背景:LL-37(也称为鼠CRAMP)是一种人类抗微生物肽,通过各种机制在针对败血症的先天免疫防御中起关键作用。然而,其是否参与脓毒症诱导的肺损伤尚不清楚.
目的:这项工作研究了LL-37对LPS在肺泡上皮细胞中产生的焦亡的影响。该研究利用体内和体外脓毒症相关急性肺损伤(ALI)模型来了解潜在的分子途径。
方法:体内,通过气管内给予LPS建立C57BL/6J小鼠脓毒症急性肺损伤模型,随后用低剂量CRAMP(重组鼠cathelicidin,2.5mg。kg-1)和高剂量CRAMP(5.0mg。kg-1)。体外,通过用LPS和ATP刺激,在人肺泡上皮细胞系(A549)中诱导了焦亡。用重组人LL-37进行处理,或者使用小干扰RNA(siRNA)在A549细胞中敲除LL-37。随后,苏木素和伊红染色观察对照组和脓毒症肺损伤组的肺组织病理学变化。用TUNEL和PI染色观察不同组小鼠肺组织和细胞的DNA断裂和焦亡。进行乳酸脱氢酶(LDH)测定以测量细胞死亡率。NLRP3、caspase1、caspase1p20、GSDMD、NT-GSDMD,使用蛋白质印迹在小鼠和细胞中检测到CRAMP,qPCR,和免疫组织化学。ELISA法检测小鼠血清中白细胞介素(IL)-1β和IL-18水平,支气管肺泡灌洗液(BALF)和肺组织和细胞培养上清液。
结果:NLRP3,caspase1p20,NT-GSDMD的表达,脓毒症肺损伤小鼠肺组织IL一18和IL一1β升高,这表明经典的焦亡途径的激活,并且与CRAMP表达的增加相吻合。重组CRAMP治疗可改善肺损伤小鼠的焦亡。体外,用LPS和ATP治疗上调了这些经典的焦亡分子,LL-37敲低加剧了焦亡,重组人LL-37治疗可减轻肺泡上皮细胞的焦亡。
结论:这些发现表明,LL-37通过调节经典的促性腺激途径成分的表达来保护脓毒症肺损伤,包括NLRP3、caspase1、GSDMD和肺泡上皮细胞下游炎症因子。
BACKGROUND: LL-37 (also known as murine CRAMP) is a human antimicrobial peptide that plays a crucial role in innate immune defence against sepsis through various mechanisms. However, its involvement in sepsis-induced lung injury remains unclear.
OBJECTIVE: This work investigates the impact of LL-37 on pyroptosis generated by LPS in alveolar epithelial cells. The research utilizes both in vivo and in vitro sepsis-associated acute lung injury (ALI) models to understand the underlying molecular pathways.
METHODS: In vivo, an acute lung injury model induced by sepsis was established by intratracheal administration of LPS in C57BL/6J mice, which were subsequently treated with low-dose CRAMP (recombinant murine cathelicidin, 2.5 mg.kg-1) and high-dose CRAMP (5.0 mg.kg-1). In vitro, pyroptosis was induced in a human alveolar epithelial cell line (A549) by stimulation with LPS and ATP. Treatment was carried out with recombinant human LL-37, or LL-37 was knocked out in A549 cells using small interfering RNA (siRNA). Subsequently, haematoxylin and eosin staining was performed to observe the histopathological changes in lung tissues in the control group and sepsis-induced lung injury group. TUNEL and PI staining were used to observe DNA fragmentation and pyroptosis in mouse lung tissues and cells in the different groups. An lactate dehydrogenase (LDH) assay was performed to measure the cell death rate. The expression levels of NLRP3, caspase1, caspase 1 p20, GSDMD, NT-GSDMD, and CRAMP were detected in mice and cells using Western blotting, qPCR, and immunohistochemistry. ELISA was used to assess the levels of interleukin (IL)-1β and IL-18 in mouse serum, bronchoalveolar lavage fluid (BALF) and lung tissue and cell culture supernatants.
RESULTS: The expression of NLRP3, caspase1 p20, NT-GSDMD, IL 18 and IL1β in the lung tissue of mice with septic lung injury was increased, which indicated activation of the canonical pyroptosis pathway and coincided with an increase in CRAMP expression. Treatment with recombinant CRAMP improved pyroptosis in mice with lung injury. In vitro, treatment with LPS and ATP upregulated these classic pyroptosis molecules, LL-37 knockdown exacerbated pyroptosis, and recombinant human LL-37 treatment alleviated pyroptosis in alveolar epithelial cells.
CONCLUSIONS: These findings indicate that LL-37 protects against septic lung injury by modulating the expression of classic pyroptotic pathway components, including NLRP3, caspase1, and GSDMD and downstream inflammatory factors in alveolar epithelial cells.