| Staphylococcus aureus(S.aureus)is often parasitized in the nasal cavity and on the skin surface of healthy humans,or adheres to the surface of medical devices.It is an important Gram-positive micrococcus for community-acquired,hospital-acquired pneumonia and secondary infections.S.aureus infection has become a global problem.It can cause nearly 20,000 deaths each year in the United States alone and generate medical insurance costs of 14.5 billion.The number of deaths caused by S.aureus infection is even higher than the number of deaths caused by AIDS in the same period.S.aureus infections mainly cause local purulent inflammation,skin and soft tissue damage,and pneumonia,and threaten public health security heavily.In addition,S.aureus is one of the major pathogens of cows mastitis.Dairy cow mastitis is often caused by poor management,microbial infections,and environmental changes.It is a worldwide disease that threatens the rapid development of the dairy industry.The elimination rate of milk cows suffering from mastitis increases,with decreased milk yield and inferior milk quality.Microbial metabolites and secreted toxins accumulate in milk and endanger human health.Due to the special structure of the breast tissue,mastitis in dairy cows is confined to one or more breast milk areas.In severe cases,cow mastitis can cause systemic symptoms or bacteremia,and it is also difficult to treat.Cow mastitis caused by Staphylococcus aureus is one of the more common types of bacterial infections.With the increase of infection time,the difficulty of curing is increasing,so early detection and early prevention of mastitis are particularly important.The practice of adding antibiotics to prevent bacterial infections during feeding and management often promotes the evolution of bacteria and the development of drug resistance spreads between bacteria.S.aureus is susceptible to drug resistance due to its own characteristics.An anti-infection method that can develop milder stress on bacteria survival can be used to treat S.aureus mastitis,and the anti-virulence strategy is one of them.S.aureus is able to secrete a-hemolysin(Hla),which is one of the Pore-forming toxins(PFTs),which can destroy the host immune barrier,activate the host inflammatory response,produce cytotoxicity,and cause skin necrosis and lethality.It plays an important role in the process of infection and is a key target for determining the virulent and antivirulence strategies of S.aureus.Inula is a sunburning root of the tall plant Aquilaria of the genus Inula,a hard,curved,cylindrical or conical shape,usually a piece of 5 to 20 centimeters long.The gas is slightly fragrant,hard-tasting,sexually temperate,liver,spleen lung three classics,able to spleen and lungs,gas analgesic,Jieyu tocolysis,in the 2005 version of the "Chinese Pharmacopoeia" widely replace Aoki(containing aristolochic acid,has been abolished pharmaceutical standards)has been more widely used in the prescription of Guanxin Suhe Pills to be used.According to the principle of antivirulence strategy,in this study,the aqueous extract method was used to prepare the crude extract of Inula.The MIC,MBC detection,growth curves and bactericidal curves were used to determine the susceptibility test.Methicillin-resistant Staphylococcus aureus(MRSA)strain US A300 produced almost no antibacterial activity,and the MIC value was greater than 1024 ?g/mL.The anti-hemolytic assay platform was established using Staphylococcus aureus alpha-hemolysin as a target,and the hemolytic activity of supernatant of S.aureus-drug co-cultivation system was significantly inhibited by detecting 32 ?g/mL crude extract of Inula ulmoides(p<0.01),but when the crude extract of Inula was only incubated with the S.aureus supernatant,the hemolytic activity of the supernatant was not impaired.The Western blotting assays showed that the incubation of crude extracts of Inula was not directly responsible for the degradation of Hla in the supernatant,but the co-culture with S.aureus significantly inhibited the content of Hla in the supernatant.The bacterial total RNA of the co-cultured system with different concentrations of the drug was extracted.Then the reverse-transcribed and real-time quantitative PCR was performed and prove that the crude extract of Inula purpurea can inhibit the transcriptional process of both global regulator Agr and hla genes in S.aureus.As an invasive virulence factor secreted by S.aureus,Hla can directly or indirectly promote lung epithelial cell and epidermal barrier damage.In the model of cell infection established by human lung cancer epithelial cell line A549,the addition of crude extract of Inula fragrans can significantly inhibit the cell death caused by MRSA strain USA300(p<0.01)without affecting the survival of S.aureus in the supernatant.Experimental therapeutics experiments on pneumococcal models of mice infected with S.aureus have shown that 200 mg/mL crude extract of Inula purpurea can significantly increase the survival rate of lethal dose-infected mice.Crude extract of Inula purpurea will also inhibit the sublethal doses caused the wet/dry weight ratio of lung tissue and the release of three pro-inflammatory cytokines(p<0.01)in the lungs:TNF-?,IL-1? and IL-6,and significant inhibit the colonization of S.aureus in lung tissue.It was proved that the inhibitory activity of the crude extract of Inula sinensis on Hla,one of the virulence factors of Staphylococcus aureus,can still be exerted in animal body.In the treatment of clinical mastitis-infected cows with mastitis,twice daily,5 days of infusion of Inula purpurea extracts can significantly improve the infection status of mastitis cows,with a cure rate of 40%.Compared with pretreatment,the number of somatic cells in milk decreased,the number of bacteria detected in milk decreased.The main scavenger enzymes for superoxide and peroxide in whey and serum:superoxide dismutase(SOD)and Valley GSH-PX enzyme activity was significantly increased,and the content of nitric oxide(NO),the main inflammatory media,was significantly lower than before(p<0.01).Based on the experimental results above,it can be shown that Inulina extract can inhibit the production of hemolysin and cytotoxicity caused by S.aureus infection via inhibiting the transcription process but without inhibiting the growth of S.aureus.The inhibit activity was also obvious in vivo experiments.The above results demonstrated the potential of the crude extract of Inula citri as a strong virulence factor inhibitor of S.aureus in the clinical treatment of S.aureus infection,providing a candidate drug for the clinical treatment of mastitis in dairy cows,and provide experimental basis for the development of anti-Hla drugs. |
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