Forsythiaside inhibits bacterial adhesion on titanium alloy and attenuates Ti-induced activation of nuclear factor-κB signaling-mediated macrophage inflammation

Forsythiaside inhibits bacterial adhesion on titanium alloy and attenuates Ti-induced activation... Background: Inflammation and biofilm formation by Staphylococcus aureus (S. aureus) are common causes of periprosthetic infection and loosening. Recently, we identified that forsythiaside is bacteriostatic for S. aureus and methicillin-resistant S. aureus (MRSA). The purpose of the present study was to examine the effect of forsythiaside on S. aureus and MRSA adhesion and biofilm formation on the surface of titanium alloy, which is a popular material for orthopedic joint prostheses. Methods: Two strains of S. aureus and MRSA were used for in vitro experiments. The spread plate method, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) were used to characterize antimicrobial activity of forsythiaside. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to investigate the inhibitory level of forsythiaside required for titanium-associated inflammation. Results: Direct colony counting showed that 16 μg/mL forsythiaside significantly inhibited S. aureus and MRSA adhesion on titanium alloy discs in 2 h. CLSM and SEM showed that higher concentrations (> 30 mg/mL) of forsythiaside effectively inhibited the adhesion of S. aureus and MRSA on the surface of the titanium disc in 24 h. Forsythiaside was capable of attenuating Ti-induced activation of nuclear factor-κB signaling, targeting IκB kinase-α (IKKα) kinases of macrophages, and influencing the expression of NF-κB downstream cytokines. Conclusions: These observations suggest that forsythiaside is a potential agent for the treatment of Ti implant- associated infection and inflammation. Keywords: Titanium, Forsythiaside, Anti-bacteria, Anti-inflammation, Nuclear factor-κB signaling pathway Background infections. One is that Ti is bio-inert and an easy substra- Titanium (Ti) and its alloys are widely used in orthopedic tum for bacterial surface adhesion and biofilm formation. implants such as hip and knee prostheses, fixations, and Therefore, Ti implant-associated infections can be inhibited dental implants. The main disadvantages of using ortho- by immobilizing antibacterial agents on the Ti surface. The pedic implants are the chances of developing aseptic loos- other reason is that the host factors required for implant- ening and infection [1]. Once implanted, orthopedic ation of the Ti prosthesis belong to a relatively immune prostheses may develop microbial infections, especially fade zone and a small bacterial load can lead to the severe those used in open fracture fixation and arthroplasty [2, 3]. Ti implant-associated infection [4]. Since Ti surfaces are Two main reasons may contribute to Ti implant-associated susceptible to bacterial adherence and biofilm formation, surface modification of Ti is an important approach for fabricating antibacterial Ti implants. In the past decade, ef- * Correspondence: YTB01975@163.com forts on biofunctionalization of Ti were mainly focused on Haifeng Li and Dongmei Tang contributed equally to this work. immobilization of antibacterial agents on the Ti surface; Ti Department of Sports Medicine, The Affiliated Hospital of Qingdao University, No. 16 Jiangsu Road, Qingdao 266003, China surface-immobilized antibiotics, antimicrobial peptide Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 2 of 10 (AMP), or inorganic ions are either bactericidal on contact forsythiaside concentration that inhibited bacterial or locally release antibacterial agents. However, cytotoxicity growth. and local burst release of the antibacterial agents and bac- terial resistance are common problems associated with Bacterial adhesion on Ti such biofunctional Ti surfaces [5]. The spread plate method was used to investigate the ef- Forsythiaside has been widely used in traditional medi- fect of forsythiaside on bacterial adhesion on titanium cines in Asia to treat gonorrhea, erysipelas, inflammation, [10]. Briefly, bacteria were diluted to 1.0 × 10 CFU/mL pyrexia, ulcer, and other diseases. Forsythiaside possesses with fresh tryptic soya broth (TSB) medium containing strong antioxidative, antibacterial, anti-inflammatory, and 10 or 50 μM forsythiaside and added to a standard cyclic adenosine activity [6, 7] and monophosphate flat-bottomed 96-well culture plate such that the final phosphodiesterase (cAMP) inhibitory effects. Further, for- volume of the solution in the well was 200 μL. The sythiaside exhibited anti-oxidative abilities, since it can culture was incubated statically at 37 °C for 24 h, after counter the adverse effects of endotoxins by decreasing the which the broth was carefully decanted and the adhered percentage of regulatory T cells (Tregs) and inhibiting the cells were washed thrice gently with 0.01 M TLR4/MyD88/NF-κB signaling pathway [8]. In addition, phosphate-buffered saline (PBS) (pH 7.4) to eliminate forsythin also triggered weight loss and inhibited cigarette free planktonic bacteria. Following ultrasonic cleaning of smoke-induced NF-κB activationinadose-dependent the Ti surface, serial dilutions of the bacterial culture manner with upregulation of Nrf2 and HO-1 expression were made and the spread plate method was used to [9]. Therefore, forsythiaside can be used as an alternative quantitatively characterize the number of viable bacteria antioxidative and antibacterial agent of natural origin. In this adhered to the flat Ti surface in the presence of different study, we investigated the antibacterial activity of forsythia- concentrations of forsythiaside at 6 and 24 h. side on bacterial adhesion on Ti. The anti-inflammatory role of THP-1-deprived macrophages toward Ti was also examined. Bacterial morphology After disinfection at high temperature and pressure, the titanium plates (1 mm × 5 mm) were placed in 24-well Methods plates and 1 mL fresh TSB bacterial suspension (S. aureus The following reagents were used in this study: for- and MRSA), diluted to 1.0 × 10 CFU/mL with fresh TSB sythiaside (Solarbio, China), Ti6AL4V (Titanium alloy, medium containing 10 or 50 μM forsythiaside, was added NaOH, Tris–HCl buffer (pH = 8.6), cDNA synthesis kit, in each well of each group. The cultures were incubated at real-time PCR kit (SYBR Premix EX Taq, TaKaRa), 37 °C for 24 h, after which they were gently removed from Pierce™ bicinchoninic acid (BCA) protein assay kit the titanium plates and washed thrice with PBS to elimin- (ThermoFisher), RNA mini kit (Qiagen), alpha minimum ate non-adherent planktonic bacteria. The Live/Dead essential medium (α-MEM, Hyclone), fetal bovine serum Baclight™ viability kit was used to enumerate viable and (FBS, Gibco, Australia), trypsin–EDTA (0.5%), Alexa dead bacteria. Briefly, the Ti substrates were stained for Fluor 488 secondary antibody, IMMULITE®/IMMULI- 15 min at room temperature per manufacturer’s instruc- TE®1000TNF-α and IMMULITE®/IMMULITE®1000 tions, followed by aspiration of the dye solution and gentle IL-1β, (SIEMENS), and the NF-κB signaling pathway kit washing with PBS to remove non-specific staining. Live or (Cell Signaling Technology, USA). dead bacteria were observed under a laser confocal micro- scope (Leica TCS, SP2, Germany). The live bacteria fluo- Antibacterial activity assay of forsythiaside resced green in the presence of the fluorescent dye The minimum inhibitory concentration (MIC) was eval- SYSTO9, whereas the dead bacteria fluoresced red in the uated by the broth microdilution method recommended presence of PI. by the National Committee for Clinical Laboratory Stan- For characterizing bacterial morphology by SEM, each dards (NCCLS) using the Mueller–Hinton broth (MHB) group was removed from the small titanium plate after medium. In brief, the bacterial cell counts were adjusted 24 h of culture and gently rinsed thrice with PBS to to approximately 2 × 10 colony-forming units (CFU)/ eliminate non-adherent floating bacteria. Glutaraldehyde mL, and the forsythiaside solution was diluted to 2048, solution (2.5%) was used for initial fixation at 4 °C for 1024, 512, 256, 128, 64, 32, 16, and 8 μg/mL. A 100-μL 2 h, followed by rinsing thrice with PBS for 1 h. Cover volume of each bacterial suspension was added to the glass slides were fixed in 0.1% osmium tetroxide solution wells of a sterile 96-well plate containing 100 μL of each for 1 h. The cells were dehydrated using an alcohol gra- concentration of forsythiaside; the final volume in each dient (30, 50, 70, 80, 90, 95, and 100%; 10 min for each well was 200 μL. Controls were prepared using the cul- concentration) and sprayed with gold at critical point ture medium. The MIC was defined as the minimum dry time. The bacterial morphology and numbers were Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 3 of 10 observed under a scanning electron microscope (SEM, kits, respectively (Siemens Healthcare Diagnostics Inc.), JEOL JSM-6360LV, Japan). according to the manufacturer’s instructions. Immunofluorescence Macrophage viability on Ti After 3 days of incubation of the Ti substrates with The human macrophage cell line THP-1 (Chinese THP-1-deprived macrophages in the presence of differ- Academy of Sciences, Shanghai, China) was cultured in ent concentrations of forsythiaside, the Ti substrates Roswell Park memorial Institute (RPMI) 1640 medium were washed with PBS to detach the non-adherent cells supplemented with 10% FBS at 37 °C in a humidified 5% and fixed with 4% paraformaldehyde overnight. Then in- CO incubator. The macrophages were induced with cubated with 0.1% Triton X-100 for 15 min, gently phorbol-12-myristate-13-acetate (PMA) at the concen- washed with PBS, the cytoskeleton was stained with tration of 100 ng/mL for 24 h. The Ti substrates were fluorescent phalloidin for 30 min according to the man- plated in 24-well plates prior to seeding of cells at the ufacturer’s protocol, and the macrophage cell morph- density of 5 × 10 /mL. After incubation with Ti sub- ology was visualized by fluorescence microscope (Nikon, strates containing 0, 10, and 50 μM forsythiaside for Japan). 24 h, macrophage cell viability on each substrate was determined using the CCK-8 kit. Western blotting After 2 and 4 h of incubation of Ti substrates with Real-time PCR THP-1-deprived macrophages in the presence of differ- The Ti substrate size used for RNA extraction was ent concentrations of forsythiaside, total proteins were 34 mm × 2 mm. THP-1-deprived macrophages were extracted from cultured cells using radioimmunoprecipi- seeded at 5 × 10 cells per Ti discs and cultured in tation assay (RIPA) lysis buffer containing 0.1% phenyl- six-well plates containing the Ti substrates. Low concen- methane sulfonyl fluoride (PMSF). Lysates were tration (10 μM) and high concentration (100 μM) of for- centrifuged at 15,000×g for 15 min, and the supernatants sythiaside were added to the plates and incubated for were collected. Protein concentration was determined 24 h. Total RNA was isolated from each Ti group using using the BCA assay. Then, each protein lysate was the RNA Mini kit after 3 days of co-culture. Five resolved using sodium dodecyl sulfate–polyacrylamide hundred nanograms RNA from each Ti sample was gel electrophoresis (SDS–PAGE) on 10% gels and reversed-transcribed using the PrimeScript™ reverse transferred to polyvinylidene difluoride membranes transcription kit. The expression of the housekeeping gene (Millipore, USA). The membranes were blocked with 5% glyceraldehyde-3-phosphate dehydrogenase (GAPDH) skimmed milk in Tris-buffered saline–Tween 20 (TBST) was used as an internal control to normalize the results solution for 1 h and then incubated with primary anti- obtained using the 2-ΔΔCT method. The sequences of bodies IKK-α, phosphorylated IKK-α,IκB-α, phosphory- primers used for real-time PCR analysis are shown in lated IκB-α, MyD88, and GAPDH (Cell Signaling Table 1. Nos2 (iNOS and CCR-7) was selected as a marker Technology, USA) diluted in 1% (w/v) skimmed milk for the M1 inflammatory phenotype, and CD206 and powder in TBST overnight at 4 °C. Membranes were CD163 were selected as markers for the M2 regenerative washed and incubated with the appropriate secondary phenotype [11]. Further, after 3 days of incubation of the antibodies. Antibody reactivity was detected by exposure Ti substrates with THP-1-deprived macrophages and dif- in the Taton imaging system. ferent concentrations of forsythiaside, the levels of the pro-inflammatory cytokines (TNF-α,IL-1β, IL-6, and Statistical analysis IL-8) in the cell supernatant was determined using the Data were expressed as mean ± standard error of mean IMMULITE/IMMULITE 1000 TNF-α,IL-1β,and IL-8 (SEM), and experiments were performed in triplicate. The SPSS (version 19.0) was used to analyze the data. One-way ANOVA is followed by an S-N-K test to evalu- Table 1 MIC of forsythiaside toward S. aureus,methicillin-resistant ate the differences between groups. p values < 0.05 were S. aureus (MRSA), Staphylococcus epidermidis, and methicillin-resistant considered statistically significant. S. epidermidis (MRSE) μg/mL 256 128 64 32 16 8 4 Blank Results S. aureus (ATCC 25923) –– – +++ + + MIC of forsythiaside S. epidermidis (ATCC 35984) –– – +++ + + The antimicrobial activity of forsythiaside was tested MRSA (ATCC 43300) –– ++++ + + against four Gram-positive bacteria that are the common MRSE –– ++++ + + causes of orthopedic implant-related infections, namely, + muddy, − clear S. aureus (ATCC 25923), methicillin- resistant S. aureus Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 4 of 10 Fig. 1 Spread-plate method to quantitatively characterize the number of viable bacteria adhered on the flat titanium with different concentration of forsythiaside at 6 and 24 h. a and b: Spread-plate method and quantitatively characterize the number of S’aureus. c and d: Spread-plate method and quantitatively characterize the number of MRSA.The asterisk denotes the significant differences in the comparison of Ti, Ti + low, and Ti + high (p < 0.05) Fig. 2 Confocal laser scanning microscopy images of bacterial (A: S. aureus, B: MRSA) adhesion and colonization on the surface of Ti in the presence of forsythiaside after staining with the Live/Dead Baclight™ bacteria viability kit Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 5 of 10 Fig. 3 Scanning electron microscopy images of bacterial (A: S. aureus, B: MRSA) adhesion and colonization on the surface of Ti in the presence of forsythiaside (MRSA, ATCC 43300), Staphylococcus epidermidis Macrophage cell viability on the Ti surface in the (ATCC 35984), and methicillin-resistant S. epidermidis presence of forsythiaside (MRSE). The MIC was 64 μg/mL for S. aureus and S. The cell proliferation of the samples at 24 h did not epidermidis and 128 μg/mL for MRSA and MRSE differ. No significant differences in cell attachment or (Table 1). proliferation were observed in the early time points between the Ti, Ti + low, and Ti + high groups in the presence of forsythiaside (Fig. 4). Characterization of the viable bacteria on Ti samples in the presence of forsythiaside The number of viable S. aureus and MRSA on the Ti + Expression of inflammatory cytokines and M1 low and Ti + high discs were significantly lower than macrophage polarization those on the control Ti surfaces at 6 and 24 h (p < 0.05, We determined the mRNA levels of inflammation-related Fig. 1). The control Ti discs showed higher numbers of and macrophage polarization-related genes, including viable S. aureus and MRSA than Ti + low and Ti + high TNF-α,IL-1β, IL-6, IL-8, iNOS, CCR-7, CD163, and discs (p < 0.05, Fig. 1). The antibacterial activity of Ti, Ti CD206 on day 3 of incubation. The expression of TNF-α, + low, and Ti + high concentration of forsythiaside was IL-1β, IL-6, IL-8, iNOS, and CCR-7 decreased in the Ti + > 90% for S. aureus and MRSA at each time point. Thus low and Ti + high groups compared in the Ti group, while forsythiaside could significantly inhibit the viable bac- CD163 and CD206 increased in the Ti + low and Ti + high teria on Ti surface. groups compared in the Ti group (Fig. 5). To assess whether changes in the expression of inflammation-related CLSM to detect the live or dead bacteria on Ti samples in the presence of forsythiaside Live/dead cell staining assays revealed that many viable bacteria were present on the Ti surface after 24 h of co-culture of each Ti sample with S. aureus and MRSA in the presence of different concentrations of forsythia- side. However, the Ti + low and Ti + high groups showed significantly reduced S. aureus and MRSA adhe- sion and growth (Fig. 2). The bacteria adhering to the Ti surface at 24 h were predominantly dead, as indicated by red staining (Fig. 2), showing that forsythiaside could eliminate bacteria on the Ti surface. SEM was further used to characterize the antibacterial properties. The control Ti harbored many adherent bacteria and Fig. 4 Effects of different concentrations of forsythiaside on viability multiple small bacterial colonies at 24 h, whereas of THP-1-deprived macrophages on Ti. Cell viability was measured in the Ti + low and Ti + high groups had very low THP-1-deprived macrophages in Ti, Ti + low, and Ti + high groups numbers of adherent bacteria at 24 h (Fig. 3). Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 6 of 10 Fig. 5 TNF-α, IL-1β, IL-6 and IL-8 mRNA levels of THP-1-deprived macrophages were used for reverse transcription real-time PCR analysis at 24 h. M1 related gene iNOS, CCR-7 and M2 related gene CD206, CD163 were investigated. The expression levels were normalized to that of GAPDH.The data are presented as the mean ± SD. *p < 0.05 genes might be a result of forsythiaside-induced cell inflammation caused by forsythiaside were not related death, macrophage viability was determined using the to the cell death during the culture time. CCK-8 assay after 1 and 3 days of culture. CCK-8 analysis showed no statistically significant changes Secretion of inflammatory cytokines during the early stages of differentiation. Therefore, We examined the inflammatory cytokine secretion on we conclude that the concentrations of forsythiaside the third day of culture. The expression of inflammatory used in this study (1–100 μM) did not exert cytotoxic cytokines TNF-α, IL-1β, IL-6, and IL-8 decreased in the effects on macrophages and that the effects on presence of low or high forsythiaside concentration Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 7 of 10 Fig. 6 Secreted concentrations of TNF-α,IL-1β, IL-6, and IL-8 in the culture supernatant of macrophages were measured via chemiluminescence cytokines analysis at 24 h. The data are presented as the mean ± SD. *p <0.05 (Fig. 6), demonstrating that macrophages in contact with Macrophage morphology Ti can trigger an inflammatory microenvironment that The immunofluorescence images showed that the mac- increases macrophage infiltration and release of inflam- rophages on the blank Ti surface were in an active state, matory mediators, whereas the addition of forsythiaside as was evident from the numerous elongated pseudopo- could inhibit the release of inflammatory mediators. dia (Fig. 7). In contrast, the macrophages on the Ti Fig. 7 Fluorescence microscopy to evaluate the morphology of macrophages and activated macrophages on each Ti sample. The cytoskeleton is stained red. The scar bar is 50 μm Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 8 of 10 surfaces that were pre-treated with low or high concen- boosted treatment technology. However, these innova- tration of forsythiaside retained their overall native tions act as “double-edged swords,” as their widespread round shapes. application is accompanied by new medical problems, such as implant infection. Reports show that the inci- NF-κB signaling pathway dence of surgical infections of orthopedic implants is ap- Toll-like receptor 4 (TLR-4), a key pathogen recognition proximately 5%, of which the rate of postoperative receptor in the innate immune system, plays an import- closure infection is 3.6–8.1%, whereas that of the open ant role in the progression of inflammation. TLR-4 acti- fractures is as high as 17.5–21.2% [12]. Infection may vation triggers an intracellular signaling pathway leading occur locally after an orthopedic implantation surgery, to NF-κB and inflammatory cytokine production, which which might affect wound healing. In addition, it can is responsible for activating the innate immune system. also lead to destruction of bone structure and loss of The activation of NF-κB signaling pathway occurs pri- bone mass, eventually resulting in loosening of the marily via activation of a kinase called the IκB kinase implant. (IKK), which is composed of a heterodimer of the cata- Infection and implant-associated inflammation are the lytic IKKα and IKKβ subunits, then increase the levels of main complications that arise after an orthopedic im- p-IκBα and decrease the production of the inhibitor of plantation. Once the implant contacts the tissue, it is at NF-κB(IκB). With the degradation of IκB, the NF-κB risk of bacterial contamination, which may result in complex is then freed to enter the nucleus where it can implant failure. Further, the host immune system may turn on the inflammation-related gene. Forsythiaside trigger an inflammatory response post-implantation administration decreased NF-κB activation in a [13, 14]. Theinnateimmuneresponsetothe implant dose-dependent manner (Fig. 8). Moreover, treatment and secretion of inflammatory cytokines play a crucial with forsythiaside affected the MyD88 proteins levels, role in determining the in vivo performance of the whereas it decreased the levels of p-IκBα which in- implant. TNF-α,IL-1β, and IL-6 are pro-inflammatory creased the production of the inhibitor of NF-κB(IκBα) cytokines that promote apoptosis of osteoblasts, inhibit (Fig. 8). These observations suggested that forsythiaside osteoblast differentiation and the expression of osteo- probably targeted IKK-α and IKK-β kinases, thereby blast differentiation-related genes, and inhibit osteogen- inhibiting downstream activation of NF-κB and secretion esis of mesenchymal stem cells. Studies have reported of inflammatory cytokines. that TNF-α and IL-1β inhibit the osteogenesis of MSCs and suppress osteoblast-related gene expression. IL-1β Discussion stimulates bone resorption by promoting osteoclast Improvements and innovation in medical implant mater- activation and mediates the osteoclastogenic effects ial have considerably aided orthopedic diagnosis and of TNF-α by enhancing the expression of RANKL Fig. 8 Western blot assays were conducted to detect the molecular mechanism by which FA inhibit Ti-associated inflammation at 2 and 4 h. Immunoblots displaying the phosphorylation of IκB-α in THP-1 after treatment with a series of concentration of FA for 2 and 4 h under the co-culture of Ti Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 9 of 10 [15–17]. IL-6 can promote osteoclast differentiation forsythiaside was capable of attenuating Ti-induced activa- and inhibit osteoblast differentiation and mineralization of tion of NF-κB signaling, targeting IκB kinase-α (IKKα)of the ECM of osteoblasts [18]. In addition, IL-8, an inflam- macrophages, and influencing the expression of NF-κB matory cytokine that is mainly produced by macrophages, downstream inflammation cytokines. Thus, our study neutrophils, and endothelial cells, shows the potential to demonstrates that forsythiaside may be used as a potential activate osteoclastic differentiation and bone resorption agent for the treatment of Ti implant-associated infection [19–21]. Therefore, it is critical to reduce the secretions of and inflammation. such inflammatory cytokines when macrophages come in Abbreviations contact with the implant. Inflammatory response-induced CFU: Colony-forming units; CLSM: Confocal laser scanning microscopy; bone destruction occurs during orthopedic infection. In FA: Forsythiaside; FBS: Fetal bovine serum; IKKα:IκB kinase-α/β; MHB: Mueller–Hinton broth; MIC: Minimum inhibitory concentration; addition, bone destruction by local bacterial metabolism is MRSA: Methicillin-resistant Staphylococcus aureus; NF-κB: Nuclear factor-κB; a pathological process accompanying bone and joint infec- RT-PCR: Real-time polymerase chain reaction; S. aureus: Staphylococcus tions, which cannot be ignored. Persistent infection and aureus; SEM: Scanning electron microscopy; Ti: Titanium inflammatory response can activate a large number of os- Funding teoclasts, resulting in bone degradation and absorption This study was funded by Qingdao Livelihood Science and Technology [22–24]. Project (173314NSH). Forsythiaside, an active constituent isolated from the Availability of data and materials Chinese medicinal herb Forsythia suspensa, exhibits The datasets used and/or analyzed during the current study are available anti-infective and anti-inflammatory effects. Our study from the corresponding author on reasonable request. demonstrated that forsythiaside inhibited S. aureus Authors’ contributions adhesion on Ti surface. Therefore, forsythiaside can be HL, DT, CQ, and YZ carried out the experiments. HL, XZ, and GW prepared used locally to suppress S. aureus adherence to the pros- the manuscript. YT and HL designed the experiments. YT revised the manuscript. All authors reviewed the manuscript. All authors read and thetic surface. In addition, the expression of inflamma- approved the final manuscript. tory cytokines TNF-α,IL-1β, IL-6, and IL-8 decreased in the presence of low or high forsythiaside concentration. Ethics approval and consent to participate Not applicable In view of the postoperative bacterial infection of osteoblasts and the osteoclast-mediated activation of Competing interests inflammatory environment [25], the dual efficacy of The authors declare that they have no competing interests. forsythiaside (broad-spectrum antibacterial activity and inflammation inhibition) makes it an ideal candidate for Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in postoperative infection control. We observed that published maps and institutional affiliations. dose-dependent forsythiaside treatment attenuated IL-1β, IL-6, TNF-α, and IL-8 expression via NF-κB sup- Author details Department of Sports Medicine, The Affiliated Hospital of Qingdao pression. TLR-4 activation upon macrophages contact University, No. 16 Jiangsu Road, Qingdao 266003, China. Department of with Ti. Subsequently, the activated kinase called the IκB Anesthesiology, The Affiliated Hospital of Qingdao University, Qingdao kinase (IKK), which is composed of a heterodimer of the 266003, China. Department of Orthopedics, Changhai Hospital, the Second Military Medical University, Shanghai, China. catalytic IKKα subunits, increased the levels of p-IκBα and decreased the production of the inhibitor of NF-κB Received: 5 February 2018 Accepted: 15 May 2018 (IκB) [26]. 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Forsythiaside inhibits bacterial adhesion on titanium alloy and attenuates Ti-induced activation of nuclear factor-κB signaling-mediated macrophage inflammation

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Copyright © 2018 by The Author(s).
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Medicine & Public Health; Orthopedics; Surgical Orthopedics
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Abstract

Background: Inflammation and biofilm formation by Staphylococcus aureus (S. aureus) are common causes of periprosthetic infection and loosening. Recently, we identified that forsythiaside is bacteriostatic for S. aureus and methicillin-resistant S. aureus (MRSA). The purpose of the present study was to examine the effect of forsythiaside on S. aureus and MRSA adhesion and biofilm formation on the surface of titanium alloy, which is a popular material for orthopedic joint prostheses. Methods: Two strains of S. aureus and MRSA were used for in vitro experiments. The spread plate method, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) were used to characterize antimicrobial activity of forsythiaside. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to investigate the inhibitory level of forsythiaside required for titanium-associated inflammation. Results: Direct colony counting showed that 16 μg/mL forsythiaside significantly inhibited S. aureus and MRSA adhesion on titanium alloy discs in 2 h. CLSM and SEM showed that higher concentrations (> 30 mg/mL) of forsythiaside effectively inhibited the adhesion of S. aureus and MRSA on the surface of the titanium disc in 24 h. Forsythiaside was capable of attenuating Ti-induced activation of nuclear factor-κB signaling, targeting IκB kinase-α (IKKα) kinases of macrophages, and influencing the expression of NF-κB downstream cytokines. Conclusions: These observations suggest that forsythiaside is a potential agent for the treatment of Ti implant- associated infection and inflammation. Keywords: Titanium, Forsythiaside, Anti-bacteria, Anti-inflammation, Nuclear factor-κB signaling pathway Background infections. One is that Ti is bio-inert and an easy substra- Titanium (Ti) and its alloys are widely used in orthopedic tum for bacterial surface adhesion and biofilm formation. implants such as hip and knee prostheses, fixations, and Therefore, Ti implant-associated infections can be inhibited dental implants. The main disadvantages of using ortho- by immobilizing antibacterial agents on the Ti surface. The pedic implants are the chances of developing aseptic loos- other reason is that the host factors required for implant- ening and infection [1]. Once implanted, orthopedic ation of the Ti prosthesis belong to a relatively immune prostheses may develop microbial infections, especially fade zone and a small bacterial load can lead to the severe those used in open fracture fixation and arthroplasty [2, 3]. Ti implant-associated infection [4]. Since Ti surfaces are Two main reasons may contribute to Ti implant-associated susceptible to bacterial adherence and biofilm formation, surface modification of Ti is an important approach for fabricating antibacterial Ti implants. In the past decade, ef- * Correspondence: YTB01975@163.com forts on biofunctionalization of Ti were mainly focused on Haifeng Li and Dongmei Tang contributed equally to this work. immobilization of antibacterial agents on the Ti surface; Ti Department of Sports Medicine, The Affiliated Hospital of Qingdao University, No. 16 Jiangsu Road, Qingdao 266003, China surface-immobilized antibiotics, antimicrobial peptide Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 2 of 10 (AMP), or inorganic ions are either bactericidal on contact forsythiaside concentration that inhibited bacterial or locally release antibacterial agents. However, cytotoxicity growth. and local burst release of the antibacterial agents and bac- terial resistance are common problems associated with Bacterial adhesion on Ti such biofunctional Ti surfaces [5]. The spread plate method was used to investigate the ef- Forsythiaside has been widely used in traditional medi- fect of forsythiaside on bacterial adhesion on titanium cines in Asia to treat gonorrhea, erysipelas, inflammation, [10]. Briefly, bacteria were diluted to 1.0 × 10 CFU/mL pyrexia, ulcer, and other diseases. Forsythiaside possesses with fresh tryptic soya broth (TSB) medium containing strong antioxidative, antibacterial, anti-inflammatory, and 10 or 50 μM forsythiaside and added to a standard cyclic adenosine activity [6, 7] and monophosphate flat-bottomed 96-well culture plate such that the final phosphodiesterase (cAMP) inhibitory effects. Further, for- volume of the solution in the well was 200 μL. The sythiaside exhibited anti-oxidative abilities, since it can culture was incubated statically at 37 °C for 24 h, after counter the adverse effects of endotoxins by decreasing the which the broth was carefully decanted and the adhered percentage of regulatory T cells (Tregs) and inhibiting the cells were washed thrice gently with 0.01 M TLR4/MyD88/NF-κB signaling pathway [8]. In addition, phosphate-buffered saline (PBS) (pH 7.4) to eliminate forsythin also triggered weight loss and inhibited cigarette free planktonic bacteria. Following ultrasonic cleaning of smoke-induced NF-κB activationinadose-dependent the Ti surface, serial dilutions of the bacterial culture manner with upregulation of Nrf2 and HO-1 expression were made and the spread plate method was used to [9]. Therefore, forsythiaside can be used as an alternative quantitatively characterize the number of viable bacteria antioxidative and antibacterial agent of natural origin. In this adhered to the flat Ti surface in the presence of different study, we investigated the antibacterial activity of forsythia- concentrations of forsythiaside at 6 and 24 h. side on bacterial adhesion on Ti. The anti-inflammatory role of THP-1-deprived macrophages toward Ti was also examined. Bacterial morphology After disinfection at high temperature and pressure, the titanium plates (1 mm × 5 mm) were placed in 24-well Methods plates and 1 mL fresh TSB bacterial suspension (S. aureus The following reagents were used in this study: for- and MRSA), diluted to 1.0 × 10 CFU/mL with fresh TSB sythiaside (Solarbio, China), Ti6AL4V (Titanium alloy, medium containing 10 or 50 μM forsythiaside, was added NaOH, Tris–HCl buffer (pH = 8.6), cDNA synthesis kit, in each well of each group. The cultures were incubated at real-time PCR kit (SYBR Premix EX Taq, TaKaRa), 37 °C for 24 h, after which they were gently removed from Pierce™ bicinchoninic acid (BCA) protein assay kit the titanium plates and washed thrice with PBS to elimin- (ThermoFisher), RNA mini kit (Qiagen), alpha minimum ate non-adherent planktonic bacteria. The Live/Dead essential medium (α-MEM, Hyclone), fetal bovine serum Baclight™ viability kit was used to enumerate viable and (FBS, Gibco, Australia), trypsin–EDTA (0.5%), Alexa dead bacteria. Briefly, the Ti substrates were stained for Fluor 488 secondary antibody, IMMULITE®/IMMULI- 15 min at room temperature per manufacturer’s instruc- TE®1000TNF-α and IMMULITE®/IMMULITE®1000 tions, followed by aspiration of the dye solution and gentle IL-1β, (SIEMENS), and the NF-κB signaling pathway kit washing with PBS to remove non-specific staining. Live or (Cell Signaling Technology, USA). dead bacteria were observed under a laser confocal micro- scope (Leica TCS, SP2, Germany). The live bacteria fluo- Antibacterial activity assay of forsythiaside resced green in the presence of the fluorescent dye The minimum inhibitory concentration (MIC) was eval- SYSTO9, whereas the dead bacteria fluoresced red in the uated by the broth microdilution method recommended presence of PI. by the National Committee for Clinical Laboratory Stan- For characterizing bacterial morphology by SEM, each dards (NCCLS) using the Mueller–Hinton broth (MHB) group was removed from the small titanium plate after medium. In brief, the bacterial cell counts were adjusted 24 h of culture and gently rinsed thrice with PBS to to approximately 2 × 10 colony-forming units (CFU)/ eliminate non-adherent floating bacteria. Glutaraldehyde mL, and the forsythiaside solution was diluted to 2048, solution (2.5%) was used for initial fixation at 4 °C for 1024, 512, 256, 128, 64, 32, 16, and 8 μg/mL. A 100-μL 2 h, followed by rinsing thrice with PBS for 1 h. Cover volume of each bacterial suspension was added to the glass slides were fixed in 0.1% osmium tetroxide solution wells of a sterile 96-well plate containing 100 μL of each for 1 h. The cells were dehydrated using an alcohol gra- concentration of forsythiaside; the final volume in each dient (30, 50, 70, 80, 90, 95, and 100%; 10 min for each well was 200 μL. Controls were prepared using the cul- concentration) and sprayed with gold at critical point ture medium. The MIC was defined as the minimum dry time. The bacterial morphology and numbers were Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 3 of 10 observed under a scanning electron microscope (SEM, kits, respectively (Siemens Healthcare Diagnostics Inc.), JEOL JSM-6360LV, Japan). according to the manufacturer’s instructions. Immunofluorescence Macrophage viability on Ti After 3 days of incubation of the Ti substrates with The human macrophage cell line THP-1 (Chinese THP-1-deprived macrophages in the presence of differ- Academy of Sciences, Shanghai, China) was cultured in ent concentrations of forsythiaside, the Ti substrates Roswell Park memorial Institute (RPMI) 1640 medium were washed with PBS to detach the non-adherent cells supplemented with 10% FBS at 37 °C in a humidified 5% and fixed with 4% paraformaldehyde overnight. Then in- CO incubator. The macrophages were induced with cubated with 0.1% Triton X-100 for 15 min, gently phorbol-12-myristate-13-acetate (PMA) at the concen- washed with PBS, the cytoskeleton was stained with tration of 100 ng/mL for 24 h. The Ti substrates were fluorescent phalloidin for 30 min according to the man- plated in 24-well plates prior to seeding of cells at the ufacturer’s protocol, and the macrophage cell morph- density of 5 × 10 /mL. After incubation with Ti sub- ology was visualized by fluorescence microscope (Nikon, strates containing 0, 10, and 50 μM forsythiaside for Japan). 24 h, macrophage cell viability on each substrate was determined using the CCK-8 kit. Western blotting After 2 and 4 h of incubation of Ti substrates with Real-time PCR THP-1-deprived macrophages in the presence of differ- The Ti substrate size used for RNA extraction was ent concentrations of forsythiaside, total proteins were 34 mm × 2 mm. THP-1-deprived macrophages were extracted from cultured cells using radioimmunoprecipi- seeded at 5 × 10 cells per Ti discs and cultured in tation assay (RIPA) lysis buffer containing 0.1% phenyl- six-well plates containing the Ti substrates. Low concen- methane sulfonyl fluoride (PMSF). Lysates were tration (10 μM) and high concentration (100 μM) of for- centrifuged at 15,000×g for 15 min, and the supernatants sythiaside were added to the plates and incubated for were collected. Protein concentration was determined 24 h. Total RNA was isolated from each Ti group using using the BCA assay. Then, each protein lysate was the RNA Mini kit after 3 days of co-culture. Five resolved using sodium dodecyl sulfate–polyacrylamide hundred nanograms RNA from each Ti sample was gel electrophoresis (SDS–PAGE) on 10% gels and reversed-transcribed using the PrimeScript™ reverse transferred to polyvinylidene difluoride membranes transcription kit. The expression of the housekeeping gene (Millipore, USA). The membranes were blocked with 5% glyceraldehyde-3-phosphate dehydrogenase (GAPDH) skimmed milk in Tris-buffered saline–Tween 20 (TBST) was used as an internal control to normalize the results solution for 1 h and then incubated with primary anti- obtained using the 2-ΔΔCT method. The sequences of bodies IKK-α, phosphorylated IKK-α,IκB-α, phosphory- primers used for real-time PCR analysis are shown in lated IκB-α, MyD88, and GAPDH (Cell Signaling Table 1. Nos2 (iNOS and CCR-7) was selected as a marker Technology, USA) diluted in 1% (w/v) skimmed milk for the M1 inflammatory phenotype, and CD206 and powder in TBST overnight at 4 °C. Membranes were CD163 were selected as markers for the M2 regenerative washed and incubated with the appropriate secondary phenotype [11]. Further, after 3 days of incubation of the antibodies. Antibody reactivity was detected by exposure Ti substrates with THP-1-deprived macrophages and dif- in the Taton imaging system. ferent concentrations of forsythiaside, the levels of the pro-inflammatory cytokines (TNF-α,IL-1β, IL-6, and Statistical analysis IL-8) in the cell supernatant was determined using the Data were expressed as mean ± standard error of mean IMMULITE/IMMULITE 1000 TNF-α,IL-1β,and IL-8 (SEM), and experiments were performed in triplicate. The SPSS (version 19.0) was used to analyze the data. One-way ANOVA is followed by an S-N-K test to evalu- Table 1 MIC of forsythiaside toward S. aureus,methicillin-resistant ate the differences between groups. p values < 0.05 were S. aureus (MRSA), Staphylococcus epidermidis, and methicillin-resistant considered statistically significant. S. epidermidis (MRSE) μg/mL 256 128 64 32 16 8 4 Blank Results S. aureus (ATCC 25923) –– – +++ + + MIC of forsythiaside S. epidermidis (ATCC 35984) –– – +++ + + The antimicrobial activity of forsythiaside was tested MRSA (ATCC 43300) –– ++++ + + against four Gram-positive bacteria that are the common MRSE –– ++++ + + causes of orthopedic implant-related infections, namely, + muddy, − clear S. aureus (ATCC 25923), methicillin- resistant S. aureus Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 4 of 10 Fig. 1 Spread-plate method to quantitatively characterize the number of viable bacteria adhered on the flat titanium with different concentration of forsythiaside at 6 and 24 h. a and b: Spread-plate method and quantitatively characterize the number of S’aureus. c and d: Spread-plate method and quantitatively characterize the number of MRSA.The asterisk denotes the significant differences in the comparison of Ti, Ti + low, and Ti + high (p < 0.05) Fig. 2 Confocal laser scanning microscopy images of bacterial (A: S. aureus, B: MRSA) adhesion and colonization on the surface of Ti in the presence of forsythiaside after staining with the Live/Dead Baclight™ bacteria viability kit Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 5 of 10 Fig. 3 Scanning electron microscopy images of bacterial (A: S. aureus, B: MRSA) adhesion and colonization on the surface of Ti in the presence of forsythiaside (MRSA, ATCC 43300), Staphylococcus epidermidis Macrophage cell viability on the Ti surface in the (ATCC 35984), and methicillin-resistant S. epidermidis presence of forsythiaside (MRSE). The MIC was 64 μg/mL for S. aureus and S. The cell proliferation of the samples at 24 h did not epidermidis and 128 μg/mL for MRSA and MRSE differ. No significant differences in cell attachment or (Table 1). proliferation were observed in the early time points between the Ti, Ti + low, and Ti + high groups in the presence of forsythiaside (Fig. 4). Characterization of the viable bacteria on Ti samples in the presence of forsythiaside The number of viable S. aureus and MRSA on the Ti + Expression of inflammatory cytokines and M1 low and Ti + high discs were significantly lower than macrophage polarization those on the control Ti surfaces at 6 and 24 h (p < 0.05, We determined the mRNA levels of inflammation-related Fig. 1). The control Ti discs showed higher numbers of and macrophage polarization-related genes, including viable S. aureus and MRSA than Ti + low and Ti + high TNF-α,IL-1β, IL-6, IL-8, iNOS, CCR-7, CD163, and discs (p < 0.05, Fig. 1). The antibacterial activity of Ti, Ti CD206 on day 3 of incubation. The expression of TNF-α, + low, and Ti + high concentration of forsythiaside was IL-1β, IL-6, IL-8, iNOS, and CCR-7 decreased in the Ti + > 90% for S. aureus and MRSA at each time point. Thus low and Ti + high groups compared in the Ti group, while forsythiaside could significantly inhibit the viable bac- CD163 and CD206 increased in the Ti + low and Ti + high teria on Ti surface. groups compared in the Ti group (Fig. 5). To assess whether changes in the expression of inflammation-related CLSM to detect the live or dead bacteria on Ti samples in the presence of forsythiaside Live/dead cell staining assays revealed that many viable bacteria were present on the Ti surface after 24 h of co-culture of each Ti sample with S. aureus and MRSA in the presence of different concentrations of forsythia- side. However, the Ti + low and Ti + high groups showed significantly reduced S. aureus and MRSA adhe- sion and growth (Fig. 2). The bacteria adhering to the Ti surface at 24 h were predominantly dead, as indicated by red staining (Fig. 2), showing that forsythiaside could eliminate bacteria on the Ti surface. SEM was further used to characterize the antibacterial properties. The control Ti harbored many adherent bacteria and Fig. 4 Effects of different concentrations of forsythiaside on viability multiple small bacterial colonies at 24 h, whereas of THP-1-deprived macrophages on Ti. Cell viability was measured in the Ti + low and Ti + high groups had very low THP-1-deprived macrophages in Ti, Ti + low, and Ti + high groups numbers of adherent bacteria at 24 h (Fig. 3). Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 6 of 10 Fig. 5 TNF-α, IL-1β, IL-6 and IL-8 mRNA levels of THP-1-deprived macrophages were used for reverse transcription real-time PCR analysis at 24 h. M1 related gene iNOS, CCR-7 and M2 related gene CD206, CD163 were investigated. The expression levels were normalized to that of GAPDH.The data are presented as the mean ± SD. *p < 0.05 genes might be a result of forsythiaside-induced cell inflammation caused by forsythiaside were not related death, macrophage viability was determined using the to the cell death during the culture time. CCK-8 assay after 1 and 3 days of culture. CCK-8 analysis showed no statistically significant changes Secretion of inflammatory cytokines during the early stages of differentiation. Therefore, We examined the inflammatory cytokine secretion on we conclude that the concentrations of forsythiaside the third day of culture. The expression of inflammatory used in this study (1–100 μM) did not exert cytotoxic cytokines TNF-α, IL-1β, IL-6, and IL-8 decreased in the effects on macrophages and that the effects on presence of low or high forsythiaside concentration Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 7 of 10 Fig. 6 Secreted concentrations of TNF-α,IL-1β, IL-6, and IL-8 in the culture supernatant of macrophages were measured via chemiluminescence cytokines analysis at 24 h. The data are presented as the mean ± SD. *p <0.05 (Fig. 6), demonstrating that macrophages in contact with Macrophage morphology Ti can trigger an inflammatory microenvironment that The immunofluorescence images showed that the mac- increases macrophage infiltration and release of inflam- rophages on the blank Ti surface were in an active state, matory mediators, whereas the addition of forsythiaside as was evident from the numerous elongated pseudopo- could inhibit the release of inflammatory mediators. dia (Fig. 7). In contrast, the macrophages on the Ti Fig. 7 Fluorescence microscopy to evaluate the morphology of macrophages and activated macrophages on each Ti sample. The cytoskeleton is stained red. The scar bar is 50 μm Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 8 of 10 surfaces that were pre-treated with low or high concen- boosted treatment technology. However, these innova- tration of forsythiaside retained their overall native tions act as “double-edged swords,” as their widespread round shapes. application is accompanied by new medical problems, such as implant infection. Reports show that the inci- NF-κB signaling pathway dence of surgical infections of orthopedic implants is ap- Toll-like receptor 4 (TLR-4), a key pathogen recognition proximately 5%, of which the rate of postoperative receptor in the innate immune system, plays an import- closure infection is 3.6–8.1%, whereas that of the open ant role in the progression of inflammation. TLR-4 acti- fractures is as high as 17.5–21.2% [12]. Infection may vation triggers an intracellular signaling pathway leading occur locally after an orthopedic implantation surgery, to NF-κB and inflammatory cytokine production, which which might affect wound healing. In addition, it can is responsible for activating the innate immune system. also lead to destruction of bone structure and loss of The activation of NF-κB signaling pathway occurs pri- bone mass, eventually resulting in loosening of the marily via activation of a kinase called the IκB kinase implant. (IKK), which is composed of a heterodimer of the cata- Infection and implant-associated inflammation are the lytic IKKα and IKKβ subunits, then increase the levels of main complications that arise after an orthopedic im- p-IκBα and decrease the production of the inhibitor of plantation. Once the implant contacts the tissue, it is at NF-κB(IκB). With the degradation of IκB, the NF-κB risk of bacterial contamination, which may result in complex is then freed to enter the nucleus where it can implant failure. Further, the host immune system may turn on the inflammation-related gene. Forsythiaside trigger an inflammatory response post-implantation administration decreased NF-κB activation in a [13, 14]. Theinnateimmuneresponsetothe implant dose-dependent manner (Fig. 8). Moreover, treatment and secretion of inflammatory cytokines play a crucial with forsythiaside affected the MyD88 proteins levels, role in determining the in vivo performance of the whereas it decreased the levels of p-IκBα which in- implant. TNF-α,IL-1β, and IL-6 are pro-inflammatory creased the production of the inhibitor of NF-κB(IκBα) cytokines that promote apoptosis of osteoblasts, inhibit (Fig. 8). These observations suggested that forsythiaside osteoblast differentiation and the expression of osteo- probably targeted IKK-α and IKK-β kinases, thereby blast differentiation-related genes, and inhibit osteogen- inhibiting downstream activation of NF-κB and secretion esis of mesenchymal stem cells. Studies have reported of inflammatory cytokines. that TNF-α and IL-1β inhibit the osteogenesis of MSCs and suppress osteoblast-related gene expression. IL-1β Discussion stimulates bone resorption by promoting osteoclast Improvements and innovation in medical implant mater- activation and mediates the osteoclastogenic effects ial have considerably aided orthopedic diagnosis and of TNF-α by enhancing the expression of RANKL Fig. 8 Western blot assays were conducted to detect the molecular mechanism by which FA inhibit Ti-associated inflammation at 2 and 4 h. Immunoblots displaying the phosphorylation of IκB-α in THP-1 after treatment with a series of concentration of FA for 2 and 4 h under the co-culture of Ti Li et al. Journal of Orthopaedic Surgery and Research (2018) 13:139 Page 9 of 10 [15–17]. IL-6 can promote osteoclast differentiation forsythiaside was capable of attenuating Ti-induced activa- and inhibit osteoblast differentiation and mineralization of tion of NF-κB signaling, targeting IκB kinase-α (IKKα)of the ECM of osteoblasts [18]. In addition, IL-8, an inflam- macrophages, and influencing the expression of NF-κB matory cytokine that is mainly produced by macrophages, downstream inflammation cytokines. Thus, our study neutrophils, and endothelial cells, shows the potential to demonstrates that forsythiaside may be used as a potential activate osteoclastic differentiation and bone resorption agent for the treatment of Ti implant-associated infection [19–21]. Therefore, it is critical to reduce the secretions of and inflammation. such inflammatory cytokines when macrophages come in Abbreviations contact with the implant. Inflammatory response-induced CFU: Colony-forming units; CLSM: Confocal laser scanning microscopy; bone destruction occurs during orthopedic infection. In FA: Forsythiaside; FBS: Fetal bovine serum; IKKα:IκB kinase-α/β; MHB: Mueller–Hinton broth; MIC: Minimum inhibitory concentration; addition, bone destruction by local bacterial metabolism is MRSA: Methicillin-resistant Staphylococcus aureus; NF-κB: Nuclear factor-κB; a pathological process accompanying bone and joint infec- RT-PCR: Real-time polymerase chain reaction; S. aureus: Staphylococcus tions, which cannot be ignored. Persistent infection and aureus; SEM: Scanning electron microscopy; Ti: Titanium inflammatory response can activate a large number of os- Funding teoclasts, resulting in bone degradation and absorption This study was funded by Qingdao Livelihood Science and Technology [22–24]. Project (173314NSH). Forsythiaside, an active constituent isolated from the Availability of data and materials Chinese medicinal herb Forsythia suspensa, exhibits The datasets used and/or analyzed during the current study are available anti-infective and anti-inflammatory effects. Our study from the corresponding author on reasonable request. demonstrated that forsythiaside inhibited S. aureus Authors’ contributions adhesion on Ti surface. Therefore, forsythiaside can be HL, DT, CQ, and YZ carried out the experiments. HL, XZ, and GW prepared used locally to suppress S. aureus adherence to the pros- the manuscript. YT and HL designed the experiments. YT revised the manuscript. All authors reviewed the manuscript. All authors read and thetic surface. In addition, the expression of inflamma- approved the final manuscript. tory cytokines TNF-α,IL-1β, IL-6, and IL-8 decreased in the presence of low or high forsythiaside concentration. Ethics approval and consent to participate Not applicable In view of the postoperative bacterial infection of osteoblasts and the osteoclast-mediated activation of Competing interests inflammatory environment [25], the dual efficacy of The authors declare that they have no competing interests. forsythiaside (broad-spectrum antibacterial activity and inflammation inhibition) makes it an ideal candidate for Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in postoperative infection control. We observed that published maps and institutional affiliations. dose-dependent forsythiaside treatment attenuated IL-1β, IL-6, TNF-α, and IL-8 expression via NF-κB sup- Author details Department of Sports Medicine, The Affiliated Hospital of Qingdao pression. TLR-4 activation upon macrophages contact University, No. 16 Jiangsu Road, Qingdao 266003, China. Department of with Ti. Subsequently, the activated kinase called the IκB Anesthesiology, The Affiliated Hospital of Qingdao University, Qingdao kinase (IKK), which is composed of a heterodimer of the 266003, China. Department of Orthopedics, Changhai Hospital, the Second Military Medical University, Shanghai, China. catalytic IKKα subunits, increased the levels of p-IκBα and decreased the production of the inhibitor of NF-κB Received: 5 February 2018 Accepted: 15 May 2018 (IκB) [26]. 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Journal

Journal of Orthopaedic Surgery and ResearchSpringer Journals

Published: Jun 5, 2018

References

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