原花青素对离体肺动脉血管张力的影响

doi:10.3969/j.issn.2095-4344.2016.18.013

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (18): 2668-2676.doi: 10.3969/j.issn.2095-4344.2016.18.013

• 心脏及血管损伤动物模型 Animal models of heart and vascular damage • 上一篇下一篇

原花青素对离体肺动脉血管张力的影响

牛彩琴¹，孙安²，雷枭¹，张团笑³

川北医学院，1中西医临床医学系，3生理学教研室，四川省南充市 637007；2解放军兰州军区空军机关医院，甘肃省兰州市 370020

收稿日期:2016-02-14 出版日期:2016-04-29 发布日期:2016-04-29
通讯作者: 张团笑，硕士，教授，川北医学院生理学教研室，四川省南充市 637007
作者简介:Niu Cai-qin, Master, Associate professor, Clinical Department of Chinese and Western Medicine, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China
基金资助:
四川省教育厅重点项目(14ZA0182)

Vasorelaxant effects of procyanidins on pulmonary artery in vitro

Niu Cai-qin¹, Sun An², Lei Xiao¹, Zhang Tuan-xiao³

1 Clinical Department of Chinese and Western Medicine, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China
2 Lanzhou Military Region Air Force Hospital Authority, Lanzhou 730020, Gansu Province, China

3 Department of Physiology, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China

Received:2016-02-14 Online:2016-04-29 Published:2016-04-29
Contact: Zhang Tuan-xiao, Master, Professor, Department of Physiology, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China
About author:牛彩琴，女，1970年生，汉，甘肃省会宁县人，2005年甘肃中医学院毕业，硕士，副教授，主要从事中西医基础与临床方面的研究。
Supported by:
the Key Program of Sichuan Department of Education, No. 14ZA0182

摘要/Abstract

摘要：

文章快速阅读：

文题释义：

血管张力：大血管(主动脉和肺动脉等)属于多单位紧张性平滑肌，是以张力改变为主的紧张性收缩。血管张力就是一种持续性的、紧张性收缩活动的结果，其大小决定着血管的口径，而后者又影响着血流阻力和血流量。实验发现原花青素可显著舒张内皮完整的肺动脉，且呈剂量依赖性。

亲和力指数(pD2)：pD2=-logK_D= log(1/K_D)。K_D是游离药物的浓度，即当药物效应达到最大效应的一半时所需的浓度(EC50)，它表示受体结合激动剂的亲和力且与之成反比。但以pD2代表亲和力则成正比。

背景：原花青素是广泛存在于植物界中的一类多酚化合物，由不同数量的儿茶素或表儿茶素缩合而成，有保护血管内皮、清除自由基、抗血小板聚集、降低毛细血管通透性、起到降低血压、抗氧化活性、抗水肿、防止冠状动脉粥样硬化性心脏病和动脉粥样硬化等作用。

目的：观察原花青素对肺动脉环的舒张作用及机制。

方法：家兔离体肺动脉环为标本，以去甲肾上腺素(1×10^-6 mol/L)预收缩肺动脉环后给予0.625，1.25，2.5 mg/L 原花青素观察其张力变化。再将不同浓度原花青素干预的家兔离体肺动脉平滑肌去除血管内皮、给予一氧化氮合酶抑制剂左旋硝基精氨酸(1×10^-⁴mol/L)、甲烯蓝(1×10^-⁵ mol/L)、环氧酶抑制剂吲哚美辛(1×10^-⁵ mol/L)和β肾上腺素能受体阻断剂普萘洛尔(1×10^-⁵ mol/L)，探讨它们对血管张力的影响。

结果与结论：①原花青素对肺动脉环静息张力无明显影响，但不同剂量的原花青素可使1×10^-6 mol/L去甲肾上腺素预收缩血管产生明显舒张，并呈量效依赖性(r=0.69，P < 0.001)。②去除血管内皮、左旋硝基精氨酸和甲烯蓝均可减弱原花青素舒张血管的作用，但吲哚美辛和普萘洛尔无影响。③20 mg/L原花青素能明显压低去除内皮细胞肺动脉血管的去甲肾上腺素、KCl和CaCl₂的量效收缩反应曲线，且去甲肾上腺素、KCl和CaCl₂的亲和力指数降低(P < 0.01)。④原花青素能抑制去甲肾上腺素引起的第Ⅰ时相收缩，而对CaCl₂引起的第Ⅱ时相收缩无影响。⑤实验结果表明原花青素对肺动脉的舒张效应是内皮依赖性的，与NO/cGMP介导有关，可拮抗受体依赖性钙通道和电压依赖性钙通道而降低细胞内Ca²⁺引起血管舒张。
中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程
ORCID: 0000-0002-0618-3755(Niu Cai-qin)

关键词: Pulmonary Artery, Endothelial Cells, Vasodilation

Abstract:

BACKGROUND: Procyanidins is a kind of polyphenol compounds in regnum vegetable, which is composed of different quantities of catechin and epicatechin. Studies show that procyanidins plays a role on protecting vascular endothelium, scavenging free radicals, resisting platelet aggregation, and reducing capillary permeability. Thus, procyanidins has obviously functions of reducing blood pressure, anti-oxidant activity, anti-edema, preventing coronary heart disease and atherosclerosis.

OBJECTIVE: To study the vasorelaxant effect of procyanidins on pulmonic rings and its mechanisms.

METHODS: Rabbit thoracic pulmonary arteries were isolated. Pre-contracted with noradrenalin (1×10^-6 mol/L) and their responses to different concentrations of procyanidins (0.625, 1.25, 2.5 mg/L) were investigated. After removal of the endothelium of pulmonary artery smooth muscle, the effects of different signaling pathway inhibitors on procyanidins-induced relaxation, including nitric oxide synthase inhibitor Nω-Nitro-L-arginine (1×10^-4 mol/L), methylene blue (1×10^-5 mol/L), prostaglandin synthase inhibitor indomethacin (1×10^-5 mol/L) and blockage of the adrenergic β-receptor propranolol (1×10^-5 mol/L), were also assessed.

RESULTS AND CONCLUSION: (1) Procyanidins did not change the resting tension of rabbit’s pulmonic rings, but caused an obvious dose-dependent relaxation in 1×10^-6 mol/L noradrenalin-precontracted pulmonic rings (r=0.69, P < 0.001). (2) The relaxant effect of procyanidins was significantly reduced by removal of endothelium or by treatment with either

Nω-Nitro-L-arginine or methylene blue, but not by treatment with prostaglandin synthase inhibitor or blockage of the adrenergic β-receptor. (3) Procyanidins (20 mg/L) dropped the dose-effect curves of noradrenalin, KCl and on pulmonic rings denuded endothelium. Moreover, affinity index of noradrenalin, KCl and CaCl₂ decreased (P < 0.01). (4) Procyanidins also inhibited the vasoconstriction caused by noradrenalin in the first phase, but had no impact on the constriction induced by CaCl₂ in the second phase. (5) Procyanidins has an endothelium-dependent vasorelaxation on isolated rabbit’s pulmonic rings, which is possibly mediated by nitric oxide/cyclic guanosine monophosphate pathways. Procyanidins blocked receptor-operated and voltage-dependent calcium channels to reduce intracellular Ca²⁺, and induced vasorelaxation.
中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Pulmonary Artery, Endothelial Cells, Vasodilation

牛彩琴，孙安，雷枭，张团笑. 原花青素对离体肺动脉血管张力的影响[J]. 中国组织工程研究, 2016, 20(18): 2668-2676.

Niu Cai-qin, Sun An, Lei Xiao, Zhang Tuan-xiao. Vasorelaxant effects of procyanidins on pulmonary artery in vitro[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(18): 2668-2676.

图/表（结果） 7

Quantitative analysis of experimental animals

Twelve rabbits of clean grade were fed in regular way to remove their thoracic pulmonary artery after anesthesia. In the final analysis, all results of the pulmonary artery were from the 12 rabbits without loss.

PC relaxes NA-precontracted pulmonic rings

To investigate the effect of PC on the contraction intact pulmonic rings, the rings were pre-treated with 1×10^-⁶ mol/L NA followed by treatment with different concentrations of PC. As expected, the pulmonic rings were contracted by NA and surprisingly the contraction was significantly reduced by PC in a dose-dependent manner. The relaxation percentages for 0.625, 1.25 and 2.5 mg/L PC were (15.42±12.86)%, (41.43±13.24)% and (52.63±10.59)% respectively (r=0.69, t=7.52, P < 0.01; Figure 1). In addition, the highest concentration of PC (2.5 mg/L) used in the study appears to relax the muscle to the same extent as 1×10^-⁵ mol/L ACh which has a vasorelaxation of (65.23±10.88) % (P > 0.05; Figure 2) .

Vasorelaxant effect of PC is mediated through the cyclic guanosine monophosphate/nitric oxide pathway

We proceeded to study the possible mechanisms by which PC relaxes NA-precontracted pulmonic rings. Pulmonic rings were pretreated with different signaling pathway inhibitors or depleted of endothelium followed by treatments with NA and PC. As shown in Figure 3, treatment of 1×10^-⁴ mol/L L-NNA (nitric oxide synthase inhibitor), 1×10^-⁵ mol/L MB (guanylyl cyclase inhibitor) or deletion of endothelial cells could abolish the vasorelaxation of PC on pulmonic rings, suggesting that endothelium/cyclic guanosine monophosphate /nitric oxide pathways were at least involved in the relaxant process. Inhibition of prostaglandin pathway by 1×10^-⁵ mol/L Indo or blockage of β-receptor with 1×10^-⁵ mol/L Prop has no effect of PC induced relaxation (Table 1), suggesting neither PG nor adrenergic β-receptor is involved.

Effects of PC on the concentration-response curves of NA and KCl

PC had a significant negative effect on the concentration-response curve of NA and KCl. After incubation with PC (20 mg/L), the contractile peak of NA decreased to (55.94±18.32) % (n=10, P < 0.01) and the pD₂ decreased from 7.57±2.41 to 6.47±1.42 (Figure 4). In addition, the contractile peak of KCl decreased to (73.28±15.93)% (n=10, P < 0.01) and the pD₂ decreased from 1.74±1.19 to 1.07±1.12 (Figure 4).

Effect of PC on the CaCl₂concentration- response curve

The concentration-response curve of CaCl₂ could be shifted to right significantly by incubation with 20 mg/L PC. The maximum response dropped to (61.75±9.81)%, (n=10, P < 0.01) and the pD₂ dropped from 3.72±1.88 to 3.17±1.55 (Figure 5).

Effects of PC on contraction induced by NA and CaCl₂

In calcium-free (0.25 mmol/L ethylenediamine tetraacetic acid) Krebs’ solution, 1×10^-⁶ mol/L NA led a transient contraction which could be reduced by PC [The blood tension decreased from (2.48±0.24) g to (1.60±0.16) g, n=12, P < 0.01] significantly. However, PC had no effect on the second contractile response induced by 10 mmol/L CaCl₂ [The tension decreased from (1.20±0.11) g to (1.52±0.14) g, n=12, P > 0.05; Figure 6].

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引言

材料方法

Design

A controlled observational experiment of animal tissue in vitro.

Time and setting

The experiments were carried out in the Medical College of Lanzhou University of China between January 2003 and January 2005.

Materials

Animals

Healthy New Zealand white rabbits (7-8 months, specific-pathogen-free), weighing (2.5±0.5) kg, either male or female, were provided by Animal Experimental Center of Lanzhou University (Certification No. Medical Animal 14-004). Twelve rabbits of clean grade were fed in regular way, and then pulmonary artery was taken out from the rabbits after anesthesia. All animal experiments were approved by the Committee on Use and Care of Animals of Medical College of Lanzhou University.

Drugs

PC was purchased from Jianfeng company of Tianjin in China (Batch number: 2002093; polyphenol concentration > 95.6%), and was diluted in double distilled water and used at final concentrations of 0.625, 1.25 and 2.5 g/L in the study. Krebs’ buffer (K-H) was composed of (mmol/L): NaCl 120, 0.25 ethylenediamine tetraacetic acid, KCl 5.9, NaH₂PO₄ 1.2, MgCl₂ 1.2, NaHCO₃ 15.4, CaCl₂ 2.5 and glucose 11.5.

Methods

Preparation of rabbit thoracic pulmonic rings

Rabbits were sacrificed after anesthesia and the thoracic pulmonary artery was dissected out and the connective tissues were denuded gently, then it was cut into rings of 2.5-3.0 mm^[22]width and some of the rings were removed of the endothelium by gently rubbing the luminal surface with a cotton swab (viability verified by 1×10^-5 mol/L acetylcholine, ACh) ^[23-25]. Each pulmonic ring was suspended in homeothermic organ baths filled with 5 mL Krebs’ buffer (37 ℃) and bubbled with 95 % O₂ and 5 % CO₂^[26]. The rings were equilibrated under a resting tension of 2.0 g for 90 minutes^[27-28], during which K-H solution was changed every 20 minutes. Before data collection, the rings were contracted with NA (1×10^-6 mol/L; Shanghai He Feng Pharmaceutical Co., Ltd.) to confirm that the endothelium was viable and only those with a good response were used for experiments. The isometric tension of the rings was recorded continuously through the tension transducer. All the data was input into the computer and processed with biological information handling system (Taimeng Electronic Limited Company, China).

Effect of PC on NA pre-contracted pulmonic rings

Following equilibration, the pulmonic rings with endothelium were treated with 1×10^-6 mol/L NA to reach a maximal contraction, which was referred to as 100 % contraction. Then the thoracic pulmonic rings were treated with PC with final concentrations of 0.625, 1.25 and 2.5 mg/L respectively. The concentration-response curves were obtained and the relaxation rate was calculated. The addition of the same amount of 0.9 % sodium chloride (normal saline) to the precontracted rings was a negative control while 1×10^-⁵ mol/L ACh was used as a positive control.

Observation of possible vasorelaxation mechanism of PC on pulmonic rings pre-contracted by NA

The rings with endothelium were washed with Krebs’ buffer several times, and incubated respectively with Nω-Nitro-L-arginine (1×10^-⁴ mol/L; Sigma, USA), methylene blue (1×10^-⁵ mol/L; Merck, Germany), indomethacin (1×10^-⁵ mol/L; Jingsu Taicang Pharmaceutical Company, China) or propranolol (1×10^-⁵ mol/L; The Second Beijing Pharmaceutical Company, China) for 15 minutes. Then the rings were incubated with 1×10^-6 mol/L NA to reach maximal contractions followed by treatment with PC with cumulative concentrations of 0.625, 1.25 and 2.5 mg/L respectively. In some experiments, instead of inhibitor pretreatment, the pulmonic rings were denuded of endothelial cells (DEN) before NA and PC treatments.

Effects of PC on the concentration-response curves of DEN pulmonic rings to NA and KCl

After removal of endothelium, the pulmonic rings were treated with cumulative NA (1×10^-8–1×10^-5 mol/L) or KCl (6.3-100 mmol/L). The maximal contraction was referred to as 100 % and the dose response curves were plotted. After washing with Krebs’ buffer, the pulmonic rings were incubated with 20 mg/L PC for 15 minutes followed by the NA or KCl concentration response experiments.

Vasodilate rate (%)=(control group effect value-drug effect value)/control group effect value. Vascular constriction rate (%)=(effect value–preload value)/(maximum effect value–preload value). vasodilate contraction tension (g)=effect value–preload value. Affinity index (pD₂)=-logK_D=log(1/K_D). K_D is the free drug concentration, it is the drug effect of half the maximum effect (EC₅₀), and mole is the concentration unit, which represents the affinity of receptor activator. The relation between affinity index value and free drug concentration value is inverse, but it is direct ratio with pD₂^[29].

Effects of PC on concentration-response curve of CaCl₂

After incubation with KCl (40 mmol/L) in Ca²⁺ free Krebs’ solution for 30 minutes, a cumulative dose CaCl₂ (1×10^{-5^-² mmol/L) was administered and the concentration-response curve was drawn. The contractile effects of 100 % were recorded as the control. After being washed with Krebs’ solution, the rings were incubated with PC (20 mg/L) for 15 minutes and the concentration-response of CaCl₂was observed repeatedly.}-1×10

Effects of PC on NA and CaCl2 concentration dependent contractile response in rabbit pulmonic rings

To study the possible effect of PC on NA-induced calcium release and calcium influx through receptor-operated calcium channels, pulmonic rings DEN were incubated in calcium-free solution containing 0.25 mmol/L ethylenediamine tetraacetic acid for 30 minutes, then treated with 1×10^-6mol/L NA. When the contractile response reached a stable plateau, CaCl₂ (10 mmol/L) was added and a further contraction was observed. Rings were washed with Ca²⁺-free solution and left to return to baseline tone for 15 minutes before 20 mg/L PC was added and the contractile effects of NA and CaCl₂ were repeated^[25].

Main outcome measures

PC has an obvious vasorelaxation effect and can low blood pressure through promoting endotheliocyte release nitric oxide and decreasing influx of extracellular Ca²⁺, which provides a theoretical basis for applying PC in cardiovascular disease’s treatment.

Statistical analysis

All data were expressed as mean±SD. The data were analyzed statistically using the SPSS 11.0 for Windows statistical program by one-way analysis of variance or Student’s t-test. P < 0.05 was set as significant difference. Correlativity analysis of drug dose-effect relationship was determined with Linear(r).

讨论

As a plant polyphenol, PC is widely recognized to have various biological effects that could be beneficial in prevention and treatment of human diseases. Zhang et al ^[1-5] reported that the extract of PC had no effect on serum vitamins C and E levels but increased serum total antioxidant activity. Many studies show that PC has vasorelaxational effects on pulmonic rings of rat or human^[30-33]. The investigation reported in this paper focused mainly on the effects of PC on endothelium-dependent relaxation. Conflicting result was produced among previous studies reported in the literature relating the effect of PC on blood vessels, such as, in the rat aorta. These studies were carried out with pure PC and aimed to test the endothelium-dependent relaxation of PC. An experiment in vivo showed that PC overcame the hypertension of the age-increasing rat. Similar to ACh, PC had a dose-dependent vasorelaxation on pulmonic rings with intact endothelium precontracted by NA^[34]. ACh activated nitric oxide synthase via M1^[35-36] and M3^[37-40] receptor, which can catalyze the substrate L-arginine to produce endothelium-derived relaxing factor^[41-42] i.e., nitric oxide. Then the guanylate cyclase was activated through soluble guanylate cyclase receptor of the vascular smooth muscle cell and the intracellular concentration of cyclic guanosine monophosphate increased which led to a decrease of free Ca²⁺, thus, having vasorelaxant effect^[43]. In our experiment, we found that relaxing effect of the PC was reduced significantly by L-NNA or MB. The relaxing effect also was reversed through DEN of the pulmonic rings. Therefore, these results indicated that the vasorelaxation was endothelium-dependent and nitric oxide played an important role, which is consistent with Fitzpatrick’s study^[44-46].

The vascular endothelial cells can pick up circulated arachidonic acid and convert arachidonic acid to the unstable intermediate endoperoxide PGH₂ by the cyclooxygenase enzymes. PGH₂ is finally converted into PGI₂ by prostaglandin synthase enzymes. PGI₂ activates adenylyl cyclase, increases cyclic adenosine monophosphate and therefore has a vasodilation effect^[47-49]. However, in our study, the prostaglandin synthase inhibitor indomethacin (10^-5 mol/L) had no significant effects on the vasorelaxation of PC on pulmonic rings precontracted with NA, which suggested that vasorelaxation of PC had nothing to do with the prostaglandins. Adrenergic β-receptors^[50-51] also play an important role in vasorelaxation through activating adenylate cyclase which converts adenosine triphosphate into cyclic adenosine monophosphate. Increasing concentration of cyclic adenosine monophosphate activated the protein kinase A which induced the relaxing effects of the vascular. However, propranolol, a selective antagonist of adrenergic β-receptors, did not affect PC-induced vasorelaxation. These results indicate that the release of vasodilator prostaglandins and adrenegic β-receptor are not associated with the rabbit pulmonic rings relaxation induced by PC.

Free Ca²⁺ in cytoplasm is one of the key factors inducing vascular constriction. Potential-dependent calcium channels are activated by depolarization of the plasma membrane when the extra-cellular K⁺concentration is increased^[52-55], while receptor-operated calcium channels are activated by NA. Opening of receptor-operated calcium and potential-dependent calcium channel increase concentrations of the free Ca²⁺in cytoplasm through promoting the inflow and delivery of Ca²⁺ from sarcoplasmic reticulum, and therefore lead to constriction. In our study, incubation with PC

(20 mg/L) significantly shifts the concentration-response curves of NA and KCl to the right on pulmonic rings DEN. The results indicated that PC blocked potential-dependent calcium and receptor-operated calcium noncompetitively and inhibited both inflow and delivery of Ca²⁺from sarcoplasmic reticulum, thus decreasing intracellular concentration of Ca²⁺ and having vasorelaxant effects. Kim et al found that procyanidins in crataegus extract may be responsible for the endothelium-dependent nitric oxide-mediated relaxation in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K⁺ channels^[56-57]. This result is not exactly consistent with ours, which is probably caused by the difference in PC source, experimental animals as well as experimental conditions. Another reports^[58-59] showed that similar to verapamil, grape seeds PC might inhibit influx of Ca²⁺ and decrease overloading Ca²⁺ caused injury in cardiac myocytes, suggesting a function of PC to antagonize Ca²⁺ channel and decrease intracellular Ca²⁺. However, it is not clear whether the Ca²⁺ channels in cardiocytes are same as those in smooth muscle cells or whether PC inhibits similar type of Ca²⁺ channels on both cells. The detailed mechanism remains to be further clarified.

In conclusion, the results indicated that the PC had a direct relaxant effect on isolated rabbit pulmonic rings, which is an endothelium dependent effect and nitric oxide plays an important role. In addition, inhibition of the inflow and delivery of Ca²⁺ from sarcoplasmic reticulum through blocking potential-dependent calcium and receptor-operated calcium is at least partially involved in the PC- induced vasorelaxant effects.
中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

原花青素对离体肺动脉血管张力的影响

Vasorelaxant effects of procyanidins on pulmonary artery in vitro

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