TNFα-induced IP3R1 expression through TNFR1/PC-PLC/PKCα and TNFR2 signalling pathways in human mesangial cell.

BACKGROUND: Little information is available regarding the mechanisms involved in cytokine-induced type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) expression in human mesangial cells (HMCs) in the occurrence of hepatorenal syndrome (HRS). Over-expression of IP(3)R1 would enhance both IP(3)-binding activity and sensitivity. We hypothesize that it is possible that increased IP(3)R1, induced by TNFα, would lead to increased IP(3) sensitivity in response to a variety of vasoconstrictors, and promote HMC contraction and thus lead to reduced GFP, promoting HRS occurrence and development. METHODS: Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNFα on IP(3)R1 mRNA and protein expression. Several inhibitors of kinases, depletion PKC, over-expression of dominant-negative mutant of PKC and non-radioactive PKC assay were used to examine the mechanism of signal transduction of TNFα-regulated IP(3)R1 in HMCs. RESULTS: TNFα increased IP(3)R1 mRNA and protein expression in HMCs, an effect that was blocked by prolonged incubated chronic PMA, D609, safingol and also by transfection with domain-negative PKCα construct. TNFα activated and promoted autophosphorylation of the PKCα. In addition, both anti-TNFR1 and anti-TNFR2 antibodies blocked TNFα-induced IP(3)R1 protein expression, while only anti-TNFR1 antibodies but not anti-TNFR2 antibodies attenuated TNFα-induced PKCα activity. CONCLUSIONS: TNFα increased the expression of IP(3)R1, and this was mediated, at least in part, through the TNFR1/PC-PLC/PKCα and TNFR2 signalling pathways in HMCs.

Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure.

AIM: To determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal permeability (IP) in mice with fulminant hepatic failure (FHF), and the expression of tight junction proteins. METHODS: We selected D-lactate as an index of IP, induced FHF using D-galactosamine/lipopolysaccharide and D-galactosamine/TNF-α, assessed the results using an enzymatic-spectrophotometric method, transmission electron microscopy, immunohistochemistry, Western blotting and real-time quantitative polymerase chain reaction. The effect of the administration of anti-TNF-α immunoglobulin G (IgG) antibody, before the administration of D-galactosamine/lipopolysaccharide, on TNF-α was also assessed. RESULTS: IP was significantly increased in the mouse model of FHF 6 h after injection (13.57 ± 1.70 mg/L, 13.02 ± 1.97 mg/L vs 3.76 ± 0.67 mg/L, P = 0.001). Electron microscopic analysis revealed tight junction (TJ) disruptions, epithelial cell swelling, and atrophy of intestinal villi. Expression of occludin and claudin-1 mRNA was significantly decreased in both FHF models (occludin: 0.57 ± 0.159 fold vs baseline, P = 0.000; claudin-1: 0.3067 ± 0.1291 fold vs baseline, P = 0.003), as were the distribution density of proteins in the intestinal mucosa and the levels of occludin and claudin-1 protein (occludin: 0.61 ± 0.0473 fold vs baseline, P = 0.000; claudin-1: 0.6633 ± 0.0328 fold vs baseline, P = 0.000). Prophylactic treatment with anti-TNF-α IgG antibody prevented changes in IP (4.50 ± 0.97 mg/L vs 3.76 ± 0.67 mg/L, P = 0.791), intestinal tissue ultrastructure, and the mRNA levels of occludin and claudin-1 expression (occludin: 0.8865 ± 0.0274 fold vs baseline, P = 0.505; claudin-1: 0.85 ± 0.1437 fold vs baseline, P = 0.1), and in the protein levels (occludin: 0.9467 ± 0.0285 fold vs baseline, P > 0.05; claudin-1: 0.9533 ± 0.0186 fold vs baseline, P = 0.148). CONCLUSION: Increased in IP stemmed from the downregulation of the TJ proteins occludin and claudin-1, and destruction of the TJ in the colon, which were induced by TNF-α in FHF mice.

Decreased IgA+ plasma cells and IgA expression in acute liver necrosis mice.

AIM: To investigate the number of intestinal immunoglobulin A (IgA+) plasma cells and expression of intestinal IgA in mice with acute liver necrosis. METHODS: A model of acute liver necrosis was established by intraperitoneal injection of galactosamine (GalN) and lipopolysaccharide (LPS). Sixty mice were randomly divided into one of 4 equal groups: normal control, acute liver necrosis, LPS, or GalN. Hematoxylin and eosin staining, immunohistochemistry, and an enzyme-linked immunosorbent assay were employed to assess liver and intestinal injury, count intestinal IgA+ plasma cells, and measure the expression level of IgA and interferon gamma (IFN-gamma) in the small intestinal mucosa of mice. RESULTS: Injured intestinal mucosa was observed in the acute liver necrosis group but not in the normal, LPS or GalN groups. Compared with the normal group, intestinal IgA+ plasma cells were slightly decreased in the LPS and GalN groups [429 +/- 20 per high power field (HPF), 406 +/- 18/HPF, respectively], whereas they were markedly decreased in the acute liver necrosis group (282 +/- 17/HPF vs 495 +/- 26/HPF in normal group, P < 0.05). The expression of intestinal IgA was also slightly decreased in LPS and GalN groups, but was markedly reduced in the acute liver necrosis group as determined by enzyme-linked immunosorbent assay (P < 0.05). In contrast, the level of IFN-gamma was slightly increased in LPS, GalN and acute liver necrosis groups, but with no statistical significance (P > 0.05). CONCLUSION: Intestinal IgA+ plasma cells and IgA expression levels indicating that mucosal immune barrier dysfunction, does exist in acute liver necrosis.