The American Journal of Chinese Medicine, Vol. 33, No. 6, 945–955
© 2005 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine
Effects of the Extract of a Chinese Herb
Tripterygium wilfordii Hook f on
Rat Pituitary Gland
Long Chen,* Huijun Wang† and Ziqin Zhao*
*Departments of Forensic Medicine and †Pathology
Shanghai Medical Center, Fudan University, Shanghai, 200032, China
Yihu Zhang and Guangzhao Huang
Department of Forensic Medicine, Tongji Medical College
Huazhong Science and Technology University, Wuhan, 430030, China
Abstract: In China, the ethylacetate extract of the herb Tripterygium wilfordii Hook f (TWEE),
containing the major active ingredient triptolide, is often used with favorable effect on
rheumatoid arthritis patients, in alternation with the use of prednisone. The mechanism of
this therapeutic effect, however, has not been completely delineated. In this study, we studied
how TWEE and prednisone affect the pituitary and adrenal glands in rats. Thirty normal
male Sprague-Dawley rats (ten per group) were randomly assigned to receive: (1) TWEE
(25 mg/kg, twice a day), (2) prednisone (2 mg/kg, twice a day), or (3) vehicle (control)
(0.5% sodium carboxymethyl cellulose 1 ml, twice a day), orally for 30 days. Pituitary and
trunk blood were collected on day 31. Adrenocorticotropic hormone (ACTH) expression
in the pituitary gland was assessed morphologically by immunohistochemical techniques.
Plasma ACTH concentrations and serum corticosterone concentrations were quantitatively
measured by radioimmunoassay. We found that TWEE significantly increased plasma ACTH
concentration and serum corticosterone concentration and dramatically increased the number
of ACTH-positive cells in the pituitary. Our findings indicate that TWEE can promote the
synthesis and secretion of ACTH cells — in the pars distalis of the rat pituitary gland and the
production of corticosterone in the zone fasciculata of the adrenal cortex. Our results indicate
that TWEE has a cortical hormone-like function and can promote adrenal cortex function by
activating the hypothalamus-pituitary-adrenal axis.
Keywords: Ethylacetate Extract of Tripterygium wilfordii Hook f; Prednisone; Pituitary;
ACTH; Corticosterone; Immunohistochemistry; Radioimmunoassay.
Correspondence to: Dr. Huijun Wang, Department of Molecular Pathology, Box: 89, University of Texas, M.D.
Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. Tel: (+01) 713-794-5507, Fax: (+01)
713-792-4851, E-mail: firstname.lastname@example.org (Current address: Department of Pathology and Pathology Center,
Shanghai Medical College of Fudan University, Shanghai, 200032, P.R. China.)
L. CHEN et al.
The ethylacetate extract of the Chinese herb Tripterygium wilfordii Hook f (TW), a vine-
like plant common in a wide area of south China, has been reported to be effective in
the treatment of several autoimmune diseases, including rheumatoid arthritis, and to have
potent anti-leukemia properties and antitumor activity (Tao et al., 2002; Ho and Lai,
2004; Shu et al., 1981; Qin et al., 1982; Li and Wu, 1991; Yang et al., 2003; Kupchan
and Schubert, 1974). Experimental and clinical studies conducted over the last 20 years
have shown TW affects the neuroendocrine system, especially the hypothalamus-pituitary-
adrenal axis (HPAA) (Chen et al., 1999; Chen and Huang, 2001; Hu and Lin, 1997; Zhang
et al., 1994; Li and Wei, 1989; Li et al., 1983; Zhou et al., 1983). However, the research
has mainly focused on the adrenal glands and not the pituitary gland.
The ethylacetate extract of Tripterygium wilfordii Hook f (TWEE) contains triptolide,
the major active ingredient of TW. Our aim was to observe how TWEE affects the
morphology and function of the pituitary gland and the function of the adrenal gland in
Materials and Methods
Animals and Treatments
Thirty male Sprague-Dawley rats (seven to eight weeks old) were purchased from the
Department of Experimental Animals of Shanghai Medical University (Shanghai, China).
Rats were housed in wire-bottomed, stainless steel cages (five rats per cage) at 23 ± 2ºC and
a relative humidity of 40 ± 8%, with a 12-hour light/dark cycle (lights on at 7:00am). All
animals were fed a standard rat diet and water ad libitum. After 3 days of acclimatization,
the rats were randomly assigned to three groups. The TWEE group received TWEE
(dry powder containing 0.1% triptolide, suspended in 0.5% sodium caboxylonethyl
cellulose), 25 mg/kg; the prednisone group received prednisone suspended in 0.5% sodium
caboxylonethyl cellulose, 2 mg/kg; and the control group received 1 ml of 0.5% sodium
caboxylonethyl cellulose. All treatments were administered orally twice a day for 30 days.
Ten rats were used per group. To adjust the quantity of the medicine, all rats were weighed
every 3 days. After 30 days, the rats were weighed, and then killed under general anesthesia
with an intraperitoneal injection of sodium pentobarbital solution (45 mg/kg) on the 31st
day between 9:30 and 11:30am. To control for body weight differences, the calculation of
relative pituitary weight = (pituitary weight/body weight) × 100% was used. Pituitary and
trunk blood were collected immediately and handled according to the requirements for each
relevant examination as indicated below. Adrenocorticotropic hormone (ACTH) in plasma
and corticosterone in serum were measured quantitatively by radioimmunoassay (RIA).
The pituitary glands were observed following hematoxylin and eosin (H&E) staining and
immunohistochemistry staining with ACTH antibody.
T. WILFORDII EFFECT ON RAT PITUITARY
ACTH and Corticosterone Radioimmunoassay Analysis
Trunk blood was collected in cool EDTA plastic tubes (coated with 14% sodium EDTA)
immersed in an ice bath at 2–8ºC during collection. The plasma was separated from the
cells by centrifugation (3000g, 4ºC, 20 minutes). The samples were stored at −30ºC.
Before assay, samples were thawed in an ice bath and kept continually at or below 4ºC.
Plasma ACTH was estimated by using a double-antibody 125I RIA kit (Diagnostic Products
Corporation, Los Angeles, CA). Serum corticosterone levels were determined by RIA
(Coat-A-Count, Diagnostic Products Corporation). The Coat-A-Count rat corticosterone
procedure is a solid-phase RIA in which rat 125I-labeled corticosterone competes for a fixed
time with corticosterone in the sample for antibody sites. The antibody is coated on the
wall of a polypropylene tube; simply decanting the supernatant terminates the competition
and isolates the antibody-bound fraction of the radiolabeled corticosterone. Counting the
tube in a gamma counter then yields a number that converts by way of a calibration curve
to a measure of the corticosterone present in the sample.
The pituitaries were removed, weighed, and fixed in fresh Bouin’s fluid for 6 hours and
then embedded in paraffin. Serial sections (5 μm) were cut from the paraffin blocks
and mounted on APES-coated slides. ACTH expression was evaluated by using the
SABC Kit according to the kit instructions (Strept Avidin-Biotin Complex, a sensitive
indirect immunohistochemistry technique, Boster, Wuhan, China). Briefly, sections
were deparaffinized in xylene and hydrated through graded ethanol to deionized water.
Endogenous peroxidase activity was blocked by 5 minutes incubation in 3% hydrogen
peroxide-methanol buffer. Non-specific binding was blocked by incubation for 20 minutes
with a dilution of 1:50 normal goat serum. Primary antibody (rabbit anti-human 18-39ACTH
antibody, Sigma) was diluted to 1:200 with 0.01M PBS and incubated over the sections in a
humidified chamber for 2 hours at 37ºC. The slides were washed three times for 3 minutes
with 0.01M PBS and then incubated with a biotinylated goat anti-rabbit antibody and
SABC reagent (Boster). The reaction product was visualized with 3,3′-diaminobenzidine-
tetrahydrochloride (Sigma). Sections were counterstained with Mayer’s hematoxylin,
dehydrated, and mounted. Negative controls were analyzed using normal goat serum,
omitting the primary antibody.
Image Analysis Technology and Statistical Analysis
Image analysis of the pituitary sections was performed by ACTH immunohistochemical
staining using the TJTY-400 multimedia color cell image analysis system (Wuhan, China).
All results were expressed as group arithmetic means with their standard errors. The data
were put into a computer database, and statistical analysis was performed using analysis of
variance followed by Student’s t-test (p < 0.05 criteria).
L. CHEN et al.
Effects of TWEE and Prednisone on Rat Body Weight, Pituitary Weight, and
the Relative Pituitary Weight
The effects of TWEE and prednisone on body weight, pituitary weight, and the relative
pituitary weight are shown in Table 1. Values for the relative pituitary weight were
significantly greater for the TWEE group than for the prednisone group (p < 0.05) or
controls (p < 0.05). In addition, the pituitary weight was significantly greater in the TWEE
group than in the prednisone group (p < 0.05), with no difference between the TWEE
and control groups. Final body weights did not differ among the three rat groups. These
results indicate that rat pituitary weight was significantly greater in the TWEE group after
eliminating the effect of body weight.
Table 1. Comparison of Rat Body Weight, Pituitary Weight, and Organ Coefficient Among the Three Groups
(Mean ± SD)
Group Body Weight (g)
213.56 ± 23.29
213.90 ± 21.79
213.60 ± 16.29
298.67 ± 35.02
290.20 ± 29.13
276.50 ± 47.86
9.02 ± 1.70
8.87 ± 1.27
9.89 ± 2.68‡
3.02 ± 0.38
3.05 ± 0.23
3.60 ± 0.83†,‡
Male Sprague-Dawley rats (seven to eight weeks old, ten rats/group) were given vehicle (control), TWEE
(containing 0.1% triptolide, 25 mg/kg, twice a day), or prednisone (2 mg/kg, twice a day) orally. All rats
were killed on day 31. Statistical analysis was performed using analysis of variance followed by Student’s t-test
(p < 0.05 criteria). *Relative pituitary weight = (pituitary weight/body weight) × 100%. †Significantly different
from the control group, p < 0.05. ‡Significantly different from the prednisone group, p < 0.05.
(% × 10−3)
Table 2. Comparison of ACTH and Corticosterone Concentrations Among the
Three Groups (Mean ± SD)
Group Plasma ACTH
50.92 ± 10.94
32.53 ± 8.49*
74.75 ± 12.00*,†
112.79 ± 19.27
77.06 ± 14.43*
142.56 ± 31.34*,†
Male Sprague-Dawley rats (seven to eight weeks old, ten rats per group) were given
vehicle (control), TWEE (containing 0.1% triptolide, 25 mg/kg, twice a day), or
prednisone (2 mg/kg, twice a day) orally. All rats were killed on day 31. Statistical
analysis was performed using analysis of variance followed by Student’s t-test (p <
0.05 criteria). *Significantly different from the control group, p < 0.05. †Significantly
different from the prednisone group, p < 0.05.
T. WILFORDII EFFECT ON RAT PITUITARY
Quantitative Measurement of Plasma ACTH and Serum Corticosterone
with the Double-Antibody 125I Radioimmunoassay
ACTH and corticosterone concentrations in the blood increased in the TWEE group and
decreased in the prednisone group compared with the controls (Table 2). Analysis of
variance indicated that significant differences of plasma ACTH and serum corticosterone
were respectively found among three groups (all p < 0.05). Our results indicated that TWEE
stimulates ACTH secretion in the rat pituitary and corticosterone secretion in the zone
fasciculate of the adrenal cortex, whereas prednisone suppresses ACTH and corticosterone
Morphological Changes in ACTH Cells in the Pars Distalis of the Rat Pituitary Gland
Under the light microscope, the pars distalis, pars intermedia, and posterior lobe of the
pituitary gland were clearly shown with H&E staining. There were no notable differences
among the three groups (data not shown), except for a slight dilation in the capillary vessels
and venous sinusoids in the pars distalis (PD) and posterior lobe (LP) of the pituitary gland
in the TWEE group (Fig. 1), which was not seen in the control and prednisone groups.
Figure 1. Light microscopic view of the pars distalis (PD), pars intermedia (PI), and posterior lobe (LP) of the
rat pituitary gland in the TWEE group. There was a slight dilatation of the capillary vessels and venous sinusoids
in the PD and LP of the pituitary gland in the TWEE group but not in the control or prednisone group (H&E