Abstract
BACKGROUND: Recent research has shown that Gastrodia elata Bl (GEB) has a cardiovascular protective effect and relaxes blood vessels with important therapeutic implication to treat coronary heart disease and hypertension. However, the mechanism is still unclear.
OBJESTIVE: To study the effect of the water decoction of GEB on noradrenaline (NA) or KCl precontracted aortic rings and the possible mechanisms.
DESIGN: A grouping observational experiment of animal tissue in vitro.
SETTING: Department of Physiology, North Sichuan Medical College.
MATERIALS: Twelve health New Zealand White rabbits (2.5-3.0 kg, 7-8 months, SPF, either gender) were provided by the Experimental Animal Center, North Sichuan Medical College. The water decoction of GEB was prepared by Huirentang Drugstore and diluted into 10%, 20%, 40% and 80% solution. The following drugs were used: Nω-nitro-L-arginine (L-NNA, Sigma, USA); Methylene blue (MB, Merck, Germany); Acetylcholine (ACh) and Propranolol (Prop) (The Second Beijing Pharmaceutical Company, China); Indomethacin (Indo, Jingsu Taicang Pharmaceutical Company, China).
METHODS: The experiment was performed in the Institute of Materia Medica, North Sichuan Medical College between January 2006 and January 2007. Rabbit smooth muscles of aortic rings were isolated and precontracted with NA. The thoracic aortic rings were treated with GEB with cumulative concentrations of 1.0, 2.0, 4.0, 8.0 and 16 g/L respectively. The aortic rings were treated with one of the following signaling inhibitors for 15 minutes, including 1×10-4 mol/L L-NNA, 1×10-5 mol/L MB, 1×10-5 mol/L Indo and 1×10-5 mol/L Prop. The changes of tension in aortic rings were recorded using a force transducer and processed by BL-410 Experimental System of Biological Function. The aortic rings were incubated with 8 g/L GEB followed by the NA and KCl dose response experiments.
MAIN OUTCOME MEASURES: Blood vessel tension ex vivo.
RESULTS: GEB did not change the resting tension of rabbit aortic rings, but GEB treatment resulted in an obvious relaxation in NA-precontracted aortic rings (r =0.85, t=18.45, P < 0.01) and the relaxant effect was dose-dependent. The relaxant effect of GEB was significantly reduced by removal of endothelium and by treatment with 1×10-4 mol/L nitric oxide synthase inhibitor L-NNA (1×10-4 mol/L), or 1×10-5 mol/L guanylyl cyclase inhibitor methylene blue (1× 10-5 mol/L MB), but not by treatment with prostaglandin synthase inhibitor or blockage of the adrenergic β-receptor (1×10-5 mol/L Prop). In addition, GEB (8 g/L) decreased the dose response curves of aortic rings to NA or KCl in the absence of endothelial cells, and changed the PD2 values for NA from (6.90±0.93) mol/L in control group to [(5.61±0.70) mol/L, t =2.41, P < 0.05] and for KCl from (1.53±0.55) mmol/L in control group to (1.10±0.25) mmol/L, t =3.82, P < 0.05] respectively.
CONCLUSION: GEB can relax isolated rabbit aorta not only in an endothelium-dependent, nitric oxide involved manner, but also is related to blockage of receptor-operated and potential-dependent calcium channels.

INTRODUCTION

Gastrodia elata Bl (GEB) is one of the Chinese traditional medicines and is commonly used to calm and expel liver wind as well as release convulsion. Recent research has shown that GEB also has a cardiovascu-lar protective effect and relaxes blood vessels with important therapeutic implication to treat coronary heart disease, high blood pressure[1-2]. However, the mechanism of the relaxant effect of GEB is still unknown. In this study, we investigated the effect of the water decoction of GEB on noradrenaline (NA) or KCl precontracted aortic rings and the possible mechanisms were addressed.

MATERIALS AND METHODS

Materials
The experiment was performed in the Institute of Materia Medica, North Sichuan Medical College between January 2006 and January 2007. Twelve health New Zealand White rabbits (2.5-3.0 kg, 7-8 months, SPF, either gender) were provided by the Experimental Animal Center, North Sichuan Medical College (certification: Medical Animal 14-004).
Preparation of the water decoction of the GEB: The water decoction of GEB was prepared by Huirentang Drugstore with concentration of 1 g/mL, which was used as 100% solution in our following experiments. Since the GEB decoction was acid with pH 5.4, it was titrated by NaOH to get neutral solution with pH 7.4 and then diluted into 10%, 20%, 40% and 80% solution. The following drugs were used: Nω-nitro-L-arginine (L-NNA, Sigma, USA); Methylene blue (MB, Merck, Germany); Ace-tylcholine (ACh) and Propranolol (Prop) (The Second Beijing Pharmaceutical Company, China); Indomethacin (Indo, Jingsu Taicang Pharmaceutical Company, China). Krebs-Henseleit (K-H) solution contains (mmol/L): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 2.5, CaCl2 2.5 and glucose 11.1.

Methods
Preparation of rabbit thoracic aortic rings
Rabbits were sacrificed by stunning and exsanguinations. The thoracic aortic was rapidly removed and incubated in K-H solution with 95% O2 and 5% CO2. After complete removal of the surrounded connective tissues, the thoracic aortic was cut into 2.5-3.0 mm rings[3] 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 aortic rings were treated with 10-6 mol/L NA and only those with good contraction were used in the following experi-ments.
In some thoracic aortic rings, the endothelial cells were re-moved from the aortic rings by gentle rubbing the inner wall with a cotton stab and the complete depletion of endothelial cells were demonstrated by unresponsiveness to 1×10-5 mol/L ACh. The changes of tension in aortic rings were recorded using a force transducer and processed by BL-410 Experimental System of Biological Function (TME, China). Resting tension was set to 2 g.

Effect of GEB treatment on NA pre-contracted aortic rings
After aortic rings reached the equilibrium in K-H solution, the resting tension was recorded. The thoracic aortic rings were then treated with 1×10-6 mol/L NA to get maximal contrac-tion, which was referred to as 100% contraction. Then the thoracic aortic rings were treated with GEB with cumulative concentrations of 1.0, 2.0, 4.0, 8.0 and 16 g/L respectively. The dose response curve to GEB was observed. The changes of tension were recorded and vasodilatation rate was calcu-lated. The addition of same amount of 0.9% sodium chloride (normal saline, NS) to the precontracted rings was a negative control while 1×10-5 mol/L ACh was used as a positive con-trol.

Effects of signaling inhibitors and endothelia cells on GEB-induced vasodilation
After maximal contraction (100%) by 1×10-6 mol/L NA, the aortic rings were treated with one of the following signaling inhibitors for 15 minutes, including 1×10-4 mol/L L-NNA, 1×10-5 mol/L MB, 1×10-5 mol/L Indo and 1×10-5 mol/L Prop, followed by treatment with different cumulative con-centrations of GEB. The changes of tension were recorded and calculated as mentioned above in 1.3. In some experiments, instead of inhibitor pretreatment, the aortic rings were de-pleted of endothelial cells followed by GEB treatment and the change of contraction was recorded.

Effects of GEB on the dose response curves of aortic rings to NA and KCl
After removal of endothelium, the aortic 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 washout, the aortic rings were incubated with 8 g/L GEB for 15 minutes followed by the NA and KCl dose response ex-periments.

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

RESULTS

GEB relaxes NA-precontracted thoracic aortic
To investigate the effect of GEB on the contraction of aortic smooth muscles, thoracic aortic rings were pre-treated with 1×10-6 mol/L NA followed by treatment with different con-centrations of GEB. As expected, the aortic rings were con-tracted by NA and surprisingly the contraction was signifi-cantly reduced by GEB in a dose-dependent manner (r=0.85, t =18.54, P < 0.01, Figure 1). In addition, as shown in Table 1, the highest concentration of GEB (16 g/L) used in the study appears to relax the muscle to the same extent as 1×10-5 mol/L ACh, with (45.36±1.48)% vasodilation for GEB and (43.67±1.23)% (t =0.89, P > 0.05) for ACh respectively.

Vasorelaxant effect of GEB was mediated through cGMP/NO pathway
Next, we proceeded to study the possible mechanisms by which GEB relaxes NA-precontracted aortic muscles. Tho-racic aortic rings were pretreated with 1×10-4 mol/L L-NNA (nitric oxide synthase inhibitor), 1×10-5 mol/L MB (guanylyl cyclase inhibitor) followed by treatment with different con-centrations of GEB. As shown in Table 1, the vasodilation effect of GEB on NA-precontracted aortic is significantly re-duced by the inhibitors, suggesting that cGMP and NO are at least partially involved in the relaxant process. In addition, removal of endothelium also reverses GEB-induced vasodila-tion. On the other hand, inhibition of prostaglandin pathway by 1×10-5 mol/L Indo or blockage of β-receptor with 1×10-5 mol/L Prop had no effect of GEB relaxation (Table 1), suggesting neither PG nor adrenergic β-receptor is in-volved.

GEB decreases the dose response curves of aortic rings to NA and KCl
To study if GEB has any effect on the membrane calcium channel, aortic rings were treated with NA or KCl in the pres-ence or absence of GEB pretreatment. As expected, both NA and KCl contract aortic muscles in a dose dependent manner. More importantly, GEB significantly inhibits NA and KCl induced contraction and this inhibition was observed at all the NA and KCl concentrations (Figure 2). Treatment with 8 g/L GEB decreases the NA induced maximal contraction to (57.76±2.29)% (t=29.24, P < 0.01) and decreases the PD2[4] from (6.90±0.93) in absence of GEB to (5.61±0.70, t=2.41, P < 0.05) in the presence of GEB (Figure 2a). Similarly, The maximal contraction by KCl after GEB treatment decreases to (69.49±1.87)% (t=20.11, P < 0.01) and the PD2[4] goes down

from (1.53±0.55) to (1.10±0.25, t=3.82, P < 0.05) (Figure 2b).

 

DISCUSSION

GEB has been reported to have vasorelaxant effect and de-crease peripheral resistance[1,2]. For example, in vivo injection of GEB can rapidly decrease blood pressure in rabbits and rats. Experiments from dog indicate that GEB can decrease the vessel resistance and increase blood flow. In this study, we observed that similar to ACh, GEB has a significant relaxation on 1×10-6 mol/L NA-precontracted aortic rings and this re-laxation is dose dependent. ACh functions by binding to M2[5], M3[6] receptors on endothelium and activates nitric oxide syn-thase, which acts on L-arginine to generate EDRF[7-8]. EDRF, chemically related to NO[9-11], can activate guanylyl cyclase through soluble guanylyl cyclase receptor to increase intra-cellular cGMP and decrease free cytosol Ca2+ and therefore relaxes the vessel[12]. To study if GEB relaxes aortic rings through the NO/cGMP pathway as ACh, we pretreated aortic rings with L-NNA (NOS inhibitor) and MB (guanylyl cy-clase inhibitor) followed by GEB treatment. The relaxant effect of GEB was significantly reduced by the inhibitors. In addition, removal of endothelium cells also decreases the vasodilation effect of GEB. All the results suggest that the effect of GEB is at least partially mediated through endothelium and related to NO/cGMP pathways.
Endothelial cells can pick up circulated arachidonic acid (AA) and convert AA to the unstable intermediate endoperox-ide PGH2 by the cyclooxygenase (COX) enzymes. PGH2 is finally converted into PGI2 by PG synthase enzymes. PGI2 activates adenylyl cyclase, increases cAMP and therefore has vasodilation effect[13]. However, in this study, prostaglandin synthase inhibitor does not affect GEB function, which sug-gests the relaxative effect of GEB is not mediated through COX/PGI2 pathway. On the other hand, adrenergic β-receptor is also involved in vasodilation by increasing intra-cellular cAMP and activating PKA[14]. However, in our study, blockage of the adrenergic β-receptor (Prop) has no effect on GEB, suggesting this effect is not mediated through adrener-gic β-receptor.
It is well known that cytosol free Ca2+ is the key player to control smooth muscle contraction. Ca2+ binds to calmodulin to form 4 Ca2+·CaM complex, which activates myosin light chain kinase (MLCK). MLCK phosphorates myosin light chain and change its conformation to cause muscle contrac-tion[15]. NA as well as high concentration of K+ can increase cytosol Ca2+ by influx Ca2+ from extracellular space and/or release Ca2+ from ER through different mechanisms. NA acti-vates receptor-operated calcium channels (ROC) while KCl activates potential-dependent calcium channels (PDC). In this study, in the absence of endothelium, 8 g/L GEB pretreatment significantly reduces NA and KCl induced contraction, indi-cating that GEB antagonizes ROC and PDC in a non-competitive manner, therefore relaxes muscle by inhibit-ing increase in intracellular Ca2+.
Taken together, we have found that GEB can relax isolated rabbit aorta. The relaxant effect is mediated not only through direct blockage of receptor-operated and potential-dependent calcium channels, but also partially through endothelium and NO/cGMP dependent pathways.

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家兔离体主动脉血管对天麻水煎剂干预的反应

张团笑1,牛彩琴2,敬华娥1 ,刘 红1 (川北医学院，1生理学教研室，2中西医临床系，四川省南充市 637007)
张团笑，男，1968年生，甘肃省静宁县人，2005年兰州大学毕业，副教授，主要从事心血管药理与生理的研究。
摘要
背景：现代医学研究发现天麻（Gastrodia elata Bl）具有明显地心肌保护和血管扩张作用，可用于冠心病、高血压的治疗，但其作用机制仍需明确。
目的：观察天麻水煎剂对去甲肾上腺素、KCl预收缩血管反应的舒张作用，并分析其作用途径。
设计：分组设计、离体动物组织对照实验。
单位：川北医学院生理学教研室。
材料：选用健康家兔12只，兔龄七、八个月，雌雄不拘，由川北医学院实验动物中心提供。实验所用药品天麻水煎剂由慧仁堂药店鉴定提取，将其配制成10%，20%，40%和80%浓度备用。β肾上腺素能受体阻断剂普萘洛尔为北京第二制药厂产品；一氧化氮合酶抑制剂左旋硝基精氨酸为Sigma公司产品；甲烯蓝、环氧酶抑制剂吲哚美辛为江苏太仓制药厂产品。
方法：实验于2006-01/2007-01在川北医学院药物研究所完成。采用家兔离体主动脉平滑肌标本，以去甲肾上腺素(1×10-6 mol/L)预收缩主动脉后给予1.0，2.0，4.0，8.0和16.0 g/L天麻水煎剂观察其张力变化，并观察去除血管内皮、给予1×10-4 mol/L左旋硝基精氨酸、1×10-5 mol/L甲烯蓝、1×10-5 mol/L吲哚美辛和1×10-5 mol/L普萘洛尔对血管张力的影响；血管张力经张力换能器输入BL-410智能型生物信号处理系统进行处理。另外观察8 g/L天麻水煎剂对无内皮细胞血管环NA和KCl的量效收缩反应。
主要观察指标：离体血管张力。
结果：天麻对血管环静息张力无明显影响，但不同剂量的天麻可使1×10-6 mol/L 去甲肾上腺素预收缩血管产生明显舒张（r=0.85, t=18.45, P < 0.01）。去除血管内皮、1× 10-4 mol/L 左旋硝基精氨酸或1×10-5 mol/L 甲烯蓝可减弱天麻的舒张作用，但吲哚美辛和普萘洛尔对其无影响。另外，8 g/L天麻水煎剂对无内皮细胞血管环去甲肾上腺素和KCl的量效收缩反应明显降低，且去甲肾上腺素和KCl的PD2（-log游离药物半数有效浓度）分别由温育前（6.90±0.93）mol/L和（1.53± 0.55） mmol/L降低为（5.61±0.70）mol/L和(1.10±0.25）mmol/L, (t=2.41，3.82, P < 0.05)。
结论：天麻水煎剂对主动脉的舒张是内皮依赖性的，并与内皮一氧化氮的释放有关；也可通过拮抗ROC和PDC通道，抑制Ca2+的内流和释放等机制有关。
关键词：主动脉平滑肌；内皮细胞；血管舒张；天麻
中图分类号: R329.41 文献标识码: A 文章编号: 1673-8225(2008)20-03988-04
张团笑, 牛彩琴, 敬华娥, 刘红. 家兔离体主动脉血管对天麻水煎剂干预的反应[J].中国组织工程研究与临床康复,2008,12(20): 3988-3991 [www.zglckf.com/zglckf/ejournal/upfiles/08- 20/20k-3988(ps).pdf]
(Edited by Yu XP/Ji H/Wang L)