Neuroscience Letters 319 (2002) 45–48
www.elsevier.com/locate/neulet
Antipyretic effects of acupuncture on the lipopolysaccharideinduced fever and expression of interleukin-6 and interleukin-1b
mRNAs in the hypothalamus of rats
Yang-Sun Son a, Hi-Joon Park a, Oh-Bin Kwon b, Sung-Cherl Jung b,
Hyung-Cheul Shin b, Sabina Lim a,*
a
Department of Meridianology and Acupoint, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-gu,
Seoul 130-701, South Korea
b
Department of Physiology, College of Medicine, Hallym University, 1 Ockcheon-dong, Chunchon, Kangwon-do 200-702, South Korea
Received 25 October 2001; received in revised form 15 November 2001; accepted 4 December 2001
Abstract
We observed the changes of body temperature and the cytokine expressions in the hypothalamus of rats to investigate
the effect and mechanism of antipyretic action of acupuncture. Lipopolysaccharide (LPS, i.p., 2.5 mg/kg) was injected
into rats and manual acupuncture was performed on Shaofu (HT8), Zutonggu (BL66) or Xingjian (LR2), respectively. The
results showed that fever induced by LPS-injection was recovered significantly by acupuncture on each acupoint. LPS
increased hypothalamic mRNA levels of interleukin-6 and interleukin-1b which, on the contrary, were also reduced to
normal levels by acupuncture stimulation on BL66. These results suggest that the acupuncture stimulation may be
effective for reducing elevated body temperature induced by bacterial inflammation, and part of its action may be
mediated through the suppression of hypothalamic production of pro-inflammatory cytokines. q 2002 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Acupuncture; Lipopolysaccharide; Thermoregulation; Hypothalamus; Interleukin-6; Interleukin-1b
Acupuncture has long been used as various therapeutic
tools in Oriental medicine and researches on acupuncture
are vigorously pursued in both eastern and western medicine
fields. There have been a few reports regarding thermoregulatory effects of acupuncture. Lin et al. [13] reported that
acupuncture on Neiguan (PC6) or Zusanli (ST36) induced
hyperthermia, whereas acupuncture on Dazhui (GV14)
produced hypothermia in normal adult. Acupuncture on
Quchi (LI11) or Hegu (LI4) has also been acknowledged
to produce hypothermia in normal subject [12]. However,
the mechanism of this thermoregulatory action of acupuncture has not been fully understood.
Our present study was designed to investigate the possible involvement of pro-inflammatory cytokines – interleukin-6 (IL-6) and interleukin-1b (IL-1b) – in this
mechanism, which have been known to act as potential
mediators of endotoxin-induced fever in the central and
peripheral organs.
* Corresponding author. Tel.: 182-2-961-0338; fax: 182-2-9617831.
E-mail address: lims@khu.ac.kr (S. Lim).
Adult male Sprague–Dawley rats (200–250 g body
weight) were housed in light-controlled (lights on from
07:00 to 19:00 h) and air-conditioned (25 ^ 28C) animal
rooms with rat chow and water ad libitum provision. The
rats were subsequently injected with lipopolysaccharide
(LPS, i.p., 2.5 mg/kg), widely used endotoxin inducing
fever.
Stainless needles (0.25 mm diameter) were inserted bilaterally into each acupoint 5 mm in depth and were twisted at
the speed of once a second for 10 s. Three acupoints, anatomically corresponding to the acupoints in human, were used
in this experiment. Shaofu (HT8) is located between the 4th
and the 5th metacarpal bones in the palm of forepaw.
Zutonggu (BL66) is located in the depression anterior to
the 5th metatarsophalangeal joint of hindpaw and Xingjian
(LR2) is located between the 1st and the 2nd toe on the
dorsal surface of hindpaw. These acupoints were selected
as they are traditionally used for the treatment of feverrelated diseases. Non-acupoint was also selected on the
part of proximal tail for the control treatment to the acupoint
groups. During the acupuncture treatment, rats were lightly
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S03 04 - 394 0( 0 1) 02 53 8- 1
Y.S. Son et al. / Neuroscience Letters 319 (2002) 45–48
46
Table 1
Sequences of primers, fragment sizes and annealing temperatures a
cDNA
IL-6
IL-1b
GAPDH
a
Sequence of primers
S
A
S
A
S
A
0
0
5 -GACTGATGTTGTTGACAGCCACTGC-3
5 0 -TAGCCACTCCTTCTGTGACTCTAACT-3 0
5 0 -TTCTTTTCCTTCATCTTTGAAGAAG-3 0
5 0 -TCCATCTTCTTCTTTGGGTATTGTT-3 0
5 0 -TCCCTCAAGATTGTCAGCAA-3 0
5 0 -AGATCCACAACGGATACATT-3 0
Fragment size (bp)
Annealing temperature (8C)
Amplification cycle
509
55
35
362
58
38
309
55
21
S: sense; and A: anti-sense.
immobilized using hands to minimize stress. The LPSinjected control group was also lightly immobilized with
the same method for 10 s. The acupuncture treatment was
performed at 20.5 h after LPS injection, at the time when the
fever was maintained constantly.
Rectal temperature was monitored using a rat rectal probe
attached to a digital thermometer (TD-300, Japan).
Temperature measurements were recorded before the
experiment, right after acupuncture treatment and every
10 min, thereafter, for the next 30 min.
Animals were sacrificed and their brains were removed at
the end of the observation period. The hypothalamus was
isolated as quickly as possible and was immediately homogenized in 0.5 ml of pre-cooled Trizol reagent (Gibco BRL).
The total RNA was extracted according to the manufacturer’s instruction and then subjected to electrophoresis in
1% agarose gel for the confirmation of the structural integrity. The absorbance at 260 nm was measured for estimation
of the RNA concentration. Three mg of total RNA was
reverse transcribed (RT) with oligo-dT primers by using
MMLV reverse transcriptase (Promega) in a total volume
of 12 ml. PCRs were performed in 50 ml reaction volume
containing 10 mM dNTP 0.8 ml, 2.5 unit Taq polymerase
(Promega), 10 £ buffer 5 ml, 20 pmol of primer pairs and 2
ml cDNA. The amplification was subsequently performed in
a thermo-cycler (Corebio system, Germany), beginning
with a 5 min pre-incubation at 948C, followed by optimal
cycles of 45 s at 948C, 60 s at optimal annealing temperature
and 80 s at 728C, ending with a 5 min incubation at 728C
(Table 1). The amplified products were electrophoresed on
1.5% agarose gel, visualized with ethidium bromide for
fragment size estimation. The images were scanned and
analyzed by gel analysis software (BIO-RAD) for quantification of band intensities.
All values of RT-PCR data were normalized to GAPDH
which served as the internal control, to correct subtle
sample-to-sample differences in the amounts of starting
material. Differences between groups were analyzed using
one way analysis of variance (ANOVA) followed by posthoc Dunnett test for multiple comparisons, if needed. P ,
0:05 was considered as the level of significance.
Rectal temperature (8C; mean ^ SEM) in normal animals
were 38.13 ^ 0.068C, and LPS injection produced a significant increase in the core body temperature at 20.5 h after
LPS (38.91 ^ 0.108C) and kept it elevated until the end of
the observation period. Acupuncture stimulation on each
acupoint, with LPS injection, significantly reduced rectal
temperature. Changes of rectal temperature in each
acupuncture group on HT8, BL66 and LR2 were
0.42 ^ 0.13, 0.58 ^ 0.19 and 0.32 ^ 0.138C at 30 min
post-acupuncture, respectively. Most effective acupoint
was BL66, which induced significant suppression of rectal
temperature after 10, 20 and 30 min of acupoint stimulation.
Acupuncture treatment at proximal tail was proved to have
no effect on LPS-induced fever (Table 2).
RT-PCR analysis of mRNA purified from the hypothalamus after 35 cycles showed that IL-6 transcripts were
detected at the positions corresponding to the predicted
size. The levels of IL-6 mRNA were increased in the
hypothalamus after 20.5 h post-LPS injection, and they
were returned to normal state after 30 min post-acupuncture
treatment (only on BL66) (Fig. 1). IL-1b mRNA levels in
Table 2
Changes of body temperature in LPS-injected rats after acupuncture stimulation (the data represents mean ^ SEM, n ¼ 6) a
Group
LPS
LPS 1 HT8*
LPS 1 BL66*
LPS 1 LR2*
LPS 1 non-acupoint
a
Changes of body temperature (8C)
Right after
After 10 min
After 20 min
After 30 min
2 0.10 ^ 0.20
2 0.05 ^ 0.13
2 0.50 ^ 0.11
2 0.10 ^ 0.17
0.02 ^ 0.06
0.12 ^ 0.20
2 0.18 ^ 0.10
2 0.50 ^ 0.15**
2 0.25 ^ 0.12
0.10 ^ 0.09
0.32 ^ 0.20
2 0.33 ^ 0.10**
2 0.55 ^ 0.18***
2 0.28 ^ 0.16**
2 0.02 ^ 0.10
0.20 ^ 0.24
2 0.42 ^ 0.13
2 0.58 ^ 0.19**
2 0.32 ^ 0.13
2 0.28 ^ 0.17
*BL66, HT8 and LR2 represent the name of acupoints. **Means P , 0.05 and ***means P , 0.01 versus LPS group at the same time
point (ANOVA followed by post-hoc Dunnett test).
Y.S. Son et al. / Neuroscience Letters 319 (2002) 45–48
Fig. 1. The interleukin-6 (IL-6) mRNA levels of hypothalamus
after 21 h LPS (2.5 mg/kg, i.p.) injection. (N) normal group without LPS; (C) control group with LPS; (A) acupuncture group on
BL66 with LPS; (NA) non-acupoint group with LPS. IL-6 mRNA
levels increased after LPS-injection, and those were suppressed
and returned to normal states by acupuncture stimulation on
BL66, not by non-acupoint stimulation. Values are represented
as mean ^ SEM (bar). aMeans P , 0:05 versus normal group;
b
means P , 0:05 versus control group (ANOVA followed by
post-hoc Dunnett test).
the hypothalamus showed similar pattern with those of IL-6
(Fig. 2).
LPS is an endotoxin derived from cell wall of gram-negative bacteria and systemic injection of LPS results in various
symptoms of bacterial infection including fever and inflammation [11]. Other studies have applied quite dramatically
different doses of LPS to induce fever (i.p., ranging from 1.0
ng/kg to 5.0 mg/kg). The doses (2.5 mg/kg) we used in this
study exhibited similar biphasic changes (initial hypothermia for 2 h followed by long lasting hyperthermia up to 72
h, data not shown) of body temperature, as reported recently
by Tollner et al., with 5.0 mg/kg of LPS [17]. Their results
showed 5 h of initial hypothermia and up to 3 days of
hyperthermia.
It has been known that, in the activation of cytokine
network, peripheral LPS injection stimulates tumor necrosis
factor-alpha (TNF-a) and IL-1, primary messenger [14]. IL1b subsequently, activates IL-6, which functions as key
mediator in acute phase response and progresses further to
stimulate central thermoregulatory organ, hypothalamus
[2,9]. The increased expression of mRNA of both IL-6
and IL-1b in the hypothalamus, following peripheral (i.p.)
LPS administration shown in this study, basically confirmed
the results of previous studies. IL-1b gene expression has
been reported in the paraventricular nucleus (PVN) of
47
hypothalamus after LPS administration [18]. LPS-induced
fever production is also blocked by the injection of IL-1b
receptor antagonist to specific areas of the hypothalamus,
such as PVN or anterior hypothalamus [3]. IL-1b causes the
increase in IL-6 and its mRNA during LPS-induced fever in
rats [9,15]. Elevated hypothalamic concentrations of IL-6
are also known to be involved in the induction of fever
elicited by peripheral (i.p.) injection of LPS [10]. Administration of lower doses of LPS which do not disrupt the
blood–brain barrier results in the expression of IL-1b, IL6 and TNF-a in the hypothalamus in parallel to stimulation
of the hypothalamus-pituitary-adrenal axis [4,8].
Several studies have reported the changes of body
temperature by acupuncture stimulation [7,12,13,16].
Stimulation on acupoints such as Dazhui (GV14), Quchi
(LI11), or Hegu (LI4) is reported to induce hypothermia,
whereas Neiguan (PC6) or Zusanli (ST36) is related to the
induction of hyperthermia. However, study on acupunctureinduced antipyretic effect is scarce. Electroacupuncture on
Quchi (LI11) acupoint for 30 min exhibited an antipyretic
effect [7]. However, we used different acupoints such as
Shaofu (HT8), Zutonggu (BL66) and Xingjian (LR2) in
our study and stimulation was applied manually for 10 s.
These acupoints have been extensively used in the practice
of Oriental medicine, especially for the regulation of both
body temperature and immune system [1]. Although none of
Fig. 2. The interleukin-1b (IL-1b) mRNA levels of hypothalamus
after 21 h LPS (2.5 mg/kg, i.p.) injection. (N) normal group without LPS; (C) control group with LPS; (A) acupuncture group on
BL66 with LPS; (NA) non-acupoint group with LPS. IL-1b mRNA
levels increased after LPS-injection, and those were suppressed
and returned to normal states by acupuncture stimulation on
BL66, not by non-acupoint stimulation. Values are represented
as mean ^ SEM (bar). aMeans P , 0:05 versus normal group;
b
means P , 0:05 versus control group (ANOVA followed by
post-hoc Dunnett test).
48
Y.S. Son et al. / Neuroscience Letters 319 (2002) 45–48
three acupoints used in this study had been reported
previously, the results of this study clearly indicates the
antipyretic action of these loci.
Even though several studies have demonstrated the
effects of acupuncture on peripheral immune system, only
few examined the influence of acupuncture on central
immune system in the brain. Yu et al. [19] reported that
electroacupuncture on Zusanli (ST36) activated natural
killer cell activity and interferon-g production of the spleen
in mice. Du et al. [5] also suggested that acupuncture stimulation on Zusanli and Lanwei increased the peripheral IL-2
activation in normal rats. The levels of IL-6 and IL-1b
mRNA in the hypothalamus were significantly decreased
by acupuncture stimulation on BL66 with simultaneous
attenuation of fever in our results. These suggest that antipyretic action of acupuncture on BL66, in particular, may be
related to the suppression of hypothalamic gene expression
of IL-6 and IL-1b in rats. Although the other two acupoints
(HT8, LR2) were effective for antipyretic action, their
mechanism may not involve the inhibition of the expression
of these two cytokines. In fact, Fang et al. [6,7] reported the
involvement of prostaglandin E2 on the antipyretic action
induced by electroacupuncture on Quchi (LI11). Their study
indicated that neither IL-6 nor IL-1b mediated the antipyretic effect.
We have come to conclude that effective acupoints for
antipyretic actions have been discovered and some of these
actions may be mediated with down-regulation of two specific cytokines, IL-6 and IL-1b.
This work was supported by Research Grant from the
Ministry of Health and Welfare of Korea to Sabina Lim
(HMP-99-O-11-0007-B).
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