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【关键词】 Isoliquiritigenin
Isoliquiritigenin, one of the major constituents of Glycyrrhiza uralensis (licorice), is a natural pigment with a simple chalcone structure 4,2',4'-trihydroxychalcone. In this study, isoliquiritigenin showed selective H2 histamine receptor (H2R) antagonistic effect and remarkably reduced several H2R-mediated physiological responses. Preincubation of U937 and HL60 hematopoietic cells with isoliquiritigenin significantly inhibited H2R agonist-induced cAMP response in a concentration-dependent manner without affecting the viability of cells. Isoliquiritigenin also blocked the binding affinity of [3H]tiotidine to membrane receptors in HL-60 cells. Isoliquiritigenin did not affect the elevation of cAMP levels induced by cholera toxin, forskolin, or isoproterenol, indicating that the action site of isoliquiritigenin is not Gs protein, effector enzyme, adenylyl cyclase, or 2-adrenoceptor. Isoliquiritigenin affected neither H1R-nor H3R-mediated signaling. In molecular docking studies, isoliquiritigenin exhibited more favorable interactions with H2R than histamine. Isoliquiritigenin prominently inhibited H2R selective agonist dimaprit-induced cAMP generation in MKN-45 gastric cancer cell. Moreover, isoliquiritigenin reduced gastric acid secretion and protected gastric mucosal lesion formation in pylorus-ligated rat model. Taken together, the results demonstrate that isoliquiritigenin is an effective H2R antagonist and provides the basis for designing novel H2R antagonist.
Histamine is one of the aminergic neurotransmitters and plays an important role in the regulation of several pathophysiological processes (Jutel et al., 2005b). Histamine is found in every human tissue and can act as a local hormone, a mediator in processes related to allergy and inflammation, or a neurotransmitter (Jutel et al., 2005a). Histamine exerts its effect through H1, H2, H3, and H4 receptors (Gutzmer et al., 2005). In particular, the H2 histamine receptor (H2R) is coupled to the Gs protein/adenylyl cyclase system in a variety of tissues (e.g., brain, stomach, heart, gastric mucosa, lung) and produces intracellular cAMP (Alewijnse et al., 1998). Gantz et al. (1991) were the first to clone a cDNA encoding a 359-amino acid H2R. Using degenerate primers based on the known sequence similarity of various G protein-coupled receptors, the H2R sequence was obtained from canine gastric parietal cDNA by PCR. Soon thereafter, the intronless genes encoding the rat, human, guinea pig, and mouse H2R were cloned by means of homology screening (Gantz et al., 1991). Many reports demonstrated the cellular function of H2R and its importance. In immune system, the responses of T helper cells (Th1 and Th2) are negatively regulated by H2R through the activation of different biochemical intracellular signals (Jutel et al., 2001). The presence of H2R in U937 (promyelocytic lymphoma cell line; Shayo et al., 2004), HL-60 (promyelocytic leukemia cell line; Suh et al., 2001), and MKN-45 (gastric cancer cell line; Arima et al., 1991) cells has been reported.
Licorice root (Glycyrrhiza uralensis) is used as a harmonizing ingredient in many traditional herbal formulations. It is used in more formulations than any other herb in oriental medicine. Although it is considered to be the quintessential "servant" herb, it is often referred to as the "King of Herbs". Although the flavonoid-rich fraction from the extract of licorice has been used as a gastrointestinal disorder and an antiulcer medicine (Fukai et al., 2002), the mechanism is still elusive.
Since Black et al. (1972) first defined the H2R and its involvement in gastric acid secretion, H2R antagonists (e.g., cimetidine, ranitidine, famotidine) have been developed and used clinically as antiacid secretagogues. These histamine H2R antagonists have revolutionized the treatment of peptic ulcers with their prominent therapeutic effects. The components of G. uralensis have a strong antipeptic ulcer effect (Shibata, 2000). Constituents of G. uralensis include glycyrrhizic acid, glycyrrhetic acid, and isoliquiritigenin (Shibata, 2000). Fukai et al. (2002) reported that the flavonoid-rich fraction of licorice extract had an antiulcer effect; therefore, from among the major constituents of licorice, we selected the isoliquiritigenin (4,2',4'-trihydroxychalcone), which has basic chemical structure of a flavonoid, as a novel H2R antagonist and examined its effect on H2R activity. Isoliquiritigenin has been reported as a potent antioxidant, has cancer-preventing properties, and inhibits platelet aggregation (Baba et al., 2002). Isoliquiritigenin also reduce nitric oxide and suppress aberrant crypt foci development (Takahashi et al., 2004). In present study, we used U937 and HL-60 hematopoietic cell lines and the rat pylorus-ligated model (Shay et al., 1954) to evaluate the potential activity of isoliquiritigenin as H2R antagonist and antipeptic ulcer agent against gastric ulcer. Through this study, we provide evidence that isoliquiritigenin negatively regulates most of the H2R's activity and clearly acts as a specific, competitive H2R antagonist in terms of blocking secretion of gastric acid and ulcer formation similar to the action of ranitidine.
Materials. Histamine, ranitidine, isoliquiritigenin, forskolin, cholera toxin, and Ro 20-1724 were obtained from Sigma (St. Louis, MO). Tiotidine was obtained from Tocris Cookson Inc. (Ballwin, MO). Dimaprit was purchased from Tocris (Bristol, UK). [3H]cAMP and [3H]tiotidine were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). RPMI-1640 and penicillin-streptomycin were purchased from Life Technologies (Gaithersburg, MD). Bovine calf serum was obtained from HyClone Laboratories (Logan, UT).
Cell Culture. U937, HL-60 cell line (American Type Culture Collection, Manassas, VA), and MKN-45 cell (Korean Cell Line Bank, Seoul, Korea) was cultured in suspension at 37°C in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated bovine calf serum and 1% (v/v) penicillin-streptomycin in a humidified atmosphere of 95% air and 5% CO2. Primary cultures of bovine adrenal cortex cells were prepared according to the method of Reichenstein et al. (2004).
Measurement of [3H]cAMP. Intracellular cAMP generation was determined by [3H]cAMP competition assay in binding to cAMP binding protein using a cAMP kit (Neuronex, Pohang, Korea), according to the manufacturer's instructions.
RT-PCR for Evaluation of H2R mRNA Expression. Total RNA was isolated from the U937 and HL-60 cells using Centrizol (Neuronex). Ten micrograms of total RNA was reverse-transcribed with the use of Superscript II reverse transcriptase (Life Technologies). cDNA was amplified with 20 pmol of specific oligonucleotide primers (Bioneer, Daejeon, Korea) using Pfu polymerase (Stratagene, La Jolla, CA).
The primer sequences are 5'-GCCCTCGAGACCATGGCACCCAATGGCACAGC and 3'-GCCGGGCCCACCCCTGTCTGTGGCTCCCTCGG. The PCR products were separated by 1% agarose gel electrophoresis. The products were subcloned into a pGEM-T Easy Vector (Promega, Madison, WI) and sequenced with a dideoxynucleotide termination method.
[Ca2+]i Measurement. Cytosolic free Ca2+ concentration ([Ca2+]i) was determined with the help of the fluorescent Ca2+ indicator fura-2/AM. In brief, the U937 cell suspension was incubated with fresh serum-free RPMI medium containing fura-2/AM (3 µM) for 40 min at 37°C with continuous stirring. The cells were then washed with Locke's solution and left at room temperature until use. Sulfinpyrazone (250 µM) was added to all solutions to prevent dye leakage. Fluorescence ratios were measured by an alternative wavelength time-scanning method (dual excitation at 340 and 380 nm; emission at 500 nm).
H3 Histamine Receptor Expressing Human Embryonic Kidney 293 Cell. The full-length human histamine H3 receptor gene was kindly provided by Dr. Liu Changlu (Johnson and Johnson Pharmaceutical Research and Development). H3R genes were subcloned into the pCIneo expression vector. Four million cells detached with trypsin were mixed together with 10 µg of DNA in gene pulser cuvettes (0.4 cm; Bio-Rad, Hercules, CA) and electroporated at 260 V, , 960 µFD using a Bio-Rad gene pulser. The cells from up to six electroporations were pooled subsequently on a cell culture dish (10 cm), trypsinized after 24-h incubation in normal culture medium, and seeded on 12-well plates for binding experiments performed the next day.
[3H]Tiotidine Binding. The binding of [3H]tiotidine to intact HL-60 cells was quantified by a method described previously (Monczor et al., 2003) with some modification. Triplicate assays were performed in polyethylene tubes in 50 mM Tris-HCl, pH 7.4. [3H]Tiotidine were incubated with 106 cells/tube in the absence or presence of isoliquiritigenin in a total volume of 200 µl. After 40 min at 4°C, incubation was stopped by dilution with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.4; rapid filtration onto GF/B glass-fiber filters (Whatman, Clifton, NJ) was performed under reduced pressure, followed by three washes with 3 ml of ice-cold buffer. Specific binding was defined as the difference in the amount of radioactivity bound in the absence and presence of 1 mM unlabeled tiotidine.
Three-Dimensional Model Building. All the simulations were performed on Linux workstations using Insight II 2005 software package (Accelrys, San Diego, CA). The primary sequence of the human H2R (Swiss-Prot ID P25021) was used. Modeler program (Fiser et al., 2000), which is an implementation of an automated approach to comparative modeling by satisfaction of spatial restraints and derives final model by optimization process consisting of applying the variable target function method with conjugate gradients optimization as well as molecular dynamics refinement, was used to build the three-dimensional structure of human histamine H2R by taking the structure of bovine rhodopsin (Protein Data Bank code 1U19) as template. The refined model thus obtained from Modeler was checked for the stereochemical parameters by the program PROCHECK.
Binding Site Analysis. Potential binding sites in the protein were identified with the help of Active_Site_Search program in Binding_Site module of Insight II 2005 software. Active_Site_Search characterizes protein active sites and binding sites by locating cavities in three-dimensional protein structures. The sites identified can be used to guide the protein-ligand docking experiments.
Ligand Preparation. Histamine and isoliquiritigenin were sketched with the help of Builder module in Insight II 2005. Then the charges and potentials were assigned using CHARMM forcefield and the minimization was done using the CHARMM module in Insight II 2005 with 1000 steps of steepest descents followed by 10,000 steps of conjugate gradients. To the minimized conformers, potentials and charges were once again assigned using CVFF forcefield and were saved in Sybylmol2 format.
Molecular Docking. The program GOLD (Genetic Optimization for Ligand Docking, Cambridge Crystallographic Data Centre, Cambridge, UK) was employed to dock the histamine and isoliquiritigenin into the histamine binding site of human histamine H2 receptor. A homology model of human histamine H2 receptor was used for molecular docking studies. Active site radius of 10.0 Å was defined from the binding site coordinates obtained from Active_Site_Search program in Insight II 2005 software. The root-mean-square deviation was considered within 1.5 Å, and the annealing parameter of van der Waals' interaction was 4.0; hydrogen bond interaction was 2.5.
Fig. 1. Basic chemical structure of a flavonoid (A). Chemical structure of isoliquiritigenin (B), histamine (C), ranitidine (D), cimetidine (E), and famotidine (F).
Gastric Secretion in Pylorus-Ligated Rat. The pylorus-ligated rat model first described by Shay et al. (1954) was used with some modification. Male Sprague-Dawley rats weighing 200 to 250 g were used. Rats were deprived of food, but not water, for 18 to 24 h before each experiment. The test substances (vehicle, ranitidine, isoliquiritigenin) dissolved in saline were administered orally. After 1 h, rats were anesthetized by light ether, a small abdominal incision was made, the pylorus was ligated, and then indomethacin (40 mg/kg) was injected through the duodenum. The animals were sacrificed 8 h after ligation of the pylorus, the stomach was clamped at the esophageal and duodenal junctions, filled with 15 ml of 4% formalin, and then rapidly removed. After 24 h, the fixed stomach was opened along the greater curvature, gently rinsed in saline, and then pinned open to expose the gastric mucosa. The hemorrhagic and ulcerative lesions were counted and measured with a light microscope by an observer who was blinded to the treatment. The ulcer index was then calculated as the sum of the diameter of all lesions. The ulcer index was determined in six animals in each animal group.
Histological Examination. The histological study of the stomach was performed after evaluation of the ulcer index. Samples of the corpus were excised and transferred to fresh formalin and later processed by routine techniques before embedding in paraffin. Sections (5 µm thick) were mounted on glass slides and stained with hematoxylin and eosin (HE). Coded slides were examined by an experienced pathologist blinded to the treatment.
Statistical Analysis. All Quantitative data are expressed as mean ± S.E.M. Comparisons between two groups were performed using Student's unpaired t test. Differences were considered to be significant when the degree of confidence in the significance was 95% or better (P < 0.05).
cAMP Production by H2R Agonist in U937 and HL-60 Cells. Histamine and dimaprit are known as highly selective histamine H2R agonists (Bakker et al., 2002). U937 and HL-60 cells express H2R (Fig. 2A, inset), which is coupled to Gs protein and mediates the cAMP pathway (Shayo et al., 1997). As shown in Fig. 2A, histamine and dimaprit induced cAMP increase in a concentration-dependent manner, and half-maximally effective concentrations (EC50) are 1.2 ± 1.3 and 4.7 ± 0.2 µM, respectively.
Fig. 2. Isoliquiritigenin attenuates H2R signaling. A, H2R agonists (histamine and dimaprit) induced cAMP production in U937 and HL-60 promyelocytes (HA, histamine; Dima, dimaprit). H2R expression in U937 and HL-60 cells was confirmed by RT-PCR, inset. cAMP generation and RT-PCR were measured as described under Materials and Methods.B, U937 Cells were preincubated with indicated concentration of ranitidine (Rani) or ILG for 5 min, then cells were stimulated with 10 µMHAor Dima for 20 min. Rani or ILG was not removed during the H2R agonist stimulation. The results are the mean ± S.E.M. of assay triplicates.
Effect of Isoliquiritigenin on H2R-Mediated cAMP Production. We preliminarily investigated the inhibitory effect of licorice constituents (10 µM each) on cAMP production induced by histamine and dimaprit (10 µM) induced in U937 cell line (data not shown). Among the components, isoliquiritigenin (Fig. 1B) exhibited the strongest inhibitory effect. Figure 2B shows that isoliquiritigenin inhibited histamine and dimaprit-induced cAMP production in a concentration-dependent manner similar to that of ranitidine [IC50 values of ranitidine = 1.0 ± 0.7 µM and isoliquiritigenin (ILG) = 2.3 ± 0.1 µM, respectively], suggesting that isoliquiritigenin inhibits H2R-mediated signals with similar effect of ranitidine.
Fig. 3. Selective effect of isoliquiritigenin on H2R. A, U937 cells were preincubated with indicated concentration of ILG for 5 min, then cells were stimulated with 10 µM dimaprit (), 1 µM isoproterenol (), or 1 µM forskolin (), respectively. B, bovine adrenal cortex cells were preincubated with 10 µM ILG, then cells were stimulated with 100 ng/ml cholera toxin (CTX). The results are the mean ± S.E.M. of assay triplicates. C, the intracellular [Ca2+]i rise induced by 100 µM histamine (HA) or HTMT (H1R-selective agonist) was measured in the absence or presence of 10 µM ILG in U937. Data are the means ± S.E.M. (bars) values of triplicate measurements. D, H3R-transfected human embryonic kidney 293 cell were stimulated with 500 nM forskolin (Fsk) in the absence or presence of (R)--methylhistamine (R, H3R-selective agonist), clobenpropit (Cloben, H3R-antagonist) + R, ILG + R, or ILG, respectively. The results are the mean ± S.E.M. of assay triplicates.
Lack of Direct Effects of Isoliquiritigenin on Signaling Mediated by Gs Protein, Adenylyl Cyclase, and 2-Adrenoceptor. As we mentioned above, H2R-mediated signaling activates Gs protein/adenylyl cyclase pathway (Klinker et al., 1996). To clarify whether the isoliquiritigenin affects only the H2R, we treated the cells with forskolin (which directly activates adenylyl cyclase), isoproterenol (which activates Gs protein coupled 2-adrenoceptor), and cholera toxin (which evokes Gs protein activity). Figure 3 shows that the addition of forskolin, isoproterenol, and cholera toxin caused a significant increase of cAMP level, and their cAMP generation were not attenuated by isoliquiritigenin pretreatment, suggesting that the site of the inhibition by isoliquiritigenin is neither the adenylyl cyclases, Gs protein, or Gs protein-coupled 2-adrenoceptor. The result indicates that isoliquiritigenin selectively inhibits H2R activation.
Selective Effect of Isoliquiritigenin on H2R Subtype. As mentioned above, histamine exerts its effect not only through H2Rs but also H1Rs and H3Rs. The H1Rs are coupled to Gq proteins, which increase intracellular Ca2+ concentration, and H3Rs are linked to Gi proteins, which down-regulate the activity of adenylyl cyclase. Thus, we checked whether isoliquiritigenin affects H1R- and H3R-mediated signaling. HTMT and (R)--methylhistamine are used as H1R- and H3R-selective agonists, respectively. Clobenpropit is used as H3R antagonist. As shown in Fig. 3, C and D, isoliquiritigenin affect neither H1 R-mediated [Ca2+]i nor H3R-mediated signaling, suggesting that isoliquiritigenin is a selective H2R antagonist.
Fig. 4. Isoliquiritigenin inhibits binding of [3H]tiotidine to H2R. HL-60 cells were preincubated with indicated concentration of ILG for 10 min; then cells were treated with [3H]tiotidine at 4°C for 80 min. Data were calculated as the mean ± S.E.M. of assay triplicates. Similar results were obtained in at least three independent experiments.
[3H]Tiotidine Binding Assay with Isoliquiritigenin. To determine whether the effects of ILG were due to its ability to block histamine binding to H2R, we tested the effects of ILG on [3H]tiotidine binding to H2R in undifferentiated HL-60 cells (Monczor et al., 2003). Through RT-PCR and calcium measurements, we could not find H1 receptor expression in our HL-60 system. In addition, [3H]tiotidine is highly H2R selective, suggesting that we can exclude the involvement of H1 receptors with [3H]tiotidine binding analysis. We found that isoliquiritigenin significantly blocked [3H]tiotidine binding to undifferentiated HL-60 cells in a concentration-dependent manner (Fig. 4), indicating that isoliquiritigenin selectively and competitively inhibited histamine binding to the H2R.
Three-Dimensional Model Building. The refined model obtained from the Modeler program was then checked for the stereochemical parameters by the program PROCHECK at 2.2 Å. For the modeled receptor, 87.3% of the backbone conformations fall within the most favored region of the Ramachandran plot, whereas the value for the A chain of bovine rhodopsin was only 79.9%. In addition, when analyzing the structural alignment of the C-trace between the human H2R model and the A-chain of bovine rhodopsin, the root-meansquare deviation of the C-trace was 0.672 Å. This suggests that the homology model we obtained is reliable. To the human H2R model thus obtained were added hydrogens at pH 7 to mimic the biological fluid environment. Then the potentials and charges were assigned using the CVFF forcefield and the receptor model was saved in Sybylmol2 format.
Binding Site Analysis and Docking. Then docking was performed with histamine and isoliquiritigenin. Goldscores of 39.08 and 48.91 were observed for histamine and isoliquiritigenin, respectively. The docked poses of both the ligands in the human H2R model can be seen in Fig. 5, A and B, respectively. From our docking results, we demonstrated that histamine can form three hydrogen bonds with the receptor, which involve those from ethylamino group of histamine to side-chain carboxyl group of Asp 98, from ethylamino group of histamine to main chain carbonyl group of Lys 166, and from N1 proton of histamine to main chain carbonyl group of Leu 274, respectively (Fig. 6A), whereas isoliquiritigenin can also forms three hydrogen bonds with the receptor, which involve those from 4-OH-benzoyl group of isoliquiritigenin to the main chain carbonyl of Leu 274, from the 2-OH-benzoyl group of isoliquiritigenin to the main chain carbonyl of Lys 166, and from the carbonyl group of isoliquiritigenin to the main chain amino group of Asn 168.
Fig. 5. Model of human H2R complexed with histamine (green) and isoliquiritigenin (pink). The model was created by Insight II 2005 (Accelrys). A, top view of the docked poses of histamine and isoliquiritigenin. B, side view of the docked poses of histamine and isoliquiritigenin.
Fig. 6. Hydrogen bonding at the histamine binding site of the human H2R model. A, hydrogen bonding interactions of histamine at the histamine binding site of human H2R model. B, hydrogen bonding interactions of isoliquiritigenin at the histamine binding site of human H2R mode. Hydrogen bonding is indicated by the dashed lines.
Effect of isoLiquiritigenin on H2R in MKN-45 Gastric Cancer Cell Line. MKN-45 gastric cancer cells express H2Rs and increase H2R expression upon retinoic acid treatment (Nakata et al., 1996). We treated MKN-45 with 5 µM retinoic acid for 72 h and tested the effect of isoliquiritigenin on dimaprit-induced cAMP generation. Similar to U937 cells, isoliquiritigenin also prominently inhibited dimaprit-induced cAMP production in MKN-45 gastric cancer cells. Isoliquiritigenin, however, did not affect forskolin-induced adenylyl cyclase activation (Fig. 7). These data indicate that isoliquiritigenin has an inhibitory effect on H2Rs expressed not only in immune cell systems but also in gastric cells. For this reason, we extended our investigation whether isoliquiritigenin can reduce the gastric ulceration in the stomach.
Fig. 7. Effect of isoliquiritigenin on H2R-mediated signaling in MKN-45 gastric cancer cells. MKN-45 cells were preincubated with 10 µM ILG for 5 min; then cells were stimulated with 10 µM dimaprit (Dima) or 1 µM forskolin (Fsk) for 20 min. ILG was not removed during the H2R agonist stimulation. The results are the mean ± S.E.M. of assay triplicates. *?, P < 0.05 compared with Dima alone.
Effect of Isoliquiritigenin on Gastric Acid Secretion and Gastric Mucosal Lesion Formation. There are many reports about the inhibitory effect of H2R antagonists on gastric acid secretion in animal model systems (Lamers, 1999). H2R antagonists reduced volume of gastric acid production in pylori-ligated animal stomach and also significantly protected gastric mucosal lesion formation. In line with the above, we tested the effect of isoliquiritigenin on gastric acid-induced ulcer formation. In our experiment, isoliquiritigenin significantly prevented the gastric acid secretion (Fig. 8A) and gastric-ulcer formation (Fig. 8, B-D), similar to the ranitidine effect. Our results demonstrate that isoliquiritigenin effectively inhibits gastric acid secretion and protects gastric mucosal layer in a manner similar to that of other H2R antagonists.
Fig. 8. Protection effect of isoliquiritigenin on the formation of gastric mucosal lesion. Test substances [vehicle (Cont), ranitidine (RA), and ILG] were administered orally, and then rats were killed 8 h after the surgery as described under Materials and Methods. A, effects of ILG and of ranitidine (RA) on the gastric acid secretion after pyloric ligature in SD rats. Each column represents the mean ± S.E.M. (n = 4-9 per group), and the difference between groups was determined by t test. **, P < 0.01 compared with control. B, each bar represents lesion score of Shay modelinduced gastric damage. The hemorrhagic and ulcerative lesions were counted and measured with a light microscope by an observer who was blinded to the treatment. The ulcer index was then calculated as the sum of the length of all lesions. The ulcer index was determined in six animals in each animal group. ***, P < 0.001 compared with control. Representative pictures of 24 h pylorus-ligated Shay-model gastric walls (C) and HE-stained gastric mucosal layer (D). Vehicle-treated control group (a and d), ranitidine-treated group (b and e), and isoliquiritigenin-treated group (c and f).
Antagonism of histamine's action at H2R has been the cornerstone of an immense market for pharmacological treatment of acid-peptic disorders of the gastrointestinal tract. Through this study, we provide evidence that isoliquiritigenin directly inhibited the H2R activity and acted as an antipeptic-ulcer drug. First, we confirmed the existence of H2Rin U937 and HL-60 cells through RT-PCR analysis, and isoliquiritigenin significantly blocked H2R-mediated cAMP production. In addition, in MKN-45 gastric cells, isoliquiritigenin also inhibited H2R-mediated signaling, indicating that isoliquiritigenin does not depend on the cell type. Second, isoliquiritigenin inhibits dimaprit-induced cAMP production via blocking of histamine binding to H2R, rather than acceleration of degradation, because the inhibition was observed in the presence of the phosphodiesterase inhibitor, Ro 20-1724. Third, isoliquiritigenin did not affect the activity of other histamine subtype receptors (H1R or H3R), cholera toxin, the adenylyl cyclase- or 2 adrenoceptor-mediated signaling pathway, suggesting that isoliquiritigenin is highly H2R-selective. Fourth, isoliquiritigenin reduced [3H]tiotidine binding on the H2R, strongly suggesting that isoliquiritigenin inhibits only the binding affinity between H2R and its corresponding ligand.
H3 receptor agonists and antagonists, such as clobenpropit, imetit, (R)--methylhistamine, and thioperamide, show various degrees of cross-reactivity with the H4 receptor. The H4 receptor is a recently identified member of the histamine receptor family. Its closest relative in the histamine receptor family is the H3 receptor; the similarity in the transmembrane region is even higher (68% sequence identity). Antagonists specific for the H3 or H4 receptor have been generated, and they are valuable tools for dissecting the biological roles of H3 and H4 receptors. As shown in Fig. 3D, isoliquiritigenin did not inhibit clobenpropit's antagonistic action on H3 receptors, indicating that isoliquiritigenin does not interfere between the H3/H4 receptor ligand and receptor. The major physiological roles of gastrin include stimulation of acid secretion and the enterochromaffin-like cell is the principal cellular transducer of the gastrin-acid signal. Activation of its gastrin receptors results in synthesis and release of histamine with consequent activation of the parietal cell H2Rs (Schubert, 2004). Histamine stimulates the parietal cells to secrete HCl (Lindstrom et al., 2001). Gastric acid secretion is under nervous and hormonal control (Lindstrom et al., 2001). The H2 subclass of histamine receptors mediates gastric acid secretion, and antagonists for this receptor have proven to be effective therapy for acid peptic disorders of the gastrointestinal tract. It has been shown that H2R activation mediates the gastric acid secretion via a G protein linked to adenylate cyclase activation and cellular cAMP generation. In the present study, 1-h pretreatment with isoliquiritigenin and ranitidine significantly inhibited the gastric acid secretion and gastric mucosal lesions in pylorus-ligated rat model (Fig. 8). Previous reports demonstrated that drugs that are unable to inhibit acid secretion cannot suppress the lesion formation in this model, because these lesions are related mainly to the significant increase in acidity of gastric secretions, and this correlates well with the severity of erosions (Shibata et al., 1998), showing that isoliquiritigenin can be very useful for peptic ulcer treatment.
Even though both histamine and isoliquiritigenin have three hydrogen bonds to the human histamine H2R model, the higher goldscore for the latter arises from the more favorable hydrophobic interactions within the receptor. In addition, the isoliquiritigenin does not seem to have a hydrogen bond with Asp 98, which is crucial for histamine binding. From our studies, we can speculate that isoliquiritigenin has more favorable interactions with histamine H2R because of the more hydrophobic interaction compared with histamine. In addition, suitable substitutions on the isoliquiritigenin that facilitates hydrogen bonding with the side-chain carboxyl group of Asp 98 may contribute to the antagonistic activity of isoliquiritigenin on the human H2R.
As shown in Fig. 1, most H2R antagonists [ranitidine (Fig. 1D), cimetidine (Fig. 1E), and famotidine (Fig. 1F)] are optimized through histamine-structure lead (Fig. 1C). However, the chemical structure of isoliquiritigenin is not optimized from histamine, suggesting that isoliquiritigenin can be used as novel H2R antagonist. Mild diarrhea, neuropsychiatric disorders, gynecomastia, impotence, dizziness, rashes, confusion, headaches, and reversible liver damage have been reported by some people using histamine-derived H2R antagonists as antiulcer treatments (Zimmerman, 1984). Because isoliquiritigenin is purified from a natural product, licorice (Ma et al., 2005), it may be considered a safe antiulcer treatment.
In previous reports, activation of H2R has led to cell proliferation and has also activated transcription of the gene encoding c-fos in a protein kinase C-dependent manner (Wang et al., 1997). Cell proliferation often requires a series of signaling steps that act in a coordinated manner to regulate nuclear events responsible for controlling cell division. Previous reports proved that H2R regulates c-Fos and c-Jun mRNA and protein level in numerous systems (Shayo et al., 1997). As in the previous reports, dimaprit transiently elevated c-Fos levels (Supplemental Data). In addition, we observed that the levels of c-Jun protein treated with H2 agonist showed a similar pattern (Supplemental Data). However, in the presence of 10 µM isoliquiritigenin, dimaprit-induced elevation of c-Fos and c-Jun protein level was completely inhibited (Supplemental Data). These data demonstrate that isoliquiritigenin negatively regulates H2R mediated c-Fos/c-Jun protein expression.
In conclusion, our results show that isoliquiritigenin is a novel, specific, and competitive histamine H2R antagonist with antisecretory and antiulcer effects of similar potency to those of ranitidine. It also induces gastroprotective effects and might provide the basis for designing novel H2R antagonists.
Acknowledgements
We thank Dr. Liu Changlu (Johnson and Johnson Pharmaceutical Research and Development) for offering full-length human histamine H3 receptor gene.
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作者单位：Division of Molecular and Life Science, System Bio-Dynamics-National Core Research Center, Pohang University of Science and Technology, Pohang, Korea (D.-C.K., S.-H.K., K.-T.K.); Division of Research and Development, Neuronex, Inc., Pohang, Korea (D.-C.K.); Department of Physiology, Seoul National U