Inhibition of α-toxin production by subinhibitory concentrations of naringenin controls Staphylococcus aureus pneumonia


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Yu Zhang a,1, Jian-feng Wang a,1, Jing Dong a, Jing-yuan Wei b, Ya-nan Wang a, Xiao-han Dai a, Xin Wang c, Ming-jing Luo a, Wei Tan a, Xu-ming Deng a,⁎, Xiao-di Niu c,⁎⁎ a Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China b Liaoning Province Academy of Analytic Science, Shenyang 110015, PR China c College of Quartermaster Technology, Jilin University, Changchun 130062, PR China a r t i c l e i n f o a b s t r a c t Article history: Received 29 June 2012 Accepted in revised form 24 January 2013 Available online 17 February 2013 Staphylococcal pneumonia provoked by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening infection in which α-toxin is an essential virulence factor. In this study, we investigate the influence of naringenin on α-toxin production and further assess its therapeutic performance in the treatment of staphylococcal pneumonia. Remarkably, the expression of α-toxin was significantly inhibited when the organism was treated with 16 μg/ml of naringenin. When studied in a mouse model of S. aureus pneumonia, naringenin could attenuate the symptoms of lung injury and inflammation in infected mice. These results suggest that naringenin is a promising agent for treatment of S. aureus infection. © 2013 Elsevier B.V. All rights reserved. Keywords: Staphylococcus aureus α-Toxin Pneumonia Naringenin 1. Introduction Staphylococcus aureus is a commensal pathogen that evokes human and animal diseases ranging from atopic dermatitis to soft-tissue infections, and even more invasive illnesses such as bacteremia, pneumonia and osteomyelitis, which have high morbidity and mortality [1,2]. In 1961, only 1 year after the introduction of methicillin into clinical practice, methicillinresistant S. aureus (MRSA) was first described in the United Kingdom as a hospital-acquired pathogen [3]. Over 50% of staphylococcal pneumonia isolates are classified as MRSA, and more than one-half of the patients who contract this pathogenic bacteria struggle to get well, even in this day and age. In recent years, due to the limited therapeutic options available for the treatment of MRSA infections as well as the decline in efficiency of development of new antibiotics with novel mechanisms of action, there is a clear need for novel therapeutic strategies against staphylococcal infections. The ability of S. aureus to cause diseases depends on its production of a series of surface-related and secreted virulence factors. The 33.2-kDa α-toxin, one of the major exotoxins of S. aureus, is a member of the β-barrel pore-forming toxin (PFT) family, members of which are secreted as water-soluble monomeric proteins [4]. The monomer assembles into a stable homoheptameric transmembrane pore that creates a 2-nm internal diameter hole through the membranes of susceptible cells (e.g., lymphocytes, macrophages, alveolar epithelial cells, pulmonary endothelium, and erythrocytes) [5,6]. This new pore leads to the rapid egress of vital molecules, thereby causing cell lysis and death. Bubeck Wardenburg et al. [7] reported that α-toxin is essential for the pathogenesis of S. aureus infections, especially in staphylococcal pneumonia. S. aureus mutant strains lacking hla (the gene encoding α-toxin) cannot induce Fitoterapia 86 (2013) 92–99 ⁎ Correspondence to: X.-M. Deng, Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Xi'an Road 5333#, Changchun 130062, China. Tel.: +86 431 87836161; fax: +86 431 87836160. ⁎⁎ Correspondence to: X.-D. Niu, College of Quartermaster Technology, Jilin University, Xi'an Road 5333#, Changchun 130062, PR China. Tel.: +86 431 87836161; fax: +86 431 87836160. E-mail addresses: (X. Deng), (X. Niu). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: neutrophil-mediated inflammatory lung injury in the murine model of this disease [8]. Naringenin (Fig. 1) is one of the flavonoids present in grapefruits and tomatoes, and it has a wide range of pharmacological properties, including anti-oxidant, anti-fibration, anti-cancer, anti-atherogenic and anti-proliferative activities [9]. Kanno S. et al. have shown that naringenin can induce cytotoxicity in various human cancer cell lines as well as apoptosis in human colon cancer Caco-2 cells and promyelocytic leukemia HL-60 cells [10]. Naringenin has also been shown to dose-dependently inhibit the assembly and long-term production of infectious hepatitis C virus particles [11]. Additionally, Bodet C. has employed two different models (both in vitro and ex vivo experiments) to investigate the capacity of naringenin to inhibit the LPS-induced inflammatory response [12]. In our current research, we elucidate the influence of naringenin on the α-toxin expression of S. aureus in vitro and further examine the in vivo performance of naringenin in the treatment of S. aureus pneumonia. 2. Materials and methods 2.1. Bacterial strains, growth conditions, and reagents S. aureus strains involved in this study are described in Table 1. For haemolysis, western blot and real-time RT-PCR assays, the strains were grown in tryptic soy broth (TSB) at 37 °C, supplemented with naringenin as required, and harvested at the post-exponential phase with OD600 nm of 2.5, 2.0, 2.7, 2.5 and 2.5 for strains ATCC 29213, wood 46, USA 300, 8325-4 and DU 1090 respectively. For cytotoxicity and in vivo studies, 8325-4 and DU1090 were grown in TSB at 37 °C to an OD600 nm of 0.5. Five-milliliters of the culture described above was resuspended in 10 ml of DMEM medium (Invitrogen, CA) for cytotoxicity studies, and fifty-milliliters of culture aliquots was centrifuged, washed with PBS and resuspended in 1 ml PBS (2×108 CFU per 30 μl) for in vivo assay. Naringenin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and it was dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich) to make a stock solution for in vitro studies. Naringenin was suspended in PBS for in vivo assay. 2.2. Minimal inhibitory concentrations (MICs) determination The minimal inhibitory concentrations of naringenin for S. aureus were determined using the broth microdilution method described by the CLSI (2005) [13]. Oxacillin was used as a positive control. 2.3. Haemolysis assay Haemolytic activity was measured as described previously [14]. Briefly, culture supernatants were collected by centrifugation (5,500 ×g at 4 °C for 1 min) and filter sterilized with a 0.22 μm (pore-size) acetate syringe filter. The reaction mixture was incubated at 37 °C including 100 μl supernatant described above, 25 μl defibrinated rabbit erythrocytes (v/v 2.5%) and 875 μl phosphate-buffered saline (PBS) for 30 min. The supernatants were collected by centrifugation (5,500 ×g at room temperature for 1 min). Then the haemolytic activity was evaluated with the values of cell-free supernatants at the OD543nm. 2.4. Western blot assay A 20 μl volume of boiled culture supernatants were applied to SDS–PAGE. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After blocked in 5% bovine serum albumin (Wako) for 2 h, the membranes were incubated with the rabbit polyclonal antibody to α-toxin (Sigma-Aldrich) diluted 1:4000 overnight at 4 °C. Then the membranes were stained with horseradish peroxidase-conjugated anti-rabbit antiserum (1:5000 dilution; Sigma–Aldrich) used as the secondary antibody. The blot was then developed using Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK). 2.5. Real-time RT-PCR S. aureus 8325-4 was cultured in TSB with or without various dose of naringenin to the post-exponential growth phase (OD600nm of 2.5). Bacterial cells were pelleted (5,000×g 5 min, 4 °C) and immediately resuspended in TES buffer containing 100 μg/ml lysostaphin (Sigma-Aldrich) and Table 1 Bacterial strains used in the study and their MICs to naringenin. S. aureus strains Description Source MIC (μg/ml) Oxacillin Naringenin ATCC 29213 MSSA, α-toxin and β-lactamase producing strain ATCC 0.25 256 ATCC 10832 Wood 46, a natural isolate that produces high levels of α-toxin ATCC 0.125 256 BAA-1717 USA 300, isolated from adolescent patient with severe sepsis syndrome in Texas Children's Hospital, α-toxinproducing strain ATCC 128 512 8325-4 A high-level α-toxin-producing strain derived from NCTC 8325 Timothy J. Foster 0.125 256 DU 1090 8325-4 defective in α-toxin, prepared by insertion of a transposon in the hla gene Timothy J. Foster 0.125 256 Fig. 1. Chemical structure of naringenin. Y. Zhang et al. / Fitoterapia 86 (2013) 92–99 93 incubated at 37 °C for 10 min. The method of isolating RNA was described by Sambanthamoorthy [15]. Total bacterial RNA was isolated using Qiagen RNeasy Maxi columns and contaminating DNA was removed using the RNase-free DNase I (Qiagen, Hilden, Germany). The quality, integrity, and concentration of the purified RNA were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), according to the manufacturer's protocol. The primer pairs used in real-time RT-PCR are listed in Table 2. RNA was reverse transcribed into cDNA using the Takara RNA PCR kit (AMV) version 3.0 (Takara, Kyoto, Japan). The PCRs were performed using the model 7000 sequence detection system (Applied Biosystems, Courtaboeuf, France) in a 25 μl volume contained SYBRPremix Ex TaqTM (Takara) as recommended by the manufacturer. All samples were analysed in triplicate, and the housekeeping gene 16S rRNA was used as an endogenous control. In this study, expression of the target gene relative to 16S rRNA was used to determine changes in transcription levels between samples. 2.6. Live/dead and cytotoxicity assays A549 human lung epithelial cells (ATCC CCL 185) were cultured in DMEM medium supplemented with 10% heatinactivated fetal bovine serum (Invitrogen, CA, USA) and 100 μg/ml of penicillin–streptomycin. For live/dead assay, the cells were seeded 2.0×104 cells per well in 96-well over 12 h before co-cultured with 100 μl of staphylococcal suspension per well in DMEM medium in triplicate wells. After incubated at 37 °C for 8 h, cells were treated with live/dead (green/red) reagent (Invitrogen). For LDH activity detection, the cells were seeded 1.5×104 cells per well for 12 h and incubated with bacterial suspension at 37 °C for 6 h in DMEM without fetal bovine serum. Cell viability was determined by measuring lactate dehydrogenase (LDH) release using Cytotoxicity Detection Kit (LDH) (Roche, Basel, Switzerland) according to the manufacturer's directions. Microscopic images of stained cells were captured using a confocal laser scanning microscope (Nikon, Tokyo, Japan). LDH activity was measured on a microplate reader (TECAN, Salzburg, Austria). 2.7. Pharmacokinetics study Mice were handled according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University. Eight-week-old C57BL/6J mice were obtained from the Experimental Animal Center of Jilin University (Changchun, China). For pharmacokinetics study, mice were treated with subcutaneous injection of 100 mg/kg of naringenin in sterile PBS. Mice were sacrificed in a CO2 chamber 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h after dosing. Blood samples were collected by cardiac puncture. Serum concentrations were determined using the WINNONLIN program (Pharsight, Mountain View, CA). 2.8. Mouse model of intranasal lung infection For lung infection, 8-week-old female C57BL/6J mice were anaesthetized with ketamine and xylazine before infection and each experimental group contained 20 mice. Anesthetized mice were held in the vertical position, and 30 μl of S. aureus suspension was deposited in the left nare until the inoculum was aspirated into the lungs. Mice were treated with subcutaneous injection of 100 mg/kg of naringenin 2 h after infection and then at 12 h intervals. The control mice were treated with 100 μl sterile PBS on the same schedule. Infected animals were killed with anaesthesia followed by cervical dislocation 24 h post-infection. The lungs were placed in 10% formalin. Formalin-fixed tissues were embedded in paraffin, stained with hematoxylin-eosin, and analysed by light microscopy. 2.9. Statistical analysis Statistical significance was determined using independent Student's t-test with spss 12.0 statistical software (SPSS Inc., Chicago, IL, USA), and a P-valueb0.05 indicated statistically significant. 3. Results 3.1. Naringenin inhibits the haemolysis by decreasing the production of α-toxin In this study, the antibacterial activity of naringenin against S. aureus was evaluated and listed in Table 1. The MICs of naringenin against S. aureus strains were 256–512 μg/ml, which indicated that naringenin has little anti-S. aureus activity. Haemolysis assay was employed to elucidate the effect of naringenin on α-toxin production. When cultured with 16 μg/ml naringenin, the haemolytic activities of the culture supernatants from S. aureus strain ATCC 29213, ATCC 10832, and 8325-4 were degraded to 5.79, 9.27 and 4.91% of their control group grown without drug respectively (Table 3). Remarkably, when S. aureus strain USA 300 was treated with 4 μg/ml naringenin, there is no detectable haemolytic activities in the supernatant. Western blot analysis was performed to examine whether the concentration-dependent decreasing of the haemolytic activity in the supernatant caused by naringenin were attributed to the reduced expression of α-toxin. As expected, the results of western blot were coincident with the haemolysis assay analysis, which there was a measurable reduction of α-toxin from supernatant treated by naringenin. Furthermore, no immunoreactive α-toxin antigen could be detected in the supernatant of the S. aureus strain 8325-4 and USA 300 with 16 μg/ml and 2 μg/ml naringenin, respectively (Fig. 2A and B). To evaluate the transcriptional level of hla from S. aureus 8325-4 in the presence of increasing concentrations of Table 2 Primers used in real-time RT-PCR. Primer Sequence Location within gene 16S rRNA-forward 5′-GCTGCCCTTTGTATTGTC-3′ 287–305 16S rRNA-reverse 5′-AGATGTTGGGTTAAGTCCC-3′ 446–465 hla-forward 5′-TTGGTGCAAATGTTTC-3′ 485–501 hla-reverse 5′-TCACTTTCCAGCCTACT-3′ 569–586 agrA-forward 5′-TGATAATCCTTATGAGGTGCTT-3′ 111–133 agrA-reverse 5′-CACTGTGACTCGTAACGAAAA-3′ 253–274 94 Y. Zhang et al. / Fitoterapia 86 (2013) 92–99 naringenin, real time RT-PCR was performed to quantify the mRNA levels of the investigated genes. Moreover, due to the expression of α-toxin was positively regulated by the Agr two-component regulatory system, the transcript level of agrA also determined. As shown in Fig. 2C, When exposed to of 16 μg/ml naringenin, the transcriptional levels of hla and agrA were reduced by 11.9 and 7.9, respectively. 3.2. Naringenin attenuate α-toxin-mediated injury of human alveolar epithelial cells Bubeck Wardenburg and Schneewind have previously demonstrated the significance of α-toxin in human alveolar cell injury, as S. aureus strains lacking α-toxin do not cause cellular injury [7]. According to the above mentioned results that naringenin reduced the production of staphylococcal α-toxin, we further investigated the effects of naringenin on protecting A549 cells from α-toxin-mediated injury in the A549 cell and S. aureus co-culture system. As shown in Fig. 3A, upon co-cultured with S. aureus 8325-4, A549 cell injury was apparent, as displayed by an increased number of red fluorescent cells. Remarkably, the addition of 16 μg/ml naringenin in the system provided significant protection of A549 injury and death, as indicated by a significant reduction in red fluorescence (Fig. 3D). The protection of naringenin against A549 from injury was further quantified via detecting the LDH release in the co-culture system. As shown in Fig. 3G, a dose-dependent reduction in LDH levels was observed following the treatment of increasing concentrations of naringenin, and the calculated 50% inhibitory concentration of naringenin was 9.22 μg/ml. 3.3. Naringenin protects mice from S. aureus pneumonia Given that naringenin protected A549 cells from α-toxinmediated injury, we further tested its therapeutic performance in treatment of S. aureus pneumonia. Time-concentration profiles of plasma for the subcutaneous naringenin dose are shown in Fig. 4. After treated with 100 mg/kg naringenin, the maximum concentration of naringenin in plasma (Cmax) was 26.04 μg/ml. The area under the concentration-time curves (AUC) for plasma calculated from 0.25 to 24 h was 391.78 mg/kg. The mice were infected intranasally (i.n.) with S. aureus strains. Following receiving naringenin or PBS control, the lung tissue was detached for histopathological analysis. As shown in Fig. 5A, the lung tissues of infected mice that received PBS revealed extreme sign of lung injury with tight texture and widespread hyperemia; while treated with 100 mg/kg naringenin, the lung tissues are resemble to normal light pink and spongy except for few focal destruction and tissue inflammation. The lung tissue sections of PBS-treated mice indicated widely consolidation, destruction of alveolar structure, and infiltration of numerous inflammatory cells in pulmonary alveolus or bronchioles. In contrast, the lung tissue of mice treated with naringenin resulted in a marked alleviation of pulmonary inflammation as indicated by less inflammatory cells infiltration (Fig. 5B). 4. Discussion Unquestionably, community and healthcare-associated infections caused by bacterial strains resistant to single or multiple antibiotics are increasing worldwide. Antimicrobial resistance has definitely become the greatest challenge of the 21st century. There are complex and multifactorial causes that have contributed to the current dilemma, of which the overuse and abuse of antibiotics in humans and animals are among the most important [16,17]. Traditional antibiotics are bactericidal or bacteriostatic agents targeting the key components that are essential for the growth of bacteria (such as cell wall synthesis, DNA replication and protein synthesis). Such agents create significant evolutionary pressure for the bacteria, which may ultimately result in the development of antibiotic resistance. Even more worrisome, the increase in antimicrobial resistance has been coupled with a decrease in the resources dedicated by pharmaceutical companies to discovering novel antibiotics [18]. In fact, only a few new antimicrobials have been successfully applied in the clinic during the past three decades [19,20]. The battles between humans and bacterial resistance are ongoing, and alternative strategies are urgently needed. Our increasing understanding of bacterial pathogenesis has revealed many novel anti-infective strategies that could be used to control bacterial infections, including the antivirulence strategy. It is well-known that the pathogenicity of bacteria depends largely on their virulence factors. Consequently, this class of anti-virulence drugs aims to lessen bacterial virulence and, therefore, pathogenicity. Rasko D.A. and Sperandio V. [21] review the common anti-virulence targets and approaches as well as the chemical structures of anti-virulence compounds in development. MRSA, one of the “superbugs,” is among the most harmful antimicrobial-resistant pathogens. The fact that MRSA has grown resistant to almost all conventional antibiotics impels the development of an anti-virulence drug for the treatment Table 3 Heamolytic activity of S. aureus culture supernatants grown in the presence of increasing concentrations of naringenin. S. aureus strains Heamolysis (%) of rabbit erythrocytes by culture supernatanta 0 1 μg/ml 2 μg/ml 4 μg/ml 8 μg/ml 16 μg/ml ATCC 29213 100% 97.67%±1.76 79.23%±1.97 43.83%±2.22⁎ 25.46%±3.78⁎⁎ 5.79%±3.51⁎⁎ ATCC 10832 100% 96.96%±2.25 83.95%±1.96 56.82%±1.45⁎ 24.96%±1.71⁎⁎ 9.27%±2.29⁎⁎ 8325-4 100% 80.60%±1.17 65.57%±3.26⁎ 43.00%±1.77⁎⁎ 13.13%±2.73⁎⁎ 4.91%±2.91⁎⁎ USA 300 100% 60.62%±2.39⁎ 13.72%±1.81⁎⁎ Noneb Noneb Noneb Values represent means±SD (n=3). * indicates pb0.05 and ** indicates pb0.01 compared to the corresponding control. a Haemolytic activity in untreated S. aureus culture supernatants was set to 100%. b No haemolytic activity was observed. Y. Zhang et al. / Fitoterapia 86 (2013) 92–99 95 of these infections. The determination of virulence targets is of primary importance. α-Toxin is one of the major exotoxins secreted by most S. aureus strains. Previous studies have addressed the importance of α-toxin in S. aureus infections, including peritonitis, septic arthritis, mastitis, and many other diseases [6,22,23]. In particular, α-toxin has been proven to play an essential role in the pathogenesis of S. aureus pneumonia, as strains lacking the ability to produce α-toxin are avirulent in a mouse model of that disease [24]. Based on these findings, α-toxin has been suggested to be a promising anti-virulence target of S. aureus. Recently, Bubeck Wardenburg and colleagues demonstrated the validity of treating staphylococcal pneumonia by blocking the function of α-toxin [25]. Newly discovered synthetic or natural Fig. 2. The production of α-toxin was reduced by naringenin. Western blotting was employed to determine the α-toxin production by S. aureus strain 8325-4 (A) and USA 300 (B) after growth with various concentrations of naringenin (lanes 1–5); lane 6, 10 ng of purified α-toxin as control. (C) Relative gene expression of hla and agrA in S. aureus strain 8325-4 exposed to different concentrations of naringenin. Data are presented as the average±SD of three independent experiments. * indicates Pb0.05 and ** indicates Pb0.01. 96 Y. Zhang et al. / Fitoterapia 86 (2013) 92–99 compounds that are demonstrated to possess anti-virulence properties will impel the development of anti-virulence drugs [26,27]. Our previous findings have also shown that some natural compounds can protect mice from S. aureus pneumonia by reducing the production of α-toxin. Comparing to the traditional antibiotics, these natural compounds supposed to fight against the virulence factors of the bacteria offer a differential traits which could not directly kill the bacteria to presumably apply a milder evolutionary pressure on the development of resistance [21]. Meanwhile, the reduction of α-toxin expression after treatment with the compounds could weaken the ability to colonize the host and potentially lighten the pathological changes which are mainly induced by this toxin. Theoretically, this strategy could make host immune system's profit of clearance of S. aureus, and, furthermore, Todd M. Jarry has certified the essentiality of α-toxin of S. aureus to escape from the endocytic vesicle into the cytosol of CFT-1 cells [28]. In this study, little antibacterial activity of naringenin against S. aureus was observed, while the haemolytic activity of supernatant was significantly inhibited after treatment with naringenin. Based on these data, the secretion of α-toxin in supernatant and the transcriptional level of the hla gene were further investigated. Coincident with attenuating the Fig. 3. Naringenin attenuate α-toxin-mediated injury of human alveolar epithelial cells. Human A549 cells were imaged with confocal laser scanning microscopy 8 h after infection with S. aureus 8325-4. Cells were co-cultured with S. aureus 8325-4(A); co-cultured with S. aureus 8325-4 in various concentrations of naringenin (B, C, D and E); uninfected (F). (G) LDH release by A549 cells was determined using cells co-cultured with S. aureus 8325-4 supplemented with the indicated concentrations of naringenin. The values in (G) represent the average±SD of three independent experiments. * indicates Pb0.05 and ** indicate Pb0.01 as compared with the naringenin-free co-culture. Y. Zhang et al. / Fitoterapia 86 (2013) 92–99 97 haemolytic activity in supernatant, naringenin could dosedependently reduce the production of α-toxin and inhibit the transcription of the hla and agrA gene. Previous studies have noted that the expression of several exotoxins, such as α-toxin, enterotoxin B, and toxic shock syndrome toxin-1, is decreased whereas expression of many surface proteins is increased in agr mutant [29]. The agr locus, which consists of five genes (agrA, agrC, agrD, agrB, and hld), codes for two divergent transcripts (RNAII and RNAIII) initiated by two distinct promoters (P2 and P3). RNA II transcript encodes four proteins, AgrB, AgrD, AgrC and AgrA. AgrD, an auto inducer propetide (AIP), is recognized by membrane receptor AgrC. And phosphorylated AgrC may subsequently phosphorylate agrA. P2 and P3 are both thought to be autocatalytically activated by phosphorylated agrA. RNAII and RNAIII are driven by the P2 and P3 promoters, respectively. AgrB, produced by RNAII, is a membrane protease which could support AgrD to be processed and secreted. The translation of the RNA III effector messenger exerts both stimulatory and inhibitory effects on the expression of various other genes [30]. Consequently, the transcription of agrA and hla was inhibited by naringenin in a dose-dependent manner and the naringenin-induced decrease in α-toxin production may be partially dependent on this molecular mechanism. Given that the pharmacokinetics and toxicology of potential agents are important considerations when treating human infections, we first assessed the pharmacokinetics of naringenin in mice, which revealed that after treatment with subcutaneous injection of 100 mg/kg naringenin, the maximum serum Fig. 4. Serum concentrations of naringenin in mice after a single subcutaneous dose of naringenin. Blood samples were collected at different time points after subcutaneous dose of 100 mg/kg naringenin. The serum concentrations were fit to a standard curve using the WinNonlin program. Fig. 5. Naringenin protection against S. aureus pneumonia. (A) Gross pathological changes of S. aureus-infected lung tissue from mice treated with PBS or naringenin. (B) Histopathology of S. aureus-infected lung tissue from mice: aggregates of purple-stained immune cells and dense accumulation of bacteria were seen in lung tissue infected with S. aureus 8325-4; open alveolus with significant decreases of immune cells in naringenin-treated or S. aureus DU 1090-infected lung tissue. 98 Y. Zhang et al. / Fitoterapia 86 (2013) 92–99 concentration of naringenin in plasma (Cmax) was 26.04 μg/ml, which is higher than the activity value of naringenin (16 μg/ml). Furthermore, R. R. Ortiz-Andrade have showed a oral LD50 >5000 mg/kg in rats and, therefore, naringenin can be considered as a non-toxic candidate for future drug development [31]. Following treatment with naringenin, gross and histopathological changes were remarkably alleviated, as indicated by light pink and fungous on the surface of lung and less accumulations of inflammatory cells (dark blue or purple) and cellular infiltrates in alveolar space. Although the clinical relevance of naringenin is limited by its low solubility and minimal bioavailability owing to its largely hydrophobic ring structure, Maria Shulman has reported that complexation with hydroxypropoyl-b-cyclodextrin, the bioavailability of naringenin could be enhanced by strengthening the solubility and enteral uptake of the flavonoid [32]. The data suggested that naringenin could be a potential agent in the treatment of S. aureus infections. Anti-virulence drugs are recommended to be used in combination with conventional antimicrobials to extend the useful lifespan of the antibiotics [33,34]. 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