嗜热栖热菌发酵产物通过抗炎作用干预人成纤维细胞衰老研究

Protective effects of Thermus thermophilus fermentation product against cellular senescence in human fibroblasts through anti-inflammatory effects

关键词:  嗜热栖热菌发酵产物  抗炎  细胞衰老  NF-κB 信号通路

Key words:  Thermus thermophilus fermentation product  anti-inflammatory  cellular senescence  NF-κB signaling pathway

摘要:

摘要：目的 通过博莱霉素诱导人皮肤成纤维细胞（HFF-1）的衰老模型，探索嗜热栖热菌发酵产物（Thermus thermophilus fermentation product，TTFP）抗衰老作用及可能的调控机制。方法 博莱霉素诱导HFF-1细胞衰老，用CCK-8法测定细胞活力确定0.0625%和0.2500%两个剂量TTFP用于后续实验。生化染色技术、流式细胞术、酶联免疫吸附反应、实时荧光定量和蛋白免疫印迹等实验手段检测TTFP对衰老细胞模型的衰老标志物β-半乳糖苷酶（SA-β-gal）的表达，I型前胶原蛋白（Pro-COL1）的分泌的影响，以及对细胞凋亡、活性氧（ROS）、线粒体膜电位（MMP）、细胞周期调控、周期调控相关因子P16、P21、TP53和促炎因子TNF-α的mRNA表达、NF-κB通路相关蛋白表达的干预。结果 与模型组相比，经TTFP干预后SA-β-gal阳性细胞数有效降低、Pro-COL1分泌显著增加，流式结果显示凋亡细胞比例和ROS阳性细胞比例显著降低，炎症相关的TNF-α和NF-κB通路相关蛋白的表达显著下调。结论 嗜热栖热菌发酵产物能干预多个细胞衰老标志，包括降低SA-β-gal活性，促进胶原分泌，抑制细胞ROS、抗细胞凋亡，可能与调控抗炎信号通路作用相关。

Abstract:

Abstract: Objective The anti-aging effects and possible regulatory mechanisms of Thermus thermophilus fermentation product (TTFP) are explored using the bleomycin-induced senescence model in human skin fibroblasts (HFF-1). Methods Senescence was induced in HFF-1 cells by bleomycin, and two doses of TTFP, 0.0625% and 0.2500%, were selected for subsequent experiments based on cell viability determined by the CCK-8 assay. The effect of TTFP on the expression of the senescence marker β-galactosidase (SA-β-gal) and the secretion of procollagen type I (Pro-COL1) in a senescent cell model was examined using biochemical staining techniques and enzyme-linked immunosorbent assay. As well as detecting changes in apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cell cycle, mRNA expression of Cell cycle regulator p16\ p21\ TP53 and the pro-inflammatory factor TNF-α, and expression of proteins related to the NF-κB pathwayflow cytometry, using real-time fluorescence quantification and protein immunoblotting. Results Compared to the model group, the number of SA-β-gal-positive cells was significantly reduced and the secretion of procollagen type I (Pro-COL1) was notably increased following TTFP intervention. Flow cytometry results indicated that the proportions of apoptotic cells and ROS-positive cells were significantly decreased, while the expression of inflammation-associated TNF-α and NF-κB pathway-associated proteins was markedly down-regulated. Conclusion The TTFP can interfere with several cellular senescence markers, including reducing the activity of SA-β-gal, and promoting collagen secretion, inhibiting cellular ROS, and anti apoptosis. These effects may be associated with the modulation of anti-inflammatory signaling pathway.