Subinhibitory Concentrations of Prim-O-Glucosylcimifugin Decrease the Expression of Alpha-Hemolysin in Staphylococcus aureus (USA300)

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 7579808, 8 pages https://doi.org/10.1155/2018/7579808 Research Article Subinhibitory Concentrations of Prim-O-Glucosylcimifugin Decrease the Expression of Alpha-Hemolysin in Staphylococcus aureus (USA300) Ouyang Ping,1 Yang Ruixue,1 Deng Jiaqiang,1 Wang Kaiyu,1 Fang Jing,1 Geng Yi,1 Huang Xiaoli ,2 Chen Defang,2 Lai Weimin,1 Tang Li,1 and Yin Lizi 1 1 2 College of Veterinary Medicine, Sichuan Agriculture University, Chengdu, China College of Animal Science and Technology, Sichuan Agriculture University, Chengdu, China Correspondence should be addressed to Yin Lizi; Received 9 March 2018; Accepted 9 May 2018; Published 19 July 2018 Academic Editor: José L. Rios Copyright © 2018 Ouyang Ping et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Staphylococcus aureus (S. aureus), an important opportunistic pathogen in human and animal, causes a series of diseases in the impairing of immunity of host and even then death. Alpha-hemolysin (Hla), a primary virulence factor, plays a major role in the pathogenic progress of S. aureus, especially in pneumonia. Prim-O-glucosylcimifugin (POG), a nature chromone compound, is an active ingredient in many Chinese Medicines. In this study, POG investigated the inhibitory effect of the secretion of Hla in S. aureus strain USA300 at the subinhibitory concentrations. The hemolysis assays and western blotting assays showed that POG can decrease the production of Hla in the USA300 growth cell cultures in a dose-dependent manner. The results of RT-PCR revealed that reduction of Hla was related to inhibit the transcription of hla and RNAIII. In the cells experiment, POG was proved to protect A549 cells from Hla-medicated injury. In conclusion, POG was shown the capacity of decreased the production of S. aureus Hla. POG can be developed as a candidate agent to treat S. aureus infections against Hla. 1. Introduction Staphylococcus aureus (S. aureus), a common Gram-positive bacterium pathogen in clinic, can cause various infectious diseases in skin, respiratory system, and bloodstream in human and animals [1]. S. aureus infection is an important zoonosis which impacts both human and animals. USA300 strain, a strain of community-associated methicillin-resistant S. aureus (MRSA), was first identified in 1998 in USA [2]. USA300 strain is causing diseases in many countries and regions around the world over the last decade [3–5]. S. aureus USA300 has become one of the most globally spread MRSA strain and caused a worldwide epidemic such as skin infection, soft tissue infections, and severe pneumonia [4]. In general, conventional antimicrobials are used to treat the bacterial diseases by killing the bacteria or inhibiting bacteria generation. Antibiotics increase selective pressure in sensitive bacteria, screen out resistant strains, and accelerate the spreading on global [6]. With widespread application and abusing of antibiotics, bacterial resistances are becoming a growing problem. With the widespread MRSA epidemics, MRSA is listed as a “serious threat” in CDC (Center for Disease Control and Prevention) [7]. Consequently, novel agents and therapeutic strategies are urgently needed to treat bacterial infections, especially of antibiotic-resistant bacteria. It was proved that the pathogenicity of S. aureus is related to the virulence factors of S. aureus [1]. Montgomery et al. (2008) have proved that USA300 isolates had stronger pathogenicity and caused more severe pneumonia in rat pneumonia model than USA400 isolates [8]. Virulence factors, like Panton-Valentine leukocidin and alpha-hemolysin (Hla), were increasingly secreted in USA300 isolates [8, 9]. Hla is an important virulence protein that is secreted by S. aureus into the extracellular. Hla causes disease by damaging various cells and tissues [10, 11]. Pneumocytes are effective target cells of Hla [11, 12]. Many researches have proved that 2 Evidence-Based Complementary and Alternative Medicine Glc O O O OH CH CH O OCH3 Figure 1: The chemical structure of prim-O-glucosylcimifugin (CAS No: 80681-45-4). Hla plays an important role in the pathogenesis of S. aureus infections, especially in pneumonia [10, 13, 14]. Consequently, Hla is considered a candidate drug target for the treatment of MRSA infections such as deadly staphylococcal pneumonia [15]. Due to the indispensable character of Hla in the pathogenicity of S. aureus, drugs reacting on the Hla will be novel medicines in S. aureus infection. As a familiar chromone, prim-O-glucosylcimifugin (POG, chemical structure shown in Figure 1) is one of Saposhnikovia divaricata (Turcz) Schischk’s major effective components. In Chinese Pharmacopoeia, POG has been defined as index component and active ingredient standardized of Saposhnikovia divaricata (Turcz) Schischk [16, 17]. Previous researches showed that POG exhibited many potent pharmacological activities including anticancer, analgesic, anticonvulsant, antipyretic, antinociceptive, and anti-inflammatory effects [18–21]. Saposhnikovia divaricata (Turcz) Schischk was commonly used for treatment of common colds and headache in traditional Chinese medicine. To our knowledge, no study had shown the effects of POG on the expression of Hla in S. aureus. In this study, we evaluated the effect of POG on the inhibition of Hla secretion in S. aureus USA300 using the hemolysis assay, western blotting, RT-PCR, and cell experiments. 2. Materials and Methods 2.1. Bacterial Strains, Cell Line, and Reagents. The communityassociated methicillin-resistant S. aureus (CA-MRSA) strain USA300 (ATCC BAA-1717), a Hla-producing strain, was used in this study. This strain was purchased from the American Type Culture Collection (ATCC). In this cells experiment, A549 cells (human alveolar epithelial cell line, ATCC CCL185) were used and commercially obtained from ATCC. Prim-O-Glucosylcimifugin (POG, purity≥98%) was obtained from Chengdu Herbpurify Co., Ltd., (Chengdu, China). The solution (40960 𝜇g/mL) of POG was prepared by dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored in 4∘ C for in vitro study. 2.2. Susceptibility Testing. The minimal inhibitory concentrations (MICs) of POG against S. aureus strain were determined by the broth microdilution method which recommended by the Clinical and Laboratory Standards Institute. Shortly, POG was diluted to the concentration range of 2-512 𝜇g/mL in a 96-well plate by double dilution method. Bacteria (5 × 105 CFU/mL) were inoculated to each well. The plate was inoculated at 37∘ C for 24 h. The MIC was defined as the lowest drug concentration that inhibited bacteria growth and was repeated for three times. 2.3. Growth Curve Assay. Bacteria were cultured in TSB at 37∘ C to OD600 = 0.3 and equally (100 ml) divi (...truncated)