Effects of wrist-ankle acupuncture on associated factors in uterus tissue and serum in rats with primary dysmenorrhea

Liu Wei-xing (刘卫星), Zhao Yan (赵岩), Yu Yue-yue (虞跃跃)

1 Acupuncture and Tuina Department, Tangshan Hospital of Traditional Chinese Medicine, Hebei 063000, China

2 College of Traditional Chinese Medicine, Hebei United University, Hebei 063000, China

Effects of wrist-ankle acupuncture on associated factors in uterus tissue and serum in rats with primary dysmenorrhea

Liu Wei-xing (刘卫星)1, Zhao Yan (赵岩)2, Yu Yue-yue (虞跃跃)2

1 Acupuncture and Tuina Department, Tangshan Hospital of Traditional Chinese Medicine, Hebei 063000, China

2 College of Traditional Chinese Medicine, Hebei United University, Hebei 063000, China

Objective:To observe effects of wrist-ankle acupuncture (WAA) on β-endorphin (EP), nitric oxide (NO) in uterus tissue and prostaglandin F2α (PGF2α), substance P (SP) in serum of rats with primary dysmenorrhea.

Wrist-ankle Acupuncture; Dysmenorrhea; Acupuncture Analgesia; Endorphins; Nitric Oxide; Prostaglandins F; Substance P; Rats

Primary dysmenorrhea refers to cramping pain in the lower abdomen occurring before or during menstruation without genital abnormalities. In severe cases, patients may experience severe pain, a bluish face, cold limbs, vomiting, sweating, weakness and even fainting. Dysmenorrhea seriously affects quality of life of women. Acupuncture therapy has been widely recognized in the field of analgesia[1-4]. This study tried to reveal the mechanism of wrist-ankle acupuncture (WAA) for primary dysmenorrhea by measuring β-endorphin (β-EP) and nitric oxide (NO) in uterine tissue and prostaglandin F2α (PGF2α) and substance P (SP) in serum. Now, the results are reported as follows.

1 Materials and Methods

1.1 Animals

A total of 45 healthy and clean female non-pregnant Wistar rats, weighing (220 ± 20) g, were provided by Experimental Animal Center of Hebei United University (Certificate No. SCXKll-00-0010).

1.2 Drugs and reagents

Oxytocin injection (Shanghai Hefeng Pharmaceutical Co., Ltd., China); diethylstilbestrol (Beijing Shuguang Pharmaceutical Co., Ltd., China); PGF2α ELISA kit (Beijing Dongge Biotechnology Co., Ltd., China); β-EP ELISA kits, SP and NO ELISA kits assay kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., China).

1.3 Equipments

721 spectrophotometer (Shanghai INESA Analytical Instruments Co., Ltd., China); MR18.12 high-speed refrigerated centrifuge (Beijing Era Beili Centrifuge Co., Ltd., China); electronic analytical balance (Shanghai Bending Weighing Apparatus Co., Ltd., China); 2 L pipette, 20 L pipette, 100 L pipette (Shanghai Aiyan Biotechnology Co., Ltd., China); cryogenic refrigerator; homogenizer; Hwato acupuncture needles, 0.35 mm in diameter, 25 mm in length.

1.4 Model preparation and grouping

Forty-five rats were randomly divided into a control group, a model group and a WAA group according to random number table, with 15 in each group. Rats in the model group and WAA group were modeling. The rat model of dysmenorrhea was prepared referring to literature[5]. Subcutaneous injection of Diethylstilbestrol to abdomen was applied once a day for 10 d (0.5 mg/d on the 1st and 10th day; and 0.3 mg/d from the 2nd day to 9th day). On 11th day, 2 U Oxytocin was intraperitoneally injected. After modeling, rats in the WAA group received acupuncture at Point Lower 1 and Point Lower 2, once a day for 10 d. According to theAcupuncture and Moxibustion Techniques[6]andPractical Handbook of Experimental Animal Science of Acupuncture and Moxibustion[7], Point Lower 1 locates at 3 cun above the ankle and next to inner edge of achilles tendon (Figure 1); Point Lower 2 locates at 3 cun above the ankle and on rearward edge of the tibia. The needles were quickly penetrated at a 30° angle with the skin, and the needle was located in the shallow skin for 30 min each time. Rats in the control group and the model group received no treatment. No animals died during the experiment.

1.5 Detection parameters

1.5.1 Writhing

After intraperitoneal injection of Oxytocin on the 11th day, the writhing latency (time from injection to the first writhing) and frequencies were observed within 30 min.

Figure 1. Point Lower 1

Figure 2. Point Lower 2

1.5.2 Parameters detected in uterine tissue and sera

After writhing test, rats were sacrificed. The uterus was quickly removed without surrounded tissue and weighed. Saline was added according to uterine weight to prepare for a 10% uterus homogenate. After 3 500 r/min centrifugation for 10 min, the supernatant was separated and stored at －70 ℃[8]. β-EP and NO concentrations were measured in strict accordance with the ELISA kit instructions. At the same time the uterus was removed, 5 mL blood was drawn from abdominal aorta and injected to a pressurized tube, stewing for 2 h then 3 000 r/min centrifugation for 10 min, stewing 30 min then centrifuged for another 30 min. The serum was drawn and stored at －20 ℃. PGF2α and SP were measured in strict accordance with ELISA kit manual procedures[9].

1.6 Statistical Methods

The SPSS 17.0 version statistical software was used for data analysis and statistical processing, measurement data were presented as mean ± standard deviationand independent samplest-test was used. APvalue of ＜0.05 expresses a statistically significant difference.

2 Results

2.1 Influence of WAA on writhing reaction in rats with primary dysmenorrhea

Except for the control group, rats in the model group and the WAA group have suffered different degrees of writhing. In the model group, the writhing reaction times were the most, and the latencies were the shortest. The writhing response times in the WAA group were significantly decreased and the latencies were significantly increased as compared with those in the model group (both P＜0.05), (Table 1).

Table 1. Comparison of withering responses in each group

Note: Compared with the control group, 1) P＜0.05; compared with the model group, 2) P＜0.05

Group n Latency (minute) Withering number (time) Control 15 30.00 0 Model 15 6.28±2.571) 18.50±8.861)WAA 15 14.77±7.071)2) 5.65±3.221)2)

2.2 Influence of WAA on β-EP and NO levels in uterus of rats with primary dysmenorrhea

Uterus β-EP and NO levels in the model group were the lowest, with statistically significant differences compared with those in the control group (P＜0.05). β-EP and NO levels in the WAA group were higher than those in the model group with statistically significant differences (P＜0.05), (Table 2)

Table 2. Comparison of β-EP and NO levels in uterine tissue in each group

Table 2. Comparison of β-EP and NO levels in uterine tissue in each group

Note: Compared with the control group, 1) P＜0.05; compared with the model group, 2) P＜0.05

Group n β-EP (pg/mL) NO (µmol/L) Control 15 8.30±0.71 73.92±8.67 Model 15 6.15±0.371) 45.44±5.151)WAA 15 9.07±0.221)2) 72.48±12.612)

2.3 Influence of WAA on serum PGF2α and SP in rats with primary dysmenorrhea

Serum PGF2α levels in the model group were the highest and SP level was the lowest, with statistically significant differences compared with those in the control group (both P＜0.05). PGF2α level in the WAA group was lower than that in the model group; and SP level was higher, with statistically significant differences respectively (both P＜0.05), (Table 3).

Table 3. Comparison of PGF2α and SP levels in uterus tissue in each group

Table 3. Comparison of PGF2α and SP levels in uterus tissue in each group

Note: Compared with the control group, 1) P＜0.05; compared with the model group, 2) P＜0.05

Group n PGF2α (pg/mL) SP (pg/mL) Control 15 125.85±23.52 38.36±21.81 Model 15 201.22±35.721) 13.64±2.891)WAA 15 141.35±21.911)2) 34.65±25.481)2)

3 Discussion

Common in young women, primary dysmenorrhea is categorized as menstrual abdominal pain in traditional Chinese medicine (TCM). It can be diagnosed as excess due to obstruction or deficiency due to malnourishment[10]. The excess syndrome includes blood stasis due to cold, qi stagnation or damp-heat, affecting the flow of blood in the uterus; deficiency syndrome includes qi-blood insufficiency, internal cold due to yang insufficiency or lack of nourishment in uterus due to liver-kidney deficiency. It is reported that WAA had a significant analgesic effect[11-14], by activating qi and blood circulation, dredging the meridians and regulating internal organs. It is simple, convenient, rapid onset without soreness or pain or adverse side effects, and is accepted by patients.

In modern research, the pathogenesis of primary dysmenorrhea is complex[15], mainly related to β-EP and NO in uterine tissue. β-EP is a morphine-like polypeptide in hypothalamic-pituitary axis and is targeting uterus with endogenous analgesic effect[16-17]. NO is a gas molecule of various biological functions, and plays an important role in the nervous, immune and circulation systems, in particular the pain modulation process. It targets different organs involved in peripheral pain modulation. NO increase will inhibit nociceptive transference to achieve analgesia; otherwise its decrease will cause pain[18-19]. In addition, serum PGF2α and SP contents are closely related to primary dysmenorrhea. PGF2α can excite uterus smooth muscle resulting in spastic contraction, reduced uterine blood flow, muscle ischemia and pain[20-21]. As a neurotransmitter, SP is distributed in the blood and tissues and involved in pain transmission and regulation. The uterus is innervated by T11-L2spinal segment ganglions. NO is released through the segment causing vasodilation and inhibiting uterus smooth muscle contraction to achieve analgesia[22-23].

The present study shows that in the model group, the β-EP and NO levels were the lowest, the serum PGF2α content was the highest, SP was the lowest; the rats writhing times were the highest, and the latencies werethe shortest; in the WAA group, PGF2α was reduced, β-EP, NO and SP contents increased compared with those in the model group with statistically significant differences. This study shows that WAA treatment may inhibit PGF2α expression, promote β-EP, NO and SP contents to relieve uterus cramps, increase blood flow, and improve uterus function to achieve analgesic effect.

Conflict of Interest

The authors declared that there was no conflict of interest in this article.

Acknowledgments

This work was supported by Hebei Tangshan Science and Technology Project (河北省唐山市科技计划项目, No.121302118b).

Statement of Informed Consent

The treatment of animals conformed to the ethical criteria in this experiment.

Received: 27 October 2014/Accepted: 30 November 2014

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Translator: Feng Xiao-ming (丰晓溟)

腕踝针对原发性痛经大鼠子宫组织及血清相关因子的影响

目的：观察腕踝针对原发性痛经大鼠子宫组织匀浆中β-内啡肽(β-endorphin, EP)、一氧化氮(nitric oxide, NO)及血清中前列腺素F2a (Prostaglandin F2α, PGF2α)、P物质(Substance P, SP)的影响。方法：将45只未孕Wistar大鼠按随机数字表法分为空白对照组、模型组、腕踝针组，每组15只。除空白对照组外，其余两组在大鼠腹部皮下连续注射已烯雌酚建立痛经模型, 腕踝针组造模成功后第1天起针刺下1区、下2区，每日1次，连续10天。空白对照组和模型组不予任何治疗。观察大鼠的扭体潜伏期与扭体次数，检测子宫组织匀浆中β-EP、NO及血清中PGF2α、SP含量。结果：模型组大鼠子宫组织匀浆中β-EP、NO水平最低，血清中的PGF2α水平最高，SP含量最低，与空白对照组比较, 组间差异有统计学意义(P＜0.05)。腕踝针组PGF2α低于模型组, β-EP、NO、SP水平高于模型组, 组间差异有统计学意义(P＜0.05)。结论：腕踝针可能通过抑制PGF2α, 上调β-EP、NO及SP缓解子宫痉挛, 增加血流量, 促进子宫组织功能改善, 达到止痛的效果。

腕踝针; 痛经; 针刺镇痛; 内啡呔; 一氧化氮; 前列腺素F类; P物质; 大鼠

R2-03 【

】A

Author: Liu Wei-xing, associate chief physician

Zhao Yan, master degree candidate.

E-mail: tsslw110@126.com

Methods:A total of 45 non-pregnant Wistar rats were randomly divided into a control group, a model group and a WAA group, 15 rats in each group. Rats in the model group and the WAA group received continuous abdominal subcutaneous injection of Diethylstilbestrol to establish dysmenorrhea rat models. On the first day after modeling, rats in the WAA group began to receive acupuncture on Point Lower 1 and Point Lower 2, once a day for 10 d. The control group and the model group didn’t receive any treatment. Writhing latencies and frequencies were recorded. β-EP and NO in uterus tissue homogenates and PGF2α, SP in serum were detected.

Results:In the model group, β-EP and NO levels were the lowest among the groups, the serum PGF2α level was the highest, and serum SP level was the lowest. These measurements showed significantly difference between the model group and the control group (P＜0.05). PGF2α in the WAA group was lower than that in the model group; β-EP, NO and SP levels were higher than those in the model group, with inter-group statistically significant differences (P＜0.05).

Conclusion:WAA may achieve analgesic effect through decreasing PGF2α, increasing β-EP, NO and SP to relieve uterine cramps, increase blood flow and promote functional improvement.