[Lipopolysaccaride influenced peritoneal mesothelial cells on the growth and expression of tumor necrosis factor through nuclear factor kappaB pathway].
OBJECTIVE
To investigate whether lipopolysaccharide (LPS) influence peritoneal mesothelial cells (PMCs) on their apoptosis, growth and expression of tumor necrosis factor (TNF) through IkappaBalpha, nuclear factor p65 pathway.


METHODS
PMCs were isolated, cultured and passaged by enzymatic disaggregation, then identified by phase contrast inverted microscope, transmission electron microscope and scanning electron microscope, with immunocytochemistry method. The PMCs were treated under conditions, which included different concentration LPS (normal control group, 0.1 mg/L, 1.0 mg/L, 10 mg/L, 50 mg/L, 100 mg/L) and different time (0, 1, 3, 6, 12, 24 hours). Hoechst 33258 fluorescence dye method was used to detect the apoptosis of PMC. The inhibitory effect of LPS on cell growth was measured with MTT method. Immunofluorescence dye was used to observe the activity of p65. Western blot method was used to detect the expression of IkappaBalpha. L929 cell line was used to detect the activity of TNF. RT-PCR was used to measure the expression of TNFalpha mRNA.


RESULTS
LPS could inhibit growth of PMC and induce apoptosis. LPS could lead IkappaBalpha to degration and p65 into nucleus. LPS could also induce the expression of TNF. The expression of TNF-alpha mRNA reached the peak at the 1st hour and highest TNF activity at the 3rd hour.


CONCLUSIONS
LPS inhibit the growth of PMC and induced the apoptosis. LPS induced PMC to express TNF-alpha in a dose-dependent manner. In the early state, PMC expressed a great deal of TNF-alpha, then began to fall quickly. During the period that LPS influence PMC on apoptosis, growth and expression of TNF, IkappaBalpha-NF-kappaB p65 pathway played the important role.
Li
Zhi-Ming
ZM
Department of Nephrology, the First Affiliated Hospital of China Medical University, Shenyang 110001, China.
Ma
Jian-Fei
JF
Wang
Li-Ning
LN
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002467:Cell Nucleus; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D000072000:NF-KappaB Inhibitor alpha; D010529:Peritoneal Cavity / Q000166:cytology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D000276:Adjuvants, Immunologic; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; C000606206:Nfkbia protein, rat; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha