Study On E.coli-induced Apoptosis Of Human Monocytic U937 And Its Mechanisms

Study on E. coli - induced Apoptosis of human monocytic U937 and its MechanismsApoptosis is a highly regulated and ordered process that plays a central role in eliminating unnecessary cells in normal development and homeostasis. Increasing numbers of pathogens have been found to modulate host cell apoptosis and thereby influence the progression of diseases. It has been found that one of the Escherichia coli(E. coli)stains, ATCC25922 can trigger apoptosis in neutro-phils.Many studies have demonstrated the important role of apoptotic cell death in different disease states and physiologic cell death, and many factors involved with the death signaling have been identified. Bcl - 2 and other members of the family play an important role in embryogenesis, tissue remodeling, and the immune response through their actions as either inhibitors or promotes of apoptosis. Bcl - 2 usually act against apoptotic cell death. Bax is one of the members of Bcl — 2 family, which drive the death mechanism.Previous research has documented the pivotal roles of Bcl - 2 and Bax in programmed cell death. However, it is not known whether Bcl - 2 and Bax are involved in the E. coli - induced cytotoxicity of human monocytic U937 cells. The invasive process of pathogenic bacteria is frequently associated with Mitogen - activated protein kinases ( MAPK) signaling activity. The MAPK family has an important role in signal transduction and the pathway is activated by a variety of stimili, such as growth factors and cellular stresses. The three major MAPK pathways include the extracellular - signal regulated kinase ( ERK, also known as P42/44 MAP kinase) , c - jun N - terminal kinase (JNK, also known as stress activated protein kinase - 1 ( SAPK1) ) and p38 MAPK (also known as SAPK2/KK). The involvement of p38 MAPK kinases and ERK1/2 in E. coli invasion of U937 has not yet been demonstrated. In this study, we investigated the effect of E. coli on U937 cell apoptosis, assessed theextent of Bel - 2 and Bax by RT - PCR and the activity of p38MAPK and ERK1/2 by Western blot in the apoptotic events.Section A. Study on E. coli - induced apoptosis in U937ObjectiveIn order to study the relationship of E. coli to U937 cell apoptosis.Methods1. U937 cells were used in all experiments, and they were infected with E. coli when U937: E. coli was 1:5, 1:10, 1:20, 1: 50 and 1:100 respectively for 30min and for different lengths of time (lOmin, 20min, 30min, 60min and 90min respectively) when U937: E. coli was 1" 20.2. The U937 apoptosis induced by E. coli was detected with Annexin V FITC/PI assay, Hoechst 33258 fluorochrome staining and Giemsa staining.Results1. Annexin V FITC/PI assay and Hoechst 33258 fluorochrome staining found the apoptotic cells at U937: E. cob' =1:10 after E. coli had infected U937, The percentage of apoptotic cells raised with the increasing of the concentrations of E. coli .2. Giemsa staining showed that U937 cells in the 30min, 60min and 90min groups (U937: E. coli = 1: 20) underwent karyopykosis, condensation, swelling and apoptotic bodies were observed. In the lOmin and 20min groups, very few apoptotic cells were observed. Annexin V FITC/PI assay showed that the percentage of apoptotic cells raised with the increasing of time. The infection groups at 30min, 60min and 90min can induce significantly U937 apoptosis than in the control group ( P < 0.001).ConclusionI.E. coli can induce U937 cells apoptosis.2. U937 apoptosis was induced by E. coli in a dose - and time - dependent manner.Section B. Expression of Bel - 2 and Bax gene family during E. coli - induced apoptosis in U937 cellsObjectiveTo explore the role of Bel - 2 and Bax in the apoptosis of U937 cells induced by E. coli.Methods1. U937 cells were infected with E. coli when U937: E. coli was 1:5, 1: 10, 1: 20, 1:50 and 1: 100 respectively for 30min2. The mRNA of Bel -2 and Bax was detected by RT -PCR.ResultsThe transcription and expression levels of Bel - 2 gene was reduced, and that of Bax gene was enhanced with the increasing of the concentrations of E. coli.ConclusionThe U937 apoptosis induced by E. cob' might be the result of the difference between the expression levels of Bel - 2 and Bax genes.Section C. Involvment of p38 mitogen - activated protein kinase and ERK1/2 in E. coli - induced U937 apoptosisObjectiveTo investigate whether the effect of E. coli on U937 cell apoptosis is mediated via p38 MAPK and ERK1/2 activationMethods1. U937 cells were infected by E. coli for different lengths of time (lOmin, 20min, 30min, 60min and 90min respectively) when U937-.E. coli was 1 -.20.2. The activity of p38MAPK and ERK1/2 was detected by Western blot.3. For the evaluation of the role of MAPKs, SB203580 and PD98059 were used as MAPKs inhibitors for p38MAPK and ERK1/2.Results1. The phosphorylations of p38 was induced after lOmin infection. Maximal phosphorylation occurred at 20min and started to decline at 30min with time -dependent manner. In contrast, the level of total p38 protein was not changed in whole experimental period.2. The phosphorylated ERK1/2 forms revealed the presence of activated ERK1/2 in control unstimulated primary cells. The downregulation of ERK1/2 by E. coli was statistically significant especially that of ERK2. Hie levels of total (phosphorylation state - independent) ERK1/2 did not change in E. coli - infected U937 cells at all times examined.3. Inhibition of p38MAPK with SB203580 caused decrease in apoptosis of U937 cells, while inhibition of ERK1/2 with PD98059 caused further increase in apoptosis of U937 cells.ConclusionThe activation of the p38 MAPK and the downregulation of ERKl/2 in U937 cells by E. coli is a major pathway to mediate the apoptosis.