目的
脓毒症（Sepsis）是一种由感染引起的急性全身炎症反应综合症，伴有急性器官功能障碍，如脓毒症相关性肺损伤（SALI）。已有研究显示了铁死亡在脓毒症及其引发的多器官功能障碍病理过程中发挥着关键作用。脓毒症导致铁稳态失衡，促进炎症信号通路的激活以及组织损伤。尽管 SALI 的机制已被广泛研究数十年，但至今仍缺乏十分有效的治疗靶点和延缓疾病进展的策略。蛋白酶 3（Proteinase 3，PRTN3）是一种多功能的中性丝氨酸蛋白酶，可经脱颗粒方式被细胞释放到胞外，具有水解多种细胞外基质蛋白的能力，与病原体清除和组织损伤有关。然而 PRTN3在脓毒症诱发的急性肺损伤的作用和潜在机制仍不清楚。因此，本研究旨在阐明Prtn3 在脓毒症相关急性肺损伤中的作用及其分子机制，并为开发针对脓毒症发生发展的干预策略提供理论和实验依据。
方法
采用腹腔注射不同剂量的 LPS（50 mg/kg 和 10 mg/kg）分别诱导构建 WT 小鼠及 Prtn3 基因敲除小鼠（Prtn3-/-）脓毒症相关急性肺损伤模型；采用组织病理学方法分析 LPS 处理后 WT 小鼠、Prtn3-/-小鼠肺组织病理改变；采用血常规分析各组小鼠肺泡灌洗液中的各系血细胞的数目和比例，采用 qRT-PCR 分析小鼠肺组织炎症因子及铁死亡相关基因表达变化；采用流式细胞术分析不同浓度铁死亡激动剂（RSL3）处理后中性粒细胞的凋亡情况；通过免疫荧光、ELISA 和 qRT-PCR 分析 Prtn3 缺失后对 RSL3 处理的中性粒细胞铁死亡的影响；通过 RNA 测序分析 Prtn3 调控中性粒细胞铁死亡的潜在分子机制；通过蛋白质谱筛选和免疫共沉淀技术分析与 PRTN3
相互作用的蛋白；通过 Western blot、免疫荧光、银染分析 PRTN3 参与调控 SMAD3的蛋白表达、磷酸化和降解过程。
结果
（1）动物实验显示 Prtn3 的缺失显著提高了 LPS 诱导的脓毒症小鼠的总体生存率（P<0.05）。
（2）组织病理学和血常规检测结果表明与 WT 小鼠相比，Prtn3 的缺失显著减少了脓毒症相关急性肺损伤的病理改变和炎症反应（P<0.05）。
（3）qRT-PCR 结果显示 LPS 处理的 WT 小鼠肺组织中 Slc7a11、Gpx4 等铁死亡标志物的表达水平显著降低（P<0.05），而 Prtn3-/-+LPS 组中上述标志物表达水平与 WT+LPS 组相比明显升高。
（4）细胞实验结果显示 RSL3 处理后中性粒细胞凋亡率明显增加，铁含量、脂质过氧化产物 4-HNE 和 MDA 的水平均呈剂量依赖性升高（P<0.05）。
（5）ELISA、免疫荧光及 qRT-PCR 结果显示 Prtn3 的敲除抑制了 RSL3 诱导的铁积累，降低了脂质过氧化产物含量，升高了 Gpx4 基因表达水平（P<0.05）。
（6）RNA 测序分析结果显示：RSL3+Prtn3-/-组中与 ROS 代谢途径、炎症反应、TNF 信号通路、Toll-like receptor 信号通路、NF-kappa B 信号通路和严重的炎症感染等相关的基因表达发生了显著改变。
（7）蛋白质谱筛选、免疫共沉淀技术结果表明在中性粒细胞中，PRTN3 和SMAD3 之间存在强相互作用，Western blot、免疫荧光、银染实验结果显示 PRTN3可以酶解 SMAD3 蛋白，减少磷酸化 SMAD3 的水平。

结论
Prtn3 基因缺失可以显著减轻脓毒症相关急性肺损伤的程度，提高小鼠的生存率。机制研究结果表明 Prtn3 通过直接剪切 SMAD3 蛋白进而影响 Gpx4 表达，影响中性粒细胞铁死亡和炎症免疫反应，进而减轻组织和器官损伤程度，提示 Prtn3 基因可以作为一个新的脓毒症相关急性肺损伤治疗靶点。本研究为中性粒细胞铁死亡调控以及急性肺损伤的治疗提供了潜在的治疗靶点和策略。

Purpose:
Sepsis is an acute systemic inflammatory response syndrome triggered by infection,accompanied by acute organ dysfunction, such as sepsis-associated lung injury (SALI).
Previous studies have revealed that ferroptosis plays a critical role in the pathological progression of sepsis and its resulting multiple organ dysfunction. Sepsis disrupts iron homeostasis,
promotes the activation of inflammatory signaling pathways,
and exacerbates tissue damage. Although the mechanisms of SALI have been extensively studied for decades, highly effective therapeutic targets and strategies to delay disease
progression remain lacking.Proteinase 3 (PRTN3), a multifunctional neutral serine protease, can be released extracellularly via degranulation. It possesses the ability to
hydrolyze various extracellular matrix proteins and is associated with pathogen clearance and tissue damage. However, the role and underlying mechanisms of PRTN3 in
sepsis-induced acute lung injury remain unclear. Therefore, this study aims to elucidate the function of Prtn3 in sepsis-associated acute lung injury and its molecular mechanisms,
providing theoretical and experimental foundations for developing intervention strategies targeting the progression of sepsis.
Methods:
Intraperitoneal injection of different doses of LPS (50 mg/kg and 10 mg/kg) was used to induce sepsis induced acute lung injury models in wild-type (WT) mice and Prtn3 gene
knockout mice (Prtn3-/-), respectively. Histopathological methods were employed to analyze lung tissue pathological changes in WT and Prtn3-/- mice after LPS treatment.
Complete blood count analysis was performed to determine the number and proportion of various blood cell types in the bronchoalveolar lavage fluid (BALF) of each group. qRT-PCR was used to analyze changes in the expression of inflammatory factors and ferroptosis-related genes in mouse lung tissues. Flow cytometry was utilized to assess the apoptosis of neutrophils treated with different concentrations of the ferroptosis inducer RSL3. The impact of Prtn3 deficiency on RSL3-induced neutrophil ferroptosis was evaluated using immunofluorescence, ELISA, and qRT-PCR. RNA sequencing was conducted to explore the potential molecular mechanisms by which Prtn3 regulates neutrophil ferroptosis. Mass spectrometry and co-immunoprecipitation techniques were
employed toidentify proteins interacting with PRTN3. Western blot, immunofluorescence, and silver staining were used to analyze the role of PRTN3 in regulating the expression,
phosphorylation, and degradation of SMAD3.
Results：
(1) Animal experiments demonstrated that the absence of Prtn3 significantly improved the overall survival rate of LPS-induced septic mice (P < 0.05).
(2) Histopathological and complete blood count results indicated that, compared to WT mice, the absence of Prtn3 significantly reduced pathological changes and inflammatory
responses in sepsis-associated acute lung injury (P < 0.05).
(3) qRT-PCR results revealed that the expression of ferroptosis markers such as Slc7a11 and Gpx4 in the lung tissues of LPS-treated WT mice was significantly decreased (P <0.05), while no significant changes in the expression of these markers were observed in the Prtn3-/- group.
(4) In vitro cell experiments showed that the apoptosis rate of neutrophils significantly increased after RSL3 treatment, accompanied by dose-dependent elevations in iron content and levels of lipid peroxidation products 4-HNE and MDA (P < 0.05).
(5) ELISA, immunofluorescence, and qRT-PCR results indicated that the knockout of Prtn3 suppressed RSL3-induced iron accumulation and lipid peroxidation product levels, while Gpx4 levels were significantly increased (P < 0.05).
(6) RNA sequencing analysis revealed significant alterations in genes related to ROS metabolic pathways, inflammatory responses, TNF signaling pathways, Toll-like receptor
signaling pathways, NF-kappa B signaling pathways, and severe inflammatory infections in the RSL3 + Prtn3-/- group.
(7) Mass spectrometry screening and co-immunoprecipitation results demonstrated a strong interaction between PRTN3
and SMAD3 in neutrophils. Western blot, immunofluorescence, and silver staining experiments showed that PRTN3 could enzymatically degrade SMAD3 protein, reducing the levels of phosphorylated SMAD3.

Conclusion:
The deficiency of the Prtn3 gene significantly alleviates the severity of sepsis-associated acute lung injury and improves the survival rate of mice. Mechanistic studies indicate
that the absence of Prtn3 can inhibit ferroptosis and inflammatory immune responses in neutrophils, thereby reducing the extent of tissue and organ damage. These findings suggest that the Prtn3 gene may serve as a novel therapeutic target for sepsis-associated acute lung injury. Furthermore, Prtn3 influences GPX4 expression and neutrophil
ferroptosis by directly cleaving SMAD3 protein. This study provides potential therapeutic targets and strategies for the regulation of neutrophil ferroptosis and the treatment of acute lung injury.