Evaluation Value of AKT/mTOR Signaling Pathway Expression in Peripheral Blood Mononuclear Cells in Patients with Chronic Obstructive Pulmonary Disease
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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（PBMCs），采用定量逆转录聚合酶链反应（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（HULEC-5a）和人肺支气管上皮细胞（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，这可能在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. -
图 1 CSE对体外AKT/mTOR信号通路表达水平作用
A～B：蛋白质印迹法检查转染的HULEC-2a和HBE细胞中AKT/mTOR信号通路表达；与对照组比较，*P < 0.05；与CSE组比较，#P < 0.05；C：免疫荧光显微镜检测AKT/mTOR信号通路表达。
Figure 1. Effect of CSE on the expression levels of AKT/mTOR signaling pathway in vitro
图 2 AKT/mTOR信号通路对人肺成纤维细胞和巨噬细胞迁移的影响（×200）
Figure 2. Effect of AKT/mTOR signaling pathway on the migration of human lung fibroblasts and macrophages （×200）
表 1 COPD 患者外周血单核细胞中AKT/mTOR信号通路表达水平（$\bar x \pm s $，n = 20）
Table 1. Expression levels of AKT/mTOR signaling pathway in peripheral blood monocytes of COPD patients （$\bar x \pm s $，n = 20）
分组 对照组 R-COPD组 AE-COPD组 F/χ2 P 性别（男 / 女，例） 17/3 18/2 19/1 0.332 0.131 年龄（岁） 58.1 ± 5.8 56.5 ± 7.2 59.6 ± 6.0 0.234 0.232 体重（kg） 65.3 ± 12.3 63.5 ± 14.2 61.8 ± 11.8 0.134 0.287 身高（cm） 169.3 ± 8.3 167.8 ± 9.3 168.2 ± 10.2 0.255 0.167 BMI（kg/m2） 22.8 ± 3.6 22.3 ± 4.7 21.6 ± 3.3 0.111 0.356 用力肺活量（FVC，% pred.） 92.65 ± 7.05 75.27 ± 9.55# 72.08 ± 10.78& −7.242 0.021* 第一秒用力呼气量（FEV1，% pred.） 82.34 ± 6.57 52.31 ± 8.63# 47.21 ± 11.32& −9.313 <0.001* 第一秒用力呼气量 / 用力肺活量（FEV1/FVC，% pred.） 88.86 ± 7.82 69.49 ± 12.6# 58.67 ± 11.68& −8.891 0.012* AKT mRNA（相对表达量） 1.1 ± 0.28 1.82 ± 0.22# 2.13 ± 0.29& 10.31 <0.001* mTOR mRNA（相对表达量） 1.21 ± 0.26 2.11 ± 0.25# 2.45 ± 0.27& 13.23 <0.001* *P < 0.05；与R-COPD组比较，&P < 0.05；与对照组比较，#P < 0.05。 
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表 2 CSE对体外AKT/mTOR信号通路表达水平的影响（$\bar x \pm s $）
Table 2. Effect of CSE on the expression levels of AKT/mTOR signaling pathway in vitro（$\bar x \pm s $）
分组 AKT mRNA mTOR mRNA AKT/GAPDH mTOR/GAPDH AKT 荧光强度 mTOR 荧光强度 HULEC-5a 对照组 1.15 ± 0.25 1.31 ± 0.27 1.31 ± 0.27 1.31 ± 0.22 105.32 ± 14.28 105.32 ± 14.28 CSE组 1.93 ± 0.22# 2.21 ± 0.25# 2.31 ± 0.33# 1.98 ± 0.23# 235.64 ± 28.37# 228.47 ± 25.19 MK-2206组 1.13 ± 0.3& 1.04 ± 0.23& 1.34 ± 0.32& 1.17 ± 0.28& 120.45 ± 16.73& 115.37 ± 15.26 F 9.323 13.311 8.377 11.329 18.632 17.853 P <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* HBE 对照组 1.01 ± 0.22 1.11 ± 0.25 1.15 ± 0.28 1.03 ± 0.22 98.76 ± 12.35 92.45 ± 11.83 CSE组 1.95 ± 0.27# 2.16 ± 0.27# 2.43 ± 0.31# 1.88 ± 0.23# 210.35 ± 22.41# 195.63 ± 20.74# MK-2206组 1.08 ± 0.24& 1.17 ± 0.28& 1.33 ± 0.31& 1.11 ± 0.26& 102.68 ± 13.54& 96.82 ± 12.17& F 7.356 12.343 15.333 13.766 16.945 15.721 P <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* *P < 0.05；与对照组比较，#P < 0.05；与CSE组比较；&P < 0.05。
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表 3 AKT/mTOR信号通路对人肺成纤维细胞和巨噬细胞迁移的影响
Table 3. Effect of the AKT/mTOR Signaling Pathway on the Migration of Human Lung Fibroblasts and Macrophages
分组 人肺成纤维细胞数量（1× 105 cells/mL） THP-1巨噬细胞（1×105 cells/mL） 对照组 200.23 ± 19.24 234.15 ± 24.27 CSE 组 391.31 ± 23.21# 411.25 ± 32.65# MK-2206 组 107.13 ± 20.37& 196.01 ± 26.43& F 24.389 36.645 P <0.001* <0.001* *P < 0.05；与对照组比较，#P < 0.05；与CSE组比较；&P < 0.05。
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