Effect Of Heme Released From Red Blood Cells Lvsed By Leptospira Hemolysin On Expression Of Inflammatory Factors In Macrophages

Objective The purpose of this study was to determine weather the hemolysins Sph2,TlyA,and HlpA produced by Leptospira interrogans could lyse red blood cells to release free heme,and then study the inflammatory ractions induced by heme to further understanding the contribution of hemolysins in Leptospirosis and the pathogenic mechanisms of Leptospirosis.Methods(1)Genes fragments of Leptospira hemolysin sph2、tlyA、hlpA and Staphylococcus aureus β-hemolysin hlb were amplified by PCR and sub-cloned into pET vector to construct the prokaryotic expression system.The expressed proteins were purified and measured the concentrations;(2)Hemolytic activity of recombinant protein was detected by blood plate hemolysis circle and spectrophotometry;(3)The Heme-Quantitatives Kit was used to quantitatively measure the concentration of free heme released from lysed sheep red blood cells induced by recombinant protein rSph2,rH1pA,rT1yA,respectively;(4)The mRNA level of the inflammatory factors IL-6、IL-8(human)、KC(mouse)、IL-18、IL-33、IL-1β、TNF-a and MCP-1 in macrophages induced by heme per and post blocking of signaling pathway NF-kB,p38MAPK and JNK were determined by real-time PCR.Results The hemolysins were successfully expressed using prokaryotic expression systems;Recombinant protein rSph2,rH1pA,rTlyA could lyse sheep red blood cells to release free heme;Heme induced IL-6,IL-8,IL-18,MCP-1 mRNA level up-regulated in THP-1 cells,and IL-6,KC,IL-18,IL-33,IL-1β and TNF-a mRNA level up-regulated in J774A.1 cells by signaling pathway NF-kB、p3 8MAPK or JNK signaling pathway.Conclusion Leptospira hemolysin Sph2,H1pA,TlyA can lyse red blood cells to release free heme;Heme can up-regulate mRNA level of inflammatory factors in macrophages,which may synergism with hemolysin causing inflammatory damage to the host cells.This is contributing to better understanding the inflammatory reaction macnisams caused by Leptospira.