The Combination Of Osthole With Baicalin Protects Mice From Staphylococcus Aureus Pneumonia

Staphylococcus aureus is an important Gram-positive pathogen that causes a variety of human and animal diseases, ranging from skin and soft tissue infections to arthritis, pneumonia, endocarditis, septicemia, which are commonly associated with high incidence and mortality rates. In 1959, methicillin, the first semi-synthetic penicillinase-fast penicillin was used in clinical, mainly for the treatment of penicillin resistant S. aureus infections. However, less than two years after meticillin was marketed, the first meticillin-resistant isolates(Meticillin-resistant Staphylococcus aureus, MRSA) were reported by Jevons. For the present, the continual appearance of multiple antibiotic-resistant and the worldwide prevalence has made MRSA infections become the most difficult-to-treat pathogen. Vancomycin is regarded as the last line of defense against MRSA infections, however, MRSA with vancomycin resistance was first described in 1997 by Hiramatsu. Subsequently, MRSA showed variable vancomycin resistant were isolated from different countries, which is a dangerous signal in controlling MRSA infections.S. aureus secretes various kinds of exotoxins and surface proteins in its pathogenicity, of which alpha-hemolysin(Hla) is one of the major virulence factors. Hla is a 33.2-k D poreforming toxin which is encoded by hla gene and regulated by the Agr two-component system. Hla is primarily secreted in the post-exponential growth phase. By binding to the target cell surfaces(such as erythrocytes and epithelial cells), the Hla monomer oligomerizes to form a 232.4-k D heptamer, forming a stable transmembrane pore, leading to the leakage of cellcontents and then causing cell death. Previous studies reported that S. aureus strains lacking Hla exhibited less virulent in a mouse model of pneumonia. As the critical role of Hla in the process of S. aureus pathogenicity, Hla may be an important anti-virulence target against S. aureus infections.In this study, we reported osthole and diosmetin, two kinds of Chinese medicinal monomers with little anti-bacterial activity against S. aureus, could reduce the hemolytic activity of culture supernatants in a concentration-dependent manner, in which osthole showed a better inhibitory effect. The analysis of western-blot, semi-quantitative RT-PCR and real-time RT-PCR showed that the two compounds could decrease the expression of Hla via down-regulation the transcription levels of hla gene, and thereby inhibiting the hemolytic activity of S. aureus culture supernatants. Previous research demonstrated that baicalin could inhibit the activity of Hla directly, therefore, we expected that the combination of osthole and baicalin might improve the therapeutic effects compared to the two used separately. In cytotoxicity assays, osthole with baicalin achieved unambiguous protection against S.aureus-mediated cell injury, and the action is better than each individual treatment. A similar effect was also reflected in the animal experiments as mice in the combination group were significantly protected compared with the two separate treatment groups. Our results suggested that the combination of osthole with baicalin protects mice from S. aurues pneumonia via targeting Hla. While the study represents a minor investigation in the combination of two kinds of Chinese medicinal monomers targeting the same virulence factor in different mechanisms, the findings may be of relevance to the development of novel therapeutics against S. aurues infections.