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Acupuncture Normalizes Dysfunction of Hypothalamic-Pituitary-Ovarian
Axis
By Bo-Ying Chen M.D. Professor of Neurobiology. Institute of Acupuncture
and Department of Neurobiology, Shanghai Medical University, Shanghai
200032, P.R. China
(Received June 3, 1997; Accepted with revisions June 30,1997)
ABSTRACT
This article summarizes the studies of the mechanism of electroacupuncture
(EA) in the regulation of the abnormal function of hypothalamic
pituitary-ovarian axis (HPOA) in our laboratory. Clinical observation
showed that EA with the effective acupoints could cure some anovulatory
patients in a highly effective rate and the experimental results
suggested that EA might regulate the dysfunction of HPOA in several
ways, which rneans EA could influence some gene expression of
brain, thereby, normalizing secretion of some hormones, such
as GnRH, LH and E2. The effects of EA might possess a relative
specificity on acupoints.
KEY WORDS: Electroacupuncture, ß-Endorphin,
GnRH, LH, Estradiol, Estrogen receptor, Ovariectomized rat, Hypothalamic-pituitary-ovarian
axis
INTRODUCTON
Acupuncture is a treasure of Chinese
traditional medicine, which is employed in the treatment of different
diseases, especially in relief of all kinds of pain [1, 2] over
the world. Since 1960s we have used acupuncture with appropriate
electro-stimulation to cure patients with anovulation disorder
(sterility), the rate of EA induction of ovulation was increased
from 50% initially to 80% presently. Other authors in China also
reported that acupuncture was successfully to treat patients with
sterility [3] and the lying-in woman with subnormal contraction
of uterus [4]. All the above research demonstrates that acupuncture
may be an effective curative method of some woman's diseases. However,
many questions, such as "why", "how
to" and "which" about the mechanism of EA effect
are unknown. To address these problems we supposed that EA might
influence the production and secretion of hormones, neurotransmitters
or neuro-modulators of HPOA leading to the normalization of hormone
status. We also noticed certain artides reported that EA might
affect the blood levels of LH, FSH, estradiol (E2) and prolactin
in the female patients [4, 5, 6] and EA may be related to long
term changes in gene expression [7, 8]. These results are all significant,
yet insufficient to explain the mechanism of EA in the regulation
of the function of HPOA. To obtain more data, a series of experimental
studies in human and animal models has been performed in our laboratory.
MATERIALS AND METHODS
Selection and treatment of cases: Ten cases of chronically
anovulatatory patients including eight cases of polycystic ovarian
disease (POCA), one case of hypogonadotropic amenorrhoea and one
case of oligomenorrhea were treated with EA in 13 menstruation
cycles. They were all of productive age and the courses of disease
were 3 to 12 years. On the 10th day of each menstruation cycle,
the patients accepted the EA treatment. "Guanyuan(RN4)," "Zhongji(RN3)," "Sanyinjiao(SP6)," and
bilateral "Zigong(EXCA1)" points were stimulated for
30 min at 8:00 AM, Q.D. for 3 days. The stimulation parameters
were 7-8mA and 4-5 Hz with G6805 model generator. The electric
current of EA was bearable well for every patient. The blood
samples were collected from forearm of the patients one time
per 15 min for detection of FSH.LH and ß-endorphin (ß-E).
Five health volunteers of a productive age with normal menstruation
cycle were selected as controls, which were undergone the same
treatment as above mentioned.
Animals and treatments: Wistar female
rats weighting 200-250g were used. The half of animals were undergone
ovariectomy and fed in the same environment with the intact rats
at least for 15 days and vaginal smears were examined per day for
3 times. No exfoliative epithelium cell was found in the smears
as an index for successfill ovariectomy. The ovariectomized rats
and intact rats were randomly divided into two groups respectively:
ovariectomized rat group (OVX), ovariectomized rat accepted EA
treatment group (OVX+EA), intact rat group (INT) and intact rat
accepted EA treatment group (INT+EA). The animals in OVX+EA and
INT+EA received EA at the experimental acupoints of Guanyuan (RN4),
Zhongji (RN3), Sanyinjiao (SP6) and bilateral Zigong (EXCA1) by
EA apparatus (Model G6805-2, SMIF, Shanghai, China) with the frequency
of 3 Hz and an intensity to produce a slight twitch of the limbs.
After 3 days' treatment animals were given EA at Waiguan (SJ5)
and Huatuojiaji (EXTRA21) as the control acupoints in the same
way (Fig 1). By the end of last experiment, animals were sacrificed
and their adrenals, brains and pituitaries were taken out for detection
of nucleolar oganizer regions (AgNORs) and hormones.
Pushpull perfusion in hypothalamic preoptic
area (POA) and elution of pituitary and LH and ß-endorphin (ß-EP)
The technique of brain pushpull perfusion was processed as previously
described by our laboratory [1]. The perfusate from hypothalamic
POA was kept at -70°C for GnRX and ß-EP RIA.
The pituitaries were retrieved and put into
4°C cooled saline.
Afterward, each pituitary was homogenized with 500µl of 70%
acetone aqueous solution at 4°C. The homogenate was centrifugalized
(2,000xg for 15 min at 4°C) and the supernatant was freeze-dried
for LH and ß-EP RIA.
Radioimmunoassay (RIA) of hormones
GnRH IRA: GnRH content in the perfusate from rat hypothalamus
was determined by RIA method developed by Nett in 1973 [9]. GnRH
was iodinated by the modified chlomine-T technique[10]. Na125 I
was manufactured by Radiochemical Center, Amersham.
ß-EP RIA: The sensitive radioimmunoassay was a routine in
our laboratory [1]. The standards of human and rat ß-EP was
synthesized by Peninsula Laboratories, Inc. and the rabbit antiserum
of both ß-EP was developed in our laboratory. The cross-reaction
from human ß-EP and camel ß-EP was detected about 20%.
The sensitivity of this method was 10pg/tube.
LH, E2 and corticosterone RIA: LH, E2 and corticosterone RIA kits
were bought from Shanghai Institute of Biologic Products, the Ministry
of Health, P.R. China. All procedures of RIA were performed as
described in the kit manuals.
Fig. 1
A: Sketch of ventral view (left) and dorsal view (right) of rat
shows the acupoints we used
B: Diagram shows the electroacupuncture procedures in conscious
rat
Staining techniques: Vaginal smears were fixed
by 100% ethyl alcohol, then stained with HE method. Adrenal sections
were cut in 4µm
thickness from paraffin blocks and processed with silver nitrate
staining technique[11]. In each case, one hundred cells in zona
fascicula were examined randomly under 100-fold oil immersion lens.
Numbers and sizes of AgNOR dots were counted and measured.
C-fos protein immunohistochemistry: The inmunohistochemical analysis
of c-fos expression in rat brain was perforrned as previously described[11].
Estrogen receptor (ER) protein immunohistochemistry
(ABC method): Under sodium pentobarbital anesthesia (50 mg/kg,
ip), the animals were perfused via left cardiac ventricle with
100ml of phosphate-buffered saline (PBS), followed by 300ml ice-cold
fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer
(pH7.4). Afterwards, brain was removed with the same fixative
for one day and immersed in 0. lM phosphate buffer containing
30% sucrose for another day. The hypothalamus blocks were frozen
with dry ice and cut into 35 µM
thick section by cryostat. The brain sections were washed with
0.01M PBS for 15min x 3 and incubated in 0.01M PBS containing 0.5%
Triton 100 and 3% normal goat serum (NGS) at 37°C-for one hour.
Afterwards, the sections incubated in 1:1,000 ER monoclonal antibody
(H222, Abott Co.) at 37°C for one hour, then at 4°C for
two days. The sections, washed in PBS three times, were processed
by ABC kit (from Vecot Labs) induding sequential incubation at
20°C in the following solutions with washes between them. (1).
second antibody (dilution 1:100), 30min. (2). A+B reagents (dilutionl:100),
60min. (3). 0.05% diaminobenzidine/ 0.02% hydrogen peroxide in
0.1M Tris- HCI buffer (pH 7.2) 10min. The sections were washed
in tap water, mounted and examined under light microscope. The
certain areas of typical immunoreactive positive neurons were measured
by computer image analysis system (Vecta PC).
ER mRNA hybridization: The total mRNA of brain was eluted by the
modified phenol method [12]. ER cDNA probe (244bp) was labeled
by the DlG-labeling kit (from Bohringman Co., Germany). The dot
blot hybridization was processed as the method described by Sambrook
J and his colleagues [13]. The dot blot images were analyzed with
gray density by computer imaging analysis software (TJTY-300, from
Tong -Ji university, Shanghai, China).
Statistics: All data in this paper were treated with analysis
of variation (ANOVA), least significant difference (ISD) or student
T-test
RESULTS
Effect of EA on ovulatary induction and curing sterility in woman
After
EA the blood ß-EP level of the patients
resulting in ovulation either declined or maintain at the levels
within the range of the normal levels and the ß-EP levels
of those failing to show ovulation were significantly higher than
the normal's' (table 1). On the other hand, the blood LH and FSH
levels of the patients with ovulation after EA treatment tended
to be the normal [14].
Table 1. Change of blood ß-EP level before
and after EA (pg/ml)
Effect of EA on dysfunction of HPOA in ovariectomized
rats: For a further study of the mechanism of EA effect
on HPOA a series of experiments in the animal models was performed.
(1). EA induces maturation and exfoliation of vaginal epithelium
cell and enhances blood level of E2.
After ovariectomy two weeks late, the exfoliated epithelium cell
disappeared from the vaginal smears of the rats, but it reappeared
in the smears following EA treatment. The blood level of E2 in
OVX was increased significantly (table 2). No obvious change was
seen in INT after EA treatment and in OVX following EA treatment
with the control acupoints.
Table 2. The level of blood E2 following EA treatment
(pg/ml)
(2). EA promotes enlargement of adrenals and enhances
activity of adrenal AgNORs as well as blood level of corticosterone
We found the adrenals of OVX+EA were enlarged and the weight of the adrenals
was raised significantly. Using histochemical method, the AgNORs of the cells
in inner adrenal cortex were examined. The result shows that the activity of
AgNORs of OVX was enhanced (table 3, 4), and the level of blood corticosterone
in OVX+EA was also increased (table 5). There were no similar effects in INT
following EA treatment and in OVX after EA with control acupoints.
Table 3. AgNORs number in OVX and INT
Table 4. Weight of adrenal
Table 5. The levels of blood corticosterone in OVX
and lNT (mean ± SE, ng/ml)
(3). EA decreases the level of hypothalamic
GnRH, pituitary LH and increases the contents of hypothalamic
and pituitary ß-endorphin
After
EA treatment the levels of GnRH released from hypothalamus was
rnarkedly decreased however, the ß-endorphin (ß-EP)
secretion in hypothalamus was raised. The pituitary content of
LH was also fallen, but the ß-EP of
pituitary was increased, as well as peripheral LH and ß-EP level (Fig.2).
Fig. 2: Change of hypothalarnic GnRH
and ß-EP, pituitary LH and ß-EP,
blood LH and ß-EP before and after EA
Effect of EA on brain c-fos expression in ovariectomized
rats: The area occupied by FOS protein labeled neuron
was detected in medial preoptic nucleus (MPN), lateral preoptic
nucleus (LPN), suprachiasmatic nucleus (SCN), paraventricular nucleus
of the hypothalamus (PAVN), medial amygdala nucleus (MAN), periventricular
nucleus of the hypothaLsmus (PVN), ventromedial nucleus of the
hypothalamus (VNH) and arcuate nucleus (AR) 4 hours after ovariectomy
(fig. 3a). The C-fos immunoreactive labeled neurons disappeared
two weeks later following ovariectomy. The rats recovering for
more than two weeks after ovariectomy, were received EA treatment.
Many specific FOS labeled cells were observed in LPN, VNH, SCN
and especially in POA, ARN, and PVN, but not any labeled neuron
could be found in MAN. No obvious C-fos expression was shown in
those nuclei in INT and INT+EA (fig. 3b).
Fig. 3a: C-fos immunocytochemistry neurons distribution after ovariectomy
Fig. 3b: C-fos expression labeled neurons following
electroacupuncture
Effect of EA on expression of ER protein and
ER mRNA in rat brain: Estrogen receptor (ER) immunoreactive neurons were
observed widely in rat brain with immunohistochemical technique,
especially in MPN, ARN and VNH. The above nuclei were measured
by computer image analysis system, and the results show that the
mean gray density in OVX+EA was decreased apparently compared with
that in OVX. Whereas there were no obvious changes of gray density
levels in INT and INT+EA (fig, 4).
Fig. 4: Effect of EA on expression of ER protein in rat brain (Immunohistochernistry
of monoclonal antibody) *p < 0.01 compared with OVX
The dot blot indicated that ER mRNA expression
was increased about 48.11% in OVX compared with INT. The gray
density of OVX was 129.75 ± l2.l3 and that in OVX+EA was 199.25 ± 5.75
attenuated significantly (Fig. 5). The gray density level in INT
was 87.60 ± 5.91, and the level in INT+EA was 83.60 ± 4.83.
There was no significant difference between INT and INT+EA
Fig. 5: Effect of EA on expression of ER mRNA in rat brain (dot blot)
*** p < 0.01 compared with OVX
DISCUSSION
Since 1985 we have observed that the effect
of EA ovulatary induction might relate to the hand skin temperature
(HST) and the blood level of ß-EP [14]. On the other hand,
after EA the blood FSH and LH levels of the patients who successfully
ovulated either declined or maintained at normal. In general,
provided that body temperature was normal and the environmental
temperature was constant round 25°C, the HST may reflect
the state of sympathetic system of a patient. These results suggest
that in anovulatary cases the hyperactive sympathetic system
can be depressed by EA and the function of HPOA can be regulated
by EA through central sympathetic system. Moreover, EA may mediate
the abnormal function via the influence on the secretion of the
hormones in the different Level of HPOA.
To gain more evidences, we designed some animal experiments to
explain the mechanism of EA effects on HPOA at the whole, cellular
and molecular levels. We found that EA can induce maturation and
exfoliation of vaginal epithelium cell in OVX rat. It is known
that maturation and exfoliation of vaginal epithelium cells are
a reaction dependent on estrogen level. So we determined the level
of blood E2 in OVX and OVX+EA. The result shows the level of blood
E2 in OVX was lower than that in normal, but it was increased significantly
after OVX accepted EA treatment with the experimental acupoints.
This result suggests EA might promote the activity of the compensative
mechanism to elevate the subnormal level of E2 induced by ovariectomy
in rats.
What is this compensative mechanism? To resolve this question,
we considered that adrenal is the main organ to secrete sexual
hormones except ovarian in females and observed the adrenals of
the animals in four groups. The results show that the mean weight
of the adrenal in OVX+EA was higher than that in OVX, INT and INT+EA,
suggesting the adrenal function might be activated by EA. Subsequently,
we detected that the number of AgNORs in zona fasciculata of OVX+EA
was significantly increased. Nucleolar organizer regions (NORs)
are loops of DNA, which possess ribosomal RNA (rRNA) genes. They
are of vital significance in the ultimate synthesis of protein.
Thus, the number and configuration of AgNORs (NORs stained by silver
staining method) may reflect the activity of cell differentiation
and transcription of nucleolar rDNA [15]. In the same time we found
the content of blood corticosterone in OVX+EA was raised markedly,
but there was no change of blood corticosterone in OVX, INT and
INT+EA. This result provided a further evidence that the adrenal
cortex cells were initiated in OVX+EA.
The results including the changes of GnRH releasing
from hypothalamus and of the pituitary and blood LH contents
suggest that the effects of acupuncture in the regulation of
HPOA may be exerted via to promote the function of hypothalamic
pituitary-adrenal axis (HPAA), increasing the synthesis and secretion
of adrenal steroid horrnones, the androgen of which then be transformed
into estrogen in other tissues and thereby reset the negative
feedback of estrogen to HPOA. Moreover, EA may accelerate the
release of brain and pituitary ß-EP
to inhibit the overnormal secretion of GnRH and LH that may be
normalized.
Recently immunohistochemical analysis of the
expression of oncogene c-fos ABl was induced by variety of stimuli
[16, 17]. This represents a new method for mapping neuronal activity
at the cellular level [18] and thus functionally and systematically
tracing neuronal pathway in the nervous system (C NS) [19]. We
used this method to examine the distribution of FOS labeled neuron
in CNS for recovery of more evidences that EA may alter the neuroendocrine
function of HPOA in ovariectomized rats in cellular and gene
level. The results show that the specific FOS labeled neurons
were observed especially in POA, ARN and PVN in OVX following
EA treatment. In above nuclei there were a high concentration
of GnRH and ß-EP
neuron [20]. These results suggest this fact that the expression
of FOS labeled neurons reappeared in above mentioned areas following
EA treatment in ovariectomized rats may be related to the changes
of GnRH and ß-EP from rat hypothalamus after EA treatment.
The level of estrogen in the body may regulate the expression
of ER, which may by down-regulated following increase of estrogen
level and up-regulated after decrease of estrogen [22]. Our finding
that after decline of blood E2 induced by ovariectomy the expression
of ER was increased and the expression of ER was inhibited by EA
inducing the elevation of blood E2 are in accordance with these
reported results. ER existing in the brain, especially in POA,
ARN and VHN may mediate the function of neuroendocrine system [22,
23]. Thus, our observations suggest that the influence of EA on
the change of ER expression in brain may be one of further mechanisms
of EA normalizing the dysfunction of HPOA.
INT rats as experimental control we adopted were all of in the
stage of preestrus and estrus because the animal sexual hormes
and brain ER expressions were changed with the sexual cycle [24].
All INT rats were selected to fix in the two stages there may be
a relative constant comparability.
Our results show no same effects were seen after EA treatment
in INT and following EA with control acupoints in OVX, suggesting
that EA may possess a relative specificity on acupoint and the
effect of EA may be a kind of normalization.
CONCLUSION
Our observations reveal that acupuncture
may regulate the abnormal function of HPOA in many ways, which
means that acupuncture may activate C-fos expression of brain,
then a long term changes at molecular level would start, following
the regulation of gene expression in FOS relative gene, such as
ER mRNA and GnRH mRNA involved. On the other hand, EA may promote
the activity of the body compensative mechanisms, then the levels
of hormones, such as GnRH, LH, estrogen and so on would be normalized.
The effect of acupuncture on regulating the function of HPOA may
possess a relative specificity of acupoint. Moreover, our clinical
and animal experimental results suggest that it is necessary for
obtaining a satisfactory effect that proper stimulation should
be about thirty minutes Q.D. for three days. This suggestion provides
a successful consideration for clinical practice in curing the
woman patients with dysfunction of sexual endocrine, such as primary
ovarian dysfunction, climacteric syndrom, after-ovariectomy and
polycystic ovarian disease etc.
ACKNOWLEDGMENT
The work was supported by National Natural
Foundation of China (3880910 and 392708340) and a grant from the
State Key Laboratory of Medical Neurobiology of China (92003).
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