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Protective effects of antimicrobial peptide S-thanatin against endotoxic shock in mice introduced by LPS
Peptides 32 (2011) 353–357
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Protective effects of antimicrobial peptide S-thanatin against endotoxic shock in mice introduced by LPS Guoqiu Wu a,∗,1 , Xiaofang Li b,1 , Xuepeng Deng b , Xiaobo Fan b , Shenglan Wang b , Zilong Shen b , Tao Xi b,∗∗ a b
Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, PR China Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Tongjiaxiang 24#, Nanjing 210009, PR China
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 25 October 2010 Accepted 25 October 2010 Available online 2 November 2010 Keywords: Thanatin Antimicrobial peptide Endotoxic shock Lipopolysaccharide TNF-␣
a b s t r a c t Sepsis continues to be a major unresolved medical challenge of the present. Severe sepsis and septic shock are the leading causes of multiple organ failure and mortality in noncoronary intensive care units (ICUs). The primary reason of septic shock is the activation of host effecter cells by endotoxin and lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the key point of treatment is removing LPS. S-thanatin (Ts), an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against gramnegative and gram-positive bacteria. We have reported its LPS-binding and -neutralizing activity in vitro. The aim of this study is to examine the LPS-neutralizing activities and the protective effects of S-thanatin in vivo. Every mice was injected intraperitoneally with LPS (from Escherichia coli O111:B4) 150 g before injected intraperitoneally or vena caudalis with 3 mg/kg, 6 mg/kg and 12 mg/kg, and measured endotoxin and tumor necrosis factor alpha (TNF-␣) concentrations in plasma, as well as lethality. The results showed that S-thanatin can significantly reduce endotoxin and TNF-␣ level in plasma, at the same time resulting in the highest survival rates. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Severe sepsis, defined as sepsis with acute organ dysfunction, causes systemic inflammation and is associated with high morbidity and mortality rates [1]. It was estimated that there would be 934,000 cases per year by 2010 [2]. In the United States, approximately 750,000 cases of severe sepsis occur each year, with a mortality rate ranging from 30% to 50% despite advances in critical care management [3,4]. Pathogenesis of the development on sepsis is highly complicated and has been the object of study for many years. The best-characterized bacterial-derived antigen is endotoxin or bacterial lipopolysaccharide (LPS) [17]. LPS is a molecule derived from the outer membrane of gram negative bacteria, such as Escherichia coli and Salmonella typhimurium, and it is the bestcharacterized bacterial antigen in immunology [17]. Incidence of sepsis due to gram-positive bacteria increased from 10% in the 1960s and 1970s to 30% in subsequent years [15].
∗ Corresponding author. Tel.: +86 02583272355x808. ∗∗ Corresponding author. Tel.: +86 02583271389. E-mail addresses: guoqiu [email protected] (G. Wu), xitao [email protected] (T. Xi). 1 These two people contributed equally to this work. 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.10.029
Antibiotic therapy on septic shock is commonly used. Every patient who has sepsis and septic shock must be evaluated appropriately before the initiation of antibiotic therapy. The choice of empirical antibiotics depends on complex issues related to the patient’s history including drug intolerances, underlying disease, the clinical syndrome, and susceptibility patterns of pathogens in the community, in the hospital, and that previously have been documented to colonize or infect the patient. There is an especially wide range of potential pathogens for neutropenic patients [9]. There is ample evidence that failure to initiate appropriate therapy correlates with increased morbidity and mortality [15]. Usually antibiotics are used as medicine against bacterial infection, and they can stimulate the release of an excess amount of a bacterial outer membrane component, endotoxin [10]. The released endotoxin activates macrophages, endothelial cells and fibroblasts to produce and release potent inflammatory mediators including tumor necrosis factor-␣ (TNF-␣), interleukin 1-beta (IL-), interleukin-6 (IL-6) and nitric oxide [17]. TNF-␣ is a pro-inflammatory cytokine, which is regarded as an important mediator of sepsis. Serum levels have been shown to correlate with outcome in both animal and human models of sepsis [7,19]. At the same time they may light a cascade of uncontrolled systemic inflammatory responses that can lead to multiple organ failure and eventually to death [17]. In other words, antimicrobial therapy may promote the further release of LPS from the cell envelope of killed
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bacteria, exacerbating sepsis itself [6]. Because of that, the requirement for novel compounds which are able to neutralize the effects of endotoxin is urgently pursued. Antimicrobial peptides (AMPs) have emerged as ideal candidates to be used as adjuncts during antibiotic therapy, not only for their broad-spectrum activity against gram-positive and gram negative bacteria, yeasts, fungi, and enveloped viruses [8,13], but also for their ability to efficiently bind LPS and block LPS-dependent cytokine induction [20,21]. They are crucial humoral components of the innate immunity system of virtually all organisms, which they defend from the invasion of attacking pathogens [5,11,12]. Thanatin contains 21 amino acids (GSKKPIIYCNRRTGKCQRM), which was isolated from the hemipteran insect Podisus maculiventris [14]. It is the first inducible insect peptide that has a broad range of activity against bacteria and fungi at physiological concentrations, and an anti-parallel -sheet structure from 8th amino acid to the C-terminus [18]. S-thanatin (GSKKPVPIIYCNRRSGKCQRM), a novel thanatin analog, which was substituted threonine at position 15 with serine [25]. Our previous studies showed that S-thanatin has a broadspectrum antimicrobial activity, especially to gram-negative bacteria [27]. Previously, we have reported the antimicrobial activity against a multidrug-resistant (MDR) clinical isolate about S-thanatin and we have assessed the LPS-binding and -neutralizing activities of S-thanatin in vitro [25]. The present study was designed to further verify the LPS-neutralizing activity of S-thanatin in vivo.
2. Materials and methods 2.1. Animals Adult male ICR mice (28–31 g) were obtained from the Animal Center, Science Academy of China (Shanghai, China). All animals were housed in individual cages and acclimatized for 1 week in Animal Environmental Control Unit (temperature, 23 ± 3 ◦ C; relative humidity, 50 ± 10%; light–dark cycle, 12 h).
2.2. Reagents LPS from E. coli serotype O111:B4 was purchased from Sigma–Aldrich (St Louis, MO), prepared in sterile saline, aliquoted, and stored at −80 ◦ C for short periods. The mouse lipopolysaccharide (LPS) ELISA Kit and mouse tumor necrosis factor ␣ (TNF-␣) ELISA Kit were purchased from R&D, and all other chemicals used were of reagent grade.
2.3. Preparation of S-thanatin S-thanatin was synthesized by the solid-phase methodology with a 9-fluorenyl-methoxycarbonyl (Fmoc) protecting group [24]. Peptides were cleaved from the resin with a solution containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane. After repeating precipitation with diethyl ether, the peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using an appropriate 0–60% acetonitrile gradient in 0.05% trifluoroacetic acid. Molecular mass was determined by electrospray mass spectrometry using an API instrument (Perkin Elmer SCIEX) as a quality control of the synthesis [24]. The peptide was taken up in oxidation buffer (1 mg/1 ml) [100 mM ammonium acetate (pH 8.5)], allowed to refold for 3 days at room temperature under stirring and purified by RP-HPLC. S-thanatin was kept in −80 ◦ C after freeze-dried.
2.4. Microorganisms For antimicrobial assays of S-thanatin experiments, a commercially available quality control strain of E. coli ATCC 25922 and Klebsiella pneumoniae ATCC700603, they were obtained from the Center of Clinical Laboratory, Health Minister of PRC, were purchased and stored in 10% glycerol at −80 ◦ C. 2.5. Evaluation of antibacterial activities Constant broth dilution method was used according to the outline by the Clinical and Laboratory Standards Institute [16]. In order to avoid cationic peptides binding with polystyrene we used constant broth dilution method with glass tubes. The minimum inhibitory concentration (MIC) was taken as the lowest antibiotic concentration at which observable growth was inhibited. Exponentially growing bacteria at 108 CFU/ml, then diluted the bacterial suspension with MH broth in 1:100 dilution. They were incubated with different concentrations of S-thanatin in fresh MH broth at 37 ◦ C for 10 h. In this solution the concentration of S-thanatin was 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 g/ml in glass tubes 1–10, respectively. 2.6. LPS challenge and treatments Under the experiment condition (1), five groups, each containing 10 animals. Group 1 was negative control group without any treatment. Animals were injected intraperitoneally with 150 l sodium chloride solution twice. Group 2 were challenge control group injected intraperitoneally with 12 mg/kg of E. coli LPS in a total volume of 150 l sterile saline. Immediately after injection, animals received intraperitoneally isotonic sodium chloride solution. Groups 3–5 as the three treatment groups, the animals were injected intraperitoneally with 150 g E. coli LPS in a total volume of 150 l sterile saline followed by injection of 3 mg/kg, 6 mg/kg or 12 mg/kg of S-thanatin intraperitioneally. Under the experiment condition (2), another five groups were used for different administrations of S-thanatin via intravenous without any other difference in comparison with the first five groups. 2.7. Evaluation of in vivo treatment After treatment, the animals were returned to individual cages and thoroughly examined daily. Depending on the specific experiment, the rate of lethality, and the levels of plasma endotoxin and tumor necrosis factor alpha (TNF-␣) were evaluated. For determination of endotoxin and TNF-␣ contents in plasma, 100 l blood samples were collected from the post-global veins by glass capillary after 0, 2, 6, and 12 h after injection into a sterile syringe and transferred to tubes containing heparin. They were measured with commercially available solid-phase sandwich enzyme-linked immunosorbent assay according to the protocol supplied by the manufacturer. 2.8. Statistical analysis MICs are presented as average values from three independent measurements. Mortality rates differences between groups were compared by Fisher’s exact test (significance level fixed at 0.05). TNF-␣ and LPS mean values in plasma were presented as means ± standard deviations (SDs) of the mean; statistical comparisons between groups were made by analysis of variance (significance level was fixed at 0.05). The post hoc comparisons were performed by the Bonferroni method. Each comparison group
G. Wu et al. / Peptides 32 (2011) 353–357
Fig. 1. Survival rates in a mice model after intraperitoneal administration of 150 g of O111:B4 LPS, and injected intraperitoneally with S-thanatin, then monitored for 72 h post injection. Ts, S-thanatin.
contained 10 animals. Significance was accepted when the P value was 0.05. 3. Results 3.1. Antimicrobial assays in vitro studies
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Fig. 3. Effect of S-thanatin on TNF-␣ production in mice challenged with O111:B4. Mice were untreated, injected intraperitoneally with LPS or 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of TNF-␣ was measured at 0, 2, 6 and 12 h after LPS and/or S-thanatin injection. All data represent mean values and standard errors. P < 0.05, when compared to LPS challenge group.
in the groups treated with 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin intraperitoneally or injected intravenously. 3.3. Effect of S-thanatin on plasma TNF-˛ in mice
A preliminary screening was performed to verify whether Sthanatin has biological activity. According to the broth dilution method, E. coli ATCC 25922 and K. pneumoniae ATCC700603 showed different susceptibilities to S-thanatin. The MICs of S-thanatin were 4–8 g/ml and 8 g/ml. That proved the biological activity of S-thanatin, and the following experiments could be carried out.
The concentrations of plasma TNF-␣ in mice challenged with LPS were 167.45 ± 38.24 g/L. In contrast, those of mice challenged with LPS and treated with S-thanatin were 109.24 ± 18.56 g/L, 90.46 ± 9.87 g/L and 86.27 ± 8.64 g/L when measured at 6 h after challenge of LPS, and we give out the changing trends of TNF-␣ levels, when measured at 0, 2, 6, and 12 h after challenge of LPS (Fig. 3). When we intravenously inject S-thanatin as the route of administration we got a similar result (Fig. 4).
3.2. Effects of S-thanatin on LPS challenged mice
3.4. Effect of S-thanatin on plasma endotoxin in mice
After the mice were challenged with LPS, the control group developed the symptom of typical septic shock, and the symptom of treatment is eased. Mice of lethality at 72 h in the challenge control group were 100% and 90%, respectively, whereas treatment with S-thanatin decreased LPS-challenged lethality (Figs. 1 and 2). All groups administered S-thanatin following LPS challenge showed increased survival in comparison with the challenge control group (P < 0.05). Survival of mice at 72 h were 80%, 100%, 90% and 70%, 90%, 80%
As shown in Figs. 5 and 6, the levels of endotoxin rapidly increased within 2 h in mice challenged with LPS, and the Sthanatin, treated groups showed significant reduction in plasma endotoxin levels compared to LPS challenge groups. S-thanatin given intraperitoneally immediately after administration of 150 g E. coli serotype O111:B4 LPS resulted in significantly (P < 0.05) lower plasma endotoxin and TNF-␣ levels
Fig. 2. Survival rates in a mice model after intraperitoneal administration of 150 g of O111:B4 LPS, and treated with vein injection of S-thanatin, then monitored for 72 h post injection. Ts, S-thanatin.
Fig. 4. Effect of S-thanatin on TNF-␣ production in mice challenged with O111:B4. Mice were untreated, injected intraperitoneally with LPS or intravenous injection with 3 mg/kg, 6 mg/kg, and 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of TNF-␣ was measured at 0, 2, 6 and 12 h after LPS and/or Sthanatin injection. All data represent mean values and standard errors. P < 0.05, when compared to LPS challenge group.
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compared with LPS challenge group in Tables 1 and 2. Furthermore, the high dose S-thanatin treatment group gave the strongest anti-endotoxin activity, especially injected intraperitoneally with S-thanatin (Tables 1 and 2).
4. Discussion LPS is an integral structural component on the outer membrane of gram-negative bacteria and can be released from the bacteria during cell division, cell death, especially as a result of antibiotic treatment against bacterial infection [18]. S-thanatin prevents its ability to induce a TNF-␣ response in LPS-sensitized mice. As a consequence, it protects mice against lethal shock. The data reported here apparently illustrate the potential antiendotoxin properties of S-thanatin, not only has a good affinity on binding to LPS in vitro [25], but also interfered with its biological activities in mice models of LPS challenge septic shock, protecting the animals against lethal endotoxemia. We can get it from the apparent improvement of animal survival rate. In the present study, we found the most effective treatment in our vivo systems was obtained when 6 mg/kg of S-thanatin was administered intraperitoneally. That showed 100% survival rate and a lower endotoxin and TNF-␣ level in plasma. TNF-␣ is involved in the development of endotoxin shock and tissue injury during overwhelming bacterial infection or challenge of lethal doses of LPS [18]. Therefore, the protective effect of S-thanatin on septic shock in mice challenged with LPS was associated with inhibition of TNF-␣ production, and an obvious dose-effect and a time-effect relationship were observed. At the same time, S-thanatin has an advantage of conferring greater stability in plasma. We have studied its metabolism in vivo by radiolabel method and the results showed that S-thanatin has a longer half-life in vivo about 6–7 h, and our results proved this conclusion well. The statistical results about two routes of administration showed different pharmacodynamics. The treatment of intraperitoneally injecting S-thanatin compared with intravenous injection treating, have obviously improved curative effect, and depleted the endotoxin contents in a shorter time. That showed evidently at 6 h (Tables 1 and 2), we can see a significant difference. Indeed, it may be that LPS was injected intraperitoneally and it was
Fig. 5. Plasma endotoxin levels in a mice model after intraperitoneal administration of LPS. Mice were untreated, injected intraperitoneally with LPS or 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of endotoxin was measured at 0, 2, 6 and 12 h after LPS and/or S-thanation injection. All data represent mean values (P < 0.05), when compared to LPS challenge group.
Fig. 6. Plasma endotoxin levels in a mice model after intraperitoneal administration of LPS. Mice were untreated, injected intraperitoneally with LPS or intravenous injection with 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of TNF-␣ was measured at 0, 2, 6 and 12 h after LPS and/or S-thanation injection. All data represent mean values. P < 0.05, when compared to LPS challenge group.
Table 1 Plasma TNF-␣ and endotoxin levels in mice 6 h after intraperitoneal injection of E. coli O111:B4 LPS and treated with intraperitoneal injection of S-thanatin. Treatmenta (intraperitoneal injection of S-thanatin)
TNF-␣ level (pg/L)b
Untreated LPS challenge Ts 3 mg/kg Ts 6 mg/kg Ts l2 mg/kg
52.67 167.45 109.24 90.46 86.27
a b c d
± ± ± ± ±
6.54 38.24 18.56c 9.87c , d 8.64c , d
Endotoxin level (g/L)b 3.68 10.98 6.13 5.87 5.14
± ± ± ± ±
0.85 2.71 1.32c 1.02c , d 0.91c , d
Ts, S-thanatin. Mean ± SD. P < 0.05, when compared to LPS challenge group. P < 0.05, when compared to Ts 3 mg/kg group.
Table 2 Plasma TNF-␣ and endotoxin levels in mice 6 h after intraperitoneal injection of E. coli O111:B4 LPS and treated with intravenous injection of S-thanatin. Treatmenta (intravenous injection of S-thanatin)
TNF-␣ level (pg/L)b
Untreated LPS challenge Ts 3 mg/kg Ts 6 mg/kg Ts l2 mg/kg
52.78 167.51 120.52 116.87 98.49
a b c d
Ts, S-thanatin. Mean ± SD. P < 0.05, when compared to LPS challenge group. P < 0.05, when compared to Ts 3 mg/kg group.
± ± ± ± ±
6.37 37.24 24.87c 20.53c , d 16.23c , d
Endotoxin level (g/L)b 3.59 12.25 10.28 9.64 8.47
± ± ± ± ±
0.57 3.01 2.38c 2.06c , d 1.97c , d
G. Wu et al. / Peptides 32 (2011) 353–357
helpful for S-thanatin to bind endotoxin. However, after 12 h, the endotoxin had fall down to the same level about the administration in two ways (Figs. 5 and 6). Since inhibition of LPS-mediated TNF-␣ production was suggested by blocking the binding of LPS to LPSrelated receptors on immune cells [23]. Refer to TNF-␣ level, we found that intraperitoneal injection of S-thanatin can give a higher suppression of TNF-␣ production (Figs. 3 and 4). Some studies proved that some peptides have strong antimicrobial activity, meanwhile with cytotoxic or hemolytic activity [22]. S-thanatin was modified from previous analogs (thanatin) and without cytotoxic and hemolytic activity to mammalian cells, which we have proved [26,27]. Even though, in this study we found individual mice of treatment groups died, especially injection in tail vein of high does S-thantin group (Figs. 1 and 2). It is a big problem for applying them to use as therapeutic directly [22], and the route of injection should be considered. Acknowledgments This work was supported by the Natural Science Foundation of Jiangsu Province, China (grant no. BK2009274) and Science Foundation of Southeast University (grant no. 3290000102). References [1] Adrie C, Alberti C, Chaix-Couturier C, Azoulay E, De Lassence A, Cohen Y, et al. Epidemiology and economic evaluation of severe sepsis in France: age, severity, infection site, and place of acquisition (community, hospital, or intensive care unit) as determinants of workload and cost. J Crit Care 2005;20:46–58. [2] Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303–10. [3] Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 1997;112:235–43. [4] Brun-Buisson C. The epidemiology of the systemic inflammatory response. Intensive Care Med 2000;26(Suppl. (1)):S64–74. [5] Bulet P, Stocklin R, Menin L. Anti-microbial peptides: from invertebrates to vertebrates. Immunol Rev 2004;198:169–84. [6] Byl B, Clevenbergh P, Kentos A, Jacobs F, Marchant A, Vincent JL, et al. Ceftazidime- and imipenem-induced endotoxin release during treatment of gram-negative infections. Eur J Clin Microbiol Infect Dis 2001;20: 804–7. [7] Calandra T, Baumgartner JD, Grau GE, Wu MM, Lambert PH, Schellekens J, et al. Prognostic values of tumor necrosis factor/cachectin, interleukin-1, interferon-alpha, and interferon-gamma in the serum of patients with septic shock. Swiss–Dutch J5 immunoglobulin study group. J Infect Dis 1990;161: 982–7.
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[8] De Smet K, Contreras R. Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol Lett 2005;27:1337–47. [9] Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2008. Intensive Care Med 2008;34:17–60. [10] Goto H, Nakamura S. Liberation of endotoxin from Escherichia coli by addition of antibiotics. Jpn J Exp Med 1980;50:35–43. [11] Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156–64. [12] Hancock RE, Chapple DS. Peptide antibiotics. Antimicrob Agents Chemother 1999;43:1317–23. [13] Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new antiinfective therapeutic strategies. Nat Biotechnol 2006;24:1551–7. [14] Hancock RE, Scott MG. The role of antimicrobial peptides in animal defenses. Proc Natl Acad Sci USA 2000;97:8856–61. [15] Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH. The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest 2000;118:146–55. [16] Jiang Z, Hong Z, Guo W, Xiaoyun G, Gengfa L, Yongning L, et al. A synthetic peptide derived from bactericidal/permeability-increasing protein neutralizes endotoxin in vitro and in vivo. Int Immunopharmacol 2004;4:527–37. [17] Lopez-Bojorquez LN, Dehesa AZ, Reyes-Teran G. Molecular mechanisms involved in the pathogenesis of septic shock. Arch Med Res 2004;35:465–79. [18] Mandard N, Sodano P, Labbe H, Bonmatin JM, Bulet P, Hetru C, et al. Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data. Eur J Biochem 1998;256:404–10. [19] Marano MA, Fong Y, Moldawer LL, Wei H, Calvano SE, Tracey KJ, et al. Serum cachectin/tumor necrosis factor in critically ill patients with burns correlates with infection and mortality. Surg Gynecol Obstet 1990;170:32–8. [20] Mookherjee N, Hancock RE. Cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections. Cell Mol Life Sci 2007;64:922–33. [21] Rosenfeld Y, Papo N, Shai Y. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J Biol Chem 2006;281:1636–43. [22] Saido-Sakanaka H, Ishibashi J, Momotani E, Amano F, Yamakawa M. In vitro and in vivo activity of antimicrobial peptides synthesized based on the insect defensin. Peptides 2004;25:19–27. [23] Tsubery H, Ofek I, Cohen S, Fridkin M. Structure–function studies of polymyxin B nonapeptide: implications to sensitization of gram-negative bacteria. J Med Chem 2000;43:3085–92. [24] Wu G, Ding J, Li H, Li L, Zhao R, Shen Z, et al. Effects of cations and pH on antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC 21332. Curr Microbiol 2008;57:552–7. [25] Wu G, Fan X, Li L, Wang H, Ding J, Hongbin W, et al. Interaction of antimicrobial peptide s-thanatin with lipopolysaccharide in vitro and in an experimental mouse model of septic shock caused by a multidrug-resistant clinical isolate of Escherichia coli. Int J Antimicrob Agents 2010;35:250–4. [26] Wu G, Wu H, Fan X, Zhao R, Li X, Wang S, et al. Selective toxicity of antimicrobial peptide S-thanatin on bacteria. Peptides 2010;31:1669–73. [27] Wu G, Wu H, Li L, Fan X, Ding J, Li X, et al. Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin. Biochem Biophys Res Commun 2010;395:31–5.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Protective effects of antimicrobial peptide S-thanatin against endotoxic shock in mice introduced by LPS Guoqiu Wu a,∗,1 , Xiaofang Li b,1 , Xuepeng Deng b , Xiaobo Fan b , Shenglan Wang b , Zilong Shen b , Tao Xi b,∗∗ a b
Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, PR China Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Tongjiaxiang 24#, Nanjing 210009, PR China
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 25 October 2010 Accepted 25 October 2010 Available online 2 November 2010 Keywords: Thanatin Antimicrobial peptide Endotoxic shock Lipopolysaccharide TNF-␣
a b s t r a c t Sepsis continues to be a major unresolved medical challenge of the present. Severe sepsis and septic shock are the leading causes of multiple organ failure and mortality in noncoronary intensive care units (ICUs). The primary reason of septic shock is the activation of host effecter cells by endotoxin and lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the key point of treatment is removing LPS. S-thanatin (Ts), an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against gramnegative and gram-positive bacteria. We have reported its LPS-binding and -neutralizing activity in vitro. The aim of this study is to examine the LPS-neutralizing activities and the protective effects of S-thanatin in vivo. Every mice was injected intraperitoneally with LPS (from Escherichia coli O111:B4) 150 g before injected intraperitoneally or vena caudalis with 3 mg/kg, 6 mg/kg and 12 mg/kg, and measured endotoxin and tumor necrosis factor alpha (TNF-␣) concentrations in plasma, as well as lethality. The results showed that S-thanatin can significantly reduce endotoxin and TNF-␣ level in plasma, at the same time resulting in the highest survival rates. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Severe sepsis, defined as sepsis with acute organ dysfunction, causes systemic inflammation and is associated with high morbidity and mortality rates [1]. It was estimated that there would be 934,000 cases per year by 2010 [2]. In the United States, approximately 750,000 cases of severe sepsis occur each year, with a mortality rate ranging from 30% to 50% despite advances in critical care management [3,4]. Pathogenesis of the development on sepsis is highly complicated and has been the object of study for many years. The best-characterized bacterial-derived antigen is endotoxin or bacterial lipopolysaccharide (LPS) [17]. LPS is a molecule derived from the outer membrane of gram negative bacteria, such as Escherichia coli and Salmonella typhimurium, and it is the bestcharacterized bacterial antigen in immunology [17]. Incidence of sepsis due to gram-positive bacteria increased from 10% in the 1960s and 1970s to 30% in subsequent years [15].
∗ Corresponding author. Tel.: +86 02583272355x808. ∗∗ Corresponding author. Tel.: +86 02583271389. E-mail addresses: guoqiu [email protected] (G. Wu), xitao [email protected] (T. Xi). 1 These two people contributed equally to this work. 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.10.029
Antibiotic therapy on septic shock is commonly used. Every patient who has sepsis and septic shock must be evaluated appropriately before the initiation of antibiotic therapy. The choice of empirical antibiotics depends on complex issues related to the patient’s history including drug intolerances, underlying disease, the clinical syndrome, and susceptibility patterns of pathogens in the community, in the hospital, and that previously have been documented to colonize or infect the patient. There is an especially wide range of potential pathogens for neutropenic patients [9]. There is ample evidence that failure to initiate appropriate therapy correlates with increased morbidity and mortality [15]. Usually antibiotics are used as medicine against bacterial infection, and they can stimulate the release of an excess amount of a bacterial outer membrane component, endotoxin [10]. The released endotoxin activates macrophages, endothelial cells and fibroblasts to produce and release potent inflammatory mediators including tumor necrosis factor-␣ (TNF-␣), interleukin 1-beta (IL-), interleukin-6 (IL-6) and nitric oxide [17]. TNF-␣ is a pro-inflammatory cytokine, which is regarded as an important mediator of sepsis. Serum levels have been shown to correlate with outcome in both animal and human models of sepsis [7,19]. At the same time they may light a cascade of uncontrolled systemic inflammatory responses that can lead to multiple organ failure and eventually to death [17]. In other words, antimicrobial therapy may promote the further release of LPS from the cell envelope of killed
354
G. Wu et al. / Peptides 32 (2011) 353–357
bacteria, exacerbating sepsis itself [6]. Because of that, the requirement for novel compounds which are able to neutralize the effects of endotoxin is urgently pursued. Antimicrobial peptides (AMPs) have emerged as ideal candidates to be used as adjuncts during antibiotic therapy, not only for their broad-spectrum activity against gram-positive and gram negative bacteria, yeasts, fungi, and enveloped viruses [8,13], but also for their ability to efficiently bind LPS and block LPS-dependent cytokine induction [20,21]. They are crucial humoral components of the innate immunity system of virtually all organisms, which they defend from the invasion of attacking pathogens [5,11,12]. Thanatin contains 21 amino acids (GSKKPIIYCNRRTGKCQRM), which was isolated from the hemipteran insect Podisus maculiventris [14]. It is the first inducible insect peptide that has a broad range of activity against bacteria and fungi at physiological concentrations, and an anti-parallel -sheet structure from 8th amino acid to the C-terminus [18]. S-thanatin (GSKKPVPIIYCNRRSGKCQRM), a novel thanatin analog, which was substituted threonine at position 15 with serine [25]. Our previous studies showed that S-thanatin has a broadspectrum antimicrobial activity, especially to gram-negative bacteria [27]. Previously, we have reported the antimicrobial activity against a multidrug-resistant (MDR) clinical isolate about S-thanatin and we have assessed the LPS-binding and -neutralizing activities of S-thanatin in vitro [25]. The present study was designed to further verify the LPS-neutralizing activity of S-thanatin in vivo.
2. Materials and methods 2.1. Animals Adult male ICR mice (28–31 g) were obtained from the Animal Center, Science Academy of China (Shanghai, China). All animals were housed in individual cages and acclimatized for 1 week in Animal Environmental Control Unit (temperature, 23 ± 3 ◦ C; relative humidity, 50 ± 10%; light–dark cycle, 12 h).
2.2. Reagents LPS from E. coli serotype O111:B4 was purchased from Sigma–Aldrich (St Louis, MO), prepared in sterile saline, aliquoted, and stored at −80 ◦ C for short periods. The mouse lipopolysaccharide (LPS) ELISA Kit and mouse tumor necrosis factor ␣ (TNF-␣) ELISA Kit were purchased from R&D, and all other chemicals used were of reagent grade.
2.3. Preparation of S-thanatin S-thanatin was synthesized by the solid-phase methodology with a 9-fluorenyl-methoxycarbonyl (Fmoc) protecting group [24]. Peptides were cleaved from the resin with a solution containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane. After repeating precipitation with diethyl ether, the peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using an appropriate 0–60% acetonitrile gradient in 0.05% trifluoroacetic acid. Molecular mass was determined by electrospray mass spectrometry using an API instrument (Perkin Elmer SCIEX) as a quality control of the synthesis [24]. The peptide was taken up in oxidation buffer (1 mg/1 ml) [100 mM ammonium acetate (pH 8.5)], allowed to refold for 3 days at room temperature under stirring and purified by RP-HPLC. S-thanatin was kept in −80 ◦ C after freeze-dried.
2.4. Microorganisms For antimicrobial assays of S-thanatin experiments, a commercially available quality control strain of E. coli ATCC 25922 and Klebsiella pneumoniae ATCC700603, they were obtained from the Center of Clinical Laboratory, Health Minister of PRC, were purchased and stored in 10% glycerol at −80 ◦ C. 2.5. Evaluation of antibacterial activities Constant broth dilution method was used according to the outline by the Clinical and Laboratory Standards Institute [16]. In order to avoid cationic peptides binding with polystyrene we used constant broth dilution method with glass tubes. The minimum inhibitory concentration (MIC) was taken as the lowest antibiotic concentration at which observable growth was inhibited. Exponentially growing bacteria at 108 CFU/ml, then diluted the bacterial suspension with MH broth in 1:100 dilution. They were incubated with different concentrations of S-thanatin in fresh MH broth at 37 ◦ C for 10 h. In this solution the concentration of S-thanatin was 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 g/ml in glass tubes 1–10, respectively. 2.6. LPS challenge and treatments Under the experiment condition (1), five groups, each containing 10 animals. Group 1 was negative control group without any treatment. Animals were injected intraperitoneally with 150 l sodium chloride solution twice. Group 2 were challenge control group injected intraperitoneally with 12 mg/kg of E. coli LPS in a total volume of 150 l sterile saline. Immediately after injection, animals received intraperitoneally isotonic sodium chloride solution. Groups 3–5 as the three treatment groups, the animals were injected intraperitoneally with 150 g E. coli LPS in a total volume of 150 l sterile saline followed by injection of 3 mg/kg, 6 mg/kg or 12 mg/kg of S-thanatin intraperitioneally. Under the experiment condition (2), another five groups were used for different administrations of S-thanatin via intravenous without any other difference in comparison with the first five groups. 2.7. Evaluation of in vivo treatment After treatment, the animals were returned to individual cages and thoroughly examined daily. Depending on the specific experiment, the rate of lethality, and the levels of plasma endotoxin and tumor necrosis factor alpha (TNF-␣) were evaluated. For determination of endotoxin and TNF-␣ contents in plasma, 100 l blood samples were collected from the post-global veins by glass capillary after 0, 2, 6, and 12 h after injection into a sterile syringe and transferred to tubes containing heparin. They were measured with commercially available solid-phase sandwich enzyme-linked immunosorbent assay according to the protocol supplied by the manufacturer. 2.8. Statistical analysis MICs are presented as average values from three independent measurements. Mortality rates differences between groups were compared by Fisher’s exact test (significance level fixed at 0.05). TNF-␣ and LPS mean values in plasma were presented as means ± standard deviations (SDs) of the mean; statistical comparisons between groups were made by analysis of variance (significance level was fixed at 0.05). The post hoc comparisons were performed by the Bonferroni method. Each comparison group
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Fig. 1. Survival rates in a mice model after intraperitoneal administration of 150 g of O111:B4 LPS, and injected intraperitoneally with S-thanatin, then monitored for 72 h post injection. Ts, S-thanatin.
contained 10 animals. Significance was accepted when the P value was 0.05. 3. Results 3.1. Antimicrobial assays in vitro studies
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Fig. 3. Effect of S-thanatin on TNF-␣ production in mice challenged with O111:B4. Mice were untreated, injected intraperitoneally with LPS or 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of TNF-␣ was measured at 0, 2, 6 and 12 h after LPS and/or S-thanatin injection. All data represent mean values and standard errors. P < 0.05, when compared to LPS challenge group.
in the groups treated with 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin intraperitoneally or injected intravenously. 3.3. Effect of S-thanatin on plasma TNF-˛ in mice
A preliminary screening was performed to verify whether Sthanatin has biological activity. According to the broth dilution method, E. coli ATCC 25922 and K. pneumoniae ATCC700603 showed different susceptibilities to S-thanatin. The MICs of S-thanatin were 4–8 g/ml and 8 g/ml. That proved the biological activity of S-thanatin, and the following experiments could be carried out.
The concentrations of plasma TNF-␣ in mice challenged with LPS were 167.45 ± 38.24 g/L. In contrast, those of mice challenged with LPS and treated with S-thanatin were 109.24 ± 18.56 g/L, 90.46 ± 9.87 g/L and 86.27 ± 8.64 g/L when measured at 6 h after challenge of LPS, and we give out the changing trends of TNF-␣ levels, when measured at 0, 2, 6, and 12 h after challenge of LPS (Fig. 3). When we intravenously inject S-thanatin as the route of administration we got a similar result (Fig. 4).
3.2. Effects of S-thanatin on LPS challenged mice
3.4. Effect of S-thanatin on plasma endotoxin in mice
After the mice were challenged with LPS, the control group developed the symptom of typical septic shock, and the symptom of treatment is eased. Mice of lethality at 72 h in the challenge control group were 100% and 90%, respectively, whereas treatment with S-thanatin decreased LPS-challenged lethality (Figs. 1 and 2). All groups administered S-thanatin following LPS challenge showed increased survival in comparison with the challenge control group (P < 0.05). Survival of mice at 72 h were 80%, 100%, 90% and 70%, 90%, 80%
As shown in Figs. 5 and 6, the levels of endotoxin rapidly increased within 2 h in mice challenged with LPS, and the Sthanatin, treated groups showed significant reduction in plasma endotoxin levels compared to LPS challenge groups. S-thanatin given intraperitoneally immediately after administration of 150 g E. coli serotype O111:B4 LPS resulted in significantly (P < 0.05) lower plasma endotoxin and TNF-␣ levels
Fig. 2. Survival rates in a mice model after intraperitoneal administration of 150 g of O111:B4 LPS, and treated with vein injection of S-thanatin, then monitored for 72 h post injection. Ts, S-thanatin.
Fig. 4. Effect of S-thanatin on TNF-␣ production in mice challenged with O111:B4. Mice were untreated, injected intraperitoneally with LPS or intravenous injection with 3 mg/kg, 6 mg/kg, and 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of TNF-␣ was measured at 0, 2, 6 and 12 h after LPS and/or Sthanatin injection. All data represent mean values and standard errors. P < 0.05, when compared to LPS challenge group.
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compared with LPS challenge group in Tables 1 and 2. Furthermore, the high dose S-thanatin treatment group gave the strongest anti-endotoxin activity, especially injected intraperitoneally with S-thanatin (Tables 1 and 2).
4. Discussion LPS is an integral structural component on the outer membrane of gram-negative bacteria and can be released from the bacteria during cell division, cell death, especially as a result of antibiotic treatment against bacterial infection [18]. S-thanatin prevents its ability to induce a TNF-␣ response in LPS-sensitized mice. As a consequence, it protects mice against lethal shock. The data reported here apparently illustrate the potential antiendotoxin properties of S-thanatin, not only has a good affinity on binding to LPS in vitro [25], but also interfered with its biological activities in mice models of LPS challenge septic shock, protecting the animals against lethal endotoxemia. We can get it from the apparent improvement of animal survival rate. In the present study, we found the most effective treatment in our vivo systems was obtained when 6 mg/kg of S-thanatin was administered intraperitoneally. That showed 100% survival rate and a lower endotoxin and TNF-␣ level in plasma. TNF-␣ is involved in the development of endotoxin shock and tissue injury during overwhelming bacterial infection or challenge of lethal doses of LPS [18]. Therefore, the protective effect of S-thanatin on septic shock in mice challenged with LPS was associated with inhibition of TNF-␣ production, and an obvious dose-effect and a time-effect relationship were observed. At the same time, S-thanatin has an advantage of conferring greater stability in plasma. We have studied its metabolism in vivo by radiolabel method and the results showed that S-thanatin has a longer half-life in vivo about 6–7 h, and our results proved this conclusion well. The statistical results about two routes of administration showed different pharmacodynamics. The treatment of intraperitoneally injecting S-thanatin compared with intravenous injection treating, have obviously improved curative effect, and depleted the endotoxin contents in a shorter time. That showed evidently at 6 h (Tables 1 and 2), we can see a significant difference. Indeed, it may be that LPS was injected intraperitoneally and it was
Fig. 5. Plasma endotoxin levels in a mice model after intraperitoneal administration of LPS. Mice were untreated, injected intraperitoneally with LPS or 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of endotoxin was measured at 0, 2, 6 and 12 h after LPS and/or S-thanation injection. All data represent mean values (P < 0.05), when compared to LPS challenge group.
Fig. 6. Plasma endotoxin levels in a mice model after intraperitoneal administration of LPS. Mice were untreated, injected intraperitoneally with LPS or intravenous injection with 3 mg/kg, 6 mg/kg, 12 mg/kg S-thanatin immediately after injection with LPS. Plasma level of TNF-␣ was measured at 0, 2, 6 and 12 h after LPS and/or S-thanation injection. All data represent mean values. P < 0.05, when compared to LPS challenge group.
Table 1 Plasma TNF-␣ and endotoxin levels in mice 6 h after intraperitoneal injection of E. coli O111:B4 LPS and treated with intraperitoneal injection of S-thanatin. Treatmenta (intraperitoneal injection of S-thanatin)
TNF-␣ level (pg/L)b
Untreated LPS challenge Ts 3 mg/kg Ts 6 mg/kg Ts l2 mg/kg
52.67 167.45 109.24 90.46 86.27
a b c d
± ± ± ± ±
6.54 38.24 18.56c 9.87c , d 8.64c , d
Endotoxin level (g/L)b 3.68 10.98 6.13 5.87 5.14
± ± ± ± ±
0.85 2.71 1.32c 1.02c , d 0.91c , d
Ts, S-thanatin. Mean ± SD. P < 0.05, when compared to LPS challenge group. P < 0.05, when compared to Ts 3 mg/kg group.
Table 2 Plasma TNF-␣ and endotoxin levels in mice 6 h after intraperitoneal injection of E. coli O111:B4 LPS and treated with intravenous injection of S-thanatin. Treatmenta (intravenous injection of S-thanatin)
TNF-␣ level (pg/L)b
Untreated LPS challenge Ts 3 mg/kg Ts 6 mg/kg Ts l2 mg/kg
52.78 167.51 120.52 116.87 98.49
a b c d
Ts, S-thanatin. Mean ± SD. P < 0.05, when compared to LPS challenge group. P < 0.05, when compared to Ts 3 mg/kg group.
± ± ± ± ±
6.37 37.24 24.87c 20.53c , d 16.23c , d
Endotoxin level (g/L)b 3.59 12.25 10.28 9.64 8.47
± ± ± ± ±
0.57 3.01 2.38c 2.06c , d 1.97c , d
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helpful for S-thanatin to bind endotoxin. However, after 12 h, the endotoxin had fall down to the same level about the administration in two ways (Figs. 5 and 6). Since inhibition of LPS-mediated TNF-␣ production was suggested by blocking the binding of LPS to LPSrelated receptors on immune cells [23]. Refer to TNF-␣ level, we found that intraperitoneal injection of S-thanatin can give a higher suppression of TNF-␣ production (Figs. 3 and 4). Some studies proved that some peptides have strong antimicrobial activity, meanwhile with cytotoxic or hemolytic activity [22]. S-thanatin was modified from previous analogs (thanatin) and without cytotoxic and hemolytic activity to mammalian cells, which we have proved [26,27]. Even though, in this study we found individual mice of treatment groups died, especially injection in tail vein of high does S-thantin group (Figs. 1 and 2). It is a big problem for applying them to use as therapeutic directly [22], and the route of injection should be considered. Acknowledgments This work was supported by the Natural Science Foundation of Jiangsu Province, China (grant no. BK2009274) and Science Foundation of Southeast University (grant no. 3290000102). References [1] Adrie C, Alberti C, Chaix-Couturier C, Azoulay E, De Lassence A, Cohen Y, et al. Epidemiology and economic evaluation of severe sepsis in France: age, severity, infection site, and place of acquisition (community, hospital, or intensive care unit) as determinants of workload and cost. J Crit Care 2005;20:46–58. [2] Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303–10. [3] Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 1997;112:235–43. [4] Brun-Buisson C. The epidemiology of the systemic inflammatory response. Intensive Care Med 2000;26(Suppl. (1)):S64–74. [5] Bulet P, Stocklin R, Menin L. Anti-microbial peptides: from invertebrates to vertebrates. Immunol Rev 2004;198:169–84. [6] Byl B, Clevenbergh P, Kentos A, Jacobs F, Marchant A, Vincent JL, et al. Ceftazidime- and imipenem-induced endotoxin release during treatment of gram-negative infections. Eur J Clin Microbiol Infect Dis 2001;20: 804–7. [7] Calandra T, Baumgartner JD, Grau GE, Wu MM, Lambert PH, Schellekens J, et al. Prognostic values of tumor necrosis factor/cachectin, interleukin-1, interferon-alpha, and interferon-gamma in the serum of patients with septic shock. Swiss–Dutch J5 immunoglobulin study group. J Infect Dis 1990;161: 982–7.
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