探討細胞外熱休克蛋白抑制由脂多醣體刺激之心肌細胞肥大及巨噬細胞發炎反應的作用機轉

Extracellular HSC70 Protects Against Cardiomyocyte Hypertrophy and Macrophage Inflammation Induced by Lipopolysaccharide

吳琳祺(LIN-CHI WU)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/藥理學科研究所/碩士(2012年)

摘要

熱休克蛋白heat shock cognate protein 70 (HSC70) 以往被認為其作用僅侷限於細胞內，最近的研究發現其也可被分泌至細胞外，並且具有許多生理功能。先前有研究指出，在活體實驗上若於脂多醣體 (lipopolysaccharide, LPS) 注射前先給予recombinant HSC70 (rHSC70)，則可以減少LPS所造成的心肌功能失調。然而其詳細的分子機轉並未被探討，因此本研究的目的為評估rHSC70的心臟保護作用及抗發炎功效並探討其機轉。
在本研究中使用兩種細胞，分別是初生鼠心肌細胞以及RAW 264.7老鼠巨噬細胞株。實驗結果發現，rHSC70 (0.1, 1 和 5 μg/ml) 能有效抑制LPS誘導的心肌細胞肥大以及抑制了誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS)、環氧化酶 (cyclooxygenas-2, COX-2) 的蛋白表現和一氧化氮 (nitric oxide, NO)、腫瘤壞死因子-α (tumor necrosis factor, TNF-α)、介白素-6 (interleukin-6, IL-6) 的釋放。此外，rHSC70也減少了LPS誘導的metalloproteinase-2 (MMP-2) 和MMP-9的酵素活性及蛋白表現量。我們發現此作用主要是透過抑制LPS誘發的mitogen-activated protein kinase (MAPK) 訊息傳遞路徑及阻斷Akt、inhibitor κB (IκB)α 的磷酸化進而減少LPS誘導的nuclear factor-κB (NF-κB) 次單元p65的核轉移以及NF-κB的DNA結合活性。因此，rHSC70抑制LPS引起的發炎反應的機制主要是rHSC70會和LPS競爭相同的接受器 (toll-like receptor 4, TLR4)，進而下調節MAPK和NF-κB的訊息傳遞。總結來說，細胞外熱休克蛋白在先天性免疫調節上扮演著重要的角色且刺激了內生性保護機制。因此，期待未來rHSC70能夠做為治療敗血症的一個選擇。

關鍵字

細胞外熱休克蛋白 ； 脂多醣體 ； 心肌細胞 ； 老鼠巨噬細胞株 ； 發炎

並列摘要

Recent studies suggest that heat shock cognate protein 70 (HSC70), an intracellular protein, can also be released to extracellular space and have physiologic functions. It has been reported that preconditioning of mice with recombinant HSC70 (rHSC70) induces cardiac functional tolerance to lipopolysaccharide (LPS), but the mechanism of rHSC70 induces LPS tolerance has not been fully elucidated. Thus, the purpose of this study was to investigate the mechanism of the effect of rHSC70. In this study, we demonstrated that preconditioning of cardiomyocytes with rHSC70 (0.1, 1 and 5 μg/ml) reduced LPS-induced cardiomyocyte hypertrophy. rHSC70 preconditioning also exhibited inhibitory effects on iNOS, COX-2, NO, TNF-α and IL-6 production in LPS-induced neonatal rat cardiomyocytes and RAW264.7 cells. In addition, we found that rHSC70 preconditioning attenuated the activity and protein expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Furthermore, we found that rHSC70 preconditioning repressed the activation of mitogen-activated protein kinase (MAPK) caused by LPS. In parallel, rHSC70 preconditioning reduced LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunit p65 and the DNA binding activity of NF-κB by blocking phosphorylation of Akt and inhibitor κB (IκB)α as well as the degradation of IκBα. Thus, the mechanism of rHSC70 induces LPS tolerance appears to be associated with down-regulation of MAPK and NF-κB signaling pathway, via competition for the same receptors with LPS. Taken together, our results show that extracellular HSC70 may play an important role in innate immunity modulation and stimulation of endogenous protective mechanisms. Therefore, extracellular HSC70 may serve as a promising strategy to treat sepsis.

並列關鍵字

extracellular heat shock protein ； LPS ； cardiomyocyte ； inflammation ； RAW 264.7

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