Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan
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ABSTRACT |
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It has been proposed
that glycyrrhetinic acid (GA) enhances endogenous glucocorticoid (GC)
action by suppressing the metabolism of the steroid. We show here that
marked involution of the thymus occurred within 24 h of a single
intraperitoneal administration of GA in mice. Thymocytes from mice
treated with GA exhibited DNA cleavage and mitochondrial transmembrane
potential disruption, as demonstrated with agarose gel electrophoresis
and flow cytometric analysis. Immunocytochemical staining revealed that
CD4+CD8+
double positive cells markedly decreased after GA treatment. In
contrast to GA in vivo, GA in vitro did not induce apoptosis of
cultured thymocytes. These findings suggest that the apoptosis-inducing effect of GA on thymocytes is due to its indirect action. Because GA
has been known to inhibit 11-hydroxysteroid dehydrogenase (11
-HSD), we measured the enzyme activity in major organs and endogenous corticosterone concentration after GA treatment. The results
showed a significant decrease of 11
-HSD activity
(P < 0.0001) and an increase in
serum corticosterone concentration (P < 0.005). We concluded that the inhibition of hepatic 11
-HSD activity by GA has a serious effect on GC metabolism, which results in
a significant elevation of systemic GC levels. Apoptosis of thymocytes
occurred as a consequence of the elevation in the level of endogenous corticosterone.
11-hydroxysteroid dehydrogenase inhibitor; corticosterone; glucocorticoid metabolism
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INTRODUCTION |
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NATURAL AND SYNTHETIC glucocorticoids (GCs) are widely used to treat patients with a variety of pathological conditions. In childhood acute leukemia, the beneficial effect of GCs is known to be based on their cytocidal potency on lymphoid cells (26, 29). The cell death resulted from the GC treatment that was exhibited by the feature of apoptosis, such as cell shrinkage, nuclear condensation, and DNA fragmentation (35). GCs induce apoptosis in immature thymocytes (2, 36), bone marrow pre-B cells (21), and, under certain conditions, in peripheral mature lymphocytes both in vitro and in vivo (14, 24, 25, 37). Thus GC-induced apoptosis in specific cellular systems including immune cells might be involved in the regulation of the immune system (3, 4) and the therapeutic efficacy of GC (1).
11-Hydroxysteroid dehydrogenase (11
-HSD), which promotes
interconversion between biologically active 11
-hydroxy GCs and inactive 11-oxo GCs, plays a critical role in the homeostasis of GC
metabolism by regulating active GC concentrations in specific cells and
tissues (31). For instance, 11
-HSD is expressed at high levels in
mineralocorticoid target tissues (32), and the enzyme protects the
nonselective mineralocorticoid receptor from GC excess (33). This loss
of function of 11
-HSD causes apparent mineralocorticoid excess (8,
27), showing a hypertensive syndrome with a low level of
mineralocorticoid. In this disorder, it has been proposed that an
intrarenal concentration of corticosterone would be abnormally high as
a consequence of its deficient metabolism by 11
-HSD and saturation
of mineralocorticoid receptors by GC (34). Hennebold et al. (10) have
shown that 11
-HSD plays a functional role in specific lymphoid
organs regulating immunologic activities via regulation of GC concentrations.
Glycyrrhetinic acid (GA; Fig.1), a saponin
isolated from licorice root (Glycyrrhizae
radix), has been known to inhibit 11-HSD activity
(17, 20, 22). However, action mechanisms for clinical effects of GA on
peptic ulcers and chronic viral hepatitis (6) have not been elucidated
yet. It was reported in early literature that GA enhances endogenous GC
action by suppressing the metabolism of GCs (18), and a recent report
showed that GA administration to normal mice inhibits 11
-HSD
activities in immune tissues such as the thymus, spleen, and peripheral
lymph nodes in a dose-dependent manner (10). To our knowledge, however,
there has been no report that demonstrates GC-induced apoptosis of
immune cells via intervention of GA in GC metabolism. Our interest has
been focused on a hypothesis that GA induces thymocyte apoptosis via
the elevation of endogenous corticosterone concentration in mice.
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MATERIALS AND METHODS |
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Mice and reagents. Male C57BL/6 mice (4 wk of age) were obtained from Charles River (Tokyo, Japan). Animals were housed in a room apart from other colonies and were maintained with food and water available at liberty. GA (1.0-5.0 mg) and corticosterone (4.0 mg), both of which were purchased from Sigma (St. Louis, MO), were administered in vivo as a fine suspension in olive oil (100 µl/animal) by a single intraperitoneal injection. Control animals received vehicle (olive oil; Wako Biochemical, Osaka, Japan) alone. The mice were killed by ether anesthesia.
Thymocyte culture. The mice thymuses
were minced, gently pressed to release thymocytes in ice-cold PBS, and
passed through a nylon mesh (35 µm) and washed. The cells were
cultured according to a modified method of another report (5). The
thymocytes were briefly suspended in ice-cold RPMI 1640 medium
supplemented with 10% fetal calf serum, 100 IU/ml of penicillin, and
100 µg/ml of streptomycin. Viable cells were then counted with a
trypan blue dye exclusion test and were adjusted to 1 × 106 cells/ml for culture. The
thymocytes were then placed in 24-well flat-bottom plates (Iwaki Glass,
Tokyo, Japan) and incubated with 1 × 1011 to 1 × 10
4 M of GA
for 6 h in a 5%
CO2-95% air atmosphere at
37°C. The GA was dissolved in ethanol, and a final
ethanol concentration in culture was adjusted at 1%. Ethanol was also
used in the control wells.
Agarose gel electrophoresis of fragmented
DNA. The extraction of thymocyte DNA and gel
electrophoresis were performed by the techniques similar to previous
reports (13). In brief, 1 × 106 cells were solubilized with
100 µl of lysis buffer (10 mM Tris-hydrochloride buffer, pH 8.0, containing 0.1 mM EDTA and 150 mM sodium chloride) and placed on ice
for 10 min. The sample was centrifuged at 20,000 g for 20 min. The supernatant was placed in a 1.5-ml microtube and was treated
with 2 µl of 10 mg/ml RNase A at 37°C for 30 min, followed by
treatment with 2 µl of 50 mg/ml proteinase K at 37°C for 60 min.
The sample was then extracted with an equal volume of phenol-chloroform
with DNA isolating-gel (Iwaki Glass) and was precipitated by an
addition of 10× vol of 3 M sodium acetate and equal volume of
ethanol at 20°C. After the evaporation of the ethanol, the
DNA was resuspended in a TE buffer (10 mM Tris-hydrochloride, pH 8.0, containing 1 mM EDTA). The DNA samples (10 µl/lane) were electrophoretically separated on 2.0% agarose gels containing 0.5 µg/ml ethidium bromide (Sigma) with a Mupid-2 (Advance, Tokyo, Japan). The DNA was then visualized with an ultraviolet transilluminater.
Flow cytometric analysis. Detection of
apoptotic cells and phenotype analysis were performed in the following
two ways by flow cytometry. 1) Cell
cycle analysis: cells were fixed for more than 30 min at
20°C in 70% ethanol at a density of 2 × 106 cells/ml. After being washed
with PBS, 100 µl of cell suspension were treated with 2 µl of 10 mg/ml RNase A in PBS at 37°C for 30 min. For an assessment of DNA
fragmentation, the cells were then stained with 0.2 µg/ml of
propidium iodide in PBS for 30 min. The percentage of apoptotic cells
corresponding to the amount of fragmented DNA in the hypoploid
sub-G0/G1
cell cycle peak was calculated. 2)
Detection of mitochondria-specific dye and T cell subtype: 1,000,000 cells were stained with chloromethyl-X-rosamine (CMXRos; Molecular
Probes, Eugene, OR) at 37°C for 30 min. CMXRos (1 mM)
was prepared as a stock solution in dimethylsulfoxide and stocked at
20°C. The stock solution was then diluted in a 100-nM working solution with PBS. After being stained with CMXRos, the cells
were washed in an ice-cold PBS supplemented with 2% heat-inactivated FBS and incubated for 20 min at 4°C with a phycoerythrin-conjugated anti-CD4 monoclonal antibody (0.1 ng/106 cells in 100 µl;
PharMingen, San Diego, CA) and with FITC-conjugated anti-CD8 monoclonal
antibody (0.1 ng/106 cells in 100 µl; PharMingen).
After staining, a total of 20,000 nongated cells were analyzed with a FACSCalibur analyzer, and obtained histograms or dot plot data were calculated by CellQuest software (Becton Dickinson, Mountain View, CA).
Determination of 11-HSD activity in tissue
homogenate. Each 20 mg of tissues (liver and kidney),
thymocytes, and thymic stromal cells taken from mice killed 24 h after
administration of GA were placed in a 1.5-ml microtube poured with 400 µl of ice-cold 0.25 M sucrose. The tissues were homogenized by
sonication. The concentration of protein in the homogenate was
determined by Lowry's method. 11
-HSD activities were determined
with a method previously described (11). Next, enzyme assay tubes
containing 840 µl of 0.1 M Tris-hydrochloride buffer including 0.01%
Triton-X (pH 8.5), 50 µl of 5 mM
NADP+ (for liver) or
NAD+ (for kidney) (Sigma), and 200 µl of 0.3 mM cortisol as a substrate were incubated at 37°C. The
incubation time for assaying both the liver and kidney homogenates was
30 min and for assaying the homogenate fraction thymocytes and thymic
stromal cells was 15 h. The enzyme reaction was terminated by the
addition of 100 µl of 5% sulfuric acid. The tubes were voltexed
vigorously and placed on ice for HPLC analysis.
Concentrations of cortisol and its 11-oxo metabolite, cortisone, in the
incubation mixtures were used to calculate the enzymatic activity of
11-HSD. This was determined by HPLC with a syringe-type extraction-injection device, Extrashot (Kusano Sci., Tokyo, Japan), as
described previously (11). Briefly, 5 µl of incubation mixture were
loaded onto Extrashot, which was then attached to the sample-loading injector of the HPLC system. Subsequently, 130 µl of dichloromethane were injected into the system through Extrashot. The mobile phase was
H2O-methanol-dichloromethane-n-hexane
(0.1:8.0:30.0:61.9, vol/vol) at a flow rate of 1.5 ml/min. The ratio of
the levels of cortisol and its metabolite cortisone, i.e.,
"cortisone concentration to cortisol concentration," was
determined, and the specific 11
-HSD activity in tissue was
calculated as picomoles of cortisone generated per minute per
micrograms of protein.
The HPLC system used in this assay procedure consisted of a solvent delivery pump (TWINCLE, Jasco, Tokyo, Japan), a syringe-loading sample injector (Model-7125, Rheodyne, Cotati, CA), a silica-gel column (LiChrosorb Si-60, Merck, Darmstadt, Germany), an ultraviolet detector (UVIDEC-100-III, Jasco), and a single pen recorder (Pantos U-228, Nippon Denshi, Tokyo, Japan). The wavelength of the detector was set at 245 nm, and the sensitivities were 0.005-0.01 AUFS.
Quantitative analysis of serum
corticosterone. Extraction of serum corticosterone with
diatomaceous earth column extraction, namely rapid flow fractionation,
was performed by a procedure described in a previous report (12).
Briefly, the serum samples were spiked with 50 ng of prednisone as an
internal standard was introduced into the rapid flow fractionation
columns. The column system was treated with 7 ml of dichloromethane.
The effluent was collected in a glass tube and left to evaporate to
dryness. The residue was reconstituted with 15 µl of dichloromethane
for injection onto the same HPLC system as described in
Determination of 11-HSD activity in tissue
homogenate. The HPLC was conducted with
the mobile phase solvent of H2O-
methanol-dichloromethane-n-hexane (0.1:4.0:30.0:61.9, vol/vol).
Statistical analyses. Values are expressed as means ± SD and compared by multiple comparison (Dunnett) or unpaired Student's t-test. Calculated P values of <0.05 were considered significant.
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RESULTS |
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GA effects on thymus weight. There was
no significant difference in the body weight between mice injected with
a single dose of 2.5 mg GA and the vehicle-treated control mice (Table
1). Although a slight decrease in the
weight was observed in the 24 h after treatment in both of the groups,
there was no significant difference between them (Table 1). The mice
injected with 2.5 mg of GA showed a significant decrease in thymus
weight 24 h after the administration compared with the
control mice given the vehicle alone
(P < 0.002; Table 1).
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GA induces thymocyte apoptosis in
vivo. Apoptotic thymocytes from mice treated by GA were
examined with flow cytometry. An increase in the percentages of
apoptotic cells was seen in a dose-dependent manner as shown in Fig.
2. After 24 h of GA treatment, the number of apoptotic cells significantly increased in the mice treated with 2.5 mg GA compared with the control mice (Fig.
3; P < 0.001). The mean percentage of apoptotic cells in the GA-treated group was 36.3 ± 11.8%, whereas that in the corresponding control was 1.7 ± 0.5%. Apoptotic cells showed no signs of increase at 6 h after administration of GA (Fig. 3). The thymocytes from the mice treated with corticosterone showed significantly higher percentages of
apoptotic cells after 6 h of administration compared with the control
mice (P < 0.001), whereas apoptotic
cells had barely been detected after 24 h of corticosterone treatment
(Fig. 3).
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DNA fragmentation in the thymocytes that were prepared from the mice
treated with the vehicle, GA, or corticosterone was analyzed with
agarose gel electrophoresis. Intraperitoneal administration of 2.5 mg
GA resulted in an apparent fragmentation of thymocyte DNA compared with
that of the control 24 h after the treatment (Fig.
4). However, thymocytes from mice treated
with GA for 6 h did not show any signs of apoptosis (data not shown).
The corticosterone-treated mice also showed significant amounts of DNA
fragments after the 6 h of administration, whereas after the 24 h of
administration the fragmentation had scarcely been detected (data not
shown).
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The mitochondria transmembrane potential also decreased significantly
24 h after the GA treatment (Fig.
5). Staining for CD4 and CD8 revealed
that there was a marked reduction in the number of
CD4+CD8+
double positive cells by GA treatment, whereas the number of CD4+CD8
or
CD4
CD8+
single positive cells and double negative cells remained unchanged (Table 2).
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GA failed to induce thymocyte death in
vitro. Thymocytes prepared from agent-untreated mice
were cultured in the presence of serial concentrations of GA in vitro.
After 6 h of incubation with
1011 to
10
4 M of GA, the thymocytes
did not show any signs of apoptosis as analyzed with flow cytometry
(Fig.
6A).
The GA did not induce apoptosis in the thymocytes even after 24 h (data
not shown). Thymocyte apoptosis by corticosterone was induced dose
dependently in vitro (Fig.6B).
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Inhibition of hepatic 11-HSD activity and rise in
serum corticosterone concentration in GA-treated mice.
Finally, we examined dehydrogenation activity of 11
-HSD in tissues
and serum corticosterone concentration in mice treated with 2.5 mg of
GA. Table 3 shows the inhibitory effect of
GA on 11
-HSD activity in liver, kidney, thymocytes, and thymic
stromal cells. Maximal inhibition of activity by GA was obtained in all
of these tissues 3 h after administration. At 24 h after the
administration of GA, 11
-HSD activities in thymocytes, thymic
stromal cells, and kidney were recovered or relatively increased when
compared with those of the control animals, whereas the activity in the
liver was still suppressed significantly (P < 0.0001; Table 3). In the
thymocytes and thymic stromal cells, 11
-HSD activities significantly
increased after the 24 h of administration (P < 0.02 compared with control).
Renal 11
-HSD activity at 24 h after the administration did not show
significant differences when compared with the control mice. We also
measured the serum concentration of corticosterone after the
administration of GA. As shown in Table 3, the mean serum
corticosterone concentration in the group treated with 2.5 mg of GA was
410 ± 143 ng/ml, which was significantly higher than that in the
control group (138 ± 55 ng/ml;
P < 0.005).
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DISCUSSION |
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From the data described in RESULTS, it
could be postulated that GA administration induced thymocyte apoptosis
by elevating levels of endogenous corticosterone via inhibition of
GC-metabolizing enzyme 11-HSD. These
considerations were supported by the experimental findings that
1) thymocytes from GA-administered
animals exhibited biochemical and morphological features of apoptosis,
2) GA administration markedly
suppressed 11
-HSD activity in the liver resulting in increased
levels of circulating GC, 3)
administration of GC also induced thymocyte apoptosis not only in vivo
but also in vitro, 4) apoptosis
induced by GC occurred faster than that induced by GA, and
5) GA had no effect on cultured
thymocytes in vitro.
The induction of thymocyte apoptosis by GA was qualitatively documented by agarose gel electrophoresis of fragmented DNA extracted from thymocytes of GA-treated mice. The GA administration resulted in cleavage of cellular DNA to make a ladder structure of DNA fragments accompanied with thymus involution. To confirm the features of cell death, the thymocytes were also examined with flow cytometry. Apoptotic cells containing a relatively small amount of DNA were dramatically increased after 24 h of GA administration. Moreover, the apoptotic features of cells were accompanied by a disruption of inner mitochondrial transmembrane potential (19). We observed here that the mitochondrial transmembrane potential was disrupted in thymocytes from GA-treated mice. These results suggest that the involution of the thymus by GA occurred as a consequence of the death of the thymocytes. Phenotypic analysis of the thymocytes stained by specific antibodies revealed that CD4+CD8+ double positive cells were the main target of the GA action. It has been reported that in vivo treatment of animals with GC or anti-Fas antibody causes massive apoptosis in CD4+CD8+ thymocytes (23, 30), which coincides with our present observation of GA action. We also investigated whether GA induces thymocyte apoptosis in vitro and concluded that the apoptosis-inducing effect of GA was not detected in vitro and did not come from the direct effect of GA on the thymocytes.
In the present study, induction of apoptosis with intraperitoneal
administration of corticosterone was observed 6 h after the treatment,
and its effect disappeared after 24 h. These observations were
consistent with those of Ishii et al. (15) who reported that the number
of apoptotic cells was maximal after 8 h and disappeared after 18 h of
GC administration. In contrast, we did not detect thymocyte apoptosis 6 h after administration of GA but found a marked increase in apoptotic
thymocytes 24 h after GA administration. Thus, although GA induced DNA
fragmentation in the thymocytes in vivo, the time required was longer
than that of the corticosterone. This time lag of GA action to induce
thymocyte apoptosis may also support the hypothesis that GA action
involved two steps, i.e., 11-HSD inhibition and an elevation in
circulating corticosterone. GC has been reported to induce thymocyte
apoptosis (4, 5), and our data of Fig.
6B coincide with these observations.
It was also reported that elevated levels of corticosterone induce
apoptosis in thymocytes, and this effect of corticosterone could be
abolished by the GC-receptor antagonist RU-38486 (9, 28).
Dougherty et al. (7) reported that long-term GC treatment caused a
significant increase in the activity of 11-HSD in murine thymus. In
our experiment, the activities of 11
-HSD in thymocytes and thymic
stromal cells decreased significantly and subsequently increased within
24 h after a single administration of GA. The early decrease of
activity was well correlated with the subsequent elevation of the
corticosterone concentrations. The surviving thymocytes at 24 h after
GA treatment were thought to have escaped from apoptosis in the
circumstance of the elevated corticosterone. Other evidence was that
there was a relatively high amount of activity of 11
-HSD, which
inactivated the cytocidal corticosterone. A similar observation has
been reported by Jellink et al. (16) in the case of long-term
administration of corticosterone resulting in induction of 11
-HSD
accompanied by cell survival. Thus the elevation of the specific
11
-HSD activity in thymus after GA administration is understandable
as a result of survival of CD4+CD8
or
CD4
CD8+
single positive thymocytes, which may have relatively high activity of
11
-HSD.
11-HSD should play an important role in the steroid conversion in
thymus. We demonstrated here that administration of GA intraperitoneally resulted in a significant increase in serum corticosterone concentrations. Serum concentration of corticosterone increased up to 410 ± 143 ng/ml after 24-h treatment by GA. We also
found here that 11
-HSD activity concomitantly decreased in a
dose-dependent manner in liver by GA and that this inhibition persisted
even after 24 h. On the other hand, renal 11
-HSD activity was not
inhibited by GA administration in the present study. Therefore, inhibition of hepatic 11
-HSD activity might have a serious effect on
GC metabolism, which results in significant elevation of GC systemic levels.
In conclusion, GA-induced thymocyte apoptosis might be due, at least in
part, to inhibition of liver 11-HSD and subsequent increase in the
level of serum corticosterone, which results in the initiation of
apoptosis of thymocytes. Thus the data suggest that pharmacological or
physiological interruption of GC metabolism influences the immune
system via alteration of the thymus cell population.
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ACKNOWLEDGEMENTS |
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This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Japan (Grant no. 09672337) and Tsumura Company.
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FOOTNOTES |
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We also wish to thank T. Kido, T. Keira, S. Horie, and, especially, S. Uda for experimental and technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and correspondence: T. Hirano, Dept. of Clinical Pharmacology, School of Pharmacy, Tokyo Univ. of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan (E-mail: hiranot{at}ps.toyaku.ac.jp).
Received 28 December 1998; accepted in final form 21 May 1999.
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