HYDROGEN PREVENTS LIPOPOLYSACCHARIDE-INDUCED PULMONARY MICROVASCULAR ENDOTHELIAL CELL INJURY BY INHIBITING STORE-OPERATED Ca2+ ENTRY REGULATED BY STIM1/ORAI1
HYDROGEN PREVENTS LIPOPOLYSACCHARIDE-INDUCED PULMONARY MICROVASCULAR ENDOTHELIAL CELL INJURY BY INHIBITING STORE-OPERATED Ca2+ ENTRY REGULATED BY STIM1/ORAI1
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DOI：
10.1097/shk.0000000000002279
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发表时间：
2024-05-01
期刊：
影响因子：
3.1
通讯作者：
Yin，Yiqing
中科院分区：
文献类型：
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作者：
Li，Yuan;Chen，Hongguang;Yin，Yiqing
背景脓毒症是一种由宿主对感染的反应失调引起的危及生命的器官功能障碍。在脓毒症条件下，肺是最脆弱的靶器官。肺微血管内皮细胞（PMVEC）在严重脓毒症所致急性肺损伤（ALI）中起重要作用。脓毒症时PMVECs功能受损是一个涉及多种机制的复杂调节过程，其中内皮细胞内钙稳态失衡是其功能受损的关键因素。我们的初步研究结果表明，氢气（H2）治疗显着减轻脓毒症肺损伤，保护PMVEC的高通透性，并降低质膜基质相互作用分子1（STIM 1）的表达，但在脓毒症模型中，H2维持内皮细胞内Ca 2+稳态的潜在机制尚不清楚。因此，本研究旨在探讨H2对STIM 1和Ca 2+释放激活的Ca 2+通道蛋白1（Orai 1）调控的分子机制，以及H2对脂多糖（LPS）诱导的PMVEC和LPS攻击小鼠的Ca 2+稳态的影响。采用肺湿/干重（W/D）比值、支气管肺泡灌洗液（BAL）中总蛋白含量和伊文思蓝染色（EBD）法评价LPS对肺内皮屏障的损伤。荧光显微镜下观察STIM 1和Orai 1的表达。此外，我们还研究了富H2培养基在LPS诱导的类似脓毒症的体外损伤中对PMVEC的调节作用。结果H2通过降低肺湿重比、降低肺泡灌洗液中总蛋白含量、降低肺通透性，减轻小鼠ALI。此外，H2还降低肺微血管内皮细胞STIM 1和Orai 1的表达。在体外，LPS处理可增加PMVECs中STIM 1和Orai 1的表达水平，而H2可逆转这些变化。此外，在败血症模拟条件下，H2改善Ca 2+内流。用肌浆网/内质网Ca 2+腺苷三磷酸酶（SERCA）抑制剂毒胡萝卜素（TG）处理，导致细胞活力显著降低，以及连接蛋白（包括VE-钙粘蛋白和闭合蛋白）表达减少。用钙池操纵的钙内流（SOCE）抑制剂YM-58483（BTP 2）处理，可增加细胞活力和连接蛋白的表达。结论本研究表明，H2处理可通过抑制STIM 1和Orai 1介导的SOCE，减轻LPS诱导的PMVEC功能障碍。
BackgroundSepsis is a type of life-threatening organ dysfunction that is caused by a dysregulated host response to infection. The lung is the most vulnerable target organ under septic conditions. Pulmonary microvascular endothelial cells (PMVECs) play a critical role in acute lung injury (ALI) caused by severe sepsis. The impairment of PMVECs during sepsis is a complex regulatory process involving multiple mechanisms, in which the imbalance of calcium (Ca 2+) homeostasis of endothelial cells is a key factor in its functional impairment. Our preliminary results indicated that hydrogen gas (H 2) treatment significantly alleviates lung injury in sepsis, protects PMVECs from hyperpermeability, and decreases the expression of plasma membrane stromal interaction molecule 1 (STIM1), but the underlying mechanism by which H 2 maintains Ca 2+ homeostasis in endothelial cells in septic models remains unclear. Thus, the purpose of the present study was to investigate the molecular mechanism of STIM1 and Ca 2+-release-activated-Ca 2+ channel protein1 (Orai1) regulation by H 2 treatment and explore the effect of H 2 treatment on Ca 2+ homeostasis in lipopolysaccharide (LPS)-induced PMVECs and LPS-challenged mice.MethodsWe observed the role of H 2 on LPS-induced ALI of mice in vivo. The lung wet/dry (W/D) weight ratio, total protein in the bronchoalveolar lavage (BAL) fluid and Evans blue dye (EBD) assay were used to evaluate the pulmonary endothelial barrier damage of LPS-challenged mice. The expression of STIM1 and Orai1 were also detected using epifluorescence microscopy. Moreover, we also investigated the role of H 2-rich medium in regulating PMVECs under LPS treatment, which induced injury similar to sepsis in vitro. The expression of STIM1 and Orai1 as well as the Ca 2+ concentration in PMVECs were examined.ResultsIn vivo, we found that H 2 alleviated ALI of mice through decreasing lung W/D weight ratio, total protein in the BAL fluid and permeability of lung. In addition, H 2 also decreased the expression of STIM1 and Orai1 in pulmonary microvascular endothelium. In vitro, LPS treatment increased the expression levels of STIM1 and Orai1 in PMVECs, while H 2 reversed these changes. Furthermore, H 2 ameliorated Ca 2+ influx under sepsis-mimicking conditions. Treatment with the sarco/endoplasmic reticulum Ca 2+ adenosine triphosphatase (SERCA) inhibitor, thapsigargin (TG), resulted in a significant reduction in cell viability as well as a reduction in the expression of junctional proteins, including VE-cadherin and occludin. Treatment with the store-operated Ca 2+ entry (SOCE) inhibitor, YM-58483 (BTP2), increased the cell viability and expression of junctional proteins.ConclusionsThe present study suggested that H 2 treatment alleviates LPS-induced PMVEC dysfunction by inhibiting SOCE mediated by STIM1 and Orai1 in vitro and in vivo.