The Mechanism Of Berberine On Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) can cause the respiratory disease of swine. The bacteria is one of pleomorphic glob?Lar short bacillus and with no spore. The respiratory disease caused by this bacteria has many characteristics such as high rate of death and high rate of infection. Easy to be infected by all ages of pigs and frequent outbreaks is also showed. Often it will infect the pigs mixing with a variety of viruses and bacteria in clinical. The mian phenomenons of infection are respiratory dysfunction and acute or chronic lung inflammation. Florfenicol usally used as treatment in the clinical, but with the incerasing antibiotic resistance and accumulation, forced us to find out new treatments.Berberine hydrochloride is a broad spectrum antibacterial agent belong to isoquinoline alkaloid of quaternary ammonium extracted from rhizoma coptidis and other plants. It has been used for many years and has nice inhibitory and germicidal functions against the Gram-negative bacterium and Gram-postive bacterium. Recently, some studies indicated that the berberine has strong cytotoxicity. This research is focus on the ablity of inhibition or killing of berberine against A. pleuropneumoniae and its mechanism.1 the effects of berberine against Actinobacillus pleuropneumoniae in vitro. The antibacterial activities were investigated in vitro by 2-time dilution method. The MIC and MBC will be measured and the time-kill curve will be drawn. ResuLts showed that the berberine had strong killing function against Actinobacillus pleuropneumoniae in vitro. Both the MIC and MBC of berberine against bacteria were 0.3125 mg/mL. All the bacteria died within 8 hours when treated with MIC concentration of berberine. However when the concentration of berberine under or equal to 1/2MIC. the time-kill curve were similar with the control group which suggested that there were dose-dependent between the berberine with bacteria.2 the effects of berberine on the cell membrane of Actinobacillus pleuropneumoniae. The leakage of macromolecuLar substances such as DNA and proteins and the leakage of Ca, Mg. K ion will response the integrity of the cell membrane. The using rate of reducing sugar and NPN will be employed to detect the permeability of cell membrane. The activites of AKP and LDH represent the integrity of the cell wall. By analysising the fluorescence change of protein located on the membrane to get a futher understanding of its mechanism. ResuLts showed that in the first of 10 min. the absorbance value of extracellular DNA showed little change. From 1?12 h. the concentration of extracellular DNA and protein increased at first followed by decreasing, the leakage of Ca, Mg, K ion increased which suggested that the integrity of the cell membrane was damaged. The extracellular activites of AKP and LDH were not show much difference compared with the control group suggested that the cell wall was less damaged. Berberine co?Ld bond with the tryptophan residues of protein and changed its structure then cuased the leakage of contents and showed the quenching was static.ResuLts showed that the berberine co?Ld damage the integrity of the cell membrane and changed the structure of some protein.3 the effects of berberine on the protein expression of Actinobacillus pleuropneumoniae.By measuring the expression of total protein and SDS-PAGE to detect the difference of protein after and befor treated with berberine. Res?Lts found that after treated with berberine. the total expression of protein increased first and then decreased and be suit for the grow state of bacteria. Meanwhile. SDS-PAGE showed the expression of protein showed some difference between the control group and the tested group. The normal protein band was clear and some of the protein bands were disapper. And the protein below 44.3kDa showed much higher changes and suggested that this part might be the target for berberine. Res?Lts showed that the berberine co?Ld influence the synthesis and expression of protein.4 the effects of berberine on the DNA expression and the interact of Actinobacillus pleuropneumoniae with berberine. Using DAPI, ?Ltraviolet absorption spectrum, EB-DNA, thermal denaturation curve to detect the changes of DNA expression after treated with berberine and the interact between them in vitro. DAPI showed that the fluorescence intensity of the control group was higher than treated group suggested that the berberine co?Ld decrease the expression of DNA. Res?Lts showed that the berberine co?Ld decrease the expression of DNA. ?Ltraviolet absorption spectrum and the PAGE showed that the berberine co?Ld interact with the DNA in vitro. EB-DNA and thermal denaturation curve showed that the berberine interacted with DNA by intercalation, and the level was higher than that of EB. Res?Lts showed that the berberine co?Ld influence the synthesis and expression of DNA and co?Ld bond with the DNA by intercalation.In a word, the berberine had a good killing effect against with the APP and co?Ld affect the cell membrane, the expression of DNA and protein.