Effects of gentianine on the production of pro-inflammatory cytokines in male Sprague-Dawley rats treated with lipopolysaccharide (LPS)

Article abstract of DOI:10.1248/bpb.28.750

This study was undertaken to elucidate the mechanism of anti-inflammatory action of gentianine, a constituent of Gentiana Macrophylla. The effects of gentianine on lipopolysacharide (LPS)-induced production of pro-inflammatory cytokines were investigated in male Sprague-Dawley rats. For the first time, we found that oral administration of gentianine (10-100 mg/kg) suppressed the increases in tumor necrosis factor-α (TNF-α) (ED₅₀, 37.7 mg/kg) and interleukin (IL)-6 (ED₅₀, 38.5 mg/kg) in the sera from the rats challenged with bacterial LPS (100 μg/kg; i.p.). However, LPS induced production of other interleukins, such as IL-α, was not significantly altered by gentianine. These results suggest that the potential anti-inflammatory action of gentianine might be at least partly based on the suppressed production of TNF-α and IL-6.

Full text of DOI:10.1248/bpb.28.750

750
Notes
Biol. Pharm. Bull. 28(4) 750—753 (2005)
Vol. 28, No. 4
Effects of Gentianine on the Production of Pro-inflammatory Cytokines in
Male Sprague-Dawley Rats Treated with Lipopolysaccharide (LPS)
a,b
a
a
a
a
Wie-Jong KWAK, Joo-Hyon KIM, Keun-Ho RYU, Yong-Baik CHO, Sun-Duck JEON, and
,
b
Chang-Kiu MOON*
a
b
Life Science Research Center, SK Chemicals; 600 Jungja-1-Dong, Changan-Gu, Suwon 440–745, Korea: and College of
Pharmacy, Seoul National University; San 56–1 Shillim-Dong, Kwanak-Gu, Seoul 151–742, Korea.
Received December 14, 2004; accepted January 14, 2005
This study was undertaken to elucidate the mechanism of anti-inflammatory action of gentianine, a con-
stituent of Gentiana Macrophylla. The effects of gentianine on lipopolysacharide (LPS)-induced production of
pro-inflammatory cytokines were investigated in male Sprague-Dawley rats. For the first time, we found that oral
administration of gentianine (10—100 mg/kg) suppressed the increases in tumor necrosis factor-a (TNF-a)
(
ED , 37.7 mg/kg) and interleukin (IL)-6 (ED , 38.5 mg/kg) in the sera from the rats challenged with bacterial
5
0
5
0
LPS (100 mg/kg; i.p.). However, LPS induced production of other interleukins, such as IL-a, was not significantly
altered by gentianine. These results suggest that the potential anti-inflammatory action of gentianine might be at
least partly based on the suppressed production of TNF-a and IL-6.
Key words gentianine; anti-inflammatory mechanism; tumor necrosis factor (TNF)-a; interleukin (IL)-6; lipopolysacharide
LPS)
(
The crude extract of Gentiana Macrophylla Radix, one of (Charles River Breeding Farms, Montreal, Quebec, Canada)
Gentiana species, has been used for the treatment of inflam- weighing between 280 g and 350 g were used for the study.
matory diseases such as osteoarthritis (OA) and rheumatoid Three rats were housed in suspended polypropylene cages
arthritis (RA) in oriental medicine. Gentianine is one of the with wire grid floors and all animals had free access to tap
major components of Gentiana Macrophylla Radix and sev- water and pellet food (Agribrands, Purina, Korea). The room
eral research groups reported its various pharmacological ac- temperature and relative humidity were set at 21 °C and 50%,
1)
tivities such as anti-ulcerogenic activity, anti-inflammatory respectively. Prior to the experiment, the rats were deprived
2)
1,2)
activity and anti-analgesic activity.
To elucidate the of food for 18 h, but not of water. The study has been carried
mechanism of the anti-inflammatory action of gentianine, its out according to the “Principles of laboratory animal care”
effects on the cytokine production were investigated in male (NIH Publication no. 85-23, revised 1985).
Sprague-Dawley rats (SD-rats) treated with lipopolysaccha-
ride (LPS).
LPS, serotype 0111:B4, derived from Escherichia coli was
obtained from Sigma Chemicals (St. Louis, MO, U.S.A.).
Pro-inflammatory cytokines such as TNF-a, IL-1 and IL-6 Microarray kit was purchased from RayBiotech, Inc. (Nor-
are produced and play critical roles in the inflammation cross, GA, U.S.A.) and enzyme linked immunosorbent assay
processes. Among these pro-inflammatory cytokines, TNF-a (ELISA) kits were obtained from R & D systems (Minneapo-
has been highlighted recently as a main mediator in the in- lis, MN, U.S.A.). All other chemicals were of the highest
flammatory diseases mechanism. In placebo-controlled clini- grade from commercial sources.
cal trials, TNF-a antagonists were proven highly effective in
To secure enough sample amounts required for this study,
the treatment of some inflammatory diseases. TNF-a may gentianine was synthesized by heating 4-methyl-5-vinylnico-
have a significant role in pathogenesis of several inflamma- tinic acid with aqueous formalin in the presence of sodium
3)
9)
tory diseases such as rheumatoid arthritis, psoriatic arthri- bicarbonate, followed by acid treatment. Purification was
4)
5)
6,7)
tis, juvenile rheumatoid arthritis, and Crohn’s disease.
performed by medium pressure liquid chromatography (40%
Bacterial LPS binds to CD14 on the monocyte membrane ethylacetate/hexanes) and re-crystallization (ethylacetate/
and stimulates the production of pro-inflammatory cytokines hexanes: 4/6) to give white crystalline: mp 80—81 °C (lit.
ꢀ1
such as IL-1, IL-6 and TNF-a that are important signaling mp 80—81 °C); IR (CHCl ) 1720 and 1621 cm ; MS m/e
3
ꢁ
molecules in initiating and coordinating a large range of im- 175 (M ). Figure 1 shows the chemical structure of gentian-
8)
mune responses against invading pathogens. Based on the ine.
facts that gentianine possesses anti-inflammatory effects and
LPS Induced Production of Cytokines in Rats Treated
inflammatory cytokines play a key role in inflammation, we with Gentianine Gentianine was dissolved in distilled
assumed that gentianine might have inhibitory effects on the water and administered orally with doses ranging between
production of pro-inflammatory cytokines. Therefore, gentia- 3—100 mg/kg. Control rats received distilled water as a vehi-
nine was examined in vitro and in vivo for its effects on the cle. Two hours after the gentianine administration, rats were
production of TNF-a and IL-6, pro-inflammatory cytokines, challenged with LPS, serotype 0111:B4, derived from Es-
which are involved in the pathogenesis of inflammatory dis-
eases.
MATERIALS AND METHODS
Experimental Animals and Materials Male SD-rats Fig. 1. The Structure of Gentianine
∗
To whom correspondence should be addressed. e-mail: moonck@snu.ac.kr
© 2005 Pharmaceutical Society of Japan

April 2005
751
cherichia coli. Animals were intraperitoneally administered Table 1. Doses of Gentianine for the Inhibition of the Production of TNF-
a and IL-6 in Male SD-Rats Treated with LPS
with either LPS (100 mg/kg) or 0.89% NaCl at an injection
volume of 1 ml/kg body weight. Two hours after the chal-
Gentianine (mg/kg)
lenge, rats were sacrificed by decapitation. The dose and
route of LPS administration adopted in the study produce
quantifiable increase in circulating concentrations of pro-in-
flammatory cytokines IL-6 and TNF-a. Following the de-
capitation, trunk blood was collected into plastic tubes and
stored on ice prior to centrifuge at 800ꢂg at 4 °C for 15 min.
After centrifugation, aliquots of serum were removed for the
determination of TNF-a and IL-6. The removed sera were
frozen immediately on dry ice and later transferred to a
Inhibition-doses
TNF-a
IL-6
10)
ED10
ED50
ED90
14.48
37.658
97.94
8.56
38.52
143.72
ꢀ80 °C freezer where they remained until the assays were
performed.
LPS Induced Production of Cytokines in Whole Blood
Treated with Gentianine Human whole bloods were ob-
tained from 3 healthy volunteers (male, age of 26—30) after
getting informed consent. Heparinized whole blood was pre-
incubated with either vehicle or gentianine at 37 °C for
10 min. LPS (1 mg/ml) was then added to the cultures and in-
cubated for another 12 h. After incubation, the incubation
mixtures were centrifuged at 1500ꢂg at 4 °C for 10 min.
The supernatants obtained were applied to the assay of the
produced cytokines.
Detection of Cytokines in the Sera Using Antibody Mi-
croarray Kit Rat cytokine array system was used as a pri-
mary screening tool to detect the cytokine production. Rat
cytokine array was purchased from RayBiotech, Inc. (Nor-
cross, GA, U.S.A.). The cytokine assay was performed ac-
cording to the standard protocol suggested by the manufac-
turer. In brief, cytokine array membranes were treated with
5% BSA, and then the membranes were incubated with one
milliliter of rat serum followed by further incubation with
HRP conjugated immunoglobulin G. The signals were visu-
alized with enhanced chemoluminescence (ECL).
Assay of IL-6 and TNF-a in the Sera Serum concen-
trations of TNF-a and IL-6 were measured by using com-
mercially available ELISA kits (R & D systems, Minneapo-
lis, MN, U.S.A.); and absorbance was read at 450 nm on
ELISA plate reader (Molecular Devices, SPECTRA max
Fig. 2. Detection of Cytokines by Utilizing Antibody Microarray
The pictures represent ECL images of microarray; (A) cytokines in the serum from
PLUS 384, U.S.A.). Results were expressed as ng TNF-a/ml saline treated rats, (B) cytokines in the serum from LPS-treated rats and (C) cytokines
in the serum from rats treated with gentianine and thereafter LPS. Blue circles denote
IL-6; red circle, TNF-a; black circle, IL-1; green square, positive control of membrane;
yellow square, negative control of membrane.
serum or pg IL-6/ml serum.
Statistical Analysis of Data All data were expressed as
meanꢃS.E.M. Statistical significances among groups were
tested using Sigma Stat (Jandel Co., San Rafael, CA, U.S.A.) of two pro-inflammatory cytokines (TNF-a and IL-6) and
by one-way analysis of variance (ANOVA) followed by Dun- those increased production was specifically inhibited by gen-
nett’s test. Differences were considered significant when p tianine. Based on this result, we decided to investigate the ef-
was less than 0.05. All experiments were done at least three fects of gentianine on the production of TNF-a and IL-6.
times, each time with three independent observations.
TNF-a is a central mediator that plays a critical role in the
regulation of inflammatory processes. It is chiefly synthe-
sized and secreted by macrophages in response to pro-in-
RESULTS AND DISCUSSION
12)
flammatory stimuli such as bacterial LPS. TNF-a func-
The mechanism of anti-inflammatory action of gentianine tions its biological activities by binding to the cell surface re-
has not yet been elucidated. Thus, we investigated, in the pre- ceptors, TNF receptor I and TNF receptor II. TNF-a is as-
sent study, the effects of gentianine on the production of pro- sociated with development of serious infectious diseases and
inflammatory cytokines (TNF-a and IL-6) in male SD-rats certain inflammatory diseases.
treated with LPS.
Micro spot antibody array was performed to detect the cy- These facts suggest that TNF-a plays a central role in the
tokines, of which production was changed in the sera from cascade of cytokine production. This theory is supported by
13)
14,15)
It is well known that
TNF-a is able to stimulate the production of other cytokines.
11)
the rats treated with gentianine followed by LPS challenge.
As shown in Fig. 2, LPS markedly increased the production fective in lowering the levels of other cytokines in LPS-in-
the fact that administration of anti-TNF-a antibodies is ef-

7
52
Vol. 28, No. 4
jected animal models.
clearly understood. But we could make an assumption that
TNF-a is also known as a crucial factor in immune re- IL-1 might be regulated differently from TNF-a and IL-6 in
sponses. TNF-a production in rat blood peaked 1 h after the present experimental condition. This assumption could be
LPS-challenge, but decreased rapidly thereafter (data not supported at least in part by the following three reports; Erroi
shown). Figure 3 showed that gentianine was found to inhibit A. et al. confirmed that IL-1 production was differentially
LPS-induced TNF-a secretion in a dose-dependent manner.
regulated from that of TNF-a and IL-6 in LPS tolerant
17)
In addition, IL-6 has been also known as an important cy- mice. And O’Brien-Ladner A. R. et al. also reported that
tokine concerned to inflammation, immuno-regulation and iron differentially regulated human alveolar macrophage-de-
hematopoiesis. Continuous overproduction of IL-6 also plays rived IL-1 and TNF-a. They found that the addition of defer-
an important role in the pathogenesis of some inflammatory oxamine, a chelator of iron, increased the release of IL-1, but
diseases such as RA.
not TNF-a, from alveolar macrophages from smokers and
18)
In this study, most abundant IL-6 production was observed nonsmokers. In addition, Meldrum D. R. et al. reported
at 2—4 h after LPS challange (data not shown). It was that tissue-specific protein kinase C isoforms differentially
1
9)
confirmed that a LPS challenge increased the production mediate macrophage TNF-a and IL-1 production.
of TNF-a and IL-6, circulating pro-inflammatory cy-
Unregulated activities of inflammatory mediators can lead
15,16)
tokines,
and LPS-induced production of TNF-a (Fig. 3) to development of serious inflammatory diseases. For exam-
and IL-6 (Fig. 4) was markedly inhibited by gentianine in ple, enhanced TNF-a synthesis is associated with the devel-
vitro and in vivo. The inhibition doses are shown in Table 1: opment of rheumatoid arthritis (RA), psoriatic arthritis and
7,20)
ED50 for TNF-a and IL-6; 37.65 mg/kg and 38.52 mg/kg re- inflammatory bowel disease.
And NO production by
spectively. But the production of other inflammatory cy- iNOS may reflect the degree of inflammation and provides a
tokines including IL-1 was not affected by gentianine in measure to assess the effect of drugs on the inflammatory
21)
LPS-injected rats (Fig. 2). This differential effect is not yet process.
It was also reported that IL-1b, TNF-a and inducible ni-
tric oxide synthase (iNOS) are highly expressed in synovial
cells, infiltrating leukocytes and endothelial cells in RA. IL-1
and TNF-a play a central role in many inflammatory
processes and appear to be involved in the catabolic process
8)
of OA. These cytokines also enhance the production of 5-
LOX products and PGE . The TNF-a inhibition by antibod-
2
ies to TNF-a itself or its receptors in these inflammatory dis-
eases has been remarkably successful. In addition, the block-
age of IL-6 may also provide a new therapeutic strategy for
22)
RA. For example, Atlizumab is a recombinant humanized
anti-human IL-6 receptor antibody that blocks IL-6 binding
on its receptor. However, the current injectable protein thera-
pies have been associated with risks and limitations. There-
fore, an oral therapy with small molecule could either replace
the injectables or provide better disease control when used
Fig. 3. LPS-Induced TNF-a Production in Rats Treated with Gentianine
Rats were given gentianine (0, 10, 30, 100 mg/kg body weight, per oral). Two hours
later, the rats were intraperitoneally challenged with LPS (100 mg/kg body weight).
TNF-a was quantified by ELISA. Each bar represents meanꢃS.E.M. for six to eight
23)
mice. Asterisks indicate significant differences from LPS treated control (one-way alone or in conjunction with existing therapies. In this re-
ANOVA followed by Dunnett’s test, ∗, ∗∗ pꢄ0.05, 0.01, respectively). The average of
spect, it might be a valuable finding that gentianine inhibits
TNF-a concentrations in the sera obtained from only LPS treated rats was regarded as
100% TNF-a production.
the production of TNF-a and IL-6. In vitro treatment of
Fig. 5. LPS Induced Production of TNF-a in Whole Blood Treated with
Gentianine
Fig. 4. LPS Induced IL-6 Production in Rats Treated with Gentianine
Rats were given gentianine (0, 10, 30, 100 mg/kg body weight, per oral). Two hours
later, the rats were intraperitoneally challenged with LPS (100 mg/kg body weight). IL-
was quantified by ELISA. Each bar represents meanꢃS.E.M. for six to eight mice.
Asterisks indicate significant differences from LPS treated control (one-way ANOVA
followed by Dunnett’s test, ∗, ∗∗ pꢄ0.05, 0.01, respectively). The average of IL-6 con-
centrations in the sera obtained from only LPS treated rats was regarded as 100% IL-6
production.
Heparinized human whole blood was preincubated with either gentianine or vehicle
at 37 °C for 10 min, and then stimulated with 1 mg/ml LPS for 12 h. TNF-a was quanti-
fied by ELISA. Each bar represents meanꢃS.E.M. for six blood samples. Asterisks in-
dicate significant differences from LPS treated control (one-way ANOVA followed by
Dunnett’s test, ∗, ∗∗ pꢄ0.05, 0.01, respectively). The average of TNF-a concentrations
in the sera obtained from only LPS treated rats was regarded as 100% TNF-a produc-
tion.
6

April 2005
753
whole blood with gentianine significantly reduced the pro-
duction of TNF-a (Fig. 5). And IL-6 production seemed to
be slightly inhibited by gentianine, but it was not statistically
significant (data not shown). In vitro effects of gentianine on
cytokine production were somewhat different from its in vivo
effects. The difference between in vivo and in vitro effects
should be further investigated. In addition, the effects on the
products of arachidonic acid metabolism should be also in-
vestigated as an additional mechanism of anti-inflammatory
action of gentianine.
In summary, the experimental data provide evidences that
gentianine might express anti-inflammatory action at least
partly through the prevention of the immune cells including
macrophages from producing TNF-a and IL-6, pro-inflam-
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