| Pseudomonas aeruginosa(PA),also known as P.aeruginosa,is an opportunistic pathogen that mainly inhabits patients with low body resistance or wound infection after burns.P.aeruginosa has a high clinical morbidity and mortality rate.The main reason is that it produces a dense and neat biofilm and enhances its drug resistance.It is an important cause of many diseases.Such as sepsis,wound infection after burns,etc.Surfactant refers to a substance that can obviously change the interface state of the solution system by adding a low dose.Its molecular formula has specific hydrophilic and hydrophobic groups that can be neatly arranged on the surface of the solution,which can reduce the surface tension of the substance,reduce the friction between the two phases,and change the permeability of the cell membrane.The chemical name of Sodium houttuyfonate(SH)is sodium decanoyl acetaldehyde bisulfite.It is an artificial chemical compound of houttuyfonate and sodium bisulfite,the effective components of the saururus chinensis family.It has been widely used in my country,such as in the clinical use of it to fight infection.However,SH has a high in vitro inhibitory concentration and has no significant bactericidal effect.It may not be able to play the role of anti-pathogenic microorganisms with a direct bactericidal mechanism similar to commonly used antibiotics.The specific anti-pathogenic microorganism mechanism has not yet been elucidated.According to the chemical structure of SH,it is found that it may have the chemical properties of an anionic surfactant,and combined with SH’s extensive antibacterial and antifungal,and immunomodulatory biological activities,we speculate that SH may destroy pathogenic bacteria through its surface activity.The membrane structure,thereby regulating the activity of immune cells,plays a role in inhibiting pathogenic microorganisms.Therefore,in this thesis,SH uses surfactants as the mode of action to deeply study its mechanism of inhibiting P.aeruginosa.Objective:Based on in vitro physical property experiments,cell experiments,and animal experiments to explore the mechanism of SH as a surfactant in inhibiting P.aeruginosa.Methods:Transcriptome sequencing was used to determine the differential expression of genes in P.aeruginosa treated with SH,In vitro physical properties test,the Krafft point(when the solubility of ionic surfactant in water increases with temperature to a certain value,the solubility increases sharply)was used to determine the Krafft of SH point,the oil discharge ring experiment detects the hydrophilic and hydrophobic ability of different concentrations of SH,the solubilization experiment detects the influence of SH on the solubility of poorly soluble substances,and the membrane permeability experiment detects the penetration of SH through the cell membrane of P.aeruginosa ability to detect the effect of SH on P.aeruginosa destroying red blood cells by rabbit red blood cell experiment,and the effect of SH on P.aeruginosa bacterial membrane protein by membrane protein spectroscopy experiment.Cell experiment:In vitro establishment of P.aeruginosa-induced phagocytic cell inflammation model to further verify the mechanism of SH as a surfactant against inflammatory damage.MTT method detects the optimal concentration of drugs and the optimal concentration of P.aeruginosa.MTT and inverted microscope observe the optimal cell seeding density.ELISA detects the level of IL-17,TNF-α,IFN-γin the cell supernatant,flow cytometry was used to detect the expression of F4/80,a marker on the surface of macrophages,and transmission electron microscopy was used to detect the phagocytic ability of macrophages.Animal experiment:In this study,BALB/C male mice were randomly divided into normal group,model group,SH group,and SDBS group.P.aeruginosa was used to smear the mouse tongue to establish a bacterial infection oral model,which was verified by HE staining of the mouse tongue.The model was successfully constructed.Serum IL-17,TNF-α,IFN-γlevels were detected by ELISA,T-SOD enzyme,CAT enzyme,MDA content expression was detected by 1%liver tissue homogenate,and liver and kidney tissues were detected by q RT-PCR In IL-17,TNF-α,IFN-γgene expression.Results:(1)The transcriptome assay results show that the mechanism of SH inhibiting P.aeruginosa is not only through a single target,but multi-targets work together,and combined with the SH structural formula,we speculate that SH may have a surface active effect.The results of in vitro physical properties test show that the Krafft value of SH is 24℃,and the results of the oil discharge ring show that SH has obvious oil discharge ability.The solubilization experiment shows that SH can effectively increase the solubility of insolubles.The results of membrane permeation show that as the concentration of SH increases,the membrane permeates effect is enhanced.The red blood cell experiment shows that the higher the SH concentration,the less the destruction of P.aeruginosa on the red blood cells.The membrane protein experiment shows that high concentrations of SH can reduce the fluorescence intensity of P.aeruginosa membrane protein.The above results suggest that SH may have the active function of anionic surfactant.(2)In the cell experiment,the results of MTT and inverted microscope showed that the cell seeding density was 3x10~5/ml,the results of MTT showed that the optimal concentration of SH was 60μg/ml,and the optimal concentration of P.aeruginosa was 1.5x10~3CFU(Clonal Formation Unit)/ml.The results of ELISA showed that the levels of IL-17,TNF-α,and IFN-γin the cell supernatant were significantly higher than those in the normal group,while the levels of SH intervention were significantly lower than those in the model group.The results of flow cytometry showed that the F4/80 expression of macrophages in the P.aeruginosa PAO1 induction group was significantly higher than that of the blank group,and significantly decreased after SH intervention;the results of flow cytometry showed that the P.aeruginosa PAO1 induction group macrophages The phagocytic ability was stronger than that of the blank group,and the phagocytic ability was significantly enhanced after SH intervention.The results of transmission electron microscopy showed that the number of macrophages phagocytosing P.aeruginosa increased significantly after SH intervention compared with the model group.We found that SH-treated P.aeruginosa is more conducive to the phagocytosis of macrophages than untreated ones.Cell experiment results show that SH may change the surface structure of P.aeruginosa through surface activity,thereby improving the phagocytic efficiency and function of macrophages,and avoiding excessive inflammation of macrophages,so as to eliminate P.aeruginosa in vitro The role of spore bacteria.(3)The results of animal experiments showed that HE staining found that the tongue tissue mucosa of the model group was seriously damaged,and the damaged tongue tissue mucosa was improved after SH intervention.The results of ELISA showed that the serum levels of IL-17,TNF-α,and IFN-γin the model group were significantly higher than those in the normal group,but were significantly reduced after SH intervention.The liver tissue results showed that the T-SOD enzyme and CAT enzyme of the model group were significantly lower than that of the normal group,and the level of MDA in the model group was significantly higher than that of the model group after SH intervention.The results of q RT-PCR showed that the IL-17,TNF-α,and IFN-γgene levels in the liver and kidney of the model group were significantly higher than those in the normal group,and the levels of IL-17,TNF-α,and IFN-γgenes in the liver and kidney were significantly lower than those in the model group after SH intervention.The results in animals indicate that SH may act on P.aeruginosa through surface activity,effectively regulate the activity of the immune system,and play a role in clearing infection.Conclusion:The transcriptome assay SH may not only have a single-target pharmacological effect.In vitro physical properties test results show that SH has surfactant properties,and the animal and cell experiments indicate that SH may change the surface structure of P.aeruginosa through surface activity and enhance the immune system of mice and cells,in particular,enhances the phagocytic function of phagocytes,down-regulates the expression of related inflammatory factor genes,and plays a role in inhibiting and eliminating P.aeruginosa inflammation in the body. |
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