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\begin{document}

\title{Time-sensitive effects of Zhuang Medicated Thread Moxibustion on
Estrogen Level in female perimenopausal Model Rats.}



\author[1]{Dan Liang}%
\author[1]{Yun Zhang}%
\author[2]{Zhiyong Cao}%
\author[1]{Jingqing Chen}%
\author[2]{Zhuli Hou}%
\author[1]{Yuhan Pan}%
\author[1]{Ke Lu}%
\author[1]{Gang Fang}%
\affil[1]{GuangXi University of Chinese Medicine}%
\affil[2]{Hubei Minzu University}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Abstract Background: To ascertain the estrogenic effect of Zhuang
Medicated Thread Moxibustion (ZMTM) and explore its time-sensitive
impact on estradiol in female perimenopausal rats. Methods: Female rats
were randomized into four groups of 10 rats, each consisting of the
control, model, ZMTM, and acupuncture groups. The perimenopausal
syndrome was induced in the last three groups with a daily subcutaneous
dose of 80 mg/kg of 4- vinylcyclohexene diepoxide for 15 days.
Afterward, rats in the model and control groups were fed routinely,
while rats in the ZMTM and acupuncture groups were treated with six ZMTM
and acupuncture courses, respectively. The rats' general condition and
estradiol (E2) levels in the rats' serum were assessed. Results:
Following the six courses of treatment, the E2 level in the model group
was significantly the lowest, while the regular group was the highest (P
\textless{} 0.05). There was also a gradual increase in the E2 level of
the ZMTM group compared to the model and acupuncture groups, such that
after the 5th and 6th courses of treatment, their E2 level was
significantly higher than the model and acupuncture groups (P
\textless{} 0.05). The control group had a better condition than all
other groups, while the ZMTM group was better than the model and
acupuncture groups. Conclusion: ZMTM can improve perimenopausal induced
rats' estrogen level, and this effect becomes better with each further
treatment course.%
\end{abstract}%



\sloppy


Time-sensitive effects of Zhuang Medicated Thread Moxibustion on
Estrogen Level in female perimenopausal Model Rats.

\textbf{Abstract}

\textbf{Background:} To ascertain the estrogenic effect of Zhuang
Medicated Thread Moxibustion (ZMTM) and explore its time-sensitive
impact on estradiol in female perimenopausal rats.

\textbf{Methods:} Female rats were randomized into four groups of 10
rats, each consisting of the control, model, ZMTM, and acupuncture
groups. The perimenopausal syndrome was induced in the last three groups
with a daily subcutaneous dose of 80 mg/kg of 4- vinylcyclohexene
diepoxide for 15 days. Afterward, rats in the model and control groups
were fed routinely, while rats in the ZMTM and acupuncture groups were
treated with six ZMTM and acupuncture courses, respectively. The rats'
general condition and estradiol (E\textsubscript{2}) levels in the rats'
serum were assessed.

\textbf{Results:} Following the six courses of treatment, the
E\textsubscript{2} level in the model group was significantly the
lowest, while the regular group was the highest (P \textless{} 0.05).
There was also a gradual increase in the E\textsubscript{2} level of the
ZMTM group compared to the model and acupuncture groups, such that after
the 5\textsuperscript{th} and 6\textsuperscript{th} courses of
treatment, their E\textsubscript{2} level was significantly higher than
the model and acupuncture groups (P \textless{} 0.05). The control group
had a better condition than all other groups, while the ZMTM group was
better than the model and acupuncture groups.

\textbf{Conclusion:} ZMTM can improve perimenopausal induced rats'
estrogen level, and this effect becomes better with each further
treatment course.

\textbf{Keywords} : ZMTM, perimenopausal syndrome, estrogen,
time-sensitiveness, Chinese Medicine, Experimental.

What is already known about this topic?

Perimenopausal syndrome (PPS) is also known as climacteric syndrome,
whose main symptoms include endocrine dyscrasia and neurasthenia induced
by the degeneration of ovarian functions\textsuperscript{2}. It is also
known as a perimenopausal disorder in traditional Chinese medicine. The
primary treatment is hormone replacement therapy, which can relieve
climacteric symptoms and negative emotions, but it has side
effects\textsuperscript{4}. Other research shows that long-term hormone
therapy increases breast cancer risk, endometrial cancer, ovarian
cancer, and other related diseases\textsuperscript{4}.

What does this article add?

This study confirms that Zhuang Medicated Thread Moxibustion can improve
perimenopausal model rats' estrogen levels; this curative effect is most
effective at the 3rd cycle and tends to become better in further
treatment cycles. Although the mechanism of action of ZMTM is yet to be
explored, making further human trials necessary, this study has however,
laid a foundation for its clinical research and application.

\textbf{BACKGROUND}

Perimenopausal syndrome (PPS) is also known as climacteric syndrome,
whose main symptoms include endocrine dyscrasia and neurasthenia induced
by the degeneration of ovarian functions\textsuperscript{2}. It is also
known as a perimenopausal disorder in traditional Chinese medicine. As a
disease in women, PPS mainly occurs around the time of menopause.
According to a study by Lijuan \emph{et.a.l} \textsuperscript{3}, the
probability of having PPS in women is the highest (92.10\%) among ages
45 to 55 years, and PPS seriously affects their quality of life and
living standard\textsuperscript{3}. The perimenopausal syndrome is a
disease with high morbidity. It can have severe physical and
psychological effects on perimenopausal women. According to modern
medicine, the diseases' significant causes include the central nervous
system and the autonomic nervous system. The primary treatment is
hormone replacement therapy, which can relieve climacteric symptoms and
negative emotions, but it has side effects\textsuperscript{4}. Other
research shows that long-term hormone therapy increases breast cancer
risk, endometrial cancer, ovarian cancer, and other related
diseases\textsuperscript{4}. Therefore, the exploration of new
treatments is vital for improving the quality of life of women.

Estrogen is of great significance in the regular operation of the female
endocrine system\textsuperscript{5}. Receptors respond to estrogen for
estrogen to exert its effect. It is interesting to note that estrogen's
primary receptor, ER-\selectlanguage{greek}α, \selectlanguage{english}is closely linked to neuron growth, function,
and structure. Contemporary literature shows that estradiol (E2)'s
positive influence on gonadotropin secretion occurs primarily in the
hypothalamus; however, it negatively influences the pituitary
gland\textsuperscript{6}. The leading causes of PPS include low estrogen
levels and degeneration of ovarian function. Therefore, an increase in
estrogen levels provides a foundation for treating PPS.

Zhuang Medicated Thread Moxibustion (ZMTM) is a characteristic surgical
procedure established on the theory of ''\emph{yin-yang} basis'', the
man-nature theory featured by the synchrony of triple initial energy,
and the theory of ''\emph{Sandao lianglu} '' which is concerned with the
tract for the digestion and absorption of food, tract of water, tract of
energy, tract of blood, and sensing tract\textsuperscript{1}.

The procedure for ZMTM entails a \emph{ramie} thread (No. 2 thread with
a diameter of 70 mm), which is first soaked in Chinese herbs and then
ignited and applied on the patient's special acupoints to stimulate
those acupoints through heat and medicine. Therefore, energy can be
regulated after treatment on the body surface (or acupoints) and the
blood and the sensing tract\textsuperscript{1}.

According to the literature, the expression of specific factors can be
regulated, and cell apoptosis improved through
ZMTM\textsuperscript{7,8}. When ovariectomized rabbits are treated with
ZMTM, the interleukin-2 (IL-2) level in their serum can be increased.
The high rates of apoptosis in splenocytes can be improved, their
immunity can be enhanced, and the negative influence on the uterus shape
and ER-\selectlanguage{greek}α \selectlanguage{english}expressions improved\textsuperscript{7,8}. Based on previous
studies' results, this study was designed from the perspective of
time-sensitive effects of ZMTM on the level of estrogen in
perimenopausal model rats. Therefore, the aim is to investigate the
influence of ZMTM on the changes of estrogen in PPS and related
features, which can thus provide a reference and favorable implication
for clinical application.

\textbf{Methods}

This prospective and randomized study was conducted at the Dept. of
Medicine of Hubei Minzu University and Guangxi University of Chinese
Medicine, performed for two years from September 2018 to September 2020.
The Institutional Review Board and the local ethical committee have
approved the study after registration with the Research Center (protocol
Gui~Ke~AD1924518). This study followed the criteria as declared by the
ARRIVE guidelines 2.0.

\textbf{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 \selectlanguage{ngerman}±\selectlanguage{english} 2\selectlanguage{english} degC controlled temperature,
50\%\selectlanguage{english} +-\selectlanguage{english} 5\% air humidity, 12\selectlanguage{english} h light--12\selectlanguage{english} h dark cycle, and fed routinely
with access to tap water \emph{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
(\emph{n} \selectlanguage{english} =\selectlanguage{english} 10), the model group (n\selectlanguage{english} =\selectlanguage{english} 10), the acupuncture group
(\emph{n} \selectlanguage{english} =\selectlanguage{english} 10), and ZMTM group (n\selectlanguage{english} =\selectlanguage{english} 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 E\textsubscript{2} 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 ''\emph{Zhuang}
.'' Typically, each acupoint was acupunctured for one ''\emph{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 \emph{Xiaguanyuan, Qizhousi} acupoints, and
\emph{Beiba}acupoints, which are characteristic in \emph{Zhuang}
medicine, mainly for treating miscellaneous gynecological
diseases\textsuperscript{9}. Acupoints on rats were located according to
Experimental Acupuncture Science\textsuperscript{10}. 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.

\textbf{Model preparation and evaluation}

The model, acupuncture, and ZMTM groups received a daily subcutaneous
injection of 4-VCD at 80 mg/kg for 15 consecutive days. The 200 g rats
were given a volume of 0.2 ml (80 mg/ml), i.e., 0.001 ml/g, for 15
consecutive days to induce the perimenopausal model. After five days of
treatment, rat vaginal smears were collected from each group for five
consecutive days. The estrous cycle of rats was confirmed by microscopic
evaluation of cells in vaginal smears. In all three groups, besides the
standard control group, rat vaginal smears showed estrous symptoms,
i.e., a significant amount of leukocytes with some epithelial cells and
keratinocytes were present, which implies that the perimenopausal model
was induced successfully. No mortality was seen during the induction of
the perimenopausal phase.

\textbf{Sample collection}

2 ml venous blood from each rat's eye socket was collected in the 1st,
3rd, 5th, and 6th courses of treatment and was kept in estrogen receptor
tubes at 4degC. When blood coagulation occurred, the tubes were placed in
a centrifuge. After centrifugation, the supernatant was collected and
kept in a freezer at -20degC. Rat vaginal smears were collected
simultaneously for H \& E staining, which was done in Guangxi National
Hospital and observed by the microscope. All rats were sacrificed by
cervical dislocation under anesthesia.

\textbf{Measurement of serum Estradiol (E\textsubscript{2})}

ELISA kit was used, and the standard operation was completed according
to instructions.

\textbf{Statistical analysis}

SPSS 19.0 was the software used for analyzing results. Data calculated
as mean +- S.D.; standard deviation ( +- s). Intragroup data were compared
using a one-factor analysis of variance (ANOVA). The differences are
statistically significant when the p-value is less than 0.05.

\textbf{RESULTS}

H \& E staining of rat vaginal smears from each group is shown in Figure
1

\textbf{Comparing the general condition of rats in each group after six
courses of treatment}

: The mental state, disposition, feed intake, reaction, and general
well-being of all rats among all groups were examined at the end of the
treatment course. The standard control rats were generally stable,
mentally agile, and had a regular feed intake and disposition. On the
contrary, the model group rats were restless, with slow reaction,
inadequate feed intake, and physically weak. However, the treatment
group, i.e., ZMTM, showed the improved (p \textless{} 0.05) mental
status of the rats and feed intake. Although the rats' disposition was
docile, there was a general improvement in the condition of the rats.
The results obtained for the ZMTM group appear similar to those of the
acupuncture group. Table 1

\textbf{The effect of ZMTM on E\textsubscript{2} of perimenopausal model
rats}

The results of E 2 levels are shown in Figures 2 and 3. As seen from
Figure 2, the standard control group had the highest estradiol level
from cycle one all through to process 6. However, the model group had
significantly (p \textless{} 0.05) lower E\textsubscript{2} values,
which was the lowest among all groups, and this was maintained low from
cycle one all through to cycle six. It was interesting to observe that
as treatment with ZMTM commenced from cycle 1, a significant increase (p
\textless{} 0.05) in E 2 levels was noted. This increase was steadily
improved significantly (p \textless{} 0.05) from cycle one through to
cycle six, with the increase in E2 levels being highest on the
6\textsuperscript{th} cycle. The effect of ZMTM on estrogen levels was
similar to the acupuncture group on the 3\textsuperscript{rd} cycle but
significantly higher (p \textless{} 0.05) on the
5\textsuperscript{th}and 6\textsuperscript{th} cycles.

Figure 3 gives an insight into the E\textsubscript{2} level in the sixth
cycle. It can be seen here that the ZMTM treatment significantly (p
\textless{} 0.05) improved the level of E2 compared to the
perimenopausal model group and the E2 value seen for the ZMTM group was
comparable to the standard control group and better than the acupuncture
group. This leads to the reversal of the perimenopausal effect as
induced by 4-VCD.

\textbf{DISCUSSION}

A perimenopausal syndrome is a common female disease typified by
neurasthenia and neuropsychological symptoms, mainly caused by an
alteration in the hormone levels around menopause\textsuperscript{11}.
According to traditional Chinese medicine, PPS falls into the category
of hysteria and perimenopausal disorder. The leading causes include
deficiency of kidney qi, stagnation of liver qi, internal disturbance of
seven emotions, and stress, injuring qi and blood. There are many
therapeutic approaches to handling PPS. According to research, standard
acupuncture and electro-acupuncture can relieve the symptoms of
menopause\textsuperscript{12}. According to Zhuang medicine, ''heiq''
(qi) and ''\emph{lwed} '' (blood) play an essential role in female
growth and development. When women are in perimenopause, it means the
degeneration of their physical quality and body function. The decline in
''heiq'' (qi) and ''lwed'' (blood) will cause the disorder of qi and
blood and finally lead to PPS. As a traditional external therapy, ZMTM
can regulate qi and blood through acupoints to balance qi and blood,
relieve symptoms, improve body health, and finally show good therapeutic
efficacy\textsuperscript{9}. Essential therapy tools include medicated
thread made from many Zhuang herbs, e.g., \emph{Santalum album,} musk,
frankincense, and \emph{Argyreia acuta} . It has a delicate smell and
can achieve good therapeutic effects\textsuperscript{13}. Qizhousi
acupoints, Beiba acupoints, and other selected acupoints are
characteristic acupoints in Zhuang medicine, mainly treating various
gynecological diseases.

Perimenopausal syndrome correlates with changes in sex hormones.
Therefore, the difference in sex hormones should be given close
attention during treatment by the treating physicians. Furthermore, the
physicians should examine the levels of luteinizing hormone (L.H.),
stimulating follicle hormone (FSH), estradiol (E2), and other hormones
to assist in the diagnosis and treatment of PPS\textsuperscript{14}.
This study indicates that rat estrogen levels decreased after
perimenopause model induction and improved after ZMTM treatment. This
effect of ZMTM was significant right from the 1st to the
6\textsuperscript{th} cycle. However, its influence became notable from
the 3\textsuperscript{rd} cycle and was most meaningful and useful
between the 5th and 6th cycles. E\textsubscript{2} levels in the ZMTM
group were significantly higher (p \textless{} 0.05) than the
acupuncture group and model group, although slightly lower than the
standard group.

\textbf{Conclusion}

This study confirms that ZMTM can improve perimenopausal model rats'
estrogen levels; this curative effect is most effective at the 3rd cycle
and tends to become better in further treatment cycles. Although the
mechanism of action of ZMTM is yet to be explored, making further human
trials necessary, this study has however, laid a foundation for its
clinical research and application.

\textbf{Abbreviations}

ZMTM: Zhuang Medicated Threads; PPS: Perimenopausal Syndrome; E:
Estradiaol; IL-2: Interleukin 2.

\textbf{Ethics approval and consent to participate:} All animal
experiments were authorized by the Guangxi Zhuang Yao Medicine Center of
Engineering and Technology,~Guangxi University of Chinese Medicine and
Hubei Minzu University Animal Ethics Committee and carried out in
compliance with the institutional guidelines.

\textbf{Consent to publish} : Not Applicable

\textbf{Availability of data and materials} : The datasets used and/or
analyzed during the current study are available from the corresponding
author on reasonable request.

\textbf{Competing interests} : The authors declare that they have no
competing interests

\textbf{Funding} : This work was sponsored by the National Natural
Science Foundation of China (No.81660830)
\selectlanguage{english};Guangxi~Program~on~Science~and~Technology~Project:~Gui~Ke~AD1924518\selectlanguage{english};

Development Program of High-level Talent Team under Qihuang Project of
Guangxi University of Chinese Medicine\selectlanguage{english}(No.2018005\selectlanguage{english})\selectlanguage{english};Guangxi~first
class~discipline~construction~project~(No.~Gui~Jiao~Ke~Yan~{[}2018{]}12).
The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of manuscript.

\textbf{Authors' Contributions} : K.L. is mainly responsible for the
design and operation of the whole experiment. Y.Z., Z.C., and J.C.
primarily assisted in the process of the investigation. Z.H. and Y.P.
wrote and revised the manuscript. DL and G.F. provided the overall idea
for the experiment and revised the paper in the later stage. All authors
have read and approved the manuscript before submission.

\textbf{Acknowledgements} : Not Applicable

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\textbf{Figure Legends:~}

\textbf{Figure 1:} H \& E staining of rat vaginal smears from each group
(100x) and Vaginal smears of rats in estrous cycle.

A: Pre estrus: a great number of nucleated epithelial cells and some
cornified epithelial cells are present.

B: Estrus period: all are cornified cells without cell nucleus, with
some epithelial cells present.

C: Late estrus: mostly cornified cells and leukocytes are present.

D: Estrus interval: most leukocytes and some mucosa and epithelial cells
are present.

\textbf{Figure 2:} Time-sensitiveness of E\textsubscript{2} in rats
after perimenopausal model induction

\textbf{Figure 3:} Comparison of E\textsubscript{2} levels in rats after
perimenopausal model induction on the sixth cycle\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2-1/Figure-2-1}

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