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

Liu Wei-xing (劉衛星), Zhao Yan (趙巖), Yu Yue-yue (虞躍躍)

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 (劉衛星)1, Zhao Yan (趙巖)2, Yu Yue-yue (虞躍躍)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 (河北省唐山市科技計劃項目, 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

[1] Li XN. Clinical observation on 352 cases of acupoints line implantation treatment in dysmenorrhea. Zhongguo Xiandai Yaowu Yingyong, 2010, 20(4): 174-175.

[2] Li YM, Bu YQ, Hou WJ, Chen SZ, Gao SZ. Observation on immediate analgesic effect of acupuncture at Shiqizhui (EX-B 8) only or multi-acupoints in patients with dysmenorrhea: a randomized controlled trial. Zhongguo Zhen Jiu, 2011, 31(3): 199-202.

[3] Zhang LM, Weng SY. Acupoint safflower injection in treating 50 cases of primary dysmenorrhea. Zhongguo Zhongyi Jizheng, 2011, 20(8): 31-32.

[4] Hu YL, Lin Q, Li Y. Eye Acupuncture for primary dysmenorrhea. Shanghai Zhenjiu Zazhi, 2011, 30(6): 370-371.

[5] Ji B, Zhang LF, Zhu J, Ren XX, Guo MW, Zhao YF, Xu LL, Song XL. On building the dysmenorrhea model and assessment methods. Zhongguo Yaolixue Tongbao, 2008, 24(6): 711-714.

[6] Lu SK. Acupuncture and Moxibustion Techniques. Beijing: China Press of Traditional Chinese Medicine, 2009: 142-147.

[7] Hu YL. Practical Handbook of Experimental Animal Science of Acupuncture and Moxibustion. Beijing: Chinese Agriculture Press, 2003: 10-25.

[8] Zhang LB, Yan XQ, Duan JA, Yan FL, Lü JL. Influence of the different extracts of artemisiae argyi on primary dysmenorrheal model in mice. Zhongguo Shiyan Fangjixue Zazhi, 2012, 6(12): 205-208.

[9] Hua YQ, Hong M, Zhu Q. Research progress in primary dysmenorhea. Nanjing Zhongyiyao Daxue Xuebao, 2003, 19(1): 62-64.

[10] Liu YC, Ma BZ. Chinese medicine treatment and research progress in dysmenorhea. Zhongyiyao Xinxi, 1990, 7(4): 19.

[11] Zhou YL, Zhang SQ, Sun GS, Chen JH, Liu P, Liu YJ, Lin GP, Hu B. Clinical observation on ankle-three-needle for treatment of nerve root pain of prolapse of lumbar intervertebral disc. Zhongguo Zhen Jiu, 2006, 26(12): 847.

[12] Zhang XS. Practical Wrist-ankle Acupuncture Therapy. Beijing: People’s Medical Publishing House, 2002: 27.

[13] Li FQ, Zhang WH, Zhao Y. Experimental study on technique of wrist-ankle acupuncture mechanism. Zhongyiyao Linchuang Zazhi, 2011, 23(10): 897-899.

[14] Zhao SH, Liu ZS. Literature analysis of the timing, frequency and duration characteristics of acupuncture intervention in dysmenorrhea. Hebei Zhongyi, 2012, 34(1): 115-117.

[15] Wang MY, Liu J, Song X, Xuan ZH, Xu F. Effects ofDanggui ShaoyaoPowder in treating dysmenorrhea and its effects on contents of arginine vasopressin in rats. Anhui Zhongyi Xueyuan Xuebao, 2011, 30(2): 49-51.

[16] Song XL, Zhang LF, Li XH, Xu LL, Li CH, Ding XY, Ren XX, Zhao YF, Guo MW, Sun ZF, Zhu J. Effects of electroacupuncture of "Sanyinjiao" (SP 6) on contents of MDA and β-EP, and expression of HSP 70 in the uterus in rats with dysmenorrheal. Zhen Ci Yan Jiu, 2010, 35(5): 342-346.

[17] Li YZ, Tong JM. Pathogenesis and treatment in primary dysmenorrhea. Chengde Yixueyuan Xuebao, 2011, 28(2): 94.

[18] Ning WX, Ye JE, Gu JH, Xu YY. Effect of percutaneous electrical stimulation of acupuncture points on PGF2α and NO of primary dysmenorrheal rat. Zhejiang Zhongyiyao Zazhi, 2010, 4(4): 248.

[19] Li LF, Xie LJ, Li GF, Hao N, Zhang QZ, Lu SJ, Li X, Li YJ. Effects of compoundXiaojingtongCapsule on MDA, NO, SOD, GSH-PX in blood serum and uterus tissue of rats with dysmenorrhea induced by oxytocin. Hebei Yiyao, 2010, 32(5): 530-532.

[20] Liu F, Zheng CH, Huang GY, Wang W. Influence of Cx43 on acupuncture effect on the primary dysmenorrheal rat. Zhongguo Zhen Jiu, 2008, 28(10): 751-756.

[21] Wu YY, Li HX, Yang Z, Yang FH, Fu YJ. The effect ofTongjingshuOral Liquid on hemorheology of dysmenorrhea rats and PGF2α of uterine tissues. Zhongguo Yiyao Daokan, 2008, 10(7): 1069-1072.

[22] Du XZ, Qin XG, Yin SL, Liu SQ. Effect of warming supplement acupuncture on pain threshold and spinal content of SP in arthritis rabbit. Zhongyi Yanjiu, 2006, 19(1): 12-15.

[23] Zhang HZ, Shan SL, Wu ZX, Wei XM. Effect of electroacupuncture on SP and PGF2α in serum of dysmenorrhea rats. Nanjing Zhongyiyao Daxue Xuebao, 2014, 30(4): 389-392.

Translator: Feng Xiao-ming (豐曉溟)

腕踝針對原發性痛經大鼠子宮組織及血清相關因子的影響

目的：觀察腕踝針對原發性痛經大鼠子宮組織勻漿中β-內啡肽(β-endorphin, EP)、一氧化氮(nitric oxide, NO)及血清中前列腺素F2a (Prostaglandin F2α, PGF2α)、P物質(Substance P, SP)的影響。方法：將45只未孕Wistar大鼠按隨機數字表法分為空白對照組、模型組、腕踝針組，每組15只。除空白對照組外，其余兩組在大鼠腹部皮下連續注射已烯雌酚建立痛經模型, 腕踝針組造模成功后第1天起針刺下1區、下2區，每日1次，連續10天。空白對照組和模型組不予任何治療。觀察大鼠的扭體潛伏期與扭體次數，檢測子宮組織勻漿中β-EP、NO及血清中PGF2α、SP含量。結果：模型組大鼠子宮組織勻漿中β-EP、NO水平最低，血清中的PGF2α水平最高，SP含量最低，與空白對照組比較, 組間差異有統計學意義(P＜0.05)。腕踝針組PGF2α低于模型組, β-EP、NO、SP水平高于模型組, 組間差異有統計學意義(P＜0.05)。結論：腕踝針可能通過抑制PGF2α, 上調β-EP、NO及SP緩解子宮痙攣, 增加血流量, 促進子宮組織功能改善, 達到止痛的效果。

腕踝針; 痛經; 針刺鎮痛; 內啡呔; 一氧化氮; 前列腺素F類; P物質; 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.