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.