Acupuncture Research 针灸研究

Purines Change at Acupoints along the Pericardium Meridian in Healthy and Myocardial Ischemic Rats*
健康与心肌缺血大鼠心经穴位嘌呤变化*

ZHOU Yu-mei $^{1}$ , ZHUANG Yi $^{2}$ , CAI Ding-jun $^{1}$ , LV Pei-ran $^{1}$ , ZHOU Jie $^{1}$ , WAN Min $^{1}$ , REN Yu-lan $^{1}$ , and LIANG Fan-rong $^{1}$
周玉梅 $^{1}$ ，庄毅 $^{2}$ ，蔡定军 $^{1}$ ，吕培然 $^{1}$ ，周杰 $^{1}$ ，万敏 $^{1}$ ，任玉兰 $^{1}$ ，梁帆荣 $^{1}$

Abstract 摘要

Objective: To quantify the purine concentrations of the acupoints along the pericardium and nonpericardium meridians under healthy and myocardial ischemia conditions to investigate the relationship between acupoint purine change and body functional status in rats. Methods: A total of 70 rats underwent an operation for myocardial ischemia, while 40 of them survived. They were randomly assigned to the following 5 subgroups: Neiguan (PC 6), Quze (PC 3), Tianquan (PC 2), Quchi (LI 11), and Jianyu (LI 15). Simultaneously, another 40 healthy rats were also randomized into the same 5 subgroups as the control group. The tissue fluids at the acupoints were collected by microdialysis for 30 min . Subsequently, the concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO) were quantified using the high-performance liquid chromatography method. Results: Compared with the healthy group, the ADO at PC $6 (P = 0.012)$ , PC $3 (P = 0.038)$ , PC $2 (P = 0.024)$ , and $LI 15 (P = 0.042)$ obviously increased in the model group, while no significant difference was observed at LI 11 ( $P = 0.201$ ). However, ATP, ADP, and AMP manifested no significant changes in these areas, except for ATP at LI $15 (P = 0.036)$ . Conclusions: Myocardial ischemia could induce an increase in ADO at acupoints of the upper arm and shoulder area, suggesting that the body functional status could affect the responsiveness of acupoints. The status of these acupoints could be pathogenically activated by disease, and distribution following some specific courses.
目标：量化健康和心肌缺血条件下心包经和非心包经穴位中的嘌呤浓度，以研究穴位嘌呤变化与大鼠身体功能状态之间的关系。方法：共 70 只大鼠接受了心肌缺血手术，其中 40 只存活。它们被随机分配到以下 5 个亚组：内关（PC 6）、曲泽（PC 3）、天泉（PC 2）、曲池（LI 11）和肩俞（LI 15）。同时，另外 40 只健康大鼠也被随机分配到与对照组相同的 5 个亚组。通过微透析法收集穴位组织液，持续 30 分钟。随后，使用高效液相色谱法定量测量三磷酸腺苷（ATP）、二磷酸腺苷（ADP）、一磷酸腺苷（AMP）和腺苷（ADO）的浓度。结果：与健康组相比，模型组中 PC $6 (P = 0.012)$ 、PC $3 (P = 0.038)$ 、PC $2 (P = 0.024)$ 和 $LI 15 (P = 0.042)$ 的 ADO 明显增加，而 LI 11 ( $P = 0.201$ )没有显著差异。然而，ATP、ADP 和 AMP 在这些区域没有表现出显著变化，除了 LI $15 (P = 0.036)$ 处的 ATP。结论：心肌缺血可能诱导上臂和肩部穴位 ADO 增加，表明身体状况可能影响穴位反应性。这些穴位的状况可能因疾病而病理性激活，并按特定途径分布。

KEYWORDS acupoint, myocardial ischemia, pericardium meridian, purine, adenosine, microdialysis, highperformance liquid chromatography
关键词穴位，心肌缺血，心经，嘌呤，腺苷，微透析，高效液相色谱法

Acupoints, which are located along the skin line of the meridians, are the special areas for transporting qiblood of Zang-Fu organs bidirectionally. Recently, some scientists revealed that acupoints are dynamic.

^{(1 - 3)}

In other words, the function of acupoints could transform from physiologically “silent” to pathologically “active” when some organs are affected by diseases. This phenomenon is well known as acupoint sensitization. For instance, the Evans Blue (EB) points from exudative plasma could be observed on the skin of rats with an acute gastric mucosal injury. These points were distributed mainly at Pishu (BL 20, 88.23%), Weishu (BL 21, 82.35%), and Zhongwan (CV 12,

17.64 %)

^{(4)}

Additionally, the local area of sensitized acupoints was found to highly express mast cells and algogenic bioactivators, such as 5 -hydroxytryptamine (5-HT), substance P (SP), calcitonin gene-related peptide (CGRP), transient receptor potential vanniloid-1 (TRPV-1), histamine (HA), and bradykinin (BK), than nonsensitized areas.

^{(5)}

This theory has explained the acupoint specificity problem in part. Nevertheless, the biological basis and the characteristics of dynamic
穴位位于经络的皮肤线上，是脏腑气血双向传输的特殊区域。最近，一些科学家揭示穴位是动态的。换句话说，当某些脏腑受到疾病影响时，穴位的功能可以从生理上的“静默”转变为病理上的“活跃”。这种现象被称为穴位敏化。例如，在急性胃黏膜损伤的小鼠皮肤上可以观察到来自渗出性血浆的伊文思蓝（EB）点。这些点主要分布在脾俞（BL 20，88.23%）、胃俞（BL 21，82.35%）和中脘（CV 12，

17.64 %)

）。此外，敏化穴位的局部区域比非敏化区域更高度表达肥大细胞和致痛生物活性物质，如 5-羟色胺（5-HT）、P 物质（SP）、降钙素基因相关肽（CGRP）、瞬时受体电位香草醛-1（TRPV-1）、组胺（HA）和缓激肽（BK）。

^{(5)}

这一理论部分解释了穴位特异性问题。然而，其生物学基础和动态特征
changes for acupoint sensitization still deserve further study.
对于穴位敏感化的变化仍然值得进一步研究。

Besides the aforementioned substances and factors, the purines, including adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO), are one kind of interesting and well-known extracellular signal transmitters, which have been popularly studied in recent years in the field of acupuncture.

^{(6 - 8)}

In 2009,
除了上述物质和因素外，嘌呤，包括三磷酸腺苷（ATP）、二磷酸腺苷（ADP）、一磷酸腺苷（AMP）和腺苷（ADO），是一种有趣且广为人知的细胞外信号转导物质，近年来在针灸领域被广泛研究。

^{(6 - 8)}

2009 年，

some raised that purines and their receptors are vital in acupuncture. Some studies demonstrated that the ATP, ADP, AMP, and ADO levels in the acupoint area obviously increased in mice and humans.

^{(6, 7)}

If the A1 receptor was blocked, the acupuncture analgesia effect vanished. Moreover, the extracellular purines could also be regulated by moxibustion and low-level laser therapy.

^{(9, 10)}

All these results strongly suggested that purines may play crucial roles in the peripheral mechanism of acupuncture analgesia. Some other studies also revealed that purinergic signaling takes part in acupoint initiating effect,

^{(11)}

indicating they could be used to measure the responding ability of acupoints. Therefore, purines may possibly illustrate the potential complicated relationship between acupoints, meridians, and diseases themselves, while further revealing the essence and nature of acupoints.
有人提出嘌呤及其受体在针灸中至关重要。一些研究表明，在小鼠和人类穴位区域的 ATP、ADP、AMP 和 ADO 水平明显升高。

^{(6, 7)}

如果 A1 受体被阻断，针灸镇痛效果就会消失。此外，细胞外嘌呤也可以通过艾灸和低强度激光疗法进行调节。

^{(9, 10)}

所有这些结果强烈表明，嘌呤可能在针灸镇痛的外周机制中发挥关键作用。其他一些研究也揭示，嘌呤能信号传导参与穴位启动效应，

^{(11)}

这表明它们可以用来测量穴位的反应能力。因此，嘌呤可能有助于阐明穴位、经络与疾病之间的潜在复杂关系，同时进一步揭示穴位本质和特性。

In the present study, the purines changes in the 5 acupoints area, including Neiguan (PC 6), Quze (PC 3), Tianquan (PC 2) at pericardium meridian, Quchi (LI 11) and Jianyu (LI 15) at Large Intestine meridian, were observed in healthy and myocardial ischemic conditions. Hence, the present study aimed to observe whether myocardial ischemic could induce the purines change, and compare the concentration difference of purines at the acupoints in different meridians, in order to confirm the phenomenon of acupoint sensitization and analyze the characteristics of acupoint sensitization.
在本研究中，观察了健康和心肌缺血条件下，心包经的 5 个穴位区域（包括内关穴（PC 6）、曲泽穴（PC 3）、天泉穴（PC 2）以及大肠经的曲池穴（LI 11）和肩俞穴（LI 15））的嘌呤变化。因此，本研究旨在观察心肌缺血是否能够诱导嘌呤变化，比较不同经脉穴位处嘌呤浓度的差异，以确认穴位敏化现象并分析穴位敏化的特征。

METHODS 方法

Animals and Grouping 动物与分组

A total of 110 adult Sprague-Dawley rats (55 males and 55 females, weighing 250-300 g), supplied by the Experimental Animal Center of Sichuan Provincial People’s Hospital [Certificate No. SCXK (Chuan) 2013-15], were used for this study. They were housed in cages at a temperature of

23 - 26^{\circ} C

and humidity of

40 % - 70 %

, under a

12 - h

light/dark cycle, with free access to food and water. All operations and procedures were performed in the laboratory of Chengdu University of Traditional Chinese Medicine (CDTCM). The study was approved by the Institutional Animal Care and Use Committee of CDTCM, China, and all the experimental procedures were directed by the Guide for the Care and Use of Laboratory Animals.
本研究共使用 110 只成年斯普林格-达利耶大鼠（55 雄性和 55 雌性，体重 250-300 克），由四川省人民医院实验动物中心提供[许可证号 SCXK（川）2013-15]。它们被饲养在温度为

23 - 26^{\circ} C

、湿度为

40 % - 70 %

的笼中，在

12 - h

的光暗周期下，可自由获取食物和水。所有操作和程序均在成都中医药大学（CDTCM）实验室进行。本研究获得了成都中医药大学中国机构动物福利与使用委员会的批准，所有实验程序均遵循《实验动物福利与使用指南》进行。

Among all the rats, 70 rats were attributed to the
在所有大鼠中，有 70 只大鼠归入
model group and underwent an operation for myocardial ischemia, while 40 of them survived. They were randomly assigned into 5 subgroups: PC 6, PC 3, PC 2, LI 11, and LI 15 by a random number table. At the same time, another 40 healthy rats were also randomized into same 5 subgroups as the control group. Therefore, eight rats were present in each subgroup.
模型组接受了心肌缺血手术，其中 40 只存活。它们通过随机数字表被分为 5 个亚组：PC 6、PC 3、PC 2、LI 11 和 LI 15。同时，另外 40 只健康大鼠也被随机分为与对照组相同的 5 个亚组。因此，每个亚组有 8 只大鼠。

Reagents 试剂

The standard substances of ATP (specification: A3377-5G; batch No. 1001420716), AMP (specification: A2753-1G; batch No. 1001447709), ADP (specification: A1752-5G; batch No. 1001428843) and ADO (specification: A1752-1G; batch No. 101183447) were purchased from Sigma-Aldrich Ltd., Chengdu, China, while the methyl alcohol [high-performance liquid chromatography (HPLC) grade] was bought from Fisher scientific Ltd., Chengdu, China.
ATP（规格：A3377-5G；批号：1001420716）、AMP（规格：A2753-1G；批号：1001447709）、ADP（规格：A1752-5G；批号：1001428843）和 ADO（规格：A1752-1G；批号：101183447）的标准物质均购自中国成都 Sigma-Aldrich 有限公司，而甲醇（高效液相色谱法[HPLC]级）则从中国成都 Fisher scientific 有限公司购买。

Myocardial Ischemia Model Replication
心肌缺血模型复制

The myocardial ischemia model was created by performing permanent ligation of the left anterior descending coronary artery (LAD) as described in a previous study.

^{(12)}

The rats were anesthetized with an intraperitoneal injection of

10 %

chloral hydrate at a dose of

0.4 mL / 100 g

, and placed in a supine position in the laboratory. A tracheal cannula was inserted via the mouth, with one end connected to a mini rodent ventilator (Kent PhysioSuite, Torrington, USA). The relevant parameters were conversed automatically with animal’s weight. That is, the tidal volume ratio was set at

1 mL / 100 g

, and the inspiration and expiration ratio was

1 : 1

. Thoracotomy was performed using an ophthalmic operating set to expose the heart, and the LAD was ligated with a 4-0 nylon suture to create a permanent coronary occlusion.
心肌缺血模型是通过永久结扎左前降冠状动脉（LAD）建立的，具体方法参考了先前的研究。

^{(12)}

将大鼠通过腹腔注射

10 %

水合氯醛以

0.4 mL / 100 g

剂量麻醉，并将其置于实验室仰卧位。通过口腔插入气管导管，一端连接到小型啮齿动物呼吸机（Kent PhysioSuite，美国托林顿）。相关参数会根据动物体重自动调节。也就是说，潮气量设置为

1 mL / 100 g

，吸呼比设置为

1 : 1

。使用眼科手术套装进行开胸手术以暴露心脏，并用 4-0 尼龙缝线结扎 LAD，以建立永久性冠状动脉闭塞。

Electrocardiographic Examination
心电图检查

Electrocardiography (ECG) of the rats were monitored throughout the whole operation by AD instruments (Australia). Three needles, which punctured in the two foot and right palm, were connected with electrodes to record the lead II of the ECG. Furthermore, this information was amplified, recognized and analyzed by PowerLab system. Thus, the S-T segment elevated over 0.2 mV from the baseline was recruited as an index of myocardial ischemia.
心电图（ECG）在整个手术过程中由 AD 仪器（澳大利亚）监测。三根针分别刺入两只脚和右手中，连接电极记录 ECG 的 II 导联。此外，这些信息通过 PowerLab 系统进行放大、识别和分析。因此，S-T 段相对于基线升高超过 0.2 mV 被作为心肌缺血的指标。

Locations of Acupoints 穴位位置

Some specific acupoints at the pericardium
一些心脏经上的特定穴位
meridian, such as PC 6, PC 3, and PC 2, were selected to observe the changes of purines along the meridians. These points were frequently used for cardiac diseases. Furthermore, two acupoints at Large intestine meridian was chosen as a comparison, such as LI 11 and LI 15, whose locations were close to the pericardium meridian in the elbow and shoulder but were irrelevant to cardiac diseases. According to the textbook of experimental acupuncture,

^{(13)}

PC 6 is at the inner side of the left forelimb, 3 mm distance above the wrist joint and between the ulnar and radial side. LI 11 is at the lateral side of the elbow joint, which is nearer to the radial bone. However, PC 3, PC 2, and LI 15 are not recorded in the textbook. Therefore, these acupoints were considered to be located according to the real sites of human in the present study: PC 3 at the cubital crease, in the ulnar side of bicipital tendon; PC 2 at the inner side of the left upper arm, 3 mm distance below the anterior axillary fold; and LI 15 at the lateral side of the shoulder, between the acromion and the greater tuberosity of the humerus.
在心经上选取了 PC 6、PC 3 和 PC 2 等穴位来观察沿经脉的嘌呤变化。这些穴位常用于治疗心脏疾病。此外，还选择了大肠经上的两个穴位作为对照，如 LI 11 和 LI 15，它们的位置靠近肘部和肩部的心经，但与心脏疾病无关。根据实验针灸学教科书，

^{(13)}

PC 6 位于左前肢内侧，腕关节上方 3 毫米处，位于尺骨和桡骨之间。LI 11 位于肘关节外侧，更靠近桡骨。然而，PC 3、PC 2 和 LI 15 未在教科书中记录。因此，在本研究中，这些穴位被认为根据人体的实际位置定位：PC 3 位于肘横纹处，肱二头肌腱尺骨侧；PC 2 位于左上臂内侧，腋前线下方 3 毫米处；LI 15 位于肩外侧，喙突和肱骨大结节之间。

Microdialysis Method and Sample Collection
微透析方法和样本采集

At the 3rd day of the model replication and regular binding, each rat was anesthetized and fixed as described earlier at

37^{\circ} C

using an animal constant temperature controller (CMA/450, Holliston, USA). All the acupoints in the present study were well positioned according to the textbook of experimental acupuncture and focused on the left forelimb of rats.

^{(13)}

A microdialysis probe (CMA20, Kista, Sweden) was implanted at about

1 - 2 mm

depth in the skeletal muscle of required acupoints, with one end linked to the microdialysis pump (CMA402, Holliston, USA) and the other attached to the microscale cooling collector (MAB85, Holliston, USA). The probe was perfused with

0.9 %

sodium chloride solution at a constant rate of

1.5 μ L / \min

for balancing 1 h (the microdialysis sample not collected during the 1 h ). After the balancing, the sample collection time lasted for 30 min . These samples were stored at

- 20^{\circ} C

for analyzing the concentration of purines substances (ATP, ADP, AMP and ADO) with HPLC method.
在模型复制和常规绑定的第 3 天，每只大鼠被麻醉并按照先前描述的方式在

37^{\circ} C

使用动物恒温控制器（CMA/450，美国霍利斯顿）固定。本研究中所有穴位均根据实验针灸学教科书准确定位，并聚焦于大鼠的左前肢。

^{(13)}

将微透析探针（CMA20，瑞典基斯塔）植入所需穴位骨骼肌的约

1 - 2 mm

深度，一端连接到微透析泵（CMA402，美国霍利斯顿），另一端连接到微尺度冷却收集器（MAB85，美国霍利斯顿）。探针以

0.9 %

的恒定速率灌注氯化钠溶液，平衡 1 小时（1 小时内未收集微透析样品）。平衡后，样品收集时间为 30 分钟。这些样品在

- 20^{\circ} C

保存，用于通过 HPLC 方法分析嘌呤物质（ATP、ADP、AMP 和 ADO）的浓度。

HPLC Analysis of Purines
嘌呤的 HPLC 分析

The purine analysis was carried out using the Shimadzu LC-10AD system Chromatographic separation was achieved using a reverse-phase column (Shimadzu Intertsustain

C_{18}, 4.6 mm \times 150 mm, 5 μ m

). The mobile phase consisted of

50 mmol / L KH {KO}_{4}

buffer (pH 6.5) and methyl alcohol (HPLC grade), which were
嘌呤分析采用 Shimadzu LC-10AD 系统进行，色谱分离使用反相柱（Shimadzu Intertsustain

C_{18}, 4.6 mm \times 150 mm, 5 μ m

）。流动相由

50 mmol / L KH {KO}_{4}

缓冲液（pH 6.5）和甲醇（HPLC 级）组成，
pumped by A and B pumps, respectively. The flow rate was maintained at

1.0 mmol / L / \min

. The whole retention time was 30 min , while the injected volume of methyl alcohol turned into

0.02 %, 0.02 %, 15 %, 15 %, 32 %, 32 %

0.02 %

, and

0.02 %

, respectively, at times

0.01, 8, 11, 17

18, 24, 25

, and 30 min . Each sample injection volume was

20 μ L

(loop injection volume). The temperature of the column was

25^{\circ} C

and purine concentration was detected at an ultraviolet wavelength 254 nm .
分别由 A 泵和 B 泵泵入。流速维持在

1.0 mmol / L / \min

。整个保留时间为 30 分钟，而甲醇的注入体积在时间

0.01, 8, 11, 17

、

18, 24, 25

和 30 分钟时分别变为

0.02 %, 0.02 %, 15 %, 15 %, 32 %, 32 %

、

0.02 %

和

0.02 %

。每个样品的注入体积为

20 μ L

（环注射体积）。柱温为

25^{\circ} C

，嘌呤浓度在 254 nm 的紫外波长下检测。

Peak identification and quantification were assessed using standard solutions of purines (ATP, AMP, ADP, and ADO) dissolved in distilled water (Watsons, Chengdu, China, Figure 1A). Fresh standard solutions were prepared every week, while working standard solutions were prepared with the appropriate dilutions of stock solution. A standard curve was prepared prior to the measurement of each batch of collected samples. The concentrations

(μ g / mL)

of ATP, ADP, AMP, and ADO were determined from the chromatogram peak areas. The chromatograms of standard substances and collected samples were manifested as follows (Figure 1B).
峰识别和定量分析使用标准纯品（ATP、AMP、ADP 和 ADO）的水溶液（Watsons, 成都, 中国, 图 1A）进行。每周制备新鲜标准溶液，而工作标准溶液则通过将储备溶液进行适当稀释制备。在每批采集样品的测量前制备标准曲线。从色谱图峰面积测定 ATP、ADP、AMP 和 ADO 的浓度

(μ g / mL)

。标准物质和采集样品的色谱图表现如下（图 1B）。

Figure 1. Peaks Identification and Quantification by HPLC
图 1. 高效液相色谱法进行峰识别和定量分析
Notes: (A) is the chromatogram of 4 standard purines solutions(ATP, AMP, ADP, ADO) dissolved in distilled water (Watsons). Peak 1 is the retention time of ATP, at 3.35 min . Peak 2 is the retention time of ADP, at 4.36 min. Peak 3 is the retention time of AMP, at 6.45 min . Peak 4 is the retention time of ADO, at 18.96 min . Simultaneously, (B) is the chromatogram of 4 purines in collected samples. The retention times of 4 purines are identified well and almost in coincidence.
注：（A）是 4 种标准纯品（ATP、AMP、ADP、ADO）的水溶液（Watsons）的色谱图。峰 1 是 ATP 的保留时间，为 3.35 分钟。峰 2 是 ADP 的保留时间，为 4.36 分钟。峰 3 是 AMP 的保留时间，为 6.45 分钟。峰 4 是 ADO 的保留时间，为 18.96 分钟。同时，（B）是采集样品中 4 种纯品的色谱图。4 种纯品的保留时间识别良好，几乎完全一致。

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018
©《中国中西医结合杂志》出版社和施普林格-威利 GmbH 德国，施普林格自然公司的一部分 2018
*Supported by National Natural Science Foundation of China (No. 81590951, 81373559, 81373561 and 81573885), the State Key Program for Basic Research of China (No. 2012CB518501), and the Project of the Second Clinical Medical College of Nanjing University of Chinese Medicine (No. RLZZ201605)
*由国家自然科学基金（编号 81590951、81373559、81373561 和 81573885）、国家重点基础研究发展计划（编号 2012CB518501）和南京中医药大学第二临床医学院项目（编号 RLZZ201605）资助
1. Department of Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu (610075), China; 2. The Second Clinical Medical College of Nanjing University of Chinese Medicine, Nanjing (210046), China
  成都中医药大学针灸推拿学院，中国成都（610075）；2. 南京中医药大学第二临床医学院，中国南京（210046）
  Correspondence to: Prof. LIANG Fan-rong, Tel: 86-2869950657, E-mail: acuresearch @ 126.com
  通讯作者：梁帆荣教授，电话：86-2869950657，邮箱：acuresearch @ 126.com
  DOI: https://doi.org/10.1007/s11655-018-2932-8

Acupuncture Research 针灸研究

Purines Change at Acupoints along the Pericardium Meridian in Healthy and Myocardial Ischemic Rats* 健康与心肌缺血大鼠心经穴位嘌呤变化*