Inhibition of Acute Lung Injury by TNFR-Fc through Regulation of an Inflammation-Oxidative Stress Pathway

BackgroundAcute lung injury (ALI), characterized by disruption of the lung alveolar-capillary membrane barrier and resultant pulmonary edema, and associated with a proteinaceous alveolar exudate, is a leading cause of morbidity and mortality. Currently, inflammation-oxidative stress interaction between TNF-α and NF-κB was identified as a key pathway of ALI. We hypothesized that a TNFR-Fc fusion protein would have beneficial effects in experimental ALI, and sought to test this idea in mice by blocking TNF-α.Methods and ResultsIntratracheal instillation of lipopolysaccharide (LPS) into the lungs of ALI mice led to histiocyte apoptosis, and detection of serum and bronchoalveolar lavage fluid (BALF) cytokines, feedback between NF-κB and TNF-α, lung albumin leakage, lung damage, IκB kinase (IKK) and NF-κB activation, I-κB degradation, and oxidative injury. LPS administration raised pulmonary inflammation as reflected by increased inflammatory cytokines, alveoli protein concentration, and ALI scores. IKK is phosphorylated following LPS challenge, leading to I-κB degradation and NF-κB p65 phosphorylation. Furthermore, NF-κB is translocated into the nucleus and up-regulates TNF-α gene transcription. Infusion of TNFR-Fc 24h before LPS challenge significantly abrogated the increase of inflammatory cytokines, especially serum TNF-α concentration, as well as pulmonary alveoli protein levels, and diminished IKK and NF-κB activation and I-κB degradation. The nuclear translocation of NF-κB was inhibited, following by down-regulation of TNF-α gene transcription. In addition, LPS intratracheal instillation induced marked oxidative damage, such as a decrease in total anti-oxidation products and an increase in malondialdehyde (MDA), as well as up-regulation of oxidation enzymes. Histologic analysis and apoptosis scores revealed that the extent of tissue lesions was significantly reduced, but not abrogated, by TNF-α blockade.ConclusionTreatment with LPS alone increased inflammation and oxidative stress in ALI mice, while administration of TNFR-Fc 24h before LPS challenge broke the feedback between NF-κB and TNF-α, resulting in decreased pulmonary inflammation/oxidative damage and tissue destruction. These results suggest a potential role for TNF-α therapy to treat clinical ALI.

急性肺损伤（Acute lung injury, ALI）以肺肺泡毛细血管膜屏障破坏、继发肺水肿为特征，常伴有蛋白性肺泡渗出物，是导致发病率与死亡率升高的主要诱因之一。目前研究已确认，TNF-α与NF-κB之间的炎症-氧化应激互作是ALI的关键通路。本研究假设TNFR-Fc融合蛋白对实验性ALI具有获益效应，并尝试在小鼠模型中通过阻断TNF-α验证这一假说。

向ALI模型小鼠气管内滴注脂多糖（lipopolysaccharide, LPS）后，可观察到组织细胞凋亡，同时可检测到血清与支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）中的细胞因子变化、NF-κB与TNF-α的反馈环路、肺白蛋白渗漏、肺组织损伤、IκB激酶（IκB kinase, IKK）与NF-κB的激活、IκB降解以及氧化损伤。LPS给药可升高肺部炎症水平，具体表现为炎症细胞因子增多、肺泡蛋白浓度升高以及ALI评分升高。LPS刺激后IKK发生磷酸化，进而引发IκB降解与NF-κB p65磷酸化。此外，NF-κB会转位入核并上调TNF-α基因的转录。在LPS刺激前24小时输注TNFR-Fc，可显著抑制炎症细胞因子的升高（尤其是血清TNF-α浓度），同时降低肺泡蛋白水平，并减弱IKK与NF-κB的激活及IκB降解。NF-κB的核转位受到抑制，随之TNF-α基因转录下调。另外，气管内滴注LPS可诱导显著的氧化损伤，表现为总抗氧化产物减少、丙二醛（malondialdehyde, MDA）水平升高，以及氧化酶表达上调。组织学分析与凋亡评分显示，阻断TNF-α可显著减轻组织损伤程度，但无法完全消除损伤。

仅使用LPS处理可加重ALI小鼠的炎症与氧化应激，而在LPS刺激前24小时给予TNFR-Fc，可打破NF-κB与TNF-α之间的反馈环路，进而减轻肺部炎症、氧化损伤与组织破坏。上述结果提示，靶向TNF-α的治疗策略有望用于临床ALI的治疗。