摘要
严重创伤、烧伤、败血症(特别是革兰氏阴性菌败血症)及各种类型的休克常导致全身炎症反应综合征(systemic inflammatoryresponse syndrome,SIRS)发生。SIRS是临床各科十分常见、平时及战时导致死亡的危重病症。虽然SIRS的发生机制迄今尚未完全阐明,但是LPS所致炎症介质的表达和细胞内信号转导通路的活化,在其发病学中的重要作用已受到广泛关注。
本研究应用RNA干扰技术,抑制RAW264.7小鼠巨噬细胞的HSP70基因表达。采用RT-PCR检测发现,大肠杆菌脂多糖(LPS)可诱导转空载体的RAW264.7小鼠巨噬细胞(NEO)产生大量IL-1β和IL-6等细胞因子。而用过表达HSP70的RAW264.7细胞进行实验,发现HSP70抑制了LPS诱导的细胞因子表达。当抑制HSP70基因的表达后,再进行LPS刺激,则细胞因子表达增多,说明抑制HSP70的表达可促进LPS诱导的细胞因子表达。
由于NF-κB/IκB信号转导通路的活化在LPS所致炎症反应中发挥了重要作用,我们进一步分析了HSP70表达抑制对LPS所致NF-κB信号通路活化的影响。通过Western blot等技术检测NF-κB(p65)的核移位,我们发现抑制HSP70表达后,HSP70对NF-κB(p65)核移位(LPS刺激60 min)及LPS刺激20 min所致的IκB降解的抑制作用被解除。
由于IκBα的降解涉及IκBa的磷酸化,而IκBα的磷酸化是由IκB的激酶IKK以及相应的磷酸酯酶相互影响、协同作用的结果。因此,我们进一步分析了HSP70表达抑制对细胞内磷酸酯酶2A活性的影响。在不用LPS刺激和LPS刺激30min后,RNA干扰组细胞内的磷酸酯酶2A活性低于非干扰对照组细胞。说明抑制HSP70的表达可降低细胞内的磷酸酯酶2A的活性。
综上所述,HSP70可能通过增高磷酸酯酶2A活性,从而抑制LPS所致IκB的磷酸化及降解,减少NF-κB的核移位,从而抑制NF-κB活化以及细胞因子的表达。
Severe trauma,burns,sepsis and various types of shock often leads to systemic inflammatory response syndrome(SIRS) occurred.SIRS is a very common clinical subjects,which can usually leads to death.Although the mechanism of SIRS has not entirely clear yet,but the important role of the expression of inflammatory and the activation of cellular signal transduction by LPS has been wide concerned.
In order to understand the anti-inflammatory role of heat shock protein 70(HSP70),we investigated the effects of HSP70 knockdown on the lipopolysacchearide(LPS)-induced production of proinflammatory cytokines and the activation of NF-κB signaling pathways in RAW264.7 mouse macrophage cell line.
Many studies have shown that elevation of HSP70 caused either by activation of the heat shock response(HSR) or through forced expression of the hsp70.1 gene down-regulated cytokine expression in RAW264.7 murine macrophages.Our data showed that inhibition of HSP70 by HSP70-siRNA could increase LPS-mediated expression of proinflammatory cytokines such as interleukin-1β(IL-1β) and interleukin-6(IL-6) at mRNA levels.
Because NF-κB regulates the transcription of an exceptionally large number of genes,particularly those involved in inflammatory and acute stress response,which suggests a major role of NF-κB signaling pathway in critical diseases.We further determined the effects of HSP70 knockdown on NF-κB signaling pathway.Firstly western blot was used to analyze the subcellular distribution of NF-κB(p65),the transcriptionally active component of the NF-κB complex.It was shown that nuclear translocation of p65 induced by LPS was strongly inhibited in cells transfected with HSP70.But this effect was significantly abolished by HSP70 knockdown.
Nuclear translocation of p65 occurs subsequently to the phos-phorylation and degradation of IκBα.Therefore we next determined the effects of HSP70 knockdown on the degradation of IκBα.It was shown that the degradation of IκBαinduced by LPS was significantly increased in the cells transfected with HSP70-siRNA,as compared with the cells transfected with HSP70.
Because the phosphorylation and the consequent degradation of IκBαare also dependent on intracellular phosphatase activity apart from the effect of IκB kinase(IKK),we further investigated the effects of the inhibition of HSP70 on intracellular phosphatase 2A activity during LPS stimulation.HSP70 knockdown could down-regulate the phosphatase 2A activity in the cells at normal condition or stimulated with LPS for 30min.
In conclusion,HSP70 could up-regulate the phosphatase 2A activity, which might inhibit the phosphorylation and degradation of IκBαand the subsequently nuclear translocation and activation of NF-κB.HSP70 could inhibit LPS-induced production of inflammatory cytokines by inactivating NF-κB pathway.
引文
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