Medicated Thread Moxibustion Therapy of Zhuang Medicine and its Application in a Rat Model of Cold-Congealing Syndrome of Primary Dysmenorrhea

The animal experiments in this study were reviewed and approved by the Animal Experiment Ethics Committee of Guangxi University of Traditional Chinese Medicine (ethical license number DW20230411-45). The animal operation procedures strictly follow the content of the Guiding Opinions on Treating Experimental Animals (Guo Ke Fa Cai Zi [2006] No. 398) and the animal protection and use standards of the International Society for Pain Research (IASP).

NOTE: SPF-grade female rats, 6 weeks old, with a body weight of 200 g ± 20 g, were used in this study. Rats in the estrous phase were selected based on vaginal smears and divided into four groups using the random number method. They are divided into the vehicle group, the cold coagulation type dysmenorrhea model group (the model group), the cold coagulation type dysmenorrhea model group + Western Medicine Group (the medicine group), the cold coagulation type dysmenorrhea model group + Zhuang Medicine line moxibustion group (the moxibustion group), with 12 rats in each group.
Vehicle group: The animals were treated with physiological saline (0.9% sodium chloride).
Model group: During the modeling process of the cold coagulation syndrome dysmenorrhea model, no intervention was performed, and only the same restraint and fixation were performed as the moxibustion group.
Medicine group: From the 6^th day of sensitization, ibuprofen dissolved in 0.9% sodium chloride solution was given by gavage every day. Each rat was given 0.06 g/kg.
Moxibustion group: From the 6^th day of sensitization, daily moxibustion treatment with Zhuang medicine thread was performed. This group received treatment once daily at 8 a.m. for 7 consecutive days.
All animals are kept at the Experimental Animal Science Center of Guangxi International Zhuang Medical Hospital (clean level). The rats were fed in plastic cages with 4 rats in one cage. They ate and drank normally, kept the circadian rhythm alternating, and should be dry indoors. The room temperature was controlled at 23 ± 1 °C, and the humidity was 60% ± 5%.

1. Establishment of a rat model of dysmenorrhea with cold coagulation syndrome

1. Freezing: On days 1-12, anesthetize the rats with an intraperitoneal injection of 0.3% pentobarbital (30 mg/kg), then place them on ice for 20 min daily for 12 consecutive days.
2. Sensitization: On days 1-12, inject rats subcutaneously with estradiol benzoate to enhance the sensitivity of the uterus to pain-causing substances. On the 1^st and 12^th days, inject 0.8 mg/animal; on the 2^nd to 11^th days, inject 0.4 mg/animal.
3. Pain induction: On the 12^th day, 1 h after injection of estradiol benzoate, inject the rats intraperitoneally with 2 U of oxytocin, causing pain.
   NOTE: The model was created using the whole-body freezing method combined with estradiol benzoate and oxytocin injection¹⁰.
4. Treat each animal group as described in steps 1.4.1-1.4.4.
   1. For the rats in the vehicle group, do not perform steps 1.1-1.3. Treat them with physiological saline (0.9% sodium chloride) alone.
   2. For the rats in the model group, perform steps 1.1-1.3. Restrain the animals in the group in a manner similar to the moxibustion group but without any drug/treatment intervention.
   3. For the rats in the medicine group, perform steps 1.1-1.3. From the 6^th day of sensitization (step 1.2), dissolve ibuprofen in 0.9% sodium chloride solution and give it by gavage daily (0.06 g/kg).
   4. For the rats in the moxibustion group, perform steps 1.1-1.3. From the 6^th day of sensitization (step 1.2), perform moxibustion treatment with Zhuang medicine thread (section 4) once daily at 8 a.m. for 7 consecutive days.

2. Setting up the moxibustion simulator for Zhuang medicine threads

1. Press and hold Button 1 for a long time to enter the setting state. Press Button 2 (Plus) or Button 3 (Minus) shortly, and a 132 code will appear. Set the button input to output 0.5 cm for each press.
2. Repeat step 2.1 until code 130 appears, and the device can ignite automatically within 6 s after ignition.
3. Repeat step 2.1 until code 131 appears, and the instrument automatically performs moxibustion twice.
4. Rotate the speed control button to set the frequency to 2 times/s.
   NOTE: The button number is detailed in Supplementary Figure 1.

3. Preparation of Zhuang medicine threads

1. Preparation of medicinal liquor
   1. Select medicinal liquor configurations according to the treatment of different diseases. In this experiment, 10 g of frankincense, 10 g of sandalwood, 10 g of woody incense, 10 g of peach kernel, 10 g of safflower, 10 g of Angelica sinensis tail, 10 g of Fangfeng, 10 g of red peony, 10 g of Sumu, 10 g of dry ginger, and 10 g of turmeric were used. Soak these ingredients in 1000 mL of 75% alcohol, and filter after 10 days to form a medicinal liquor.
2. Soaking the medicinal thread: Soak the ramie thread in the above-mentioned medicinal liquor for at least 10 days (Figure 2), take out the medicinal thread for moxibustion, and seal the medicinal thread in a bottle for later use.
3. Take out a medicinal thread with a length of approximately 21 cm each time when using it (Figure 3).

4. Intervention methods of Zhuang medicine thread moxibustion

1. Acupoint selection
   NOTE: The rats are anesthetized by intraperitoneal injection of 0.3% pentobarbital (30 mg/kg).
   1. Clean the hair in the abdominal area of rats using a small animal shaving machine.
   2. Based on the experience in treating dysmenorrhea with thread moxibustion, select acupoints^11,12,13 such as Shenque, Guanyuan, Zhongji, and Uterine acupoints. Use a marker to mark the above acupoints on the lower abdominal area of the rat.
      NOTE: Refer to Figure 4 for the acupoints. Shen que (CV8, the intersection of the upper 2/3 and lower 1/3 of the line connecting the xiphoid process and the upper edge of the pubic symphysis); Guan yuan (CV4, 25 mm below Shenque acupoint); Zhong ji (CV3, 8 mm below Guanyuan acupoint); Left uterine acupoint (left, EX-CA1, opening 25 mm to the left side of Zhongji acupoint); Right uterine acupoint (Right, EX-CA1, opening 25 mm to the right side of Zhongji acupoint).
2. Fixing the rats
   1. Fix the rats on a transparent acrylic board using high elastic rubber bands (Acrylic board size 10 cm × 15 cm × 5 cm) to expose the abdomen.
   2. Place the fixed rat on the operating table of the Zhuang Medicine Line Moxibustion simulator, adjust the positioning ring to adhere to the rat's abdomen, and adjust the position of the rat to the left and right so that one marked acupoint is located at the center of the positioning ring (Figure 5).
3. Placing the medicated line
   1. Take out one soaked medicine thread and insert it into the medicine thread control device.
   2. Rotate the screw knobs on the vertical and horizontal axes of the support frame. Adjust the shell of the drug line control device up and down so that the distance between the bottom of the shell and the bottom of the positioning ring is 2 cm (Figure 6), and adjust the ignition support shaft and positioning rod shaft accordingly.
   3. Click the wire entry button to expose the medicine wire 0.5 cm from the shell (set the wire end to exit 0.5 cm for each press) (Figure 7).
4. Point moxibustion
   1. Press the ignition button, and the ignition device will automatically ignite. The default timing is 6 s. After that, the automatic moxibustion will be performed twice, with a frequency set to 2 times/s (Figure 8)
      NOTE: It has been previously repeatedly tested that at a windless environment with a temperature of 23 ± 1 °C and humidity of 60% ± 5%, bead fire could form after 6 s.
   2. After the moxibustion of an acupoint, move the transparent acrylic frame to adjust its position so that the next acupoint requiring moxibustion is located at the center of the positioning circle. Repeat steps 4.3.3 and 4.4.1.

5. Torsion scoring operation

1. Inject rats with oxytocin (2 U) and place them in empty cages.
2. Observe the twisting response of each rat within 20 min.
   NOTE: Except for the vehicle group, 6 rats were selected for each group. If a twisting reaction occurs, the primary dysmenorrhea model was successfully established in rats¹⁴. The saline group was injected with equal doses of physiological saline. Behavioral scoring proposed by Schmauss¹⁵was used in this study: For normal exploration behavior, 0 point; The torso is tilted to one side, and the abdomen is inward concave due to contraction, 1 point; Extension of hind limbs, dorsiflexion of hind paws, trunk extension with pelvic rotation, 2 points; Abdominal muscle contraction, hind limb extension, 3 points (Figure 9) The latency, the number of twists, and the twist score of the twist response were all recorded within 20 min. The latency period of rat writhing is the time (min) from oxytocin injection (causing pain) to the first appearance of the writhing reaction in rats.
   Twist score = [0 level (number of times) × 0 points + 1 level (frequency) × 1 point + 2 levels (frequency) × 2 points + 3 levels (frequency) × 3 points].

6. Infrared thermal imaging

1. Anesthetize the remaining 6 rats in each group by intraperitoneal injection of 0.3% pentobarbital 30 mg/kg, exposing the uterine projection area.
2. After the operation, place the rat in the field of view of the infrared thermal imager lens and record the temperature of the uterine projection area using the infrared thermal imager.
3. Measure each acupoint 3 times and take the average value as the temperature of that acupoint.
4. Export infrared thermal images using thermal imaging software (Figure 10).
   NOTE: On the 12^th day of modeling (i.e., the 7^th day of intervention), the remaining 6 rats in each group were subjected to infrared thermal imaging observation. Primary dysmenorrhea rats with cold coagulation syndrome will experience a decrease in the surface projection temperature of the uterus. Observing the surface projection temperature of the uterus in rats can determine the severity of the cold coagulation syndrome. The lower the temperature, the more severe the cold coagulation syndrome.