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Objective To investigate the relationship among p38 mitogen-activated protein kinase (p38MAPK), NF-KB and monocyte chemoattractant protein-1 (MCP-1), and to study the role of p38MAPK, NF-κB and MCP-1 in diabetic nephropathy. Methods Protein expressions of p38MAPK and NF-κB, and mRNA expression of MCP-1 were initially investigated in rat mesangial cell line HBZY-1, which were incubated separately with 25mmol/L glucose, 100nmol/L insulin, 100 μmol/L H2 O2 and 100mg/L advanced glycosylation end products (AGEs). The relationship among p38MAPK, NF-κB and MCP-1 expression Was observed by usingSB203580, a specific inhibitor of p38MAPK. Results The expressions of p38MAPK, NF-κB and MCP-1 were increased in HBZY-1 cells incubated separately with 25mmol/L glucose, 100nmol/L insulin, 100μmol/L H2 O2and 100 mg/L AGEs. Expressions of NF-κB and MCP-1 were significantly reduced when p38MAPK was inhibited by SB203580. Conclusion p38MAPK, NF-κB and MCP-1 are involved in development of diabetic nephropathy, and p38MAPK stimulation is essential for the expressions of NF-κB and MCP-1.
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The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.
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Objective To investigate the extracellular regulated protein kinases (ERKs) pathway of hepatitis B virus(HBV) X protein(HBx) on glomerular mesangial cell(GMC) proliferation of rat and tumor necrosis factor(TNF)-? expression.Methods The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector PCI-neo and confirmed by restrict endonuclease digestion and sequence analysis.PCI-neo contained HBV X gene (PCI-neo-X) was transfected into cultured GMC via liposome.GMC proliferation,TNF-? and its mRNA expression were investigated in the condition of with or without U0126 in the culture media.HBx,ERK1/2 and phosphorynated-ERK1/2 (p-ERK1/2) expression in GMC were assessed by Western blot.TNF-? mRNA expression was assessed by semi-quantitative reverse transcriptase-PCR (RT-PCR).TNF-? level in supernatants was measured by enzymelinked immunosorbent assay(ELISA).GMC proliferation was assessed by methyl thiazolyl tetrazolium(MTT).Results HBx expression was found in transfected GMC,and became prominent at 36 and 48 hours after transfection whether with or without U0126 in culture media.TNF-? mRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-? levels in supernatants in PCI-neo-X transfection without U0126 group were (189.0?18.1) ng/L at 36 hours and (172.3?24.3) ng/L at 48 hours after transfection,respectively.In contrast,TNF-? levels in supernatants with U0126 were (65.6?11.6) ng/L and (84.0?24.6) ng/L,respectively.The TNF-? le-vels in the latter groups were significantly lower than the formers (Pa
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Objective observe the post-radiation expression of rat mesangial cell plasminogen activator inhibitor-1 (PAI-1), and the effect of PPAR-? ligand on radiation induced PAI-1 expression. Methods After 10?Gy ?-ray radiation of cultured rat kidney mesangial cell, RT-PCR was used to observe PPAR-?expression. PAI-1 expression after 10?Gy radiation ?10?mmol/L pioglitazone (one of PPAR-? ligands) was measured by Northern blot and Western blot. Gel-shift method was used to study the effect of AP-1 of PPAR-? ligand on radiation induced PAI-1 expression. Methods Rat kidney mesangial cell expressed PPAR--? mRNA. PAI-1 over-expressed at 24 hour after 10?Gy radiation in a radiation dose dependent manner, but not at other time. When the cell was treated with 10?Gy + 10?mmol/L pioglitazone, PAI-1 upregulation was blunted at message and protein level. AP-1 activation was increased from the 2nd hour after 10?Gy radiation and reached the highest level at the 4th hour. When pioglitazone was added, the radiation induced increase in AP-1 activation was suppressed at the 4th hour. Conclusions Pioglitazone, mediated by AP-1 gene, can suppress radiation induced PAI-1 upregulation.
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AIM: To investigate whether transferrin rec ep tor (TfR) and Fc ?/? R are the major IgA 1 receptor on human mesangial cells (HMC). METHODS: Serum IgA 1 was isolated by jacalin affinity chromatog raphy and heated to aggregated form (aIgA 1). RT-PCR was performed to investiga te the expression of TfR mRNA and Fc?/?R mRNA. Binding capacity of aIgA 1 to human mesangial cell line (HMCL) was evaluated by radio-ligand binding assay. Bi nding specificity was determined by competitive inhibition assay and phosphoryla tion of extracellular signal-regulated kinase (ERK) was determined by Western bl ot. RESULTS: TfR cDNA and Fc?/?R cDNA products were amplified from HMC but not from HMCL. aIgA 1 was found to bind to HMCL in a dose-dependent, s aturable manner and the binding was inhibited by BSA. Scatchard analysis reveale d a Kd of (6.4?1.7)?10 -7 mol/L and the binding sites were (3.0?1.2) ?106/cells. Both aIgA 1 from patients with IgAN and healthy controls were ab le to induce the phosphorylation of ERK in a similar time-dependent manner, but the effect of aIgA 1 from patients with IgAN was much stronger (P