A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties

Peptide preparation

Cecropin DH and its parent peptide cecropin B were synthesized by GL Biochem Ltd. (Shanghai, China) using the solid-phase synthesis technique. And the success of peptide synthesis was identified via electrospray ionization mass spectrometry. The purity of the synthetic peptides used to evaluate biological activity was higher than 95% confirmed with analytical reverse-phase high-performance liquid chromatography.

Antimicrobial measurements and kinetics of bacteria killing

The minimum inhibitory concentrations (MICs) of cecropin DH against Escherichia coli ATCC 25922, E. coli DH5α, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923 and Micrococcus luteus NCIMB 8166 were determined using the modified classical microtiter broth dilution method (available from http://cmdr.ubc.ca/bobh/method/modified-mic-method-for-cationic-antimicrobial-peptides/) and compared with those of cecropin B and melittin purchased from (MedChemExpress, Monmouth Junction, NJ, USA). Briefly, bacteria were incubated in Mueller Hinton Broth (MHB) medium overnight at 37 °C (30 °C for B. subtilis) with shaking and diluted into fresh MHB at a cell density of 5 × 10⁵ colony-forming units (CFUs)/mL. A 100 μL volume of cell suspension was dispensed in each well of a sterile 96-well plate from column 1 to column 11, and 100 μL of MHB was dispensed in column 12 for sterility control and to blank the plate scanner. A 11 μL sample of each of the peptides (10× stocks) was dissolved separately in 0.01% acetic acid and 0.2% bovine serum albumin, and added to each well (columns 1–10) to a final concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 and 0.195 μM, respectively. Column 11 without peptides served as positive controls (untreated bacteria). The plate was incubated at 37 °C (30 °C for B. subtilis) for 18–24 h in an incubator, and the absorbance was recorded at 590 nm. Three replicates were generated for each test sample. In the present study, the MIC was taken to be the lowest concentration of peptides that prevented visible turbidity. Then 10 μl 10⁻⁶ dilution of the first three wells that showed no visible growth of above overnight cultures, were plated onto Mueller Hinton Agar (MHA) plates and incubated at 37 °C (30 °C for B. subtilis) for 18 h. The lowest concentration of peptides that prevent any residual colony formation is the minimal bactericidal concentration.

Next, the salt sensitivity tests for cecropin DH were performed at fixed concentrations of NaCl, CaCl₂ or MgCl₂. MIC values against E. coli ATCC 25922 were measured as described above in the presence of 100 mM NaCl, 2 mM CaCl₂ or 1 mM MgCl₂.

The in vitro bacteria killing kinetics of cecropin DH against E. coli ATCC 25922 were also determined. Exponentially growing E. coli cells were resuspended in MHB to a density of 4 × 10⁶ CFUs/mL. Bacterial suspensions were treated at 1× and 4× MIC with peptide. At various time periods (0, 2, 3 and 6 h), 50 μL samples were removed and diluted appropriately in MHB, and CFUs were determined by spotting 100 μL of serially diluted samples onto MHB agar plates and incubating for 18 h at 37 °C.

Measurement of hemolytic activity

The hemolytic activities of cecropin DH and cecropin B peptides were determined as the amount of hemoglobin released by the lysis of mouse red blood cells (mRBCs) (Kim et al., 2011). Fresh mRBCs were collected and centrifuged at 1,000 g for 10 min. The erythrocytes were then washed three times and resuspended in phosphate-buffered saline (PBS, pH 7.4) at 8% (v/v). Each 50 μL sample of mRBCs was incubated with 50 μL serial dilutions of peptides dissolved in PBS for 1 h at 37 °C. Samples were then centrifuged at 1,000 g for 5 min at 4 °C, and the supernatant was transferred to a 96-well microtiter plate. The release of hemoglobin was monitored by measuring the absorbance of the supernatant at 405 nm. No hemolysis (blank) and 100% hemolysis controls consisting of mRBCs in PBS and 0.1% (v/v) Triton X-100, respectively, were included. Percentage hemolysis was calculated as follows: $$\text{Hemolysis}\left(\text{\%}\right)\text{=}\left[\frac{\left({\text{OD}}_{\text{405 nm}}\text{sample}-{\text{OD}}_{\text{405 nm}}\text{zero lysis}\right)}{\left({\text{OD}}_{\text{405 nm}}\text{100\% lysis}-{\text{OD}}_{\text{405 nm}}\text{zero lysis}\right)}\right]\text{×100}.$$

Cytotoxicity assay

The cytotoxicity of cecropin DH and cecropin B peptides against RAW264.7 mouse macrophage cells (from the Cell Resource Center of the Shanghai Academy of Life Sciences, Chinese Academy of Sciences) was determined using a CCK-8 cell proliferation and cytotoxicity assay kit (BestBio, Shanghai, China). RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning Cellgro, Manassas, Virginia, USA) supplemented with antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Sangon Biotech, Shanghai, China) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C in a humidified chamber under a 5% CO₂ atmosphere. Cells were pre-seeded on a 96-well plate at a density of ∼6 × 10⁴ to 1 × 10⁵ cells/mL and cultured overnight. Increasing concentrations of the peptides (0.195–100 μM) in DMEM medium were added, and wells containing cells without peptides served as controls. After incubating plates for 1 day, 10 μL of CCK-8 solution was added to each well and plates were incubated for an additional 2.5 h at 37 °C. The absorbance at 450 nm was measured using a microplate autoreader (SpectraMax i3x; Molecular Devices, San Jose, CA, USA).

BODIPY-TR-cadaverine displacement assay

The binding affinity of cecropin DH to the lipid A portion of bacterial LPS from E. coli 055:B5 purchased from (Sigma-Aldrich, St. Louis, MO, USA) was determined using the fluorescent dye BODIPY-TR-cadaverine (BC; from ThermoFisher Scientific Inc., Waltham, MA, USA) displacement assay (Ma et al., 2015; Sautrey et al., 2014), in which the fluorescence of the probe is self-quenched upon binding to cell-free LPS, while fluorescence is emitted when LPS binds to the peptides. Briefly, 100 μL solutions in 20 mM TRIS and 100 mM NaCl (pH 7.4) containing equal volumes of LPS-probe mixture (final concentrations of 25 μg/mL for LPS and 2.5 μg/mL for BC) and 1.56–50 μM peptide were incubated in 96-microwell black plates in the dark for 0.5 h. Fluorescence was measured using a microplate autoreader with excitation at 580 nm and emission at 620 nm. Values were converted to %ΔF (AU) using the following equation: $$\text{\%ΔF}\left(\text{AU}\right)\text{=}\left[\frac{\left({\text{F}}_{\text{obs}}-{\text{F}}_{\text{0}}\right)}{\left({\text{F}}_{\text{100}}-{\text{F}}_{\text{0}}\right)}\right]\text{×100,}$$ where F_obs is the observed fluorescence of BC with LPS-peptide interaction, F₀ is the fluorescence of BC with LPS in the absence of peptide (corresponding to no displacement of the probe) and F₁₀₀ is the fluorescence upon addition of 10 μg/mL polymyxin B purchased from (Sigma-Aldrich, St. Louis, MO, USA) to the LPS-probe mixture (corresponding to maximal displacement of the probe).

Disassociation of LPS

Interactions between cecropin DH and fluorescein isothiocyanate (FITC)-conjugated LPS from E. coli 055:B5 purchased from (Sigma-Aldrich, St. Louis, MO, USA) were analyzed by exciting FITC-LPS (0.5 μM) at 480 nm in 20 mM TRIS and 100 mM NaCl (pH 7.4) in the absence and presence of different concentrations of peptide (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 μM). Fluorescence emission spectra at 515 nm were recorded using a SpectraMax i3x microplate autoreader (Molecular Devices, San Jose, CA, USA).

Static light scattering assay

To obtain information on the particle size distribution of LPS micelles in the absence and presence of cecropin DH, static light scattering (SLS) measurements were performed on a Malvern Zetasizer μV instrument (Malvern, UK). Measurements were made for LPS micelles at a final concentration of 1 mg/mL mixed with 0.25 mM peptides. LPS and peptide were dissolved in TRIS buffer (20 mM TRIS, 100 mM NaCl, pH 7.4), and all solutions were filtered and degassed prior to use. Data were acquired at 298 K using disposable cuvettes. Scattering data were collected at 90° and analyzed using the software supplied with the instrument.

Examination of LPS micelle damage by transmission electron microscopy

Transmission electron microscopy (TEM) was performed to observe the influence of peptides on the morphology of LPS micelles. A total of 50 μL solutions containing 0.25 mM cecropin DH and 1 mg/mL LPS micelles in Milli-Q water were incubated at room temperature overnight. Untreated LPS micelles were used as controls. After incubation, a drop of the mixture was deposited onto a carbon-coated grid, negatively stained with phosphotungstic acid and visualized using a Tecnai G2 Spirit BioTWIN 120 kV TEM instrument (FEI, Hillsboro, Oregon, USA).

Circular dichroism spectroscopy

Circular dichroism (CD) experiments were performed at room temperature with a Chirascan qCD spectropolarimeter (Applied Photophysics Ltd., Surrey, UK). Spectra were recorded at wavelengths ranging from 190 to 240 nm. CD spectroscopy was used to investigate the secondary structure of cecropin DH in the absence and presence of 2 mg/mL LPS and E. coli ATCC 25922 bacterial cells in 10 mM sodium phosphate buffer (pH 7.4). The peptide concentration was 0.2 mg/mL. The CD spectra of cecropin DH in sodium phosphate buffer, in LPS or E. coli cells environment were obtained by subtracting the peptide control spectrum. Spectra were collected in quartz cuvettes with a 0.5 mm path length and averaged across three consecutive scans. A 1 nm data pitch, 0.4 s time-per-point and 1 nm bandwidth were generally used. In addition, E. coli bacterial cell cultures for CD experiments were grown in MHB medium at 37 °C overnight, harvested (4,000 g, 8 min) and resuspended in 10 mM sodium phosphate buffer (pH 7.4) at OD₆₀₀ values of 4.0, 2.0, 1.0 and 0.5. Peptide solution was added to the resuspended E. coli cells to give a final OD₆₀₀ of 2.0, 1.0, 0.5 and 0.25, and samples were incubated at room temperature for 4 h (Avitabile, D’Andrea & Romanelli, 2014).

Nuclear magnetic resonance experiments

For saturation transfer difference (STD) experiments, the nuclear magnetic resonance (NMR) sample contained 1 mM cecropin DH, 0.3 mg/mL LPS, in 10 mM sodium phosphate D₂O buffer at pH 5.8. STD experiments were recorded on a Bruker 500 MHz spectrometer at a temperature of 298 K with 1,024 scans. Selective saturation of LPS resonances was performed by selective irradiation at frequency at −2.0 ppm (on-resonance) or 40 ppm for the reference spectrum (off-resonance). A cascade of 44 selective Gaussian-shaped pulses was applied for a total saturation time of 2 s. The low power Gaussian pulses had a duration of 45 ms and inter-pulse delays of 450 μs. The difference spectrum, which contains signals arising from the saturation transfer, was obtained by subtracting the off-resonance spectrum from the on-resonance spectrum by phase cycling.

³¹P nuclear magnetic resonance spectra of LPS were recorded on a Bruker 850 MHz spectrometer at 298 K. Interactions of peptides with LPS were examined by recording a series of one-dimensional ³¹P NMR spectra of LPS, whereby 4 mg/mL LPS in 20 mM TRIS (pH 7.4) and 100 mM NaCl was titrated with various peptide concentrations (0.2, 0.4 and 0.6 mM).

Reverse-transcription PCR assay

RAW264.7 murine macrophage cells were plated in six-well plates (5 × 10⁵ cells/well) and cultured overnight. Cells were then stimulated with or without 200 ng/mL LPS (negative controls) in the presence or absence of peptides (10 μM) for 3 h. Total RNA was prepared using an RNAprep Kit (TIANGEN, Beijing, China), and equal amounts of total RNA were reverse-transcribed into cDNA using a RevertAid first-strand cDNA synthesis kit (ThermoFisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Products were amplified from cDNAs by PCR using the following specific primers: interleukin-1β (IL-1β) (forward = 5′-CTGTCCTGATGAGAGCATCC-3′, reverse = 5′-TGTCCATTGAGGTGGAGAGC-3′); interleukin 6 (IL-6) (forward = 5′-ACAAGTCCGGAGAGGAGACT-3′, reverse = 5′-GGATGGTCTTGGTCCTTAGC-3′); macrophage inflammatory protein (MIP)-1 (forward = 5′-ATGAAGCTCTGCGTGTCTGC-3′, reverse = 5′-TGAGGAGCAAGGACGCTTCT-3′); MIP-2 (forward = 5′-ACACTTCAGCCTAGCGCCAT-3′, reverse = 5′-CAGGTCAGTTAGCCTTGCCT-3′); TNF-α (forward = 5′-GTTCTGTCCCTTTCACTCACTG-3′, reverse = 5′-GGTAGAGAATGGATGAACACC-3′); inducible nitric-oxide synthase (iNOS) (forward = 5′-CTGCAGCACTTGGATCAGGAACCTG-3′, reverse = 5′-GGGAGTAGCCTGTGTGCACCTGGAA-3′); glyceraldehyde-3-phosphate, used as an internal control (forward = 5′-ACCACAGTCCATGCCATCAC-3′, reverse = 5′-TCCACCACCCTGTTGCTGTA-3′). Thermal cycling consisted of an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1.5 min and extension at 72 °C for 1 min, and a final extension step of 10 min at 72 °C.

Measuring nitrite production in LPS-stimulated RAW264.7 cells

RAW264.7 murine macrophage cells (1.5 × 10⁵) cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin were plated and adhered to a 96-well culture plate. After stimulating with LPS (200 ng/mL) in the presence or absence of peptides (10 μM) for 24 h, culture medium was collected to determine nitrite levels using Griess reagent (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. The absorbance at 540 nm was measured using a SpectraMax i3x microplate reader (Molecular Devices, San Jose, CA, USA) and converted to nitrite concentration by referring to a standard curve generated using NaNO₂.

Quantification of pro-inflammatory cytokine production by LPS-stimulated RAW264.7 cells

RAW264.7 murine macrophage cells were seeded in 96-well plates (5 × 10⁴ cells/well) and incubated overnight. Cells were pretreated with peptides (10 μM) and incubated at 37 °C for 1 h before the addition of 20 ng/mL LPS. After incubation for another 6 h, supernatants were harvested for TNF-α and IL-6 analyses using commercially available mouse TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (DAKEWE, Shanghai, China) according to the manufacturer’s protocols. The absorbance at 450 nm was measured using a SpectraMax i3x microplate reader (Molecular Devices, San Jose, CA, USA).

Statistical analysis

The statistical significance of differences between samples were analyzed by one-way analysis of variance, followed by Bonferroni’s multiple comparison test (GraphPad Prism; GraphPad Software, San Diego, CA, USA). Differences with p < 0.05 were considered statistically significant.

Antimicrobial activity

The antimicrobial activities of cecropin DH were examined against three representative Gram-negative (E. coli ATCC25922, E. coli DH5α and P. aeruginosa) and three Gram-positive (B. subtilis, S. aureus and M. luteus) bacterial strains (Table 1), and compared with the activities of melittin, which is known to have profound antibacterial activities. The antimicrobial activities of the parent peptide cecropin B are also listed for comparison. Overall, as indicated in Table 1, cecropin DH peptides displayed a good inhibitory effect on E. coli, P. aeruginosa, B. subtilis and M. luteus, which was comparable to those of cecropin B and melittin. Interestingly, both cecropins in this study exhibited almost no activity against S. aureus, with MICs >100 μM.

The bacteria killing activity of cecropin DH against E. coli was also monitored by assessing cell viability loss after incubation with peptide at 1× and 4× MIC for 6 h. As shown in Fig. 1A, cecropin DH exerted an obvious dose-dependent antibacterial activity within 6 h. After 2 h of incubation, a reduction in the number of CFUs was approximately 2 log with peptide concentration of 1×MIC, and a rapid loss of cell viability (3.8 log reduction in the number of CFUs) was observed at 4×MIC. The number of CFUs recovered for the E. coli incubated with 1×MIC peptide after 6 h incubation. While cecropin DH at 4×MIC preserves its antimicrobial activity at hour 6. The salt sensitivity of the antimicrobial activity was estimated by monitoring the MICs of peptides in the presence of 100 mM NaCl, 1 mM MgCl₂ and 2 mM CaCl₂, which were chosen for their biological relevance. For example, 100 mM NaCl was reported to present in the airway surface fluid of healthy subjects and patients with cystic fibrosis (Scudiero et al., 2010). As shown in Fig. 1B, the MIC values of cecropin DH were not significantly affected by the presence of different salts. The antibacterial activity against E. coli was largely unchanged with 2 mM CaCl₂, and even doubled in the presence of 100 mM NaCl and 1 mM MgCl₂.

Hemolysis and cytotoxicity

The hemolysis activity of cecropin DH and cecropin B against mRBCs was assessed as a measurement of peptide toxicity to eukaryotic cells. Hemolysis percentage was quantified at a concentration range from 0.78 to 200 μM. As shown in Fig. 2A, although a dose-dependent hemolytic activity was observed, in general cecropin DH induced very little hemolytic activity. At concentrations of 100 or 200 μM, cecropin DH showed 2.9% and 7.8% red cell hemolysis, respectively. At a concentration below 100 μM, almost no hemolytic activity was observed. Similarly, cecropin B lacked hemolytic activity, even at concentration as high as 200 μM. As a comparison, melittin caused 10% hemolysis at concentration as low as 0.78 μM (Kim et al., 2011; Pandey et al., 2010). The cytotoxicity of these two peptides was evaluated using RAW264.7 murine macrophage cells with various peptide concentrations (0.195–100 μM), and the effects on cell growth were subsequently evaluated using CCK-8 cell viability assay reagent. As illustrated in Fig. 2B, neither of the peptides affected the viability of RAW264.7 cells at concentrations less than 25 μM, and the survival rates of RAW264.7 cells were greater than 95%. Cecropin DH showed high cytotoxicity at concentrations above 25 μM, and an analysis of dose-response curves by GraphPad Prism 7.00 software yield an IC₅₀ of 46.34 μM, much higher than its MIC value (Table 1).

Disruptions of LPS aggregates

Lipopolysaccharide forms super-molecular structures in aqueous solution, which provides a convenient in vitro model for studying the effects of cecropin DH. First, the binding ability of cecropin DH to LPS was investigated using the BC fluorescence-based displacement assay. As shown in Fig. 3A, cecropin DH induced a significant dose-dependent BC fluorescence displacement. The BC displacement percentage was 30% at a low peptide concentration of 1.56 μM, and increased to 89% at 3.125 μM. At a cecropin DH concentration above 6.25 μM, fluorescence recovered to 100%, reflecting potent binding to the lipid A region of LPS. For FITC-conjugated LPS, the fluorescence intensity of FITC was largely quenched due to the formation of LPS soluble aggregates in solution (Tobias et al., 1995). As shown in Fig. 3B, the fluorescence intensity of FITC-LPS changed with increasing peptide concentration. Again, the addition of active peptide increased the fluorescence intensity in a concentration-dependent manner, indicating possible LPS dissociation and/or structural disorganization of LPS micelles. Similar phenomena have been observed with other AMPs in which binding of proteins or peptides alleviates quenching and enhances FITC fluorescence (Bhunia et al., 2009a; Kim et al., 2011).

The effect of cecropin DH on LPS aggregates can be conveniently characterized by SLS. As shown in Fig. 3C, LPS in aqueous solution assembles into large aggregates with a broad size distribution from 50 to 200 nm. After the addition of peptides, the population of large LPS micelles decreased, and a new component of ∼30 nm with a much narrower size distribution appeared. These smaller LPS micelles were further verified by TEM imaging (Fig. 3D). These observations demonstrated that the AMP cecropin DH destabilizes LPS micelles, causing them to break into smaller assemblies.

Interactions between cecropin DH and LPS

Further experiments were performed to investigate the interactions of cecropin DH and LPS. STD-NMR spectra were recorded on 1 mM peptide samples with 0.3 mg/mL LPS (in 10 mM sodium phosphate D₂O buffer at pH 5.8). In the STD-NMR experiments, selective saturation pulses were applied at LPS resonances, and magnetic saturation was subsequently transferred to LPS-bound peptides. Saturation difference spectra were generated from the intensity difference between LPS-bound peptide and free peptide signals. Figure 4A shows the ¹H spectrum of free peptides and the STD spectrum of peptides bound to LPS. A relatively strong STD effect of cecropin DH was observed for the aromatic ring protons of Trp2 and Phe5 (6–8 ppm), as well as a number of aliphatic side chain proton resonances (0–5 ppm) (Bhunia, Ramamoorthy & Bhattacharjya, 2009b; Kim et al., 2011; Lee et al., 2015), implying that these hydrophobic side chains are closely associated with LPS micelles.

Direct evidence for the formation of α-helical structure in cecropin DH was provided by CD spectroscopy. CD spectra of cecropin DH dissolved in phosphate buffer (Fig. 4B) or water (Fig. S1) exhibit a negative peaks at 198 nm, indicating disordered conformations in aqueous buffer. By contrast, in 2 mg/mL LPS solution (Fig. 4B), and in the presence of other hydrophobic reagents such as trifluoroethanol, dodecylphosphatidylcholine and sodium dodecyl sulfate (SDS; Fig. S1), two negative minimum peaks at 208 and 222 nm appear, indicating that cecropin DH adopts an α-helical conformation in hydrophobic environments. When cecropin DH peptides were incubated with E. coli cells, α-helical structure also formed, and the α-helical content was dependent on cell concentration. This is due to the fact that more LPS molecules are available for peptide binding with the increment of cell concentration. Fourier-transform infrared (FTIR) spectra subsequently provided additional evidence; in the presence of LPS, the appearance of a peak at 1,655 cm⁻¹ in the amide I region indicates an α-helical conformation for cecropin DH (Fig. S2A). Furthermore, a peak at ∼1,675 cm⁻¹ was observed, which is characteristic of carbonyls not involved in hydrogen-bonded structures (Sun et al., 2015).

³¹P nuclear magnetic resonance signals from phosphate groups which locate within the lipid A moiety and inner sugar core region, provide a convenient probe of the local structural and environmental conditions around the LPS head group. Figure 4C shows an overlay of ³¹P NMR spectra of 4 mg/mL LPS in the absence and presence of cecropin DH peptide at different concentrations (0.2, 0.4 and 0.6 mM). The ³¹P NMR spectra of LPS micelles exhibit a strong peak close to −1.30 ppm and two weak peaks at approximately 0.8 and 1.78 ppm, corresponding to the diphosphate and monophosphate groups of LPS, respectively (Bera et al., 2015; Strain, Fesik & Armitage, 1983). As the molar ratios of peptides to LPS increased, the most significant and obvious change happens at resonance of 1.78 ppm, which became sharper and upfield-shifted to 1.48, 1.06 and 0.52 ppm respectively. This suggests that the LPS monophosphate group may be directly involved in peptide binding and undergo dynamics at fast exchange regime. The changes in strong peak at −1.30 ppm and peak at 0.8 ppm were relatively minor. The observed line broadening of ³¹P resonances is caused by the chemical or conformational exchange processes between different states of LPS-peptide complexes.

Inhibition effect of peptides on LPS-induced pro-inflammatory cytokine production

Lipopolysaccharide, also termed endotoxin, is the major component of the outer membrane of Gram-negative bacteria, and its release can cause strong immunogenicity and provoke immune responses in immunocytes. LPS can induce the expression of iNOS and the pro-inflammatory cytokines TNF-α, IL-1β, MIP-1, MIP-2 and IL-6 in macrophages. Therefore, we investigated the effects of cecropin DH on the expression of iNOS and pro-inflammatory cytokines induced in RAW264.7 macrophages by stimulating with 200 ng/mL LPS over 3 h by Reverse-transcription PCR. As shown in Fig. 5A, cecropin DH was more effective than cecropin B for suppressing iNOS expression, and was superior for suppressing all pro-inflammatory cytokine genes except IL-6.

We then evaluated LPS-stimulated NO production and cytokine production in RAW264.7 macrophages. RAW264.7 cells were stimulated with 200 ng/mL LPS in the presence of the 10 μM peptide. NO production was determined by the Griess method that detects nitrite accumulation in the culture medium. As illustrated in Fig. 5B, 200 ng/mL LPS induced 33 μM nitrite release. Compared with cecropin B, the addition of cecropin DH was more effective at inhibiting NO production, which was decreased by 85%. The inhibitory effect of cecropin DH on the release of TNF-α and IL-6 in LPS (20 ng/mL)-stimulated RAW264.7 cells was also investigated using commercially available ELISA kits. A quantitative analysis of TNF-α and IL-6 concentrations revealed that cecropin DH clearly inhibited both TNF-α and IL-6 production in LPS-simulated RAW264.7 cells (Fig. 5C). A 20 ng/mL dose of LPS alone induced 1,116 pg/mL TNF-α, and 10 μM cecropin DH blocked its release by 67.2%, whereas cecropin B blocked the release by 58.4%. With respect to IL-6, LPS (20 ng/mL) alone induced 70.5 pg/mL, cecropin DH blocked its release by 88.7% and cecropin B blocked the release by 90.1%. These data are in good agreement with the observed inhibition of the expression of iNOS, TNF-α and IL-6 genes by the peptides (Fig. 5A). In general, the novel peptide cecropin DH exhibited more potent effect on reducing the inflammatory response than its parent peptide cecropin B.