| BackgroundAtherosclerosis (AS) is a chronic inflammatory / autoimmune disease. Heat shock protein (HSP) increases under heat stress or other stress state, which can induce immune inflammatory response and natural or acquired immune response. Recently, many researches have demonstrated that HSP has positive correlation with atherosclerosis. HSP includes HSP60, HSP70, HSP90 and other small molecules, which is closely related to the occurrence and development of cardiovascular disease, especially HSP60. Now many focus on the affect of HSP70 on Atherosclerosis. Research finds that HSP70 may also as the endogenous ligand of TLR4 involves in TLRs signal transduction to induce immune responses and plays an important role in the development of immune/ inflammatory diseases. HSP70 and TLR4 receptor are both ancient and important proteins, which can mediate immune / inflammatory response and induce inflammatory mediators (IL-1, TNF-α, IL-6, IL-8, MCP-1). IL-8 participates in the pathogenesis of AS as MCP-1, which can promote adhering to vascular endothelial cells and moving to endothelium, then monocytes cells swallowed the lipid and turned into foam cells, resulted in vascular smooth muscle cell proliferation. This study intends to investigate the possible effect on the expression of MCP-1 and IL-8 by THP-1 cells.ObjectiveTo explore the mechanism and the effect of HSP70 protein on MCP-1, IL-8 expression stimulated by the heat shock protein.MethodsIn each experiment, 160nmol/L phorbol ester incubates THP-1 cells for 24h to induce their differentiation into macrophages, then change serum-free culture medium and add processing factors.1)common temperature control group:THP-1 cells grow in 10% newborn calf serum RPMI-1640 medium, 37℃and 5% CO2 incubator. 160 nmol / L PMA incubate THP-1 cells for 24 hs to induce their differentiation into macrophages.2) Heat shock treatment groups: THP-1 macrophages were heated for 1 h at 42℃water, then to 37℃resumption for 6 h.3) Heat shock + anti-TLR4 antibody group:THP-1 macrophages were added with the 10ug/ml Anti-TLR4 for 2 h and watered in 42℃water for 1 h, then to 37℃resumption for 6 h.4) heat shock + P38 inhibitor group:THP-1 macrophages were added with 20umol/ml P38 specially inhibitor (SB203580) for 30mins and watered in 42℃water for 1 h, then to 37℃resumption for 6 h.5) heat shock + NF-κB inhibitor group:THP-1 macrophages were added with 30umol/ml NF-κB specially inhibitor (PDTC) for 30min and watered in 42℃water for 1 h, then to 37℃resumption for 6 h.6) heat shock + anti-HSP70 antibody group:THP-1 macrophages were added with 2ug/ml anti-HSP70 for 2h and watered in 42℃water for 1 h, then to 37℃resumption for 6 hResults:THP-1 macrophages by heat shock can increase the expression of MCP-1, IL-8, HSP70, TLR4, P38 and NF-κB, but decrease the expression of TLR4, P38, NF-κB, MCP-1 and IL-8 by anti- HSP70 antibody. When added anti-TLR4 antibody and heat-shock treatment, HSP70 expression had no change, but the expression of p38, NF-κB, MCP-1 and IL-8 were decreased. When added P38-specific inhibitor SB203580 and heat shock treatment, the expression of HSP70 had no change, but the expression of TLR4, NF-κB, MCP-1 and IL-8 were decreased. When added NF-κB-specific inhibitor PDTC and heat shock treatment, the expression of HSP70, TLR4 and p38 had no obviously change, but the expression of MCP-1 and IL-8 were decreased. When added anti-HSP70 antibody and heat shock treatment, the expression of TLR4, p38, NF-κB, MCP-1 and IL-8 were decreased, but the extent of decrease was lower than the former three blocks.Conclusion:1. HSP70 can partly increase expression of MCP-1 and IL-8 in THP-1 macrophages by heat shock throng activated TLR4, P38 and NF-κB signaling pathway.2. Anti-TLR4 antibody can inhibit of the expression MCP-1 and IL-8 in THP-1 macrophages by heat shock, the activation of P38 and NF-κB signaling pathway has relation with TLR4.3. NF-κB specific inhibitor PDTC and P38 specific inhibitor SB203580 can decrease the expression of MCP-1 and IL-8 in THP-1 macrophages by heat shock.
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