Suppression of Infection-Induced Endotoxin Shock in Mice by a Citrus Flavanone Naringin

• Abstract
• Introduction
• Materials and Methods
• Mice
• Bacteria and inoculation
• Determination of antimicrobial activity in vitro
• Determination of lethal shock
• Preparation of blood sample
• Determination of endotoxin in plasma
• Determination of TNF titer
• Electrophoresis and Western blotting
• Measurement of blood coagulation factors
• Histology
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

The protective effect of the Citrus flavanone naringin was demonstrated in an endotoxin shock model based on Salmonella infection. Intraperitoneal (i. p.) infection with 10⁸ CFU Salmonella typhimurium aroA caused lethal shock in lipopolysaccharide (LPS) -responder but not LPS-non-responder mice. Administration of 1 mg naringin 3 h before infection resulted in protection from lethal shock, similar to LPS-non-responder mice. The protective effect of naringin was time- and dose-dependent. Treatment with naringin resulted not only in a significant decrease in bacterial numbers in spleens and livers, but also in a decrease in plasma LPS levels. In addition, naringin markedly suppressed TNF-α and normalized the activated states of blood coagulation factors such as prothrombin time, fibrinogen concentration and platelet numbers caused by infection. Interestingly, treatment with naringin suppressed high levels of soluble CD14 and high mobility group-1 molecule caused by infection.

Introduction

The pathogenesis of septic shock is initiated by lipopolysaccharide (LPS), the main constituent of the outer membrane of Gram-negative bacteria. Biological activities of LPS are transduced by signaling through the Toll-like receptor 4 (TLR4)/MD2 which is expressed on various types of cells implicated in the innate immunity and is mediated by the secretion of numerous mediators in vitro and in vivo [1]. One of the main pathogenic mediators in septic shock is TNF-α, produced by LPS-stimulated macrophages [2], [3]. Development of substances capable of antagonizing various LPS activities has been investigated. Even though it has been found that certain compounds can antagonize the pathogenic activities of LPS, especially of lipid A, in an in vitro study, their efficacy would be different in in vivo models, particularly in infection models. One of the reasons for such discrepancies is certainly the difference in availability of LPS and of antagonists in vivo due to their differential clearance from the circulation. In a previous report [4] we demonstrated the difference between the clearance rate and biological activity of different types of purified LPS preparations and of LPS released from bacilli during infection with Escherichia coli.

Among various LPS activities, attempts to screen substances capable of suppressing LPS-induced TNF-α production have been performed. An inhibitory substance from cinnamon bark could bind directly to the lipid A moiety to suppress TNF-α release and lethal shock [5]. We have been screening medicinal plants possessing suppressive activities on LPS-induced TNF-α production. Previously, we showed that naringin, known as the main flavonoid of grapefruit, suppressed LPS-induced TNF-α production. Since sensitization with d-galactosamine (GalN) makes mice hypersensitive to TNF-α produced by LPS-stimulated macrophages [6], the inhibitory effect of naringin on lethal shock has been demonstrated in GalN-sensitized mice [7].

In the present study we investigated if naringin would be able to counteract endotoxin shock in a bacterial infection with an attenuated strain, Salmonella typhimurium aroA (SL7207), in comparison with the therapeutic effects of antibiotics [8].

Materials and Methods

Mice

BALB/c and ddY mice (Clea Japan Inc., Tokyo) and BALB/lps^d mice, obtained as breeding pairs from the Max-Planck-Institute for Immunobiology (Freiburg in Breisgau, Germany) and raised in the animal facility of the Kitasato University under specific pathogen-free conditions, were used. The infectious experiments were carried out in a P-2 level room. The experiments described in this study were performed in adherence to the National Institutes of Health guidelines on the use of experimental animals. Approval of the Animal Use Committee of the Kitasato University School of Science was obtained prior to initiating the experiments.

Bacteria and inoculation

An attenuated strain of S. typhimurium aroA (SL7207) was used. Bacteria were grown in tryptic soy broth (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) for 18 h at 37 °C. After washing with phosphate-buffered saline (PBS), bacterial numbers were calculated from a standard curve which was based on turbidity and counting colony numbers. Mice were injected i. p. with a bacterial suspension (5 × 10⁸ CFU/mL) at a volume of 0.2 mL. The inoculum size was checked by plating diluted bacterial suspensions, which were used for infection, on tryptic soy agar (Difco Laboratories, Detroit, MI).

Determination of antimicrobial activity in vitro

Freshly prepared bacteria as described above were suspended in fresh tryptic soy broth at different concentrations of naringin (Tokyo Chemical Industry Ltd., Tokyo, Japan) or fosfomycin (FOF) (Meiji Seika Ltd., Tokyo, Japan). The optical density was measured at different time points during incubation.

Determination of lethal shock

Female BALB/c mice (9 weeks of age) were injected i. p. with the vehicle (saline) or different doses of naringin before or after infection. Female ddY mice (9 - 10 weeks of age) were injected i. p. with vehicle alone, 3 mg naringin or 3 mg rutin (Wako Pure Chemical Industries, Ltd., Osaka Japan) 3 h before infection. As a positive control, the antibiotic ceftazidime (CAZ: 20 mg/kg) (Tanabe Seiyaku Co., Ltd., Osaka, Japan) was injected i. p. 1 h post infection (p. i.). Animals were observed for 21 days.

Preparation of blood sample

Following deep anesthesia with diethyl ether, blood was taken from mice by cardiac puncture. The blood was immediately mixed with 20-µL heparin for estimating endotoxin units (EU), TNF-α titers and fibrinogen concentration, and then centrifuged at 10,000 × g for 1 min at room temperature. Plasma samples were used immediately or kept at -80 °C until use. For counting platelet numbers, blood samples (0.5-mL aliquots) were immediately mixed with 0.1 mL of 3.8 % sodium citrate solution (Nipro Inc., Tokyo, Japan).

Determination of endotoxin in plasma

Plasma components affecting the Limulus cascade reactions were eliminated before endotoxin determination as described previously.

Determination of TNF titer

Serum TNF titers were estimated by a cytotoxicity test using TNF-sensitive L929 (C5F6) cells in the presence of actinomycin-D-mannitol as described previously.

Electrophoresis and Western blotting

Plasma samples were mixed with sample buffer, heated in boiling water for 2 min and analyzed using a 12.5 % gel by SDS-PAGE. After blotting, soluble CD14 (sCD14) and HMG-1 were visualized using an anti-mouse CD14 mAb (rmC5 - 3, PharMingen, San Diego, CA) and polyclonal rabbit anti-HMG-1 antibody (PharMingen), and a VECTASTAIN ABC-PO kit (Vector Laboratories Inc., Burlingam, CA).

Measurement of blood coagulation factors

Prothrombin time (PT), fibrinogen concentration, fibrinogen degradation products (FDP) and platelet numbers were measured at 14 h after infection. Platelet numbers were differentially counting by utilizing a SS-3000 autoanalyzer (Sysmex Co., Tokyo, Japan). Fibrinogen concentration in plasma was measured by a Coagulex 100 autoanalyzer (International reagent Co., Kobe, Japan).

Histology

Liver samples of BALB/c mice 24 h after infection were fixed with 10 % formalin in 0.01 M phosphate buffer (pH 7.4) and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE).

Statistical analysis

Statistical significance of the data was determined by the Scheffe post hoc- and log-rank-tests. A P value of less than 0.05 was taken as significant.

Results

Although low antimicrobial activity against Pseudomonas aeruginosa has been reported [9], naringin is generally not considered as an antibiotic. To estimate whether naringin possesses antimicrobial activity in vitro, S. typhimurium aroA bacilli were incubated for 5.5 h in broth containing 1 mg/mL of naringin or 10 minimum inhibitory concentration (MIC) of FOF, which was 100 µg/mL to 10⁷ CFU/mL in the tube test. Naringin did not show any antimicrobial activity (data not shown).

To confirm whether infection-induced lethal shock is mediated by LPS, mice were infected i. p. with 10⁸ CFU of S. typhimurium aroA. As shown in Fig. [1], all LPS-responder BALB/c mice died within 2 days after infection. By contrast, none of LPS-non-responder BALB/lps^d mice died through lethal shock, suggesting that infection-induced lethal shock is mediated by LPS. More than 4 days p. i., BALB/lps^d mice gradually succumbed by liver lesions due to bacterial growth (data not shown). Administration of naringin blocked lethal shock due to infection. The time course of survivors in naringin-treated mice was similar to that in BALB/lps^d mice.

To estimate the dose effect of naringin on inhibition of lethal shock, BALB/c mice were treated i. p. with different doses of naringin 3 h before infection. As shown in Fig. [2] A, significant activity was detected in mice that were administered 0.1 to 1.0 mg naringin. The activity was highly variable at a dose of 0.03 mg. Administration of 3 mg naringin reduced the protective activity rather than the optimal dose. To determine time dependency, mice were treated with 1 mg naringin 24, 3 or 1 h before infection, simultaneously with infection or 1 h p. i. A protective effect was not observed in the latter two cases (Fig. [2] B). On the other hand, naringin was effective when mice were treated 24 to 1 h before infection. These results indicate that the protective effect of naringin on infection-induced lethal shock is time- and dose-dependent.

To estimate whether administration of naringin reduced bacterial growth, bacterial numbers in spleens and livers of BALB/c mice, treated i. p. with 1 mg naringin 3 h before infection, were measured at different time points. As shown in Fig. [3], significant decreases in bacterial numbers were observed in both organs of infection controls and naringin-treated mice from 90 min after infection, whereas bacterial numbers in both organs 30 min after infection were almost identical.

To estimate whether plasma LPS levels released during infection decreased as a result of treatment with naringin, plasma samples were collected at different time points from BALB/c mice treated i. p. with 1 mg naringin 3 h before infection. As shown in Fig. [4], significantly lower levels of plasma LPS were seen in naringin-treated groups from 90 min after infection, although no significant difference was observed in plasma LPS levels from both infection controls and naringin-treated mice at 30 min after infection.

Previously, we demonstrated that administration of naringin suppressed LPS-induced TNF-α levels [7]. Experiments were therefore performed to estimate whether treatment with naringin suppressed TNF-α secretion caused by infection. Since plasma TNF-α levels peaked at 90 min after infection (data not shown), the effect of naringin was compared at the peak. As listed in Table [1], plasma TNF-α levels were significantly suppressed by treatment with naringin. The suppressive activity of naringin was stronger in mice treated with 3-mg naringin than those with 1-mg naringin.

Since detectable levels of sCD14 were measured 6 to 9 h after administration of LPS [10], experiments were attempted to estimate whether treatment with naringin reduces plasma sCD14 levels during infection. As shown in Fig. [5] A, i. p. infection with 10⁸ CFU of S. typhimurium aroA caused secretion of detectable levels of sCD14 which peaked at 9 h p. i. and remained at high levels until 12 h p. i. Treatment with 1-mg naringin 3 h before infection down-regulated sCD14 levels at the later stage (9 to 12 h p. i.), although sCD14 levels at 6 h p. i. did not show a significant difference between infection control and naringin-treated mice.

Wang et al. reported that non-histone-type DNA-binding protein HMG-1 was a late mediator in a shock model caused by administration of LPS [11]. To estimate whether naringin reduces HMG-1 levels, plasma samples were collected at 12, 18 and 24 h after infection. As shown in Fig. [5] B, detectable HMG-1 levels were observed at 24 h after infection in controls. Plasma HMG-1 levels at 24 h p. i. were not detectable on treatment with naringin 3 h before infection.

To estimate the protective effect of naringin on histological changes, livers were removed from three groups, i. e., untreated control mice, infection controls, and naringin-treated and infected mice. As a result of infection, the blood stream stopped due to severe thrombosis following fibrin precipitation in the vessels of infection controls compared with untreated controls (Fig. [6] A). As a result, severe liver damage occurred due to the death of cells belonging to the vessel supply area (Fig. [6] B). Liver damage by infection-induced shock was by a different mechanism than the previously described shock induced in the GalN/LPS model. In the GalN/LPS model, hepatocytes in GalN-sensitized mice received death signals of TNF-α, which were produced by macrophages stimulated with LPS [7]. Treatment with naringin blocked liver damage due to decreases in fibrin precipitation following hepatocyte death by stopping the blood stream (Fig. [6] C).

To estimate whether naringin suppressed the activation of blood coagulation factors such as PT, fibrinogen concentration, FDP and platelet numbers, blood samples were collected at 14 h after infection from BALB/c mice treated with 1 mg naringin 3 h before infection. As listed in Table [2], the PT value of infected controls increased markedly (more than 60 seconds). The PT value was significantly suppressed by the administration of naringin. Although the concentration of fibrinogen was decreased by infection, plasma fibrinogen levels were inversely enhanced by treatment with naringin. No significant difference was observed in the FDP value. The decrease in platelet numbers was inhibited significantly by administration of naringin, whereas platelet numbers markedly decreased during infection. These results indicate that treatment with naringin inhibits the activation of blood coagulation system caused by infection.

The protective effect of naringin on the infection-induced endotoxin shock was compared with those of the flavonol glycoside rutin [12] and the therapeutic antibiotic CAZ as positive controls. As shown in Fig. [7], the activity of naringin was stronger (but not significant) than that of rutin. On the other hand, the post-treatment of CAZ caused complete protection from the endotoxin lethality.

Discussion

The present study is the first report that naringin acts as an inhibitor on infection-induced lethal shock, although naringin does not possess antimicrobial activity in vitro. Protective effects of naringin were time- and dose-dependent and expressed through inhibition of the activated stages of blood coagulation factors implicated in thrombosis caused by infection.

In a previous study [7], we showed that naringin had inhibitory activities in LPS-induced TNF-α production in vivo following lethal shock in GalN-sensitized mice. In a preliminary study, naringin did not neutralize the LPS action due to direct binding to LPS molecule when naringin and LPS were mixed together and incubated before the automatic kinetic chromogenic Limulus reaction. Plasma LPS levels markedly increased by infection and peaked 9 to 12 h p. i. Treatment with naringin resulted in a significant reduction of not only bacterial numbers in organs but also plasma LPS levels. Since naringin does not have any antimicrobial activities, decreases in bacterial numbers in livers and spleens seem to be expressed by host-mediated mechanisms stimulated with naringin and correlate with a reduction of plasma LPS levels. Reduced LPS levels caused down-regulation of plasma TNF-α, sCD14 and HMG-1 formation following inflammatory responses.

Treatment with naringin inhibited production of proinflammatory cytokine TNF-α caused by infection (Table [1]), although the degree of inhibition by naringin was not so strong in this model. It assumes that decreases in plasma TNF-α levels by naringin seem to down-regulate expression of adhesion molecules in the vessel and subsequent production of cytokines and chemokines. Lower levels of sCD14 would reduce formation of an sCD14/LPS complex and binding to TLR4/MD2, which is the receptor for LPS and a pivotal molecule in innate immunity. Thus lower sCD14 levels seem to decrease signal transduction for mRNA expression of cytokines and chemokines via TLR-4 expressing on endothelial cells in the vessel. Recently, HMG-1 has been identified as a late mediator of endotoxin lethality. HMG-1 release was initiated by stimulation with TNF-α and produced 6 h after administration of LPS [11]. In our shock model, detectable levels of HMG-1 were observed only at 24 h after infection. Since this time point is the late stage of lethal shock, down-regulation of HMG-1 levels by naringin seems to closely correlate to rescue from lethal shock.

In mouse models endotoxin shock causes death within 24 to 48 h. In human patients it is characterized by disseminated intravascular coagulation, resulting in multiple organ failure [13]. Recently, Yun-Choi and coworkers [14] reported that higemanine, an Aconitum alkaloid, significantly ameliorated the decrease of fibrinogen levels and platelet numbers in plasma, the increase of FDP levels, and the prolongation of PT induced by the i. v. infusion of LPS in rats. Because these findings are in line with our observations (Table [2]), the downstream effects of naringin on blood coagulation systems also appear to significantly contribute to protection from endotoxin shock. Lee et al. reported down-regulation of VCAM-1 and MCP-1 by naringin in a dietary study in rabbits [15]. To explain the lower degree of thrombosis found in naringin-treated mice in our experiments, MCP-1 down-regulation would account for the depletion of macrophages. Future studies will have to address in detail whether or not the down-regulation of VCAM-1 and MCP-1 by naringin is demonstrable in our animal model.

In in vitro studies using macrophages, flavonoids have been reported to attenuate LPS-induced TNF-α production by interfering with LPS signaling by reducing the activation of several MAPK family members (ERK, p38 and CK2) [16], [17], [18]. The following in vivo experiments have been reported; the effects of naringin [7] and woogonin [19] on LPS-induced lethal toxicity in D-GalN-sensitized mice, the activities of rutin, quercetin, baicalein, baicalin, catechin and naringenin in two types of LPS-induced shock models [12], and the protective effect of luteolin on lethal toxicity induced by a high amount of LPS [20]. Takahashi et al. demonstrated that, among rutin, quercetin, baicalein, baicalin, catechin and naringenin, the strongest activity in terms of protection from LPS-induced lethal shock was observed in a group of mice treated with rutin [12]. As shown in Fig. [7], the protective action of naringin was stronger (but not significantly) than that of rutin in our infection model. Although post-treatment with CAZ completely blocked the endotoxin lethality, antibiotics did not improve coagulation systems in our model [8]. Thus the use of naringin together with antibiotics would improve the coagulation systems and prevent the development of lethal shock.

Acknowledgements

We would like to thank Yasuaki Higuchi, Kanae Yuasa and Masayuki Itoh for expert technical assistance.

References

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• 20 Kotanidou A, Xagorari A, Bagli E, Kitsanta P, Fotsis T, Papapetropoulos A, Roussos C. Luteolin reduces lipopolysaccharide-induced lethal toxicity and expression of proinflammatory molecules in mice.  Am J Respir Crit Care Med. 2002;  165 818-23
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Dr. Yoshio Kumazawa

Department of Biosciences

School of Science

Kitasato University

1-15-1 Kitasato

Sagamihara

Kanagawa 228-8555

Japan

Phone: +81-42-778-9534

Fax: +81-42-778-9534

Email: kumazawa@jet.sci.kitasato-u.ac.jp

References

• 1 Beutler B, Poltorak A. Positional cloning LPS, and the general role of toll-like receptors in the innate immune response.  Eur Cytokine Network. 2000;  11 143-52
  PubMedSearch in Google Scholar
• 2 Beutler B, Cerami A. Cachectin/tumor necrosis factor: an endogenous mediator of shock and inflammation.  Annu Rev Biochem. 1985;  57 505-16
  PubMedSearch in Google Scholar
• 3 Old L J. Tumor necrosis factor (TNF).  Science. 1985;  230 630-2
  PubMedSearch in Google Scholar
• 4 Hasunuma R, Morita H, Tanaka S, Roland R, Freudenberg M A, Galanos C, Kumazawa Y. Differential clearance and induction of host responses by various administered or released lipopolysaccharides.  J Endotoxin Res. 2001;  7 421-9
  CrossrefPubMedSearch in Google Scholar
• 5 Azumi S, Tanimura A, Tanamato K. A novel inhibition of bacterial endotoxin derived from cinnamon bark.  Biochem Biophys Res Commun. 1997;  234 506-10
  CrossrefPubMedSearch in Google Scholar
• 6 Galanos C, Freudenberg M A, Reutter W. Galactosamine-induced sensitization to the lethal effects of endotoxin.  Proc Natl Acad Sci USA. 1979;  76 5939-43
  PubMedSearch in Google Scholar
• 7 Kawaguchi K, Kikuchi S, Hasegawa H, Maruyama H, Morita H, Kumazawa Y. Suppression of lipopolysaccharide-induced tumor necrosis factor-release and liver injury in mice by naringin.  Eur J Pharmacol. 1999;  368 245-50
  CrossrefPubMedSearch in Google Scholar
• 8 Kawaguchi K, Hasunuma R, Kikuchi S, Roland R, Morikawa K, Kumazawa Y. Time- and dose-dependent effect of fosfomycin on suppression of infection-induced endotoxin shock in mice.  Biol Pharm Bull. 2002;  25 1658-61
  CrossrefPubMedSearch in Google Scholar
• 9 Ng T B, Ling J M, Wang Z T, Cai J N, Xu G J. Examination of coumarins, flavonoids and polysaccharopeptide for antibacterial activity.  Gen Pharmacol. 1996;  27 1237-40
  CrossrefPubMedSearch in Google Scholar
• 10 Morita H, Hasunuma R, Hoshino M, Fujihara M, Tanaka S, Yamamoto S, Kumazawa Y. Difference in clearance of exogenously administered smooth-form LPS following host response among normal, sensitized and LPS-tolerant mice.  J Endotoxin Res. 1997;  4 415-23
  PubMedSearch in Google Scholar
• 11 Wang H, Bloom O, Zhang M, Vishnubhakat J M, Ombrellino M, Che J, Frazier A, Yang H, Ivanova S, Borovikova L, Manogue K R, Faist E, Abraham E, Andersson J, Andersson U, Molina P E, Abumrad N N, Sama A, Tracey K J. HMG-1 as a late mediator of endotoxin lethality in mice.  Science. 1999;  285 248-51
  CrossrefPubMedSearch in Google Scholar
• 12 Takahashi K, Morikawa A, Kato Y, Sugiyama T, Koide N, Mu M M, Yoshida T, Yokochi T. Flavonoids protect mice from two types of lethal shock induced by endotoxin.  FEMS Immunol Med Microbiol. 2001;  31 29-33
  PubMedSearch in Google Scholar
• 13 Mammen E F. The hematological manifestations of sepsis.  J Antimicrob Chemother. 1998;  41 A17-A24
  CrossrefPubMedSearch in Google Scholar
• 14 Yun-Choi H S, Pyo M K, Chang K C, Lee D H. The effects of higenamine on LPS-induced experimental disseminated intravascular coagulation (DIC) in rats.  Planta Med. 2002;  68 326-29
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Lee C H, Jeong T S, Choi Y K, Hyun B H, Oh G T, Kim E H, Kim J R, Han J I, Bok S H. Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits.  Biochem Biophys Res Commun. 2001;  284 681-8
  CrossrefPubMedSearch in Google Scholar
• 16 Kim H K, Cheon B S, Kim Y H, Kim S Y, Kim H P. Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships.  Biochem Pharmacol. 1999;  58 759-65
  CrossrefPubMedSearch in Google Scholar
• 17 Wadsworth T L, McDonald T L, Koop D R. Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced signaling pathways involved in the release of tumor necrosis factor-alpha.  Biochem Pharmacol. 2001;  62 963-74
  CrossrefPubMedSearch in Google Scholar
• 18 Xagorari A, Roussos C, Papapetropoulos A. Inhibition of LPS-stimulated pathways in macrophages by the flavonoid luteolin.  Br J Pharmacol. 2002;  136 1058-64
  CrossrefPubMedSearch in Google Scholar
• 19 Van Dien M, Takahashi K, Mu M M, Koide N, Sugiyama T, Mori I, Yoshida T, Yokochi T. Protective effect of wogonin on endotoxin-induced lethal shock in D-galactosamine-sensitized mice.  Microbiol Immunol. 2001;  45 751-6
  PubMedSearch in Google Scholar
• 20 Kotanidou A, Xagorari A, Bagli E, Kitsanta P, Fotsis T, Papapetropoulos A, Roussos C. Luteolin reduces lipopolysaccharide-induced lethal toxicity and expression of proinflammatory molecules in mice.  Am J Respir Crit Care Med. 2002;  165 818-23
  PubMedSearch in Google Scholar

Dr. Yoshio Kumazawa

Department of Biosciences

School of Science

Kitasato University

1-15-1 Kitasato

Sagamihara

Kanagawa 228-8555

Japan

Phone: +81-42-778-9534

Fax: +81-42-778-9534

Email: kumazawa@jet.sci.kitasato-u.ac.jp