Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFκB signal-transduction pathways

Abstract

Burgeoning antibiotic resistance and unfavorable outcomes of inflammatory injury after Pseudomonas aeruginosa infection have necessitated the development of novel agents that not only target quorum sensing (QS) but also combat inflammatory injury with the least risk of resistance. This study aimed to assess the anti-QS and anti-inflammatory activities of baicalein, a traditional herbal medicine that is widely used in the People’s Republic of China, against P. aeruginosa infection. We found that subminimum inhibitory concentrations of baicalein efficiently interfered with the QS-signaling pathway of P. aeruginosa via downregulation of the transcription of QS-regulated genes and the translation of QS-signaling molecules. This interference resulted in the global attenuation of QS-controlled virulence factors, such as motility and biofilm formation, and the secretion into the culture supernatant of extracellular virulence factors, including pyocyanin, LasA protease, LasB elastase, and rhamnolipids. Moreover, we examined the anti-inflammatory activity of baicalein and its mode of action via a P. aeruginosa-infected macrophage model to address its therapeutic effect. Baicalein reduced the P. aeruginosa-induced secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNFα. In addition, baicalein suppressed P. aeruginosa-induced activation of the MAPK and NFκB signal-transduction pathways in cocultured macrophages; this may be the mechanism by which baicalein inhibits the production of proinflammatory cytokines. Therefore, our study demonstrates that baicalein represents a potential treatment for P. aeruginosa infection because it clearly exhibits both antibacterial and anti-inflammatory activities.

Keywords:

• baicalein
• Pseudomonas aeruginosa
• quorum sensing
• virulence
• inflammatory cytokines
• signal transduction

Supplementary materials

Figure S1 HPLC-MS analysis of AHLs of Pseudomonas aeruginosa.

Notes: The retention times of odDHL (A) and BHL (B) were detected by HPLC, and the MS/MS fragment peaks of odDHL (C) and BHL (D) were analyzed by ESI-MS. Standard curves of odDHL (E) and BHL (F) generated from the pure compounds were drawn for quantification. Drug-free control sample (G) and treatment with 128 µg/mL (H), 64 µg/mL (I), and 32 µg/mL (J) baicalein samples were quantitated by HPLC-MS in MRM mode, respectively.
Abbreviations: HPLC, high-performance liquid chromatography; MS, mass spectrometry; odDHL, N-(3-oxododecanoyl)-l-homoserine lactone; BHL, N-butanoyl-l-homoserine lactone; ESI, electrospray ionization; MRM, multiple reaction monitoring; ES, electron spray; TIC, total iron count; std, standard; ES+, positive ionization mode of the electron spray.

Figure S2 Effects of different sub-minimum inhibitory concentration levels of baicalein on the viability of primary macrophages evaluated using the MTT assay.

Note: Data represent mean ± standard error of three independent experiments.
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NS, not significant.

Table S1 Primers used in this study for real-time polymerase chain reaction

Table S2 Precursor ions (M + H)⁺ and fragmentation ions derived from the acyl-chain moiety (M + H −101)⁺ of AHLs detected and identified by HPLC-MS

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81260663 and 81260002), the Program for the Cultivation of “139” High-Level Backbone Medical Talents in Guangxi Province (2014GWKJF14), and the Innovation Project of Guangxi Graduate Education (YCBZ2013014). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also thank Dr Liang Yang (Nanyang Technological University, Singapore) for generously donating the P. aeruginosa PAO1 wild-type strain and its mutants deficient in lasR-rhlR (ΔlasI-ΔrhlI).

Disclosure

The authors report no conflicts of interest in this work.