Zhang Wei, Kong Lingxin, Hu Feng, Li Jiyuan, Ma Shuli, Liang Hong, Wang Mingxia, Li Xingsan, Du Linlin, Li Jiping
Office of Academic Research, Qiqihar Medical University, Qiqihar, China.
Qiqihar Center for Disease Control and Prevention, Qiqihar, China.
Medicine (Baltimore). 2025 Jun 27;104(26):e43016. doi: 10.1097/MD.0000000000043016.
Long-term co-exposure to nanocarbon black particles (NCBP) and benzo[a]pyrene (B[a]P), as major components of PM2.5, may contribute to respiratory diseases by inducing oxidative damage. However, the molecular mechanisms underlying their synergistic effects remain unclear. This study aimed to investigate the impact of long-term co-exposure to NCBP and B[a]P on reactive oxygen species (ROS) levels and the protein kinase B (AKT)/cyclin-dependent kinase inhibitor 1B (P27) signaling pathway in human bronchial epithelial cells (BEAS-2B) cells.
Cytotoxicity concentrations of NCBP and B[a]P for BEAS-2B cells were screened using the cell counting kit-8 assay and applied to subsequent long-term exposure experiments. Intracellular ROS levels were measured via fluorescent probe assay. Western blot was performed to analyze the expression of AKT/P27 pathway-related proteins (phosphatidylinositol 3-kinase, phosphorylated AKT [p-AKT], total AKT, and P27). The regulatory relationship between the pathway and P27 was validated using an AKT inhibitor, and statistical analysis was conducted to evaluate significance.
Significant reductions in cell viability (P < .05) were observed at concentrations of 16 μM B[a]P and 80 μg/mL NCBP. Long-term co-exposure to NCBP and B[a]P-induced ROS accumulation (P < .05). Western blot revealed upregulated p-AKT expression (P < .05) and significant suppression of P27 (P < .05) in the co-exposure group. Inhibition of p-AKT by an AKT inhibitor reversed the downregulation of P27 induced by co-exposure (P < .05), indicating that P27 is a downstream target of the AKT pathway. Compared to single exposures, co-exposure exhibited stronger synergistic effects on oxidative stress dysregulation.
Long-term co-exposure to NCBP and B[a]P exacerbates oxidative damage in BEAS-2B cells by activating the AKT signaling pathway, suppressing P27 expression, and promoting ROS accumulation. This study elucidates potential mechanisms of multi-pollutant synergistic effects, providing new insights for health risk assessment of environmental co-exposure.
长期共同暴露于作为细颗粒物主要成分的纳米炭黑颗粒（NCBP）和苯并[a]芘（B[a]P），可能通过诱导氧化损伤导致呼吸系统疾病。然而，它们协同作用的分子机制尚不清楚。本研究旨在探讨长期共同暴露于NCBP和B[a]P对人支气管上皮细胞（BEAS-2B）中活性氧（ROS）水平及蛋白激酶B（AKT）/细胞周期蛋白依赖性激酶抑制剂1B（P27）信号通路的影响。
使用细胞计数试剂盒-8法筛选NCBP和B[a]P对BEAS-2B细胞的细胞毒性浓度，并应用于后续长期暴露实验。通过荧光探针法测量细胞内ROS水平。采用蛋白质印迹法分析AKT/P27通路相关蛋白（磷脂酰肌醇3激酶、磷酸化AKT [p-AKT]、总AKT和P27）的表达。使用AKT抑制剂验证该通路与P27之间的调控关系，并进行统计分析以评估其显著性。
在16 μM B[a]P和80 μg/mL NCBP浓度下观察到细胞活力显著降低（P < 0.05）。长期共同暴露于NCBP和B[a]P会诱导ROS积累（P < 0.05）。蛋白质印迹显示，共同暴露组中p-AKT表达上调（P < 0.05），P27受到显著抑制（P < 0.05）。AKT抑制剂对p-AKT的抑制作用逆转了共同暴露诱导的P27下调（P < 0.05），表明P27是AKT通路的下游靶点。与单一暴露相比，共同暴露对氧化应激失调表现出更强的协同作用。
长期共同暴露于NCBP和B[a]P通过激活AKT信号通路、抑制P27表达和促进ROS积累，加剧了BEAS-2B细胞中的氧化损伤。本研究阐明了多污染物协同作用机制，为环境共同暴露的健康风险评估提供了新见解。
