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Objective To evaluate the blocking effect and mechanism of Soybean-derived Bowman-Birk inhibitor (BBI)on LPS-mediated downregulation for tight junction protein(HT-29 cells)in intestinal epithelial cells(IECs). Methods The toxic effect of LPS and BBI on HT-29 cells was detected by CCK8 Kit.HT-29 cells were pretreated by BBI for 6 hours prior to LPS stimulation, the expression of tight junction protein(ZO-1 and Occludin), TLR4, and MyDD8 was detected by the quantitative real-time polymerase chain reaction(PCR)and Western Blot;activation of NF-κB was measured by Western Blot.Results LPS(1 000ng/mL)and BBI(1 000μg/mL)showed no cytotoxicity on HT-29 cells.LPS could significantly upregulate the expression of TLR4 in HT-29 cells, the up-regulation had time-dose effect, and could significantly downregulate the expression of tight junction protein, the down-regulation effect was directly proportional to the concentration of LPS, could activate NF-κB, and had dose effect, effect of LPS on HT-29 cells could be significantly inhibited by BBI.Conclusion By inhibiting the expression of TLR4 and activation of NF-κB in IECs induced by LPS, BBI can significantly block the LPS-mediated inhibitory effect on tight junction protein in intestinal epithelial cells.
ABSTRACT
Objective To investigate the inhibitory effect and mechanism of soybean-derived Bowman-Birk inhibi-tor (BBI)on LPS-induced expression of inflammatory cytokines in intestinal epithelial cells.Methods Cytotoxicity effect of LPS and BBI on intestinal epithelial cells was analyzed by MTT assay.Intestinal epithelial cells were pre-treated with BBI,followed by LPS stimulation,expression of inflammatory cytokines(TNF-α,IL-1β,IL-8,and MCP-1)was detected by quantitative real-time polymerase chain reaction;the activation of NF-κB was measured by pNF-κB-luc system and Western Blot.Results The maximum concentration of LPS (10000 ng/mL)and BBI (1000 μg/mL)had no cytotoxicity effect on intestinal epithelial cells.LPS could potently up-regulate the expression of inflammatory cytokines(TNF-α,IL-1β,IL-8,and MCP-1 ),the up-regulation was positively correlated to the concentration of LPS;LPS-induced expression of inflammatory cytokines in intestinal epithelial cells could achieve the highest level,then decreased over time.The up-regulation of LPS on inflammatory cytokines in intestinal epi-thelial cells had dose-time effect;when intestinal epithelial cells were pretreated by BBI for 6 hours,the inhibitory effect of BBI on LPS-induced expression of inflammatory cytokines in intestinal epithelial cells was most obvious, and had dose-time effect.Conclusion BBI can potently inhibit LPS-induced expression of inflammatory cytokines through inhibiting NF-κB in intestinal epithelial cells.
ABSTRACT
Objective To investigate the inhibitory effect and mechanism of soybean-derived Bowman-Birk inhibi-tor (BBI)on LPS-induced expression of inflammatory cytokines in intestinal epithelial cells.Methods Cytotoxicity effect of LPS and BBI on intestinal epithelial cells was analyzed by MTT assay.Intestinal epithelial cells were pre-treated with BBI,followed by LPS stimulation,expression of inflammatory cytokines(TNF-α,IL-1β,IL-8,and MCP-1)was detected by quantitative real-time polymerase chain reaction;the activation of NF-κB was measured by pNF-κB-luc system and Western Blot.Results The maximum concentration of LPS (10000 ng/mL)and BBI (1000 μg/mL)had no cytotoxicity effect on intestinal epithelial cells.LPS could potently up-regulate the expression of inflammatory cytokines(TNF-α,IL-1β,IL-8,and MCP-1 ),the up-regulation was positively correlated to the concentration of LPS;LPS-induced expression of inflammatory cytokines in intestinal epithelial cells could achieve the highest level,then decreased over time.The up-regulation of LPS on inflammatory cytokines in intestinal epi-thelial cells had dose-time effect;when intestinal epithelial cells were pretreated by BBI for 6 hours,the inhibitory effect of BBI on LPS-induced expression of inflammatory cytokines in intestinal epithelial cells was most obvious, and had dose-time effect.Conclusion BBI can potently inhibit LPS-induced expression of inflammatory cytokines through inhibiting NF-κB in intestinal epithelial cells.
ABSTRACT
Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences. The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.