Study On The Anti-infective Mechanism Of Fraxetin Aganist Pathogenic Bacteria Aeromonas Hydrophila

Aeromonas hydrophila is a conditional pathogen which is common in natural water,in the meantime,it is one of the important pathogens that cause diseases such as bacterial septicemia and bacteral enteritis.Nowadays,antibiotics are the main means to prevent and treat the bacterial diseases of fish.However,a large number of irregular applications in antibiotics will increase the drug resistance of pathogens.As a result,when the disease breaks out again,antibiotic treatment often fails to get effective treatment.Therefore,it is urgent to research and develop some new antibiotics with low toxicity and low resistance.There are more and more researches on Chinese herbal medicines and their extracts in the field of prevention and treatment of bacterial diseases in aquaculture.Fraxetin,also known as ash lactone,is the active ingredient of Chinese herbal medicine Ash Bark.It is a natural simple coumarin compound with anti-pathogenic microorganisms,anti-inflammatory and analgesic,anti-tumor,anti-oxidation,and neuroprotection vascular protection and other pharmacological activities.In this study,to explore the bacteriostatic mechanism of fraxetin in vitro,the effects of fraxetin on the minimum inhibitory concentration（MIC）,growth curve,biofilm and virulence-related phenotypes in non-antibacterial concentration such as lipase activity,exercise ability,protease activity and hemolysis in A.hydrophila were determined.The effect of fraxetin on the cytotoxicity of Grass carp kidney cells（CIK）was determined by MTT method.Through the establishment of a model of A.hydrophila infection,the effects of fraxetin on blood biochemical indexes and antioxidant capacity of Megalobrama amblycephala infected with A.hydrophila were detected,in order to study the protective mechanism of fraxetin on infected A.hydrophila.The main findings are as follows:1. Fraxetin had a significant inhibitory effect on A.hydrophila.The culture solution infected by A.hydrophila was clear and transparent after 24 hours,when the concentration of fraxetin was 128μg/m L and 256μg/m L.Therefore,the MIC was 128μg/m L.The inhibitory effect of fraxetin on A.hydrophila was dependent on concentration.Fraxetin significantly inhibited the growth of A.hydrophila,when its concentration was more than 16μg/m L（P<0.05）.8μg/m L fraxetin had non-significant inhibitory effect on the growth of A.hydrophila（P>0.05）.Logarithmic growth phase was apparently delayed（P<0.05）,and the concentration of bacterial solution in plateau was dramatically lower than that in control group,when the concentration of fraxetin was more than 32μg/m L.8μg/m L fraxetin observably reduced the lipase activity of A.hydrophila（P<0.05）.It had non-significant effect on the motility and hemolytic activity of A.hydrophila when the concentration of fraxetin was 2 to 8μg/m L（P>0.05）,while that could decrease the protease activity of A.hydrophila（P<0.05）.2. After 2 hours of fraxetin treatment with CIK cells,there was no significant effect on the proliferation of CIK cells（P>0.05）.When the action time on the application of fraxetin to CIK cells is prolonged to 24 h,,fraxetin more than 32μg/m L significant damaged to the cells（P<0.05）,and its IC₅₀was 83.22μg/m L.When the action time was further extended to 48 h,16μg/m L fraxetin would have a significant effect on cell（P<0.05）,with the IC₅₀was 59.72μg/m L.The fish body test results showed that fraxetin improved the survival rate of the fish,furthermore,the fraxetin has better protection effect on the fish when the concentration of fraxetin is 4μg/g and 16μg/g.The effects of fraxetin on blood biochemical indexes are as follows:fraxetin（64μg/g,16μg/g,and 4μg/g）has no significant effect on the plasma GLU content of fish（P>0.05）,but it can observably reduce TG content（P<0.05）.The TC content in the infected group increased strikingly（P<0.05）,and when fraxetin（64μg/g and 4μg/g）was administered,the TC content was significantly reduced（P<0.05）.The TP content and ALB content in the infected group increased significantly（P<0.05）;fraxetin（64μg/g and 4μg/g）could significantly reduce the content of TP,ALB,and C3（P<0.05）.The ALP activity of the fish in the infected group decreased observably（P<0.05）.Fraxetin（64μg/g,16μg/g,and4 μg/g）significantly increased the ALP activity（P<0.05）.The AST activity was significantly increased after A.hydrophila infection（P<0.05）;while after treatment with fraxetin（64μg/g,16μg/g,and 4μg/g）,the AST activity decreased significantly（P<0.05）.After administration fraxetin（64μg/g）,the ALT activity can be significantly increased,and its LDH activity can be reduced（P<0.05）.The results of liver anti-oxidation index results were as follows:A.hydrophila infection significantly reduced T-SOD activity in the liver（P<0.05）;when fraxetin was administered 16μg/g and 4μg/g,the activity of T-SOD increased,but the effect was not remarkable（P>0.05）.When fraxetin was administered with 64μg/g,the T-SOD activity was significantly reduced（P<0.05）.The CAT activity was significantly reduced after fish infection（P<0.05）;when fraxetin（64μg/g,16μg/g,and 4μg/g）was administered,it could strikingly increase the CAT activity（P<0.05）.After treatment with fraxetin（64μg/g and 4μg/g）,the GSH content was reduced,but it wasn’t significant（P>0.05）;fraxetin（16μg/g）significantly reduced the GSH content（P<0.05）.The MDA content was observably decreased when fraxetin was 16μg/g and 4μg/g（P<0.05）,and with administration of 64μg/g,fraxetin,the MDA content reduced,but was not significant（P>0.05）.Tissue gene expression results were shown below:When fraxetin（64μg/g and16μg/g）was administered,it could significantly down-regulate Nrf2,Keap1,SOD,and GSH-Px in fish liver tissues after infection with A.hydrophila（P<0.05）,andreduced CAT m RNA expression strikingly（P<0.05）.The expression levels of Nrf2,Keap1,GSH-Px,CAT and SOD in the spleen of the infected group were significantly increased（P<0.05）,and after drug treatment（fraxetin 64μg/g,16μg/g,4μg/g and enrofloxacin 20μg/g）,the expression of Nrf2,Keap1,GSH-Px,CAT and SOD in the spleen were all observably reduced（P<0.05）.What’s more,the expression of Keap1 and CAT in the kidney of infected fish were significantly down-regulated（P<0.05）with administration of fraxetin（64μg/g）.