Study On The Anti-MRSA Infection Mechanism Of Corynebacterium Pyruviciproducens

Objective According to the experiments in vivo and ex vivo to investigate the ability of anti-MRSA infection of C.pyruviciproducens and CP-PGN,find out the possible mechanisms and effective composition of CP to play the function of anti-infection.Methods(1)Using the TCA method to extraction the CP-PGN,then treated with the lysozyme to determination whether the hazel sediment wastarget product.Analysing the structures、 types and contents of amino acids of CP-PGN.(2)Establishing the mouse models of MRSA infection and therapy to test whether CP/CP-PGN could enhance the ability of anti-infection.Mice were observed and noted with survival state and time.Detecting the pathological changes of lung,CFU of MRSA inliver,lung and blood and changes of spleen from the dead mice,checking the level of cytokines(IL-6 and TNF-α by q RT-PCR)in blood and count leucocytes in peripheral blood.(3)According to the vitro experiments,RAW264.7 cultured with MRSA,then collected cells to make climbing tablets,and the phagocytosis rate was evaluated under the microscope through the gram staining to analysis whether CP/CP-PGN could increase the phagocytosis of cells.(4)Griess assay was used to detect the content of NO.Transwell assay was used to observe the migration rate.The expression levels of TNF-α,IL-12 and IL-10 in cells was detected by quantitative real-time PCR to investigate the effects of CP/CP-PGN on the activation of RAW264.7 cells and peritoneal macrophages of mice.(5)Using the quantitative real-time PCR and flow cytometry to detect the expression levels of i NOS,Arginase-1 and the percentages of CD86 to study the effect of CP/CP-PGN on the differentiation of macrophages.(6)TLR2-si RNA was used to silence TR2 gene expression in RAW264.7 cells,then quantitative real-time PCR,Griess assay,cellmigration analysis and flow cytometry were used to determine the effect of CP/CP-PGN on activation and differentiation of RAW264.7.Results(1)The lysozyme assay experiment showed that the extracted product was peptidoglycan and CP-PGN possessed the characteristics of peptidoglycan.The molecular weight of CP-PGN is 18774 Da,showed the characteristic peaks of PGN by infrared spectroscopy.GB/T14965-1994 hand detected its types and contents of amino acids.(2)CP and CP-PGN had the significant preventive and therapeutic effects on MRSA-induced bloodstream infections.(3)CP and CP-PGN could promote the phagocytosis of macrophages、migration and production of cytokines,indicating that CP and CP-PGN could effectively activate the immune function of macrophages.(4)All the data indicated that CP and CP-PGN could effectively activate macrophages and differentiation them into the classic M1 phenotypes.(5)The results of qRT-PCR and flow cytometry showed that CP and CP-PGN could induce the formation of M1 macrophages.(6)After silencing TLR2 by siRNA,the relative index was significantly decreased compared with the non-interfering group,indicating that TLR2 silencing could down-regulate the activation and differentiation of macrophages by CP and CP-PGN.Conclusion The relevant cell and animal experiments showed that CP and CP-PGN could effectively activate macrophages and induced them intothe classic M1 phenotypes via TR2 pathway,which had the significant preventive and therapeutic effects on MRSA-induced infection.After the further systematic study,CP and CP-PGN were expected to become an effective immunomodulator,the anti-infection adjuvant therapy and prevention were provided a good choice for the elderly,patients with chronic diseases and immunocompromised people.