HSPA12B inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells
HSPA12B inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells
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HSPA12B抑制脂多糖诱导的人脐静脉内皮细胞炎症反应
DOI：
10.1111/jcmm.12464
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发表时间：
2015-03-01
影响因子：
5.3
通讯作者：
Liu, Li
中科院分区：
文献类型：
--
作者：
Wu, Jun;Li, Xuehan;Liu, Li
热休克蛋白A12b(HSPA12B)是新发现的HSP70蛋白家族成员。本研究探讨HSPA12B对内毒素(LPS)诱导的人脐静脉内皮细胞(HUVECs)炎症反应的影响及其可能机制。采用脂多糖诱导人脐静脉内皮细胞炎症模型。通过感染编码绿色荧光蛋白HSPA12B的重组腺病毒实现HSPA12B在人脐静脉内皮细胞中的高表达。利用siRNA技术实现了HSPA12B基因的敲除。病毒感染或siRNA转染后24小时，用1g/mlLPS刺激HUVEC 4h。内皮细胞通透性测定采用跨膜通透性测定。用髓过氧化物酶测定法测定人中性粒细胞(PMN)与人脐静脉内皮细胞的结合率。通过创面愈合实验测定细胞迁移能力。用RT-qPCR和Western印迹分别分析目的基因的mRNA和蛋白表达水平。用双抗体夹心法测定细胞因子IL-6和肿瘤坏死因子-α的释放。HSPA12B抑制内毒素诱导的HUVEC通透性，减少PMN与HUVECs的黏附。HSPA12B还可抑制内毒素诱导的黏附分子和炎性细胞因子的表达上调。相反，HSPA12B的敲除增强了内毒素诱导的黏附分子和炎性细胞因子的表达。此外，HSPA12B激活了PI3K/Akt信号通路，并被Wortmannin药理抑制，完全取消了HSPA12B对内皮细胞炎症反应的保护作用。我们的结果表明，HSPA12B通过激活PI3K/Akt信号通路来减轻内毒素诱导的HUVECs的炎症反应。
Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein-HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1g/ml LPS for 4hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound-healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT-qPCR and Western blot, respectively. The release of cytokines interleukin-6 and tumour necrosis factor- was measured by ELISA. HSPA12B suppressed LPS-induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS-induced up-regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS-induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS-induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.