Experimental animals
Female Sprague-Dawley rats (40 in number) (180 – 220g) about six weeks old were acquired from the Changsha Topgene Biotechnology Co., Ltd; (certification: SCXK (Xiang) 2014—001. The animals had been housed under managed conditions with a 22 ± 2 °C controlled temperature, 50% ± 5% air humidity, 12 h light–12 h dark cycle, and fed routinely with access to tap water ad libitum and allowed to acclimatize for one week before the commencement of the experimental study. The maintenance and treatment of rats was carried out strictly following the Care and Use of Laboratory Animals’ guidelines published by the U.S. National Academies Press (Eighth Edition, update, 2011). The committee approved the experimental protocols on the Ethics of Animal Experiments of the School of Zhuang Medicine, Guangxi University of Chinese Medicine.
Randomization of consecutive rats was done using the slot method, and the rats were divided into four groups: the standard control group (n  = 10), the model group (n = 10), the acupuncture group (n  = 10), and ZMTM group (n = 10). A single operator performed all procedures on the groups’ rats under standard aseptic conditions and protocols to remove bias. The First operator and second operator (readings) were double-blind. And a third operator was used for evaluating the data.
Zhuang medicated thread moxibustion, alcohol lamp, glass slides, normal saline, 1 ml syringe, and cotton swabs were all provided by the School of Zhuang Medicine laboratory, Guangxi University of Chinese Medicine. 4-vinyl cyclohexene dioxide (4-VCD), Rat E2 ELISA KIT (Shanghai Meilian Biotech Co., Ltd.), inverted research microscope (DM18, Leica, Germany), multifunctional microplate reader (M200 TECAN, Switzerland), and high-speed freezing centrifuge (3K15 Sigma, Germany).
The standard control and model groups received no treatment. They were routinely fed and had access to water. However, for rats in the ZMTM group, they received ZMTM. After the thread was ignited and the acupoints were located, the operator used his wrist and fingers to press the thread’s ignited tip on the acupoints swiftly. When the glow was extinguished, the whole operation could be seen as one ”Zhuang .” Typically, each acupoint was acupunctured for one ”Zhuang .” Again, the no.2 thread was selected, which was 70 mm in diameter, and when the ignited thread was held over the acupoints, a 1 - 2 cm tail of the thread should be left intact. Disinfection of acupoints was not necessary before applying ZMTM because of the heat. Typical acupoints include Xiaguanyuan, Qizhousi acupoints, and Beibaacupoints, which are characteristic in Zhuang medicine, mainly for treating miscellaneous gynecological diseases9. Acupoints on rats were located according to Experimental Acupuncture Science10. The ZMTM was applied to acupoints once per day, 2 Zhuang per acupoint, for six courses. Rats in the acupuncture group were treated with 0.28 filiform needles with the same acupoints, and the treatment time was similar to the ZMTM group. Vaginal smears collection time was strictly based on the menstrual cycle of female rats. Typically, one course of treatment lasts for one menstrual cycle.