Research Of Combination Therapy Of LysGH15 And Apigenin For Treating Pneumonia Caused By Staphylococcus Aureus

Staphylococcus aureus is a gram-positive bacteria, which widely existed in human and animal skin surface and digestive tract. When immunity is low, Staphylococcus aureus breaks through the body’s natural immune barrier and causes infectious diseases. Staphylococcus aureus(hereinafter referred to as S. aureus) can cause a variety of infectious diseases, mastitis in dairy cows is a common S. aureus infections in clinical veterinary, while skin and soft tissue infections and life-threatening blood, respiratory infections caused by S. aureus are also very common, pneumonia attracts much attention because of its high morbidity and mortality rates in respiratory infections. Since the non-standard use of antibiotics, resistant strains wanton rebellion, even a super bacteria(Surperbug), so that the treatment of bacterial infectious diseases difficult. Phage lyase, encoded by the phage genome at the end process of phage infects bacteria, targets to hydrolyze the peptidoglycan on the cell wall of gram-positive bacteria to cause bacterial death rupture. In the growing situation of bacterial resistance, anti-toxin policy of natural medicine is a developed way to alleviate bacterial infection, at the same time, anti-inflammatory characterisics of medicine also is a hot topics on new drug development in today’s world. In this study, we choosed S. aureus phage lyase Lys GH15 and apigenin monomers to explore the combination therapeutic potential against S. aureus pneumonia.First, the S. aureus phage lyase Lys GH15 was induced expression and purification to give the size of 55 KDa and a 5mg/ml concentration of Lys GH15 protein. Then we studied the interaction of Lys GH15 with apigenin by UV spectrophotometer. 50μg/ml of Lys GH15 was added to the MRSA strain USA300 bacterial suspension and the change of OD600 value was measured within 10 min, while the OD600 value of bacterial suspension added the same concentration Lys GH15 and 8μg/ml apigenin was measured, no significantly different existed between the two groups(P> 0.05); Hemolysis and Western blot experiments explored the effect of Lys GH15 on the inhibition of S. aureus α- hemolysin by apigenin, the results showed that rabbit erythrocyte hemolysis rate of bacteria supernatants added 8μg/ml apigenin was only 2%, bacteria supernatants added apigenin and 50 or 100μg/ml Lys GH15 was not detected hemolysis; Western blot results showed that α-hemolysin content in S. aureus USA300 supernatant added 8μg/ml apigenin was significantly reduced, almost no α- hemolysis was detected in supernatant added 8μg/ml apigenin and 100 μg/ml Lys GH15. These results showed that the combination of apigenin and Lys GH15 exhibited a better result in the inhibition of α- hemolysin.Further research on the influence of Lys GH15 on the anti-inflammatory effects of apigenin, we used mice monocyte-macrophage Raw264.7 stimulated with S. aureus to produce an inflammatory model for the study. MTT experiments was used to determine the Lys GH15 and apigenin are not toxic on Raw264.7 cells within the range of 0.2-12.5μg/ml. After added Lys GH15, apigenin and Lys GH15 and apigenin into Raw264.7 cells stimulated with S. aureus, respectively, RT-PCR and ELISA results showed that apigenin significantly inhibit cytokine TNF-α, IL-6, IL-1β transcription and expression, the addition of Lys GH15 has not significant effecte on the inhibition. Western blot detection of the above-described process on Raw264.7 in MAKP and NF-κB signaling pathway, results showed that apigenin can inhibit the activation of MAKP and NF-κB signaling pathway in Raw264.7 cells stimulated. At the same time, Lys GH15 exhibited partially synergistic effect on the inhibition.In vitro, we identified the synergistic effect of Lys GH15 and apigenin in inhibition the expression of hemolysin and anti-inflammatory. In order to study the treatment effect of combination therapy on mouse S. aureus pneumonia, we intranasally established a mouse model of pneumonia by S. aureus USA300. We finded the minimum lethal dose of USA300 was 5×107CFU. Then we used Lys GH15 and apigenin alone or combined treatment respectively, the results showed that combination therapy can significantly improve the health status of the mice, reduced lung wet weight and dry weight ratio and the number of cells、 protein levels and cytokine levels in the bronchoalveolar lavage fluid compared to the untreated group(P <0.01); the therapeutic effect of combination therapy was significantly better than Lys GH15 monotherapy(P<0.05) and apigenin monotherapy(P<0.01), at 24 h after S.aureus challenge, the bacteria number of combination therapy treated mice was 3.5×101 CFU/mg in lung, the bacteria number of untreated group、apigenin alone treated group and Lys GH15 alone treated groups was 3.5×1010CFU/mg, 1×105CFU/mg and 3.0×102CFU/mg in lung, respectively. With regard to its better protective efficacy, the combination therapy of Lys GH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA.