Background

NOD-like receptor protein 3 (NLRP3) inflammasome activation has emerged as a crucial contributor to sepsis-induced lung injury. Geranylgeranyl diphosphate synthase 1 (GGPPS1) reportedly exerts the pro-inflammatory capability via activation of NLRP3 inflammasome. However, little is known about the role and mechanism of GGPPS1 in sepsis-induced lung injury.

Methods

Mice underwent cecal ligation and puncture (CLP) surgery to establish the in vivo model of sepsis. The lung injury of mice was assessed by analyzing the histological changes, the lung wet/dry ratio, PaO₂/FiO₂ ratio, myeloperoxidase (MPO) activity, total protein content, total cell, and polymorphonuclear leukocyte counts. Mouse alveolar macrophages MH-S were exposed to LPS for developing in vitro model of sepsis. The mRNA and protein expression levels of GGPPS1, beclin-1, and autophagy and inflammasome-related genes were detected using quantitative reverse transcription-polymerase chain reaction and western blot assays. Enzyme-linked immunosorbent assay was conducted to determine the levels of interleukin (IL)-1β and IL-18.

Results

We successfully established sepsis-induced acute lung injury in vivo by CLP surgery. GGPPS1 was upregulated in the lung tissues of CLP-induced septic mice. The activation of autophagy and NLRP3 inflammasome were found in the lung tissues of CLP-induced septic mice. The addition of exogenous GGPP (synthesis products catalyzed by GGPPS1) and autophagic inhibitor 3-MA aggravated sepsis-induced hypoxemia, alveolar inflammatory response, intrapulmonary hemorrhage, and pulmonary edema, as evidenced by increased lung injury score, lung wet/dry weight ratio, MPO activity, total protein content, total cell, and PMNs counts, and decreased PaO₂/FiO₂ ratio. While NLRP3 inhibitor MCC950 exerted the opposite effects. Additionally, administration of exogenous GGPP could inhibit the activation of autophagy, enhance the activity of NLRP3 inflammasome, and the production of IL-1β and IL-18. Inhibition of autophagy by 3-MA treatment also promoted the activity of NLRP3 inflammasome and the production of IL-1β and IL-18. While MCC950 restrained the activity of NLRP3 inflammasome, but did not affect the activation of autophagy. Notably, the expression of GGPPS1 was unaltered in CLP-induced mice following GGPP, 3-MA, or MCC950 treatment. Moreover, GGPPS1 was upregulated in MH-S cells stimulated with LPS, and GGPPS1 knockdown enhanced the activation of autophagy and inhibited the activity of NLRP3 inflammasome in vitro. Importantly, depletion of GGPPS1 could alleviate LPS-induced inflammatory response by inducing autophagy-dependent NLRP3 inflammasome inhibition.

Conclusion

GGPPS1 knockdown suppressed NLRP3 inflammasome activity via promoting autophagy and then attenuated sepsis-induced acute lung injury, revealing a novel target for treating sepsis-induced lung injury.

中文翻译：

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香叶基香叶基二磷酸合酶 1 敲低通过促进脓毒症引起的急性肺损伤中的自噬来抑制 NLRP3 炎症小体活性

 背景

NOD 样受体蛋白 3 (NLRP3) 炎性体激活已成为脓毒症引起的肺损伤的关键因素。据报道，香叶基香叶基二磷酸合酶 1 (GGPPS1) 通过激活 NLRP3 炎症小体发挥促炎能力。然而，关于 GGPPS1 在脓毒症引起的肺损伤中的作用和机制知之甚少。

 方法

对小鼠进行盲肠结扎穿刺（CLP）手术以建立脓毒症体内模型。通过分析组织学变化、肺湿/干比、PaO ₂ /FiO ₂比值、髓过氧化物酶(MPO)活性、总蛋白含量、总细胞和多形核白细胞计数来评估小鼠肺损伤。将小鼠肺泡巨噬细胞 MH-S 暴露于 LPS 中以建立败血症的体外模型。使用定量逆转录聚合酶链反应和蛋白质印迹法检测 GGPPS1、beclin-1、自噬和炎症小体相关基因的 mRNA 和蛋白表达水平。采用酶联免疫吸附法测定白细胞介素（IL）-1β和IL-18的水平。

 结果

我们通过 CLP 手术在体内成功建立了败血症诱发的急性肺损伤。 GGPPS1 在 CLP 诱导的脓毒症小鼠的肺组织中表达上调。在 CLP 诱导的脓毒症小鼠的肺组织中发现自噬和 NLRP3 炎性体的激活。添加外源性GGPP（GGPPS1催化的合成产物）和自噬抑制剂3-MA会加重脓毒症引起的低氧血症、肺泡炎症反应、肺内出血和肺水肿，表现为肺损伤评分、肺湿/干重比增加， MPO 活性、总蛋白含量、总细胞和 PMN 计数以及 PaO ₂ /FiO ₂比率降低。而NLRP3抑制剂MCC950则发挥相反的作用。此外，给予外源GGPP可以抑制自噬的激活，增强NLRP3炎症小体的活性以及IL-1β和IL-18的产生。 3-MA 处理抑制自噬还促进 NLRP3 炎性体的活性以及 IL-1β 和 IL-18 的产生。而MCC950则抑制NLRP3炎症小体的活性，但不影响自噬的激活。值得注意的是，在 GGPP、3-MA 或 MCC950 治疗后，CLP 诱导的小鼠中 GGPPS1 的表达没有改变。此外，LPS刺激的MH-S细胞中GGPPS1表达上调，体外GGPPS1敲低可增强自噬的激活并抑制NLRP3炎症小体的活性。重要的是，GGPPS1 的消耗可以通过诱导自噬依赖性 NLRP3 炎症小体抑制来减轻 LPS 诱导的炎症反应。

 结论

GGPPS1敲低通过促进自噬抑制NLRP3炎性体活性，然后减轻脓毒症引起的急性肺损伤，揭示了治疗脓毒症引起的肺损伤的新靶点。