Chen Yi, Wang Lei, Kang Qiuxiang, Zhang Xu, Yu Guifang, Wan Xiaojian, Wang Jiafeng, Zhu Keming
Department of Anesthesiology and Intensive Care Medicine, Changhai Hospital, the Second Military Medical University, Shanghai, China.
Department of Anesthesiology, the Third People's Hospital, Shanghai Jiaotong University, Shanghai, China.
Cell Physiol Biochem. 2017;42(1):156-168. doi: 10.1159/000477308. Epub 2017 May 25.
BACKGROUND: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI) during sepsis. Heat shock protein A12B (HSPA12B) is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. METHODS: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15): a sham operation group, a cecal ligation and puncture (CLP) group, a HSPA12B siRNA-CLP group and a negative control (NC) siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6) and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. RESULTS: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e.g., IL-1β, TNF-α, and IL-6) expression, and increased myeloperoxidase activity, neutrophil infiltration, pulmonary edema, and pulmonary endothelial cell apoptosis. Additionally, HSPA12B knockdown worsened survival after CLP surgery. The potential protective mechanisms of HSPA12B may involve the inhibition of ERK phosphorylation and caspase-3 activation in vivo and in vitro. CONCLUSION: HSPA12B protected against sepsis-induced ALI. The potential mechanism may be partly due to the inhibition of ERK phosphorylation and caspase-3 activation. These findings provide a potential therapeutic target for treating sepsis.
背景：肺内皮损伤是脓毒症期间急性肺损伤（ALI）发病机制中的关键过程。热休克蛋白A12B（HSPA12B）主要在内皮细胞中表达，并能抵御多种有害因素。然而，HSPA12B在脓毒症诱导的ALI中的作用及其潜在作用机制仍不清楚。 方法：在体内实验中，将C57BL/6小鼠随机分为四组（n = 15）：假手术组、盲肠结扎和穿刺（CLP）组、HSPA12B siRNA-CLP组和阴性对照（NC）siRNA-CLP组。通过鼻腔吸入2-OMe修饰的HSPA12B siRNA或NC siRNA对小鼠进行治疗。通过CLP诱导脓毒症。在CLP手术后24小时和48小时采集样本。使用苏木精和伊红染色监测肺组织样本的病理变化和评分。通过酶联免疫吸附测定法分析支气管肺泡灌洗液中促炎细胞因子（如白细胞介素（IL）-1β、肿瘤坏死因子（TNF）-α和IL-6）的水平以及髓过氧化物酶活性。使用湿重与干重比评估肺水肿。使用流式细胞术对中性粒细胞和肺泡巨噬细胞进行计数。通过TUNEL染色检测肺内皮细胞凋亡。通过蛋白质免疫印迹分析测量MAPK家族信号分子和半胱天冬酶-3的表达水平。此外，记录7天生存率。在体外实验中，将人脐静脉内皮细胞预先用HSPA12B siRNA或pIRES2-EGFP-HSPA12B-Flag质粒转染，并用脂多糖处理；随后，通过蛋白质免疫印迹法测量MAPK家族信号分子和半胱天冬酶-3的表达水平。 结果：鼻腔吸入纳米聚合物包裹的HSPA12B siRNA可特异性下调肺组织中HSPA12B的mRNA和蛋白质表达水平。给予HSPA12B siRNA会加重肺病理损伤，上调促炎细胞因子（如IL-1β、TNF-α和IL-6）的表达，并增加髓过氧化物酶活性以及中性粒细胞浸润、肺水肿和肺内皮细胞凋亡。此外，敲低HSPA12B会使CLP手术后的生存率降低。HSPA12B的潜在保护机制可能涉及在体内和体外抑制ERK磷酸化和半胱天冬酶-3激活。 结论：HSPA12B可预防脓毒症诱导的ALI。潜在机制可能部分归因于对ERK磷酸化和半胱天冬酶-3激活的抑制。这些发现为治疗脓毒症提供了一个潜在的治疗靶点。
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