THP-1巨噬细胞热休克处理对MCP-1和IL-8表达的影响
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摘要
研究背景:动脉粥样硬化(AS)是一种慢性炎症/免疫性疾病。热休克蛋白( heat shock protein,HSP)是机体细胞在受到热应激或其它应激状态下合成增多的一类蛋白质,可诱导免疫炎症反应,介导天然及获得性免疫反应。近来很多报道显示HSP与动脉粥样硬化的程度呈正相关。主要以HSP60、HSP70、HSP90和小分子HSP与心血管疾病的发生发展密切相关,其中对HSP60与AS之间的关系研究较多,而对HSP70在AS中的作用正日益引起人们的重视。最近又有研究发现,HSP70也可能作为TLR4的内源性配体参与TLRs信号传导机制,从而诱导免疫应答,在免疫/炎症性疾病的发生、发展中起着重要作用。HSP70和TLR4受体均是古老而重要的蛋白,均能介导免疫/炎症反应,诱导炎性介质(IL-1、TNF-α、IL-6、IL-8、MCP-1)等产生。其中,IL-8可以同MCP-1一样促使外周血单核细胞向血管内皮细胞黏附,并迁移至内皮下,吞噬脂质转化成为泡沫细胞,导致血管平滑肌细胞异常增殖,参与AS发生的病理过程。而本研究意在从热休克出发,探讨对THP-1巨噬细胞表达MCP-1和IL-8的可能影响。
目的:体外模拟热休克的应激状态,探讨热休克刺激产生的HSP70对体外培养THP-1巨噬细胞TLR4/P38MAPK、TLR4/NF-κB信号通路激活后的MCP-1、IL-8表达的影响。
方法:在每次实验前用160nmol/L佛波酯孵育THP-1细胞24h,使其诱导分化成巨噬细胞,换无血清培养基后加处理因素。
1)常温对照组:THP-1细胞生长于含有10%新生小牛血清的RPMI-1640培养液中,37℃、5%CO2培养箱中静置培养。再用160nmol/L佛波酯(PMA)孵育THP-1细胞24h,使其诱导分化成巨噬细胞。
2)热休克处理组:THP-1巨噬细胞42℃水浴受热1 h,37℃恢复6 h。3)热休克+抗TLR4抗体组:
THP-1巨噬细胞加入Anti-TLR4 10ug/ml 2h后42℃水浴受热1 h, 37℃恢复6 h。
4)热休克+P38抑制剂组:
THP-1巨噬细胞加入P38特异性抑制剂(SB203580)20umol/ml 30min后42℃水浴受热1 h, 37℃恢复6 h。
5)热休克+NF-κB抑制剂组:
THP-1巨噬细胞加入NF-κB特异性抑制剂(PDTC)30umol/ml 30min后42℃水浴受热1 h, 37℃恢复6 h。
6)热休克+抗HSP70抗体组:
THP-1巨噬细胞加入anti-HSP70 2ug/ml 2h后42℃水浴受热1 h, 37℃恢复6 h。
结果:
热休克处理THP-1巨噬细胞能够上调HSP70、TLR4、P38、NF-κBMCP-1和IL-8等表达。加入抗HSP70抗体后均可下调由热休克处理导致的TLR4、p38、NF-κB、MCP-1和IL-8表达升高。加入抗TLR4抗体热休克处理后,HSP70表达无变化,p38、NF-κB、MCP-1和IL-8表达下调。加入p38特异性抑制剂SB203580热休克处理后HSP70表达无变化,TLR4、NF-κB、MCP-1和IL-8表达下调。加入NF-κB特异性抑制剂PDTC热休克后HSP70、TLR4和p38表达无明显变化,MCP-1和IL-8表达下调。
结论:
1.热休克处理THP-1巨噬细胞后激活TLR4、p38MAPK和NF-κB信号通路、上调MCP-1和IL-8的表达是部分通过HSP70起作用。
2.TLR4抗体可以抑制热休克导致的MCP-1和IL-8表达上调被,提示p38和NF-κB两条信号通路活化与TLR4有关。
3.使用NF-κB特异性抑制剂PDTC和p38特异性抑制剂SB203580都可以抑制由热休克引起的MCP-1和IL-8表达上调。
Background
Atherosclerosis (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.
Objective
To explore the mechanism and the effect of HSP70 protein on MCP-1, IL-8 expression stimulated by the heat shock protein.
Methods
In 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 h
Results:
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.
目的:体外模拟热休克的应激状态,探讨热休克刺激产生的HSP70对体外培养THP-1巨噬细胞TLR4/P38MAPK、TLR4/NF-κB信号通路激活后的MCP-1、IL-8表达的影响。
方法:在每次实验前用160nmol/L佛波酯孵育THP-1细胞24h,使其诱导分化成巨噬细胞,换无血清培养基后加处理因素。
1)常温对照组:THP-1细胞生长于含有10%新生小牛血清的RPMI-1640培养液中,37℃、5%CO2培养箱中静置培养。再用160nmol/L佛波酯(PMA)孵育THP-1细胞24h,使其诱导分化成巨噬细胞。
2)热休克处理组:THP-1巨噬细胞42℃水浴受热1 h,37℃恢复6 h。3)热休克+抗TLR4抗体组:
THP-1巨噬细胞加入Anti-TLR4 10ug/ml 2h后42℃水浴受热1 h, 37℃恢复6 h。
4)热休克+P38抑制剂组:
THP-1巨噬细胞加入P38特异性抑制剂(SB203580)20umol/ml 30min后42℃水浴受热1 h, 37℃恢复6 h。
5)热休克+NF-κB抑制剂组:
THP-1巨噬细胞加入NF-κB特异性抑制剂(PDTC)30umol/ml 30min后42℃水浴受热1 h, 37℃恢复6 h。
6)热休克+抗HSP70抗体组:
THP-1巨噬细胞加入anti-HSP70 2ug/ml 2h后42℃水浴受热1 h, 37℃恢复6 h。
结果:
热休克处理THP-1巨噬细胞能够上调HSP70、TLR4、P38、NF-κBMCP-1和IL-8等表达。加入抗HSP70抗体后均可下调由热休克处理导致的TLR4、p38、NF-κB、MCP-1和IL-8表达升高。加入抗TLR4抗体热休克处理后,HSP70表达无变化,p38、NF-κB、MCP-1和IL-8表达下调。加入p38特异性抑制剂SB203580热休克处理后HSP70表达无变化,TLR4、NF-κB、MCP-1和IL-8表达下调。加入NF-κB特异性抑制剂PDTC热休克后HSP70、TLR4和p38表达无明显变化,MCP-1和IL-8表达下调。
结论:
1.热休克处理THP-1巨噬细胞后激活TLR4、p38MAPK和NF-κB信号通路、上调MCP-1和IL-8的表达是部分通过HSP70起作用。
2.TLR4抗体可以抑制热休克导致的MCP-1和IL-8表达上调被,提示p38和NF-κB两条信号通路活化与TLR4有关。
3.使用NF-κB特异性抑制剂PDTC和p38特异性抑制剂SB203580都可以抑制由热休克引起的MCP-1和IL-8表达上调。
Background
Atherosclerosis (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.
Objective
To explore the mechanism and the effect of HSP70 protein on MCP-1, IL-8 expression stimulated by the heat shock protein.
Methods
In 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 h
Results:
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.
引文
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[1] Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282:2085–2088.
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[7] Homeostatic role of Toll-like receptor 4 in the endothelium and heart. J Cardiovasc Pharmacol Ther. 2007 Dec;12(4):322-6.
[8] Schroder NW, Schumann RR: Single nucleotide polymorphisms of Toll-like receptors and susceptibility toinfectious disease. Lancet Infect Dis 2005;5:156–164.
[9] Kiechl S, Lorenz E, Reindl M, et al: Toll-like receptor 4polymorphisms and atherogenesis. N Engl J Med2002;347:185–192.
[10] Hamann L, Gomma A, Schroder NW, et al: A frequent toll-like receptor (TLR)-2 polymorphism is a risk factor for coronary restenosis. J Mol Med 2005;83:478–485..
[11] Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis. Front Biosci. 2008 Jan 1;13:2041-59. Med 2005;11:1173–1179.
[12] Regulation of TLR4 expression is a tale about tail. Arterioscler Thromb Vasc Biol. 2006 Dec;26(12):2582-4
[13] Berliner JA, Watson AD: A role for oxidized phospholipids in atherosclerosis. N Engl J Med 2005;353:9–11.
[14] Miller YI: Toll-like receptors and atherosclerosis: oxidized LDL as an endogenous Toll-like receptor ligand. Future Cardiology 2005;1:785–792.
[15] Schaefer L, Babelova A, Kiss E, et al: The matrix component biglycan is proinflammatory and signals throughToll-like receptors 4 and 2 in macrophages. J Clin Invest 2005;115:2223–2233.
[16] Zhou J, An H, Xu H, Liu S, Cao X. Heat shock up-regulates expression of Toll-like receptor-2and Toll-like receptor-4 in human monocytes via p38 kinasesignal pathway. Immunology 2005 Apr;114(4):522-30
[17] Multiple roles of Toll-like receptor signaling in atherosclerosis. Curr Opin Lipidol. 2006 Oct;17(5):527-33
[18] Pharmacogenomics: a tool to prevent and cure coronary heart disease. Curr Pharm Des. 2007;13(36):3726-34. Review