Pneumolysin Induces Apoptosis In THP-1Cells Involved P38MAPK Signal Pathway

ObjectiveStreptococcus pneumoniae (S.pn) is a common Gram-positive bacteriain clinical, which can cause Pneumonia, meningitis, Otitis media and sepsis.In Developing Countries, sepsis death caused by S.pn account for25%inchildren’ preventive death under5years old, resulting in about1.2millioninfants death each year. Pneumolysin (PLY) plays an important role inbacterial pathogenesis and inducing host defense responses, but the themolecular mechanisms of host defense against PLY lacks the specificunderstand in current.The immune cells in the blood will act on bacteria and productdirectly or interact with signaling molecules secreted by infected tissueindirectly after bacteria invading into the blood, causing the host immunedefense, in this process, monocytes play an important role. But at present,there is rarely reported on the mechanism of monocytes and PLYinteraction, so we used human THP-1cells (acute monocytic leukemiamononuclear cell lines) and PLY in vitro incubated way to discuss themechanism of monocytes and PLY interaction preliminarily, studied monocytes’ apoptosis and the changes of signal molecules in apoptosispathway, to lay a foundation for clarifying the mechanism of PLYtriggering host instantaneous and early innate immune response in furtherstudy.Methods1. We used the following experimental methods to study the effect ofPLY on THP-1cells’ growth condition: MTT method used for thedetermination of PLY inhibiting THP-1cells’ growth; Wright staining usedfor observation the effect of PLY on THP-1cells’ morphology;Transmission electron microscope used to observe THP-1cells’ subcellularstructure change after treating with PLY; Flow cytometry detected cellapoptosis rate; Agarose gel electrophoresis used to observe DNAfragmentation; at the same time, we used△A146PLY (The deletion ofAlanine at the146th amino acids site of peumolysin, which is nearly lostits pore-forming activity) treatment group as negative control.2. We discussed the immune mechanisms of PLY inducing THP-1cells apoptosis preliminarily from the following aspects: Fluorometricanalysis used for the determination of Caspase3,8,9activity;Immunocytochemistry used for assaying the expression ofapoptosis-related protein Bax, Bcl-xl, p38MAPK; the expression of totalp38MAPK and phosphorylative p38MAPK were further validated throughWestern-Blot; Flow cytometry detected the apoptosis rate of THP-1cells after pretreated with specific inhibitors of p38MAPK;△A146PLYtreatment group was used as negative control likewise.Results1. PLY could inhibit the proliferation of THP-1cells apparently in atime-and dose-dependent manner assayed by MTT after THP-1cellsexposure to PLY at0.05,0.1,0.5,1,2.5,5μg/ml for24,48,72,96h; Weobserved significant apoptotic morphological changes in the cells exposedto PLY after Wright staining; The ultrastructural study disclosed THP-1cells were apoptotic and characterized by condensation of nuclearfragmentation and apoptosis body; Treated with0.05μg/ml and o.1μg/mlPLY, the apoptosis rate of THP-1cells were13.15%and24.58%after6hrespectively,17.09%and60.08%after12h respectively detected by flowcytometry; A typical apoptotic ladder was identified in DNAelectrophoresis.2. Exposure to pneumolysin significantly increased the activities ofcaspase3,8,9; Pro-apoptotic protein Bax expression was increased whileanti-apoptotic protein Bcl-xl expression was decreased; Western-Blotshowed that the phosphorylation level of p38MAPK increased.Pre-exposure to p38MAPK inhibitor SB203580could decrease THP-1cells’ apoptosis rate induced by PLY.Conclusions1. THP-1cells were apoptotic when interacted with pneumolysin. 2. Pneumolysin induced THP-1cells apoptosis was via mitochondriapathway, dependent on caspase and involved the activation of p38MAPKsignal pathway.