附子水煎剂对家兔离体主动脉血管张力的影响

doi:10.3969/j.issn.2095-4344.2015.33.013

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (33): 5312-5317.doi: 10.3969/j.issn.2095-4344.2015.33.013

• 血管组织构建 vascular tissue construction • 上一篇下一篇

附子水煎剂对家兔离体主动脉血管张力的影响

李红专¹，刘行海²，牛彩琴³，张团笑²

¹甘肃省中医院骨伤科，甘肃省兰州市 730050；川北医学院，²生理学教研室，³中西医临床系，四川省南充市 637007

出版日期:2015-08-13 发布日期:2015-08-13
通讯作者: 牛彩琴，硕士，副教授，川北医学院中西医临床系，四川省南充市 637007
作者简介:李红专，男，1973年生，汉族，甘肃省静宁县人， 2014年天津中医药大学毕业，博士，副主任医师，主要从事中西医结合治疗脊柱疾患的研究和治疗。
基金资助:
四川省教育厅重点项目(14ZA0182)

Tension effect of water decoction of Aconitum carmichaeli Debx on rabbit aorta in vitro

Li Hong-zhuan¹, Liu Xing-hai², Niu Cai-qin³, Zhang Tuan-xiao²

1 Department of Orthopedics and Traumatology, Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou 730050, Gansu
Province, China 2 Department of Physiology, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China 3 Clinical Department of Chinese and Western Medicine, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China

Online:2015-08-13 Published:2015-08-13
Contact: Niu Cai-qin, Master, Associate professor, Clinical Department of Chinese and Western Medicine, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China
About author:Li Hong-zhuan, M.D., Associate chief physician, Department of Orthopedics and Traumatology, Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou 730050, Gansu Province, China
Supported by:
the Key Program of Sichuan Department of Education, No. 14ZA0182

摘要/Abstract

摘要：

背景：附子为毛莨科乌头子根的加工品，具有温肾回阳，回阳救逆和温里散寒之功效。研究发现其具有明显的强心、降压、扩张血管、镇痛、抗炎及毒性等作用。

目的：观察附子水煎剂对主动脉环的舒张作用及机制。

方法：采用家兔离体主动脉平滑肌标本，以去甲肾上腺素(10^-6 mol/L)预收缩主动脉后给予0.5，1.0，2.0，4.0，8.0 g/L附子水煎剂观察其张力变化。然后将不同浓度的附子水煎剂干预的家兔离体主动脉平滑肌加入去除血管内皮、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变化的影响。

结果与结论：附子水煎剂对血管环静息张力无明显影响，但不同剂量的附子水煎剂可使10^-6 mol/L 去甲肾上腺素预收缩血管产生明显舒张。去除血管内皮、给予左旋硝基精氨酸或给予甲烯蓝可减弱附子水煎剂对血管的舒张作用，但吲哚美辛和普萘洛尔对此无影响。另外，4 g/L附子水煎剂对无内皮细胞血管环去甲肾上腺素和KCl的量效收缩反应无明显影响。提示附子对主动脉的舒张作用是内皮依赖性，与内皮细胞释放的一氧化氮有关，而与平滑肌细胞膜上的受体依赖性Ca²⁺通道和电压依赖性Ca²⁺无关。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 组织构建, 组织工程, 附子, 血管, 内皮细胞, 血管舒张, 一氧化氮, 血管张力, 离体

Abstract:

BACKGROUND: Aconitum carmichaeli Debx (ACD) is the tuberous root of Aconium carmicgaekum, used as cardiotonic to restore yang for the treatment of collapse and shock, to warm the kidney and reinforce yang, and to expel cold and promote the flow of yang-qi. Studies have found that ACD has obviously cardiotonic, antihypertensive, vasodilatory, analgesic, anti-inflammatory and toxic effects.

OBJESTIVE: To observe the vasodilatory effects of a water decoction of ACD on rabbit’s aorta rings and its mechanism.

METHODS: Rabbits aorta arteries were isolated, pre-contracted with noradrenaline (10-6 mol/L) and their responses to different concentrations of ACD (0.5, 1.0, 2.0, 4.0, 8.0 g/L) were investigated. The effects of removal of vascular endothelium and different signaling pathway inhibitors (Nω-nitro-L-arginine: 1×10-4 mol/L, methylene blue: 1×10-5 mol/L, indomethacin: 1×10-5 mol/L, propranolol: 1×10-5 mol/L) on ACD-induced vasodilation were also assessed.

RESULTS AND CONCLUSION: ACD could not change the resting tension of rabbit aortic rings, but ACD treatment resulted in an obvious relaxation in narodrenaline-precontracted aortic rings and the relaxant effect was dose-dependent. The vasodilatory effect of ACD was significantly reduced by removal of endothelium, 1×10-4 mol/L Nω-nitro-L-arginine and 1×10-5 mol/L methylene blue but not reduced by indomethacin and propranolol. In addition, 4 g/L water decoction of ACD did not decrease the dose-response curves of artery rings to narodrenaline or KCl in the absence of endothelial cells. ACD can relax isolated rabbit’s aorta, which may be related to endothelium-released nitric oxide, but has no significant relevance with receptor-operated and voltage-dependent calcium channels.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Aconitum, Aorta, Endothelial Cells, Vasodilatio

中图分类号:

R318

李红专，刘行海，牛彩琴，张团笑. 附子水煎剂对家兔离体主动脉血管张力的影响[J]. 中国组织工程研究, 2015, 19(33): 5312-5317.

Li Hong-zhuan, Liu Xing-hai, Niu Cai-qin, Zhang Tuan-xiao. Tension effect of water decoction of Aconitum carmichaeli Debx on rabbit aorta in vitro[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(33): 5312-5317.

图/表（结果） 3

Quantitative analysis of experimental animals

Twenty rabbits of clean grade were fed in regular way to remove their thoracic aortas after anesthesia. In the final analysis, all the results of the thoracic aorta were from the 20 rabbits without loss.

ACD relaxed NA-precontracted thoracic aorta

ACD which was administrated progressively had an apparent dose-dependent effect on the aortic rings pre-contracted with NA (r=0.64, t=14.89, P < 0.01; Table 1, Figure 1). After treatment with ACD (4 g/L) and acetylcholine (10^-5 mol/L), (56.14±2.47)% (n=12) and (58.44±2.46)% (n=14) NA pre-contracted rings were relaxed, respectively, but there was no significant difference (P > 0.05; Figure 2).

The vasorelaxant effect of ACD is mediated through cGMP/NO pathway Next, we proceeded to study the possible mechanisms by which ACD relaxes NA-precontracted aortic muscles. Thoracic aortic rings were pretreated with 10^-4 mol/L N^ω-nitro-L-arginine (nitric oxide synthase inhibitor), 10^-5 mol/L methylene blue (guanylyl cyclase inhibitor) followed by treatment with different concentrations of ACD. As shown in Table 1, the vasodilation effect of ACD on NA-precontracted aortic was significantly reduced by the inhibitors, suggesting that cGMP and NO are at least partially involved in the relaxant process. In addition, the removal of endothelium also reversed ACD-induced vasodilation. On the other hand, inhibition of prostaglandin pathway by 10^-5 mol/L indomethacin or blockage of β-receptor with 10^-5 mol/L propranolol had no effect on ACD relaxation (Table 1), suggesting neither PG nor adrenergic β-receptor is involved.

ACD decreased the concentration-response curves of thoracic aorta to NA and KCl ACD (4 g/L) had no influence on the concentration-responses curves of NA and KCl. Before or after incubation with the ACD (4 g/L), the pD₂^[26] of NA was 7.07±0.90 and 6.93±0.87 (Figure 3A); the pD₂ of KCl was 1.40±0.36 and 1.25±0.21, respectively (Figure 3B).

参考文献

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 Institute of Materia Medica, North Sichuan Medical College between January 2007 and January 2008.

Materials

Animals

Healthy New Zealand white rabbits (7-8 months, SPF), weighing 2.5-3.0 kg, either male or female, were provided by the Experimental Animal Center, North Sichuan Medical College (certification No: SYXK(Chuan) 2013-076). Twenty rabbits of clean grade were fed in regular way, and then the thoracic aorta was taken out from the rabbits after anesthesia.

Drugs

The water decoction of ACD was prepared by Huirentang Drugstore with concentration of 1.0 g/mL, which was used as 100% solution in the following experiments. Since the ACD decoction was acid with pH 5.4^[18-19], it was titrated by NaOH to get neutral solution with pH 7.4^[20] and the concentration of this decoction was 1.0 g raw drug/mL.

Main reagents and instruments used for detecting the effect of water decoction of ACD on the rabbit aorta:
Drug	Source
N^ω-nitro-L-arginine	Sigma, USA
Methylene blue	Merck, Germany
Acetylcholine and propranolol	The Second Beijing Pharmaceutical Company, China
Indomethacin	Jingsu Taicang Pharmaceutical Company, China
ACD	Huirentang Pharmacy, China
NA	Shanghai He Feng Pharmaceutical Co., Ltd.
BL-410 Experimental System of Biological Function	TME, China

Methods

Preparation of rabbit thoracic aortic rings

Rabbits were sacrificed after anesthesia. The thoracic aorta was rapidly removed and incubated in Krebs-Henseleit (K-H) solution (mmol/L: NaCl 118, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 2.5, CaCl₂ 2.5 and glucose 11.1.) with 95% O₂and 5% CO₂^[21]. After complete removal of the surrounded connective tissues, the thoracic aorta was cut into 2.5-3.0 mm rings^[22-25]and incubated in K-H solution in organ baths at 37 ℃ with 95% O₂ and 5% CO₂ for 1.5-2.0 hours, during which K-H solution was changed every 20 minutes. Then the thoracic aortas were treated with 10^-6 mol/L NA, and only those with good contraction were used in the following experiments.

In some specimens, the endothelial cells were removed from the thoracic aorta by gentle rubbing the inner wall with a cotton stab and the complete depletion of endothelial cells was demonstrated by unresponsiveness to 10^-5 mol/L acetylcholine. The changes of tension in the thoracic aorta were recorded using a force transducer and processed by BL-410 Experimental System of Biological Function. Resting tension was set to 2 g.

Effect of ACD treatment on NA pre-contracted thoracic aortic rings

After the equilibration period, the thoracic aortas with endothelium were pre-contracted by adding NA 10^-6 mol/L, and then administrated with the ACD at different concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 g/L. After that, the relaxation-response curves were drawn to calculate the relaxation rate, which was compared with the same dose of normal saline and acetylcholine (10^-5 mol/L).

Effects of signaling inhibitors and endothelial cells on ACD-induced vasodilation

After washed by Krebs solution (37 ℃), the rings with endothelium were incubated respectively with N^ω-nitro- L-arginine (10^-4 mol/L), methylene blue(10^-5 mol/L), indomethacin (10^-5 mol/L), propranolol(10^-5 mol/L) for 15 minutes followed by removal of the endothelium, and then the three different doses of ACD were administrated respectively to observe the rings responses.

Inhibition of ACD concentration-response curves of NA and KCl

After rings were stabilized under 2.0 g resting tension for 90 minutes in Krebs solution, the concentration-response curves to NA (10^-8-10^-5 mol/L) or KCl (6.3-100 mmol/L) were observed in the absence and presence of ACD(4 g/L) in the rabbit aortic rings without endothelium.

Main outcome measures

Changes in vascular tension of rabbit aorta in vitro.

Statistical analysis

The data were expressed as Mean±SD and processed with SPSS 11.0. Difference comparison was determined with t test and dose-dependent manner by the Pearson test. A value of P < 0.05 was set as significant difference.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

讨论

In vivo experiments have showed that ACD relaxes the peripheral vessels of the anesthetized cats or dogs apparently. But the effects on the aorta have been not reported. In our experiments, all the results showed that ACD could relax the aortic rings with endothelium pre-contracted with NA in the same manner as acetylcholine in a dose-dependent manner. It is now recognized that vasodilator responses to acetylcholine are mainly modulated by epithelium-derived relaxing factors composing of NO and cyclooxygenase products such as PGE₂ and PGI₂ which are produced through activating the endothelium receptors of M₁^[27-29] or M₃^[30-33], even the endothelial target for acetylcholine^[34-35]. Epithelium-derived relaxing factors, chemically related to NO^[36-42], can activate guanylyl cyclase through soluble guanylyl cyclase receptor to increase intracellular cGMP and decrease free cytosol Ca²⁺ and therefore relaxes the vessel. All these products may induce relaxation of vascular smooth muscle. NO can stimulate soluble guanylyl cyclase in smooth muscle cells to increase the second messenger guanosine 3’,5’-cyclic monophosphate (cGMP), which leads to relaxation largely via voltage- independent mechanism^[43]. In our experiments, endothelium removal could reduce the ACD vasodilatation. We also discovered that N^ω-nitro-L-arginine, an inhibitor of NO synthesis, decreased the relaxation caused by ACD. Methylene blue, an inhibitor of cGMP synthesis, also reduced the relaxation induced by ACD. Our results suggest that the relaxation of the rabbit aorta caused by ACD may be dependent on endothelium and probably relate to NO and cGMP.

The endothelium may release a number of prostaglandins, either vasodilator or constrictor. Indomethacin inhibits the synthesis of the postaglandins and markedly inhibits the transient relaxation induced by arachidonic acid^[44-46]. However, in our experiments, indomethacin did not affect the ACD-induced relaxation in the endothelium-intact aorta. Propranolol, a selective antagonist of adrenergic β-receptors^[47-49], also did not affect ACD-induced relaxation. These results indicate that the release of vasodilator prostaglandins and adrenegic β-receptor are not associated with the rabbit aorta relaxation induced by ACD.

Ca²⁺ 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 increased^[50-53]. In this experiment, ACD did not affect the KCl pre-contracted aorta rings, and incubation with ACD (4 g/L) had no significant change on the concertration-response curves induced by KCl, which indicates that ACD cannot block calcium influx through the potential-dependent calcium channels. Agonists of adrenegic receptors (such as NA) that activate receptor-operated channels in cell membranes of vascular smooth muscle and increase calcium influx, can also activate G protein and phospholipase C to produce the inositol 1,4,5-triphosphate (IP₃), which can cause calcium release from the endoplasmic reticulium. In our experiment, ACD also did not affect the NA-precontracted aortic rings, which indicates that ACD may not block calcium influx through receptor-operated channels.

In conclusion, our results indicate that the water decoction of monkshood root ACD has a direct relaxant effect on rabbit isolated aorta, which is related to endothelium. The mechanism of action mainly involves NO and cGMP, and has no significant relevance to calcium influx via receptor-operated channels and potential-dependent calcium channels. However, with the increasing concentrations of ACD on rings pre-contracted with NA, the blood vessel tension of relaxation aortic rings will become stronger, and the mechanism is to study. 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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附子水煎剂对家兔离体主动脉血管张力的影响

Tension effect of water decoction of Aconitum carmichaeli Debx on rabbit aorta in vitro

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