The Research Of Autophagy On Pseudomonas Aeruginosa In Patients With Diabetic Foot Infection

Objective: 1.In order to study the interactions between PA and autophagy,the expression of autophagy-related proteins in the wound tissue with or without Pseudomonas aeruginosa(PA)infection were detectd and compared.2.Explore the influence of infection with multidrug-resistant Pseudomonas aeruginosa(MDRPA)and ischemia on autophagy.3.Discuss the effects of the different virulence proteins secreted by T3 SS of PA on autophagy.Methods: 1.24 patients hospitalized in Metabolic Hospital of Tianjin Medical University from March 2016 to February 2017 who infected PA with DF were collected.The clinical data of the patients were collected and analyzed.The patients were divided into two groups depend on ischemic or not.The patients were divided into two groups depend on infecting by MDRPA or not.Patients were divided into infection stage and infection control stage before and after infection control.2.The tissue were collected by debridement,The tissue wsa divided into three parts.The first part was used for bacterial culture and drug resistance test.The number of colonies on wound were counted and frozen in the refrigerator.The virulence genes carried by PA T3 SS were identified by PCR.The second part of the tissue were used to prepare paraffin section.By HE dyeing to compare the pathology change of tissue.Immunohistochemistry assay was used to dectected the semi quantitative of autophagy related protein LC3(Microtubule-associated protein1 light chain 3),Beclin-1 and P62.Double immunofluorescent staining assay was used to detected the level of autophagy in macrophages.The last part of the granulation tissue were quantified determination of LC3-II/I,Beclin-1 and P62 by western blotting assay.3.After the infection were cleared,the above indicators were measured as described above.Results: 1.After the infection were cleared,the inflammatory markers(white blood cell count,neutrophil percentage,erythrocyte sedimentation rate and high sensitivity C-reactive protein),bacterial colonies,the area of the wound,FMN were decreased compared with PA infection(P < 0.05),no differences were found in other general data(P > 0.05).The inflammatory markers After the infection were cleared,the inflammatory markers(white blood cell count,neutrophil percentage,erythrocyte sedimentation rate and high sensitivity C-reactive protein),bacterial colonies,the area of the wound,FMN were decreased compared with PA infection(P < 0.05),no differences were found in other general data(P > 0.05).(N%,ESR,Hs-CRP)and bacterial colonies in MDRPA group were higher than the N-MDRPA group(P < 0.05).Compared with ischemic group,the area of the wound were smaller and ABI were lower(P < 0.05).2.After the infection were cleared,the number of new blood capillary and fibroblasts in granulation tissue were increased compared with PA infection,the inflammatory cells were less.What's more the expression of the positive staining cells of the LC3 and Beclin-1 were higher,P62 were lower(P < 0.05).Immunofluorescence showed that the detection of LC3 merged with CD14 were lower.Western blotting showed that the expression of LC3-II/I and Beclin-1 were higher,P62 were significantly decreased(P < 0.05).Compared with N-MDRPA group,the number of inflammatory cells in granulation tissue were less in MDRPA group,new blood capillary and fibroblasts had no statistical difference.The expression of positive staining cells of the LC3 were higher(P < 0.05),no statistical difference were found in expression of Beclin-1 and P62(P > 0.05).Immunofluorescence showed that the detection of LC3 merged with CD14 were lower.Western blotting showed that the expression of LC3-II/I and Beclin-1 were higher,P62 were lower(P < 0.05).The number of new blood capillary,fibroblasts and inflammatory cells in granulation tissue were less in ischemia group than nonischemia group.The expression of positive staining cells of the LC3 and Beclin-1 were higher,P62 were less(P < 0.05).Western blotting showed that the expression of LC3-II/I and Beclin-1 were higher,P62 were less(P < 0.05).Immunofluorescence showed that the detection of LC3 merged with CD14 were lower.3.PCR detection showed PA carried virulence gene of exo S.Conclusion: After the infection were cleared and metabolism was improved,the level of autophagy was increased,which amplied severe metabolic disorders may downregulate autophagy.PA may downregulate or even escape autophagy by some mechanism.What's more,the level of autophagy decreases furtherly,indicating that MDRPA may be more capable of escaping autophagy.Combined with ischemia,the level of autophagy was decreased obviously.Moderate induction of autophagy may be a new candidate for the treatment of DF combined with PA infection.