidant activities and phenolic content of Berberis vulgaris and Berberis croatica Horvat. Food Chem Toxicol 48:2176–80. Zambelli V, Santaniello A, Fumagalli F, et al. (2011). Efficacy of aminaftone in a rat model of monocrotaline induced pulmonary hypertension. Eur J Pharmacol 667:287–91.
Pharmaceutical Biology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/iphb20
The promising effect of barberry (Zereshk) extract against experimental pulmonary microvascular remodeling and hypertension: A comparison with sildenafil ab
ab
Naser Mahdavi , Siyavash Joukar , Hamid Najafipour
bc
d
& Majid Asadi-Shekaari
a
Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran, b
Department of Physiology and Pharmacology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, c
Click for updates
Cardiovascular Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran, and d
Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran Published online: 02 Jun 2015.
To cite this article: Naser Mahdavi, Siyavash Joukar, Hamid Najafipour & Majid Asadi-Shekaari (2015): The promising effect of barberry (Zereshk) extract against experimental pulmonary microvascular remodeling and hypertension: A comparison with sildenafil, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1050676
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1050676
ORIGINAL ARTICLE
The promising effect of barberry (Zereshk) extract against experimental pulmonary microvascular remodeling and hypertension: A comparison with sildenafil Naser Mahdavi1,2, Siyavash Joukar1,2, Hamid Najafipour2,3, and Majid Asadi-Shekaari4 Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran, 2Department of Physiology and Pharmacology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, 3Cardiovascular Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran, and 4Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
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Abstract
Keywords
Context: Despite the beneficial effects of barberry (Berberis integerrima Berberidaceae) on decreasing systemic hypertension, its influence has not been investigated on pulmonary hypertension. Objective: The objective of this study is to examine the effect of barberry fruit, on monocrotaline-induced pulmonary hypertension. Materials and methods: Nine groups were arranged as follows: the control group, the monocrotaline (M) group, the barberry groups with doses of 50, 100, and 200 (mg/kg), the M plus barberry groups, and the M plus sildenafil group. Two weeks after a single injection of monocrotaline (60 mg/kg, s.c.), barberry water extracts or sildenafil (30 mg/kg/d) were gavaged daily for 2 weeks. At the end of the 4th week, hemodynamic, biochemical, and histopathological parameters were assessed. Results: In comparison with the M group, barberry (200 mg/kg) or sildenafil significantly reduced the right ventricular systolic pressure (RVSP) (22.95 ± 1.78 mm Hg and 30.71 ± 1.64 mm Hg, versus 41.28 ± 1.5 mm Hg), right ventricular hypertrophy (RVH) (0.39 ± 0.03 and 0.42 ± 0.02, versus 0.57 ± 0.02), and the medial wall thickness (MWT) (4.56 ± 0.15 mm and 5.97 ± 0.19 mm, versus 7.02 ± 0.43 mm). Barberry or sildenafil had no significant effect on the plasma level of endothelin-1, glutathione peroxidase, and the malondialdehide of lung. Conclusion: 200 mg/kg of barberry has an improving effect on the monocrotaline-induced pulmonary hypertension. This effect was stronger than that of the sildenafil’s and may have been mediated through mechanisms other than the modulation of the endothelin-1 or redox system.
Berberine, endothelin-1, monocrotaline, pulmonary arterial hypertension
Introduction In clinics, pulmonary arterial hypertension (PAH) is described as a mean pulmonary artery pressure 25 mm Hg at rest or 30 mm Hg with exercise and with pulmonary capillary wedge pressure (15 mm Hg) (Barst et al., 2004). The survival time for the untreated idiopathic PAH (formerly included in primary pulmonary hypertension) was reported to be 3 years (Archer et al., 2010). PAH can be associated with various other diseases including HIV infection, connective tissue diseases, portal hypertension, schistosomiasis, chronic hemolytic anemia, and congenital heart disease, and the prognosis could depend on the underlying diseases (Chan & Loscalzo, 2008). The increased vasoconstriction of the pulmonary arteries in PAH Correspondence: Siyavash Joukar, Physiology Research Center, Institute of Neuropharmacology and Department of Physiology and Pharmacology, School of Medicine, Kerman University of Medical Sciences, PO Box 7616914115, Kerman, Iran. Tel/Fax: +98 341 3220081. E-mail: [email protected], [email protected]
History Received 6 November 2014 Accepted 8 May 2015 Published online 29 May 2015
has been attributed to the reduced expression/activity of the voltage-gated potassium channels and also dysfunction of the endothelium that lead to an imbalance in the production and release of both vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) and vasoconstrictors such as endothelin-1 (ET-1) and serotonin (5-HT) (Humbert et al., 2004). Many of these regulators of vascular tone also have played roles in controlling cell proliferation and apoptosis and can contribute to the remodeling of the pulmonary vasculature. The structural changes of pulmonary vessels in PAH can include the medial thickening due to the smooth muscle cell hyperplasia and hypertrophy (Yildiz, 2009), formation of a neointima composed of smooth muscle cells or myofibroblasts embedded in a mucopolysaccharide matrix and the plexiform lesions due to endothelial and smooth muscle cell proliferation (Widerman & Hamal, 2011). Currently, there are three main classes of drugs used for the treatment of this disease: prostanoids, endothelin-1 receptor antagonists, and phosphodiesterase-type 5 (PDE5) inhibitors (Rhodes et al., 2009).
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Although, to some extent, these drugs can decelerate the PAH development and attenuate its clinical symptoms, they cannot cure the disease completely (Rhodes et al., 2009). Hence, finding of new agents for treating of PAH is necessary. There are two famous barberry species, Berberis integerrima Berberidaceae and Berberis vulgaris Berberidaceae (Zereshk), in Iran. Berberis integerrima is a thorny shrub with fragile branches to a height of 1–3 m. Its fruits are 7– 10 mm long and 3–4 mm in diameter, with a mild sour taste (Ardestani et al., 2013; Joukar & Mahdavi, 2014). The major chemical compounds of barberry fruits include coumarin, ascorbic acid, berberine, caffeic acid, b-carotene, flavonoids, malic acid, palmatine, carbohydrates, tannin, and ursolic acid (Imanshahidi & Hosseinzadeh, 2008). Overall, the amounts of ash, fiber, protein, reducing sugars, total sugar, and pH are higher in B. integerrima whereas the moisture content, brix, and color indices are higher in B. vulgaris. The amounts of P, Zn, Fe, Na, and K in B. vulgaris are higher than in B. integerrima and the amounts of Mn, Mg, and Cu, Ca are higher in fruits of B. integerrima. In addition, the total phenolic and total anthocyanin contents of fresh fruits of B. integerrima are higher than in the fresh fruits of B. vulgaris (Ardestani et al., 2013). The various parts of Zereshk plant including its root, bark, leaf, and fruit have long been used in the West and East folk medicine (Imanshahidi & Hosseinzadeh, 2008). In traditional medicine, barberry has been used for the treatment of various heart diseases such as hypertension and arrhythmia (DerMaderosian, 2001; Fatehi et al., 2005). Several pharmacological studies have indicated the cardiovascular effects of barberry and berberine, its famous alkaloid constituent, such as preventing ischemia-induced ventricular tachyarrhythmia, improving cardiac contractility and lowering peripheral vascular resistance, and blood pressure (Chun et al., 1979; Marin-Neto et al., 1988). A study demonstrated that the administration of the barberry extract, the fruit of B. vulgari (0.05–1 mg/100 g body weight), significantly decreased the systemic arterial blood pressure and the heart rate dose dependently in rats (Fatehi-Hassanabad et al., 2005). However, so far the influence of this edible and medicinal fruit has not been investigated on pulmonary hypertension. The present study was conducted to examine the effects of the water extract of barberry, a popular seasoning especially used in the eastern societies, on the monocrotaline-induced pulmonary arterial hypertension, and to compare its effect with sildenafil.
Materials and methods Sildenafil sulfide (Kharazmi Pharmaceutical CO, Tehran, Iran) and sodium thiopental (Biocheme, Kundl, Austria) were dissolved in physiological saline and monocrotaline (MCT) (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) plus saline. Barberry (Berberis integerrima fruit) was collected from Baft area of Kerman, Iran, and it was identified by the Department of Botany, Bahonar University of Kerman. A voucher specimen (KF-1194) of the plant was deposited in the Herbarium Center of the Pharmacy Faculty of Kerman University of Medical Sciences. The glutathione peroxidase (GPX) assay kit was from Randox, Northern
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Ireland, UK, and the endothelin-1 (ET-1) kit was obtained from USCN Life Science Inc., Wuhan, China. Animal groups The study procedures were approved and performed based on the national guidelines for conducting animal studies (Ethic committee permission No. 90/315-Kerman University of Medical Sciences, Kerman, Iran). Seventy-two male Wistar rats (purchased from the Physiology Research Center, Kerman, Iran) weighing between 250 and 300 g were allowed free access to standard laboratory rat chow and tap water and were housed under controlled humidity and room temperature and a 12 h light/dark cycle. Animals were divided to nine groups as follows: the CTL (control) group which received a single injection of 0.5 ml saline plus DMSO (i.p.) on the first day of study as the vehicle of monocrotaline. The M (monocrotaline) group which received equal volume of single dose of MCT (60 mg/kg, i.p.) on the first day of study. The B50, B100, and B200 groups which received saline plus DMSO (i.p.) on the first day of study. Then, at the beginning of the third week, they were treated with barberry extracts by the gavage method with doses of 50, 100, and 200 mg/kg/d, for the next 2 weeks. The MB50, MB100, and MB200 groups received monocrotaline on the first day and then were treated with barberry extracts similar to the B50, B100, and B200 groups. The Msil group was treated with sildenafil (30 mg/kg/d, orally) for 2 weeks at the beginning of the third week after monocrotaline injection. Survival At the end of the experiment, the rats which received monocrotline had significant mortality, but no rat died in the MB200 group. Therefore, we designed our study so that 7–8 rats could stay alive at the treating period (4th week). Three out of the 11 rats of the M group (n ¼ 8, surviving), two out of nine in the MB50 group (n ¼ 7, surviving), one out of nine in the MB100 group (n ¼ 8, surviving), one out of eight in the MSil group (n ¼ 7, surviving) died during the treatment period. Induction of pulmonary arterial hypertension The pulmonary arterial hypertension was induced by a single intraperitoneal injection of 60 mg/kg monocrotaline on the first day of the study (Sauvageau et al., 2009). At the end of the fourth week, the animals showed obvious pulmonary hypertension. Barberry extracts preparation Each 10 g of dried barberry was boiled with 100 ml water for 5 min. The mixture was then filtered, and the liquid evaporated under vacuum at 45–50 C. The material was then dried by exposure to hot air (40 C) for 48 h (FatehiHassanabad et al., 2005). Then, barberry extracts were kept in glass vials at 20 C prior to use. Hemodynamic measurements Four weeks after monocrotaline or vehicle injection, on the 29th day, the animals were anesthetized with an
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intra-peritoneal injection of 50 mg/kg thiopental sodium. A polyethylene cannula containing saline-heparin solution (15 l/mL) was inserted into the isolated right carotid artery and then was connected to a pressure transducer to measure the arterial blood pressure (Joukar et al., 2010). The mean arterial pressure (MAP) was calculated by ‘‘MAP ¼ Pd + (PsPd)/3 formula’’, in which Pd is the diastolic arterial pressure and Ps is the systolic arterial pressure. The right ventricle was gently cannulated with a filled catheter (saline–heparin solution, 15 l/mL) connected to a pressure transducer of a PowerLab system (Power Lab Systems, Springs, CO) to record its systolic pressure as an equivalent parameter of the pulmonary systolic pressure.
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Biochemical parameters In the evening of the 28th day, under ether light anesthesia, 2 ml of blood sample taken by retro-orbital puncture, then centrifuged to later produce a serum which was frozen and kept at 80 C for measurement of ET-1. At the end of the experimental recording, a piece of the lung tissue was cut and frozen in liquid nitrogen and then was stored at 80 C for the measurement of the ET-1, GPX, and malondialdehide (MDA) levels of the lung tissue. The ET-1 content was measured by using an ET-1 immunoassay kit by a sandwich enzyme immunoassay technique (Zambelli et al., 2011). All reagents, samples, and standards were prepared according to the manufacturer protocol. The reaction product was measured by reading the absorbance at 450 nm. The GPX level was measured by using RANDOX kit (Randox, Northern Ireland, UK) via the ultra violet (UV) method. Briefly, 0.05 ml sample supernatant was mixed with 1 ml diluting agent. After incubating for 5 min, 1 ml Drobkin’s reagent was added. Samples were thoroughly mixed and, after 20 min, they were measured by reading the absorbance at 340 nm. The MDA level was measured using TBARS assessment (Joukar et al., 2012). Histopathological assessment
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Data analysis Statistical analysis was performed by statistical software SPSS 20 (SPSS Inc., Chicago, IL). All values were expressed as means ± SEM. Comparisons were performed among different groups by one-way ANOVA and post hoc Tukey’s test. p Value less than 0.05 were considered statistically significant.
Results Hemodynamic experiments Mean systemic arterial pressure (MAP) did not show any significant difference among the animal groups (Figure 1). The right ventricular systolic pressure (RVSP) significantly increased in the M group in comparison with the CTL, B50, B100, and B200 groups (p50.001). In the presence of monocrotaline, 2-week consumption of the barberry extracts with doses of 50, 100, and 200 mg/kg significantly reduced the RVSP. This effect was prominent in the MB100 and MB200 groups (p50.001 versus the M group) compared with the MB50 group (p50.01 versus the M group). The administration of sildenafil also reduced the RVSP (p50.01 versus the M group), although this beneficial effect was insignificantly weaker than the effect of higher doses of barberry (Figure 2). RVH index RVH index significantly increased in the M group compared with the control groups (CTL, B50, B100, and B200). The animal groups that received monocrotaline plus sildenafil (Msil group) or barberry extract with dosages of 100 and 200 mg/kg (MB100 and MB200) showed a reduction in the RVH index whenever compared with monocrotaline alone (p50.05 for Msil and MB100 versus the M group and p50.01 for MB200 versus the M group) (Figure 3). Histopathological assessment Because of the stronger beneficial effect of 200 mg/kg of the barberry extract on the improvement of the
At the end of the experiment, lungs were washed by saline infusion and then infused with 40 mL of 4% paraformaldehyde solution. Thereafter, lungs were removed and fixed in 10% buffered formalin, and after tissue processing, they were embedded in paraffin. Three micron-thick sections were prepared and stained with hematoxylin and eosin and were examined under light microscopy. The medial thickness and vessel diameter of 60 pulmonary arterioles, with a size of 25–100 mm, in each lung section were measured randomly in blind manner (Cowan et al., 2000; Mathew et al., 1997). Right ventricular hypertrophy index After hemodynamic measurements, the hearts of the animals were also excised and cleaned by saline. The right ventricular hypertrophy (RVH) index was calculated using the following formula: ‘‘RVH ¼ 2 (RVFWT)/(LVFWT + IST)’’, where RVFWT is the right ventricular free wall thickness, LVFWT is the left ventricular free wall thickness and IST is the interventricular septum thickness (Nishida et al., 2004).
Figure 1. Mean systemic arterial pressure (MSAP) in different experimental groups at the end of the 4th week. Values are mean ± SEM. n ¼ 7–8. The differences in data were not significant. CTL, control group; B50, 50 mg/kg barberry extract; B100, 100 mg/kg barberry extract; B200, 200 mg/kg barberry extract; M, monocrotaline group; MB50, monocrotaline + 50 mg/kg barberry extract; MB100, monocrotaline + 100 mg/kg barberry extract; MB200, monocrotaline + 200 mg/kg barberry extract; Msil, monocrotaline + 30 mg/kg sildenafil.
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Figure 2. Right ventricular systolic pressure (RVSP) in different experimental groups (mm Hg) in all groups at the end of the 4th week. Values are mean ± SEM. n ¼ 7–8. ***p50.001 compared with CTL, B50, B100, and B200 groups. #p50.01 compared with M and MB200 groups. zp50.001 compared with the M group. p50.001 compared with the M group. *p50.01 compared with the M group. RVSP increased in the monocrotaline group compared with other groups and barberry extracts (50, 100, and 200 mg/kg) dose dependently reduced this parameter. CTL, control group; B50, 50 mg/kg barberry extract; B100, 100 mg/kg barberry extract; B200, 200 mg/kg barberry extract; M, monocrotaline group; MB50, monocrotaline + 50 mg/kg barberry extract; MB100, monocrotaline + 100 mg/kg barberry extract; MB200, monocrotaline + 200 mg/kg barberry extract; Msil, monocrotaline + 30 mg/kg sildenafil.
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Figure 4. Medial wall thickness of pulmonary arterioles (mm). Data are presented as mean ± SEM. n ¼ 7–8, ***p50.001 compared with CTL and B200 groups. *p50.05 compared with the M group. zp50.001 compared with Msil and M groups. Medial wall thickness of pulmonary arteriols significantly decreased in MB200 compared with monocrotaline and Msil groups. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
Plasma and lung tissue ET-1 level The administration of monocrotaline was associated with the increase of serum ET-1 in the M group (p50.001 versus the CTL group). The consumption of 200 mg/kg barberry extract and the sildenafil could not attenuate the effect of the monocrotaline on the serum ET-1 level (Figure 6). The lung tissue ET-1 was lowered in the M group compared with the other groups, but it was not statistically significant. The usage of sildenafil or barberry extract plus monocrotaline insignificantly elevated the lung ET-1 to values of the CTL and B200 levels, respectively (Figure 7). Lung tissue GPX and MDA Figure 3. Right ventricular hypertrophy index. Data are presented as mean ± SEM. n ¼ 7–8. *p50.05 compared with CTL and B100 groups. #p50.01 compared with the B200 group. yp50.01 compared with the M group. p50.05 compared with the M group. RVH increased in the monocrotaline group. Two weeks consumption of barberry extracts attenuated this parameter especially in dose of 200 mg/kg. CTL, control group; B50, 50 mg/kg barberry extract; B100, 100 mg/kg barberry extract; B200, 200 mg/kg barberry extract; M, monocrotaline group; MB50, monocrotaline + 50 mg/kg barberry extract; MB100, monocrotaline + 100 mg/kg barberry extract; MB200, monocrotaline + 200 mg/kg barberry extract; Msil, monocrotaline + 30 mg/kg sildenafil.
hemodynamic parameters and the RVH index, histopathological and biochemical indices were assessed only in the CTL, B200, M, MB200, and Msil groups. MWT significantly increased in the M group compared with the CTL and B200 groups. Two weeks usage of 200 mg/kg barberry extract obviously decreased the growing effect of monocrotaline on the thickness of the arterioles wall (p50.001 compared with the M group). Sildenafil also attenuated the monocrotaline induced arteriole remodeling (p50.05 versus the M group); however, this valuable effect was weaker than in the barberry extract (p50.05) (Figures 4 and 5).
The animals with monocrotaline-induced pulmonary hypertension showed a mild but non-significant reduction in GPX (Figure 8) and a mild raise in MDA content of the lung tissue (Figure 9). Barberry extract consumption recovered these effects whereas sildenafil could not improve the elevated level of MDA (Figure 9).
Discussion This study was conducted to determine the therapeutic effect of aqueous extract of barberry on monocrotaline-induced pulmonary hypertension and to compare it with the effect of sildenafil – a known drug for PAH treatment. MCT injection had no significant effect on MAP in this study. This result is in accordance with the findings of previous studies (Mei et al., 2011; Nishida et al., 2004). In addition, different groups which received barberry extract alone or plus monocrotaline and also sildenafil plus monocrotaline group did not show significant changes in systemic arterial pressure. However, higher doses of barberry (100 and 200 mg/kg) to some extent reduced the systemic blood pressure which was statistically insignificant. A study reported that barberry aqueous extract significantly reduces the systemic blood pressure in normotensive and hypertensive
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Figure 5. Arterioles of lung tissue of different animals group. (A) CTL group, (B) B200 group, (C) M group, (D) MB200 group, and (E) Msil group. Arterial wall thickness in monocrotaline group increased compared with other groups and two weeks consumption of barberry extract (200 mg/kg) attenuated this remodeling and its effect was stronger than 2 weeks consumption of sildenafil.
rats (Fatehi-Hassanabad et al., 2005). It showed that barberry caused the enhancement of K+ current outflux and this could be accounted for the reduction of systemic blood pressure through peripheral resistance vessels relaxation. The discrepancy between our findings with the results of Fatehi et al. (2005) and Fatehi-Hassanabad et al. (2005) could be due to the difference in the administration method of the barberry extract. We gavaged the extract for 2 weeks and a day after the last administration, pressure recording was done. Fatehi et al. (2005) used hypertensive animals and injected the barberry extract intravenously and immediately measured the systemic blood pressure. The other possible reason may come from this fact that barberry extract like antihypertensive drugs have stronger effects on lowering the systemic blood pressure in hypertensive conditions than in normotensive conditions.
In agreement with previous studies (Mei et al., 2011; Nishida et al., 2004), we observed a significant increase in RVSP, as an equivalent parameter of pulmonary arterial pressure, and the RVH in the M group. In addition, monocrotaline increased the medial wall thickness of the pulmonary arterioles, which is consistent with previous studies (Mei et al., 2011; Nishida et al., 2004; Todd et al., 1985). Barberry extract significantly decreased the monocrotaline-induced pulmonary arterial hypertension, the RVH, and also the medial wall thickness of pulmonary arterioles. These beneficial effects were comparable with sildenafil and even it was more prominent in the case of barberry with the dose of 200 mg/kg. Barberry contains phenolic compounds such as caffeic and chlorogenic acids, which strengthen the K+ current outflux
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Figure 6. Serum Et-1 levels in experimental groups at the end of the 4th week. Data are presented as mean ± SEM. n ¼ 6–7. zp50.001 compared with the CTL group. *p50.05 versus the B200 group, #p50.05 compared with the B200 group. p50.01 compared with the CTL group. Barberry extract (200 mg/kg) and sildenafil (30 mg/kg) could not alter serum ET-1 level in the monocrotaline group. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
Figure 7. Lung ET-1 levels at the end of the 4th week in all groups. Values are mean ± SEM. Differences were not statistically significant. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
and consequently increase the vasorelaxation (Fatehi et al., 2005; Fatehi-Hassanabad et al., 2005). The lowering effects of barberry on the pulmonary arterial hypertension and also the RVH through the reduction of the right ventricular afterload may be attributable to its phenolic compounds. On the contrary, Ivanovska and Philipov (1996) showed that the alkaloid constituents with an isoquinolinic nucleus such as berberine, berbamine, and palmatine are important compounds of the barberry root extract. An in vitro study reported that berberine inhibits the rat’s vascular smooth muscle cell migration and its proliferation. In addition, in vivo findings suggest that berberine improves the neointima formation after balloon injury (Lee et al., 2006). Therefore, a part of the preventive effect of barberry on the pulmonary arteriolar remodeling and PAH may result from the effects of its berberine components. Biochemical measurements showed the significant increase of serum ET-1, the non-significant reduction of ET-1 and GPX and also the non-significant elevation of MDA levels of the lung tissue in the monocrotaline group.
Pharm Biol, Early Online: 1–7
Figure 8. Glutation peroxidase (GPX) activity of lung tissue in different groups at the end of the 4th week. n ¼ 6. Difference in data was not statistically significant. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
Figure 9. Malondialdehide (MDA) content of lung tissue at the end of the 4th week in all groups. n ¼ 6. Difference in data was not statistically significant. CTL, control group; B200, barberry extract (200 mg/kg); M, monocrotaline group; MB200, monocrotaline plus barberry extract (200 mg/kg); Msil, monocrotaline plus sildenafil (30 mg/kg).
In agreement with our findings, others demonstrated that monocrotaline increases the plasma ET-1 (Jasmin et al., 2003; Miyauchi et al., 1993), and it has been observed the elevated levels of plasma ET-1 in patients with PAH (Frasch et al., 1999). In addition, following monocrotaline-induced PAH, reduction has been observed in the level of the lung tissue ET-1 (Miyauchi et al., 1993). Increase in the plasma level of ET-1 may originate from other organs such as the kidney and the heart (Miyauchi et al., 1993). There is some evidence in favor to significant downregulation of the ETB receptor in the pulmonary resistance arteries of PAH rats (Sauvageau et al., 2009). Therefore, the other possible reason to explain high serum ET-1 in the M group is the attenuation of ET-1 clearance by ETB receptors. Barberry and sildenafil did not show any significant influences on the monocrotalineinduced elevated serum ET-l levels. It is in accordance with the previous study that sildenafil did not alter the elevated ET1 followed by pulmonary hypertension in the rat (Dai et al., 2006). As a result, the therapeutic effect of barberry or
DOI: 10.3109/13880209.2015.1050676
sildenafil on monocrotaline-induced PAH could not be mediated through the modulation of ET-1. As shown in Figures 8 and 9, barberry to some extent insignificantly recovered the harmful effect of monocrotaline on the GPX and MDA level of the lung tissues of the PAH rats. Previous studies confirmed the antioxidant effects of barberry (Tomosaka et al., 2008; Zovko et al., 2010). Therefore, a small part of the beneficial effects of barberry in the present study may result from its effect on redox balance.
Conclusion
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Treatment with aqueous extract of barberry particularly in the dose of 200 mg/kg attenuated the pulmonary arterial hypertension and pulmonary vascular remodeling in monocrotalineinduced PAH in rat and these effects were stronger than sildenafil. The results suggest that barberry involves the mechanisms other than the modulation of endothelin and redox imbalance in the prevention of PAH development.
Acknowledgements The data presented in this article are from a Master’s thesis (Naser Mahdavi) performed in the Department of Physiology of School of Medicine and Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran.
Declaration of interest The authors report that they have no conflicts of interest. The authors are thankful to the Vice Chancellor of Research, Kerman University of Medical Sciences, for financial support.
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