枸杞环肽调控NLRP3及NF-κB信号通路减轻BaP诱导的气道上皮细胞炎性损伤

Lycium barbarum Cyclic Peptide Regulates NLRP3 and NF-κB Signaling Pathways to Reduce BaP-Induced Inflammatory Airway Epithelial Cell Injury

摘要:

该研究通过活性追踪法分离枸杞活性肽（Lycium barbarum L. Bio-active Peptide，LBP），并研究其抗苯并芘（Benzopyrene，BaP）诱导的气道上皮细胞损伤活性及潜在机制。利用CCK-8法检测LBPs细胞毒性；ELISA法检测LBP-1抑制BaP诱导的人支气管上皮16-HBE细胞炎症因子（TNF-α、IL-1β、IL-6、IL-18、NO和PGE2）的分泌；Western Blot法检测LBP-1及BaP对16-HBE细胞COX-2、iNOS及NLRP3炎症小体和NF-κB信号通路相关蛋白表达的影响。结果显示，LBP-1能显著减轻BaP暴露导致的16-HBE细胞活力降低，且浓度小于1 mmol/L时无显著细胞毒性；LBP-1降低了由BaP暴露导致的细胞炎症因子分泌增加，TNF-α、IL-1β、IL-18、IL-6、NO和PGE2的浓度分别降低了34.93%、27.41%、31.05%、35.28%、51.15%及27.46%，同时PGE2和NO的关键酶（COX-2、iNOS）的表达分别降低了81.72%及41.70%；Western Blot结果显示，LBP-1可抑制IκBα和p65的磷酸化，降低NLRP3及含有Card的凋亡相关斑点样蛋白（Apoptosis-associated Speck-like Protein containing a CARD，ASC）的表达，从而抑制了NLRP3及NF-κB信号通路的激活。以上结果证实LBP-1可通过调控炎性细胞因子及NLRP3、NF-κB信号通路相关蛋白的表达发挥抗BaP诱导的气道上皮细胞炎性损伤的作用。

Abstract:

In this study, Lycium barbarum L. bioactive peptide (LBP) was isolated by the activity tracking method, and its activity against benzopyrene (BaP)-induced airway epithelial cell injury and the potential underlying mechanism were investigated. The cytotoxicity of LBPs was examined by the CCK-8 method; The inhibitory effects of LBP-1 against BaP-induced secretion of inflammatory factors (TNF-α, IL-1β, IL-6, IL-18, NO and PGE2) in human bronchial epithelial cells (16-HBE cells) were examined by ELISA; Western Blot method was used to examine the effects of LBP-1 and BaP on the expressions of COX-2, iNOS, NLRP3 inflammasomes and NF-κB signaling pathway-related proteins in 16-HBE cells. The results showed that LBP-1 could significantly reduce the decrease of 16-HBE cell viability caused by BaP exposure, and there was insignificant cytotoxicity when the concentration was lower than 1 mmol/L; LBP-1 decreased BaP exposure-induced increase in the secretion of cytokines, and the concentrations of TNF-α, IL-1β, IL-6, IL-18, NO and PGE2 decreased by 34.93%, 27.41%, 31.05%, 35.28%, 51.15% and 27.46% respectively, while the expressions of the key enzymes (COX-2 and iNOS) of PGE2 and NO decreased by 81.72% and 41.70% respectively; Western Blot results showed that LBP-1 could inhibit the phosphorylation of IκBα and p65, reduce the expressions of NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC), thereby inhibiting the activation of NLRP3 and NF-κB signaling pathways. The above results confirmed that LBP-1 could play a role in counteracting BaP-induced inflammatory airway epithelial cell injury through regulating the expressions of inflammatory cytokines and NLRP3 and NF-κB signaling pathway-related proteins.