内质网应激在HO-1保护内毒素急性肺损伤中的作用及机制研究

Acute lung injury (ALI) is the major cause of death in critically ill patients,and treatment such as anti-infection and mechanical ventilation isn’t satisfactory. We are committed to explore the endogenous protective mechanism in the prevention and treatment of ALI induced by LPS and have achieved certain results: HO-1 plays an important role in endogenous protective effect during LPS induced ALI , but mechanism of HO-1 action in cell micro-structural level is far from clear. Endoplasmic reticulum stress (ERS) may be involved in the key point of the pathogenesis of lung injury , while the regulatory effect and mechanism about the effects of HO-1 on ERS in lung disease has not been elucidated. Combined with previous research , we hypothesize that HO-1 resist apoptosis, inflammation and other events in lung tissue by regulating ERS. We use the HO-1 knock out mouse to clarify the effect of ERS in HO-1 protection against the LPS-induced ALI in mice model and LPS challenge alveolar epithelial cell model in vitro, by observing the change of morphology of the endoplasmic reticulum, ERS markers, specific protein involved in ERS-initiated apoptosis and inflammatory factors, in addition, we use the specific blockers to seek signal pathways activating ERS, to explore the mechanism of HO-1 on ERS against LPS induced acute lung injury and try to provide new ideas and theoretical basis for the prevention and treatment of ALI induced by LPS.

内毒素急性肺损伤(ALI)是危重症患者死亡的主要原因，抗炎、机械通气等治疗效果多不理想。我们致力于探讨内毒素ALI内源性保护机制并取得一定成果：证实HO-1在内毒素ALI中发挥重要内源性保护作用，但HO-1在细胞超微结构内环境层面作用机制远未阐明。内质网应激(ERS)可能是参与肺损伤发病的关键环节，目前HO-1对肺部相关疾病ERS调控作用及机制尚不清楚。结合前期研究基础，我们提出“HO-1可能通过调控肺组织ERS以抵抗细胞凋亡、炎症反应等事件而发挥肺保护作用”假说。本课题拟利用HO-1基因敲除小鼠，通过LPS诱导小鼠内毒素ALI模型和离体LPS攻击肺泡上皮细胞模型，观察内质网形态学、ERS发生标志物、ERS特异凋亡蛋白及炎性因子的变化，并利用相关特异阻断剂探讨调控ERS的信号通路，以阐明ERS在HO-1保护内毒素ALI中的作用机制及信号分子通路，为防治内毒素ALI提供新思路。

内毒素急性肺损伤(ALI)是临床常见急危重症，其防治一直是临床难题。本项目利用HO-1基因敲除小鼠，通过成功建立LPS诱导小鼠内毒素ALI 模型和离体LPS攻击肺泡上皮细胞模型，观察了内毒素ALI时ERS发生标志物、ERS特异凋亡蛋白及炎性因子的变化，并利用相关特异阻断剂探讨了调控ERS的可能信号通路。研究发现：HO-1和ERS参与了小鼠内毒素ALI的发病过程； HO-1基因敲除后内毒素导致的肺损伤加重，在基因水平进一步验证了HO-1的肺保护作用；HO-1基因缺失小鼠给予脂多糖后GRP78、p-IRE1α 及 p-PERK mRNA及蛋白表达水平升高，ERS特异凋亡蛋白CHOP 及caspase-12水平升高，TNF-α、IL-6及IL-1β水平升高，IL-10水平下降，ATF6 mRNA及蛋白表达水平未见明显变化，而HO-1诱导剂作用相反；同样，HO-1基因沉默肺泡上皮细胞鼠给予脂多糖后GRP78、p-IRE1α 及 p-PERK mRNA及蛋白表达水平升高，ERS特异凋亡蛋白CHOP 及caspase-12水平升高，TNF-α、IL-6及IL-1β水平升高，IL-10水平下降；进一步的机制研究发现HO-1可能通过PERK/eIF2α通路抑制内质网应激介导的细胞损伤。据此可以得出结论，在动物和细胞水平，HO-1可能通过抑制内质网应激相关蛋白GRP78、ATF6、p-IRE1α、p-PERK 而减轻ERS特异凋亡蛋白CHOP 及caspase-12介导的细胞凋亡及TNF-α、IL-6、IL-1β及IL-10相关炎性反，最终发挥其内源性保护作用，减轻内毒素急性肺损伤，为内毒素肺损伤中发挥内源性保护作用的可能机制提供理论依据，为HO-1在微环境层面的分子作用机制，为防治内毒素ALI提供了重要的理论依据。