LIU Chao (刘超), LI Xinhua (李新华),2, ZHOU Chenxi (周晨曦), LIANG Yulei (梁玉磊),2,ZHANG Xuanping (张选平),2, LIU Yucai (刘昱材),2, ZHAO Zhiguo (赵志国),2, MA Xiaoshun (马小顺),2

1 Hebei University of Chinese Medicine, Shijiazhuang 050200, China

2 Key Research Office of Hebei Province Needling and Moxibustion Techniques Effect Specificity, Shijiazhuang 050200, China

Abstract

Keywords: Moxibustion Therapy; Indirect Moxibustion; Ginger-partitioned Moxibustion; Dinoprost; Estradiol;Dysmenorrhea; Rats

In this study, to explore the analgesic effect and the potential mechanism of ginger-partitioned moxibustion for model rats of PD due to cold-dampness stagnation,we observed the writhing latency, calculated the writhing score, and detected the serum PGF2α, E2, P, and related molecular receptor genes (uterine tissue PGF2αreceptor mRNA and E2receptor mRNA) after ginger-partitioned moxibustion intervention in the PD rat model.

1 Materials and Methods

1.1 Laboratory animals

Thirty-two healthy non-pregnant specific-pathogenfree grade female Wistar rats were provided by the Animal Experiment Center of Hebei Medical University[Animal production license No. SCXK (Hebei) 2018-004,body mass: (210±10) g]. According to the random number table method, the rats were divided into a normal group, a model group, a ginger-partitioned moxibustion group, and a Western medicine group,with 8 rats in each group.

The four groups of rats were fed with tap water and ordinary pelleted rat feed under the same feeding conditions. The feed production license number was SCXK (Hebei) 2018-003. Rats were allowed to eat and drink freely in the animal room with a light-dark cycle of 12 h and the room temperature of 18-26℃. All rats completed the entire experimental procedure. During the experiment, the rats were treated following the animal ethical standards and the requirements of the animal experiment center, which had been approved by the Ethics Committee of Hebei University of Traditional Chinese Medicine (Approval No. DWLL2018048).

1.2 Main drugs, instruments, and reagents

1.2.1 Preparation of moxa cone and ginger slices

Moxa cones: Small-sized moxa cones (each cone was 0.5 g with a bottom diameter of 5 mm and height of 5 mm) were uniformly made by our research group.

Ginger slices: A piece of ginger was uniformly made into round slices with a thickness of about 4 mm and a diameter of 8-10 mm, tapped twice with a plumblossom needle for later use.

1.2.2 Drug

Ibuprofen tablets (State Food and Drug Administration Approval No. H20065680, Guangdong Huanan Pharmaceutical Group Co., Ltd., China) were made into a solution (1.25 g/L) with normal saline.

1.2.3 Main experimental instruments

FJ-2021 r-radioimmunity counter (Xi’an 262 Factory,China); BIO-RAD CFX96 fluorescence quantitative polymerase chain reaction (qPCR) system (Bio-Rad,USA).

1.2.4 Main experimental reagents

Estradiol benzoate injection (veterinary medicine permission No. 163231439) and oxytocin injection(veterinary medicine permission No. 163231571)(Shanghai Quanyu Biotechnology Animal Pharmaceutical Co., Ltd., China); E2and P radioimmunoassay (RIA) kit (Tianjin Jiuding Medical Bioengineering Co., Ltd., China); PGF2αenzyme-linked immunosorbent assay (ELISA) kit (Shanghai Senxiong Technology Industrial Co., Ltd., China).

1.3 Model preparation

The model rats with PD due to cold-dampness stagnation were established by estradiol benzoate,oxytocin combined with ice-cold water bath method[10-12]. Rats in each group were adaptively fed for 2 d before the experiment. Except for the normal group,rats in the other groups were used to prepare the model after adaptive feeding. Estradiol benzoate was subcutaneously injected into the thigh for ten consecutive days, once a day, at 0.8 mg/rat on the 1st day and the 10th day, 0.4 mg/rat on the 2nd-9th days,and oxytocin (2 U/rat) was intraperitoneally given on the 11th day. After daily injection of estradiol benzoate,rats were immersed into the ice-water mixture for cold and damp stimulation (room temperature 18-26 ℃,30 min), once a day for 10 consecutive days. Rats in the normal group were injected with normal saline according to the above method. When the symptoms of cold-damp stagnation syndrome (the skin color of the mouth, nose, ears, and lips of the model rats became lighter, the claws, tail, and fur became darker, chills,shivering, or sneezing occurred, the trunk curled up,depression, decreased activities, water, and food intake,and loose stools) appeared, the rat model of the cold-damp stagnation-like syndrome was successfully established. After oxytocin administration on the 11th day of modeling, the rat trunk appeared opisthotonos,the hind limbs extended backward, and the abdomen exhibited writhing behaviors such as contraction and twitching or lateral pelvic rotation, suggesting that the rat model of PD due to cold-dampness stagnation was successfully made.

1.4 Intervention in different groups

1.4.1 Ginger-partitioned moxibustion group

Acupoints: Shenque (CV8) and Guanyuan (CV4).

Methods:From the 8th day of modeling, after modeling for 75 min, the acupoints were positioned according to theExperimental Acupuncture Science[13].A ginger slice was put on the acupoint and a moxa cone was put on the ginger slice to perform the gingerpartitioned moxibustion, with 6 cones for each acupoint,once a day, for a total of 3 d.

1.4.2 Western medicine group

Treatment was performed from the 8th day of modeling, after modeling for 75 min.

The rats were administered with ibuprofen solution(prepared with normal saline at the concentration of 1.25 g/L) by gavage, 0.8 mL/rat, once a day, for three consecutive days.

1.4.3 Normal group and model group

Rats in the normal group and the model group were raised under the same conditions without any treatment.

1.5 Observation items

1.5.1 Rat writhing response

On the 11th day of modeling, the model rats were intraperitoneally injected with oxytocin, and the normal rats were intraperitoneally injected with normal saline.Based on the four levels of writhing in the behavioral scoring scale (Table 1)[14], the rat writhing behavior was observed, the time of the first writhing behavior was recorded, and the writhing behavior within 20 min was evaluated.

Table 1. Scale of writhing response in dysmenorrhea rats

Time was recorded ever since the oxytocin injection,and the first writhing behavior time was recorded as the writhing latency period.

Writhing score = Level 0 × 0 point + Level 1 × 1 point +Level 2 × 2 points + Level 3 × 3 points.

1.5.2 Expression of serum PGF2α, E2and P

After 1 h of intraperitoneal injection of oxytocin in the model rats and normal saline in the normal rats, the

rats were anesthetized with 6% chloral hydrate[intraperitoneal injection, 300 mg/(kg·bw)], then 5 mL abdominal aorta blood was collected and centrifuged to carefully store the supernatant in a -80 ℃ refrigerator for later use. Serum E2and P were detected by RIA method, and serum PGF2αwas detected by ELISA method.

1.5.3 The mRNA expression of PGF2αand E2receptors

After abdominal aorta blood collection, rat abdomen was dissected and the uterus was removed. The uterus was placed on an ice tray, washed with normal saline,and immediately stored in liquid nitrogen. Total RNA was extracted from tissues, and real-time qPCR(RT-qPCR) was used to detect the mRNA expression of PGF2αand E2receptors. The primer sequences are shown in Table 2.

A 20 μL reaction system for RT-qPCR assay: 10 μL of PreMix, 0.4 μL of upstream primer, 0.4 μL of downstream primer, 2 μL of cDNA, and the remaining volume was made with nuclease-free H2O. The mixture was mixed slowly, centrifuged briefly, annealed (25 ℃,5 min), extended (42 ℃, 60 min), inactivated (70 ℃,15 min) in a PCR system, then cooled on ice and transferred into a -20 ℃ freezer for future use. A twostep PCR reaction was performed: pre-denaturation(95 ℃, 10 min) followed by 40 cycles of reaction[denaturation (95 ℃, 15 s), and annealing extension(60 ℃, 60 s)].

After the amplification was completed, the Cq values of the target gene and the internal control gene GAPDH in each sample were obtained, and the difference between them was ∆Cq. For each target gene, the Q value was calculated according to the formula Q=2-∆Cq,and the mean of the Q value was obtained. Then the ratio of the Q value to the mean of the Q value(Q value ÷ Q mean= RQ value) of each target gene was obtained, which was the relative quantitative value of each target gene expression. RQ values were used for data analysis, statistical processing, and comparison among groups.

Table 2. RT-qPCR primer sequences

1.6 Statistical analysis

The experimental data were analyzed using SPSS version 23.0 statistical software. The data of each group were tested for normality and homogeneity of variance.If both were satisfied, one-way analysis of variance was used for data analysis, the least significant difference(LSD) was used for post-hoc comparison, and numerical variables were presented as mean ± standard deviation(±s; if one of the two was not satisfied, theH-test was used for multi-sample comparison, and the numerical variable was presented as median (interquartile range) [M (IQR)]. All hypothesis tests were considered statistically significant atP＜0.05.

2 Results

2.1 Comparison of writhing latency and writhing score

There was no writhing response in the normal group within 20 min. Compared with the model group, rat writhing latencies in the ginger-partitioned moxibustion group and the Western medicine group were statistically significantly prolonged (P＜0.01); the writhing latency in the ginger-partitioned moxibustion group was statistically significantly longer than that in the Western medicine group (P＜0.05).

Compared with the model group, the rat writhing response scores in the ginger-partitioned moxibustion group and the Western medicine group were decreased(P＜0.01); compared with the Western medicine group,the writhing response score in the ginger-partitioned moxibustion group was decreased (P＜0.05), (Table 3).

Table 3. Comparison of writhing latency and writhing score of rats among groups ( ±s

Table 3. Comparison of writhing latency and writhing score of rats among groups ( ±s

Note: GPMG=Ginger-partitioned moxibustion group; WMG=Western medicine group; compared with the model group, 1)P＜0.01; compared with the Western medicine group, 2) P＜0.05

Groupn Writhing latency within 20 min(s)Writhing score(point)Model 893.00±26.41 41.75±17.77 GPMG 8 285.63±53.581)2) 12.25±3.371)2)WMG8189.50±53.511) 21.88±10.951)

2.2 Comparison of serum PGF2α, E2, and P levels

Compared with the normal group, the serum PGF2αand E2levels in the model group were increased(P＜0.01), while the P level was decreased (P＜0.01).Compared with the model group, the serum PGF2αlevels in the ginger-partitioned moxibustion group and the Western medicine group were significantly decreased (P＜0.05,P＜0.01), the E2levels were significantly decreased (P＜0.01), and the serum P levels were significantly increased (P＜0.05). Compared with the Western medicine group, the E2level in the ginger-partitioned moxibustion group was decreased(P＜0.05), but there was no significant difference in the serum PGF2αand P levels (P＞0.05), (Table 4).

2.3 Comparison of mRNA expression of PGF2α and E2 receptors in rat uterus

Compared with the normal group, the mRNA expression levels of uterine PGF2αand E2receptors in the model group were increased (P＜0.01,P＜0.05).Compared with the model group, the mRNA expression levels of PGF2αand E2receptors in the uterus of the ginger-partitioned moxibustion group and the Western medicine group were decreased (P＜0.05). However,there was no significant difference between the gingerpartitioned moxibustion group and the Western medicine group (P＞0.05), (Table 5).

Table 4. Comparison of rat serum PGF2α, E2, and P levels among groups ( ±s, pg/mL)

Table 4. Comparison of rat serum PGF2α, E2, and P levels among groups ( ±s, pg/mL)

Note: PGF2α=Prostaglandin F2α; E2=Estradiol; P=Progesterone; GPMG=Ginger-partitioned moxibustion group; WMG=Western medicine group; compared with the normal group, 1) P＜0.01; compared with the model group, 2) P＜0.01, 3) P＜0.05; compared with the Western medicine group, 4) P＜0.05

Group n PGF2α E2 P Normal 8 105.12±18.77 152.41±35.28 30.11±12.14 Model 8 183.64±11.071) 752.47±60.061) 8.31±2.141)GPMG 8 157.67±30.631)3) 427.17±74.631)2)4) 19.17±6.311)3)WMG 8 138.05±26.171)2) 491.13±53.411)2) 19.60±3.281)3)

Table5. ComparisonofratmRNA expression of PGF2α and E2 receptorsamong groups(±s

Table5. ComparisonofratmRNA expression of PGF2α and E2 receptorsamong groups(±s

Note: PGF2α=Prostaglandin F2α; E2=Estradiol; GPMG=Gingerpartitioned moxibustion group; WMG=Western medicine group;PGF2αR=Prostaglandin F2α receptor; E2R=Estradiol receptor;compared with the normal group, 1) P＜0.01, 2) P＜0.05; compared with the model group, 3) P＜0.05

Groups n PGF2αR E2R Normal 8 0.86±0.14 1.14±0.77 Model 8 1.25±0.241) 1.44±0.462)GPMG 8 1.00±0.293) 0.89±0.173)WMG 8 1.02±0.253) 1.13±0.333)

3 Discussion

In gynecology of traditional Chinese medicine, PD belongs to the category of “abdominal pain during menstruation”. It is mostly due to the stagnation of Qi and blood, blockage of meridians, and poor transportation and transformation, resulting in pain.The earliest literature on dysmenorrhea isJin Gui Yao Lue(Synopsis of Prescriptions of the Golden Chamber).It is easy to get dysmenorrhea due to cold-dampness stagnation if caught in the rain or walking in the water during or before menstruation or living in a cold and damp place for a long time. In this experiment, rat model establishment was based on the dysmenorrhealike model. Referring to the pathogenesis of colddampness stagnation syndrome, rats were stimulated with cold and dampness for a long time during the modeling process to establish a cold-dampness stagnation-like dysmenorrhea model.

Traditional Chinese medicine believes that dysmenorrhea is located in the uterus and the Thoroughfare and Conception Vessels, and is closely related to the spleen, liver, and kidney meridians. The Conception, Thoroughfare and Governor Vessels,originate from the uterus, ascend to the chest, pass through the lower abdomen, and intersect with the three Yin meridians of foot at Guanyuan (CV4), and can regulate many gynecological diseases. Guanyuan (CV4)and Shenque (CV8) both are the key acupoints of the Conception Vessel, located in the lower abdomen of the human body. In theZhen Jiu Da Cheng(Complete Compendium of Acupuncture and Moxibustion), the frequency of acupoints of the Conception Vessel and the Foot Taiyin Meridian ranks the highest for the treatment of menstrual diseases, and the acupoints are mostly distributed in the chest and abdomen[15].

Shenque (CV8) is located in the center of the umbilicus and is often used to treat PD. Modern anatomical studies have confirmed that the umbilicus is directly connected to the skin fascia and the abdominal wall membrane. It is the last closed place during abdominal wall development, with the periumbilical venous network of the abdominal wall distributed under it[16]. Moxibustion at Shenque (CV8) can activate Qi and blood, remove blood stasis and relieve pain.Guanyuan (CV4) is the crossing acupoint of the Conception Vessel and the three Yin meridians of the foot, with the main indication of dysmenorrhea,amenorrhea, and irregular menstruation. Gingerpartitioned moxibustion at Guanyuan (CV4) not only can regulate the Thoroughfare and Conception Vessels,but also treat many gynecological diseases. Studies have found that the L3segment of the spinal nerve is the main nerve segment that covers Guanyuan(CV4)[17-20], and it is also the main segment of the afferent spinal nerve of the uterine nerve. These associations build the neurological basis. Therefore,ginger-partitioned moxibustion at Guanyuan (CV4) can relieve dysmenorrhea symptoms mainly through neuromodulation, inhibiting the excitability of uterine autonomic nerves, relieving the abnormal spasm of uterine tissues, and increasing blood supply to ischemic sites, thereby playing an analgesic effect.Ginger-partitioned moxibustion is a commonly used moxibustion method[21]. When moxa is burned, its medicinal and warming effects can expand the local capillaries, improve the local blood circulation in the uterus, and reduce the spasm degree of uterine smooth muscles[22]. Ginger is warm in nature and spicy in taste with the effects of warming meridians and collaterals,and dissipating wind and cold, having a good effect in promoting blood circulation and removing blood stasis[23-24]. Ginger-partitioned moxibustion can integrate the above effects to dilate uterine blood vessels and improve local blood microcirculation, thus to play an analgesic effect on PD due to cold-dampness stagnation.

Modern medicine believes that PGF2αis one PG type,mainly distributed in women’s endometrium and ovary and most closely related to dysmenorrhea[25]. Women’s PGF2αlevel fluctuates periodically with the menstrual cycle. During menstruation, increased PGF2αcauses abnormal contraction of uterine smooth muscles and capillaries. It is an important factor leading to dysmenorrhea. Serum PGF2αbinds to its specific receptors in the endometrium and acts on the spiral arterioles, which causes the capillaries in the uterus to contract and break violently, resulting in the shedding of the endometrium and the formation of menstrual onset.Abnormally increased PGF2αleads to intrauterine vasospasm and ischemia and uterine smooth muscle spasm, stimulates uterine pain neurons, and causes lower abdominal contracture pain and PD[26-27].Compared with non-dysmenorrhea patients, the peripheral blood PGF2αlevel of PD patients abnormally increases, and the PGF2αchange is affected by E2and P levels. The increase in E2level will indirectly accelerate the formation and secretion of endogenous PGF2α,which will lead to the aggravation of uterine capillary spasm and ischemia. The uterine tissues will form a hypoxic environment due to insufficient blood supply,resulting in increased acidic substances and stimulated pain neurons to produce pain sensation. E2level is positively correlated with the pain degree of PD. The increase in P level can reduce the formation and secretion of endogenous PGF2αby antagonizing the secretion-promoting effect of E2on PGF2α, and produce an analgesic effect. It is negatively correlated with the pain degree of dysmenorrhea. Ibuprofen is a commonly used non-steroidal anti-inflammatory drug for PD treatment, but its oral efficacy is slow, and long-term use can cause gastrointestinal damage[28].

This study showed that the serum levels of PGF2αand E2in model rats with PD due to cold-dampness stagnation were higher than those in normal rats, and the P level was lower than that in normal rats.Ginger-partitioned moxibustion at Shenque (CV8) and Guanyuan (CV4) can exert an analgesic effect by reducing serum PGF2αand E2levels, increasing P level,prolonging the writhing response latency, and reducing the writhing response score of model rats with PD due to cold-dampness stagnation. As steroid hormones,PGF2αand E2need to bind with their specific receptors[29]. Ginger-partitioned moxibustion at Shenque (CV8) and Guanyuan (CV4) of dysmenorrhealike model rats can reduce the mRNA expression of PGF2αand E2receptors in the uterine tissues, which means that the targets of PGF2αand E2, which can exacerbate dysmenorrheal, are reduced. Therefore,uterine spasm and pain are reduced[30]. This experiment confirms that ginger-partitioned moxibustion at Shenque (CV8) and Guanyuan (CV4) can produce satisfactory immediate analgesic effect on rats with PD due to cold-dampness stagnation, and thus can be used as a common treatment method for cold-dampness stagnation type PD.

Conflict of Interest

The authors declare that there is no potential conflict of interest in this article.

Acknowledgments

This work was supported by Science and Technology Research Project of Universities in Hebei Province (河北省高等学校科学技术研究项目, No. ZD2019099).

Statement of Human and Animal Rights

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

Received: 18 February 2021/Accepted: 10 June 2021