外周血单核细胞AKT/mTOR信号通路表达在慢性阻塞性肺疾病患者中的评估价值

Evaluation Value of AKT/mTOR Signaling Pathway Expression in Peripheral Blood Mononuclear Cells in Patients with Chronic Obstructive Pulmonary Disease

摘要

目的 探讨AKT/mTOR信号通路表达在慢性阻塞性肺疾病患者中的研究价值。方法 收集2023年1月至2024年6月于鄂尔多斯市中心医院健康对照者20例、稳定期COPD（R-COPD）患者20例及急性加重期COPD（AE-COPD）患者20例。所有研究对象均采集外周静脉血样本，分离其血清及外周血单个核细胞（peripheral blood mononuclear cells，PBMCs），采用定量逆转录聚合酶链反应（auantitative reverse transcriptase polymerase chain reaction，qRT-PCR）检测PBMCs中AKT/mTOR信号通路的表达水平。体外实验中，将人肺微血管内皮细胞（human lung microvascular endothelial cells-5a，HULEC-5a）和人肺支气管上皮细胞（human bronchial epithelial cells，HBE）分为对照组、香烟烟雾提取物（CSE）组和AKT抑制剂MK-2206组，通过qRT-PCR、蛋白印迹及免疫荧光法检测各组细胞中AKT/mTOR信号通路的表达；采用条件培养基共培养实验，将含MK-2206的HULEC-5a细胞上清液加入肺成纤维细胞或人白血病THP-1细胞系衍生巨噬细胞的培养孔中，通过Transwell法分析细胞迁移能力。结果 三组受试者性别、年龄、体重、身高及BMI差异无统计学意义（P > 0.05）。肺功能方面，AE-COPD组FVC（72.08±10.78，% pred.）、FEV₁（47.21±11.32，% pred.）及FEV₁/FVC（%，58.67±11.68）低于R-COPD组和健康对照组（P ＜ 0.05）。qRT-PCR显示，AE-COPD组PBMCs中AKT mRNA和mTOR mRNA表达量高于R-COPD组和健康对照组（F = 10.31、13.23，P = 0.001）。体外实验中，HULEC-5a和HBE细胞CSE组AKT/mTOR的mRNA及蛋白表达高于对照组，而MK-2206组较CSE组降低（P ＜ 0.05）。免疫荧光定量显示，CSE组AKT/mTOR荧光强度升高，MK-2206组则明显降低（F = 18.632、17.853，P = 0.000）。Transwell实验显示，CSE组人肺成纤维细胞[（391.31±23.21）个/视野]和THP-1巨噬细胞[（411.25±32.65）个/视野]数量高于对照组，MK-2206组较CSE组减少（F = 24.389、36.645，P = 0.001）。结论 AKT/mTOR信号通路在COPD加重期间上调，AKT抑制剂MK-2206降低了AKT/mTOR信号通路表达，AKT/mTOR信号通路可影响肺成纤维细胞和单核细胞源性巨噬细胞的迁移，可能在COPD加重过程中发挥重要作用。

Abstract

Objective To investigate the research value of AKT/mTOR signaling pathway expression in patients with chronic obstructive pulmonary disease (COPD). Methods From January 2023 to June 2024, 20 healthy controls, 20 stable COPD (R-COPD) patients, and 20 acute exacerbation COPD (AE-COPD) patients were recruited from the Central Hospital of Ordos City. Peripheral venous blood samples were collected from all participants, with serum and peripheral blood mononuclear cells (PBMCs) isolated. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to assess the expression levels of the AKT/mTOR signaling pathway in PBMCs. In vitro experiments involved human lung microvascular endothelial cells (HULEC-5a) and human bronchial epithelial cells (HBE), which were categorized into control, cigarette smoke extract (CSE), and AKT inhibitor MK-2206 groups. qRT-PCR, Western blot, and immunofluorescence techniques were utilized to evaluate the expression of the AKT/mTOR signaling pathway in each group. Through conditional medium co-culture experiments, HULEC-5a cell supernatant containing MK-2206 was introduced to the culture wells of lung fibroblasts or THP-1 cell line-derived macrophages, and cell migration ability was assessed using the Transwell method. Results No statistically significant differences were observed in gender, age, weight, height, and BMI among the three groups (P > 0.05). In terms of lung function, the AE-COPD group exhibited lower FVC (72.08±10.78, % pred.), FEV₁ (47.21±11.32, % pred.), and FEV₁/FVC (58.67±11.68, %) compared to the R-COPD and healthy control groups (P ＜ 0.05). qRT-PCR analysis indicated higher AKT mRNA and mTOR mRNA expression in PBMCs of the AE-COPD group relative to the R-COPD and healthy control groups (F = 10.31, 13.23, P = 0.001). In vitro experiments revealed that the CSE group had elevated AKT/mTOR mRNA and protein expression in HULEC-5a and HBE cells compared to the control group, while the MK-2206 group exhibited lower levels (P ＜ 0.05). Immunofluorescence quantification demonstrated increased AKT/mTOR fluorescence intensity in the CSE group and significantly reduced intensity in the MK-2206 group (F = 18.632, 17.853, P = 0.000). Transwell experiments showed higher numbers of lung fibroblasts [(391.31±23.21) cells/field] and THP-1 macrophages [(411.25±32.65) cells/field] in the CSE group, with the MK-2206 group displaying reduced numbers compared to the CSE group (F = 24.389, 36.645, P = 0.001). Conclusion AKT/mTOR signaling pathway is upregulated during COPD exacerbation. AKT inhibitor MK-2206 reduces AKT/mTOR pathway expression and may affect lung fibroblast and monocyte-derived macrophage migration, potentially playing a crucial role in COPD exacerbation process.

关键词

外周血单核细胞 / Akt/mTOR / 慢性阻塞性肺疾病

Key words

Peripheral blood mononuclear cells / Akt/mTOR / Chronic obstructive pulmonary disease

参考文献

[1] 付道芳, 徐治波, 陈红, 等. 康北高原慢性阻塞性肺疾病急性加重患者发生静脉血栓栓塞症风险预测模型的构建及预测效能[J]. 临床肺科杂志, 2024, 29(2): 178-183.

[2] Vogelmeier C F, Román-Rodríguez M, Singh D, et al. Goals of COPD treatment: Focus on symptoms and exacerbations[J]. Respir Med, 2020, 166: 105938.

[3] Guo P, Li R, Piao T H, et al. Pathological mechanism and targeted drugs of COPD[J]. Int J Chron Obstruct Pulmon Dis, 2022, 17: 1565-1575.

[4] Peng Y, Wang Y, Zhou C, et al. PI3K/Akt/mTOR pathway and its role in cancer therapeutics: Are we making headway?[J]. Front Oncol, 2022, 12: 819128.

[5] Wang L, Jiang W, Wang J, et al. Puerarin inhibits FUNDC1-mediated mitochondrial autophagy and CSE-induced apoptosis of human bronchial epithelial cells by activating the PI3K/AKT/mTOR signaling pathway[J]. Aging, 2022, 14(3): 1253-1264.

[6] Crisafulli E, Torres A. COPD 2017: A year in review[J]. COPD J Chronic Obstr Pulm Dis, 2018, 15(2): 118-122.

[7] Easter M, Bollenbecker S, Barnes J W, et al. Targeting aging pathways in chronic obstructive pulmonary disease[J]. Int J Mol Sci, 2020, 21(18): 6924.

[8] Zhao Y Y, Tian Y, Zhang J, et al. Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo[J]. Drug Des Devel Ther, 2014, 8: 1827-1837.

[9] 郑琪, 秦斌斌, 沈斌, 等. Vanin-1在慢性阻塞性肺疾病患者血清中的表达及临床意义[J]. 临床肺科杂志, 2024, 29(2): 207-211.

[10] Besnek Ç, Akkok B, Sahin H, et al. Determination of the frequency of sarcopenia in patients admitted with COPD diagnosis with the SARC-F survey[J]. BMC Pulm Med, 2025, 25(1): 334.

[11] Wang C, Zhou J, Wang J, et al. Progress in the mechanism and targeted drug therapy for COPD[J]. Signal Transduct Target Ther, 2020, 5(1): 248.

[12] Chen L, Zhu D, Huang J, et al. Identification of hub genes associated with COPD through integrated bioinformatics analysis[J]. Int J Chron Obstruct Pulmon Dis, 2022, 17: 439-456.

[13] Ding T, Zhao S, Gu Y, et al. IL-17A regulates airway remodelling in COPD through the PI3K/AKT/mTOR pathway[J]. Sci Rep, 2025, 15(1): 16546.

[14] Zeng Y, Zhao Y, Chen Y, et al. PECAM EMPs regulate apoptosis in pulmonary microvascular endothelial cells in COPD by activating the Akt signaling pathway[J]. Tob Induc Dis, 2022, 20: 40.

[15] Tan A C. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer (NSCLC)[J]. Thorac Cancer, 2020, 11(3): 511-518.

[16] Li Q, Li Z, Luo T, et al. Targeting the PI3K/AKT/mTOR and RAF/MEK/ERK pathways for cancer therapy[J]. Mol Biomed, 2022, 3(1): 47.

[17] Rios-Fuller T J, Mahe M, Walters B, et al. Translation regulation by eIF2α phosphorylation and mTORC1 signaling pathways in non-communicable diseases (NCDs)[J]. Int J Mol Sci, 2020, 21(15): 5301.

[18] Fang L, Wang X, Zhang M, et al. microRNA-101-3p suppresses mTOR and causes mitochondrial fragmentation and cell degeneration in COPD[J]. Can Respir J, 2022, 2022: 5933324.

[19] Friedmann-Morvinski D, Hambardzumyan D. Monocyte-neutrophil entanglement in glioblastoma[J]. J Clin Invest, 2023, 133(1): e163451.

[20] Stewart I D, Nanji H, Figueredo G, et al. Circulating fibrocytes are not disease-specific prognosticators in idiopathic pulmonary fibrosis[J]. Eur Respir J, 2021, 58(1): 2100172.

基金资助

内蒙古自治区卫生健康委科研项目(2021N032125)