| There were a series of problems arising with the widespread use of antibiotics,such as toxicity,repeat-infection,bacterial drug resistance,especially the spread of bacterial resistance will cause no fit treatment for multi-drug resistant bacteria infection.Some research shows that some Chinese herbal drug has good bacteriostatic effect,its efficacy has obtained widely recognized in clinical treatment of infection disease,and there were little toxic and side effects,and it was difficult to induce drug resistance.Especially some traditional Chinese medicines can remove the drug-resistant bacteria resistant plasmid,decline the resistance.Rhizoma Coptis,a commonly used traditional Chinese herbal drug,has a broad spectrum of antibacterial,antiviral,but the investigation about the antimicrobial mechanism was less.In this paper,The Rhizoma Coptis was choosed as tested drug,and the Bacillus dysenteriae was choosed as tested bacteria,the antimicrobial mechanism was explored from following aspects.The experiment takes aqueous extract of Rhizoma Coptidis as the test drug,and Bacillus dysenteriae was selected as the test bacterium.The effect of the concentration of aqueous extract of Rhizoma Coptidis of the minimum inhibitory concentration of on bacillus dysenteriae and the influence on the growth curve of the cell was explored by half-proportion and absorptiometry.The the suitable concentration of Rhizoma Coptidis and incubation time were set,and the effect of Rhizoma Coptidis on cell membrane permeability,energy metabolism and cell wall of dysentery bacteria was determined by spectrophotometry.The influence of Rhizoma Coptidis on genomic DNA and protein synthesis of Bacillus dysenteriae were determined by gel retardation assay.The influence of Rhizoma Coptidis expression level of four kinds of virulence factors,including ipaH,dsbA,sodB and iutA,were evaluated by fluorescence quantitative PCR assay.The effect of Rhizoma Coptidis on the level of ROS in Bacillus dysenteriae was detected by flow cytometry DCFH-DA staining.The apoptosis of bacillus dysenteriae was detected by flow cytometry Annexin double staining of V-FITC and PI.flow cytometry PI staining to detect the changes of cell cycle.Result:?1?Investigation of the antibacterial activity of Rhizoma Coptidis on Bacillus dysenteriae The Rhizoma Coptidis has strong inhibitory effect on the pathogenic bacteria of bacillus dysenteriae.The results of MIC was 5 mg·mL-1,and logarithmic growth period was 10 h,and the inhibitory effect was increased with the increase of the time and concentration of Rhizoma Coptidis.?2?Effect of Rhizoma Coptidis on cell membrane and energy metabolism of Bacillus dysenteriae Compared with control group,leakage rate of macromolecular substance,including DNA,RNA,was 40.1% in 2.5 mg·mL-1 Rhizoma Coptidis group after 10 h culture,and the activity of succinate dehydrogenase?SDH?and malate dehydrogenase?MDH?were decreased by 21.9%,8.69%,respectively.Leakage rate of macromolecular substance,including DNA,RNA,was 65.4% in 5 mg·mL-1 Rhizoma Coptidis group,and the activity of succinate dehydrogenase?SDH?and malate dehydrogenase?MDH?were decreased by 53.9%,37.9%,respectively.Leakage rate of macromolecular substance,including DNA,RNA,was 75.3% in 10 mg·mL-1 Rhizoma Coptidis group,and the activity of succinate dehydrogenase?SDH?and malate dehydrogenase?MDH?were decreased by 68.5%,69.8%,respectively.?3?The effects of Rhizoma Coptidis on Bacillus dysenteriae bacteria cell wall Compared with control group,the content of AKP was enhanced rapidly when Rhizoma Coptidis and dysentery bacilli were cultured for 10 hours.The AKP content increased 62% in 2.5 mg·mL-1 Rhizoma Coptidis treatment group.The AKP content increased 77.6% in 10 mg·mL-1 Rhizoma Coptidis treatment group.?4?The effect of Rhizoma Coptidis on genomic DNA and protein synthesis of Bacillus dysenteriae When concentrations of Rhizoma Coptidis reached 5 mg·mL-1,the genomic DNA in cell partly combined with protein of Rhizoma Coptidis staying in the hole.The DNA synthesis was also significantly reduced with the extension of treatment time.Compared with the control group,the soluble protein content was reduced by 35% in 2.5 mg·mL-1 Coptis treatment group.the soluble protein content was reduced by 69% in 10 mg·mL-1 Coptis treatment group.?5?The effect of Rhizoma Coptidis on the expression of four virulence factors The expression levels of ipaH,dsbA,sodB and iutA of four kinds of virulence genes all decreased,which indicated that Rhizoma Coptidis could inhibit the expression of four virulence genes to reduce the damage to the body.?6?The effect of Rhizoma Coptidis on ROS content of Bacillus dysenteriae The content of reactive oxygen species?ROS?level in the Rhizoma Coptidis treated group was higher than that of the control group,which indicated that Rhizoma Coptidis broke the balance of ROS production and scavenging of ROS in vivo which caused the accumulation of ROS in bacteria affecting the normal physiological activity of the cell.?7?Effect of Rhizoma Coptidis on cell apoptosis The apoptosis rate of the treated group was higher than that of the control group,which showed that Rhizoma Coptidis induced cell apoptosis.?8?The effect of Rhizoma Coptidis on cell cycle of Bacillus dysenteriae The G1 phase in the treatment group was lower than that in the control group,and the S phase in the treatment group was higher than that of the control group,which showed that the cell cycle of the cell cycle was disturbed by the Rhizoma Coptidis.Conclusions: Rhizoma Coptidis could induce metabolic disorder of cell by changing structure of membrane protein of cell,declining the activity of SDH and MDH.And it also destroy the integrity of cell wall,disturb the synthesis of tropina by combining with the genomic DNA,increase the ROS level of thallus,cause apoptosis and derangement of cell cycle. |
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