BBI Inhibits Effects Of LPS On Inflammatiory Cytokines And Tight Junctional Proteins In Intestinal Epithelial Cells

Objective:BBI is a component of soybean products with stable biochemical properties,which is known to have anti-inflammation and anti-HIV function.Chronic and systemic inflammation is one of the key factors of HIV disease progression.Although antiretroviral therapy(ART)can effectively inhibit HIV replication,systemic inflammation in HIV-infected patients still exists.Because GI mucosal tissues contain a number of activated CD4+T cells,GI is one of the main attack target of HIV.HIV infection causes the death of CD4+T cells and GI immunity demage,resulting a repaied bacterium replication and LPS production(LPS translocation).LPS can enter into systemic circulation and induce theimmune activation/inflammation.Therefore,bacteria and LPS translocation is an important marker of HIV disease progression.The main aims of our thesis are to study:1 Wether BBI can inhibit LPS-induced inflammatory cytokines(TNF-?,IL-1?,IL-8,MCP-1)expression in intestinal epithelial cells;2 Whether BBI has the ability to block LPS-mediated tight junctional proteins down-regulation in intestinal epithelial cells;3 Whether BBI can counteract LPS-induced ROS in intestinal epithelial cells;4 The mechanism of BBI effect on inhibiting LPS-induced inflammatory expression and tight junctional proteins down-regulation in intestinal epithelial cells.MethodsUsed intestinal epithelial cells(HT-29)to perform the following experiments:I To examine the impact of BBI and/or LPS treatment on the viability of HT-29 cells(CCK8 assay);2 To measure inflammatory cytokines(TNF-?,IL-1?,IL-8,MCP-1),tight-junctional proteins(ZO-1 and Occludin),TLR4 and MyDD8 using quantitative real-time PCR and Western Blot;3 To study the inhibitory effect of BBI on LPS-induced expression of inflammatory cytokines(TNF-?,IL-1?,IL-8,MCP-1),tight-junctional proteins(ZO-1 and Occludin),TLR4 and MyDD8 using quantitative real-time PCR and Western Blot;4 To examine the effect of BBI and/or LPS on NF-?B in HT-29 cells uisng Western Blot and NF-?B-Luci System);5 To analyze the effect of LPS and/or BBI on ROS production by DCFH-DA kit.Results1 LPS and/or BBI showed no/little effect on the viability of HT-29 cells;2 LPS could induce the expression of inflammatory cytokines(TNF-?,IL-1?,IL-8,MCP-1)and TLR4 in HT-29 cells,but the BBI could block the LPS effect;3 LPS down-regulated the expression of the tight-junctional proteins in HT-29 cells.However,BBI counteracted the effect of LPS on the tight junctional proteins;4 BBI could compromise the effect of LPS-induced ROS production;5 BBI potently inhibited LPS-induced phosphorylation of NF-?B in HT-29 cells.ConclusionBBI inhibits LPS-induced inflammatory cytokines expression and ROS production through compromising LPS-induced TLR4 expression and NF-?B activation.Moreover,BBI can block LPS-mediated down-regulation of the tight junctional proteins in intestinal epithelial cells.These results indicate that BBI possesses the function to block the LPS effect on intestinal epithelial cells.Future in vivo studies are necessary in order to determine wether BBI can be used to treat HIV infection mediated systemic inflammation/immune action.