Intervention Mechanism of miR-155 in Macrophage-Derived Exosomes from Sepsis Patients on Endothelial Cell Ferroptosis
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摘要:
目的 探讨脓毒血症患者肺泡巨噬细胞（alveolar macrophage，AM）来源外泌体（Exosomal，Exos）中miR-155对内皮细胞铁死亡的干预机制。 方法 本研究为单中心研究，研究对象为邯郸市中心医院2022年1月至2023年6月收治的106例脓毒症患者。根据是否存在急性呼吸窘迫综合征（acute respiratory distress syndrome，ARDS），将患者进一步分为ARDS组（n = 21）和非ARDS组（n = 85）。体外实验中，将脂多糖（lipopolysaccharide，LPS）处理的Raw 264.7外泌体与小鼠肺微血管内皮细胞（pulmonary microvascular endothelial cells，PMVEC）一起孵育作为LPS-Exos组，从正常Raw 264.7培养物上清液中提取的外泌体作为正常对照组（NC-Exos），单独的PMVEC细胞培养物作为空白组（Con）。分别从巨噬细胞的条件培养基或脓毒症患者支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中分离Exos，通过RT-qPCR分析miR-155表达情况，蛋白质印迹分析GPX4、Nrf2蛋白表达。通过试剂盒检测PMVEC细胞中Fe2+、SOD、ROS水平变化。 结果 与非ARDS组相比，ARDS组年龄、SOFA评分、miR-155表达、乳酸水平增加（P < 0.05）。与NC-Exos相比，LPS-Exos中miR-155表达上调（P < 0.01）。与Con组相比，LPS-Exos组PMVEC细胞活力、SOD水平降低（P < 0.01），细胞中的Fe2+、ROS水平增加（P < 0.05）。与NC-Exos组相比，LPS-Exos组PMVEC细胞中经典的铁死亡抑制基因如GPX4和Nrf2的蛋白表达降低（P < 0.05）。与Con组相比，Inhibitor-NC+LPS-Exos组PMVEC细胞活力、SOD水平、GPX4、Nrf2蛋白表达降低（P < 0.001），Fe2+、ROS水平增加（P < 0.0001 ）。与Inhibitor-NC+LPS-Exos组相比，miR-155 Inhibitor+LPS-Exos组PMVEC细胞活力、SOD水平、GPX4、Nrf2蛋白表达增加（P < 0.001），Fe2+、ROS水平降低（P < 0.01）。使用双荧光素酶报告基因分析证实了miR-155和Nrf2之间的直接结合关系。结论 AM来源的Exos中miR-155水平升高预测脓毒症患者发生ARDS的能力优于SOFA评分，其与SOFA评分组合在预测脓毒症患者发生ARDS方面具有较高的能力。LPS诱导的AM来源的Exos可能通过转运miR-155促进PMVEC细胞铁死亡，其作用机制可能与抑制Nrf2表达有关。 Abstract:Objective To investigate the intervention mechanism of miR-155 in alveolar macrophage （AM）-derived exosomes （Exos） on ferroptosis of endothelial cells in septic patients. Methods This was a single-center study of 106 septic patients admitted to Handan Central Hospital from January 2022 to June 2023. Patients were further stratified into ARDS group （n = 21） and non-ARDS group （n = 85） based on the presence or absence of acute respiratory distress syndrome （ARDS）. In vitro, exosomes from lipopolysaccharide （LPS）-treated Raw 264.7 cells were co-cultured with mouse pulmonary microvascular endothelial cells （PMVEC） as the LPS-Exos group. Exosomes extracted from normal Raw 264.7 culture supernatant served as the normal control group （NC-Exos）, and PMVEC cell culture alone served as the blank control group （Con）. Exosomes were isolated from conditional culture medium of macrophages or bronchoalveolar lavage fluid （BALF） from septic patients. miR-155 expression was analyzed by RT-qPCR, and GPX4 and Nrf2 protein expression was analyzed by Western blotting. Changes in Fe2+, SOD and ROS levels in PMVEC cells were detected using assay kits. Results Compared to the non-ARDS group, the ARDS group exhibited higher age, SOFA score, miR-155 expression, and lactate levels （P < 0.05）. miR-155 expression was significantly upregulated in LPS-Exos compared to NC-Exos （P < 0.01）. Compared with Con group, the activity and SOD level of PMVEC cells in LPS-Exos group decreased （P < 0.01）, while the levels of Fe2+ and ROS in cells increased （P < 0.05）. Compared with NC-Exos group, the protein expression of classical ferroptosis suppressor genes such as GPX4 and Nrf2 in PMVEC cells was decreased in LPS-Exos group （P < 0.05）. Compared with Con group, PMVEC cell viability, SOD levels, and GPX4 and Nrf2 protein expression were decreased in the Inhibitor-NC+LPS-Exos group （P < 0.001）, while Fe2+ and ROS levels were increased （P < 0.0001 ）. Compared with the Inhibitor-NC+LPS-Exos group, PMVEC cell viability, SOD levels, and GPX4 and Nrf2 protein expression were increased in the miR-155 Inhibitor+LPS-Exos group （P < 0.001）, while Fe2+ and ROS levels were decreased （P < 0.01）. Dual-luciferase reporter analysis confirmed direct binding between miR-155 and Nrf2.Conclusion The elevated level of miR-155 in AM-derived Exos has a superior predictive ability for ARDS development in septic patients compared to the SOFA score. When combined with the SOFA score, it demonstrates high predictive capability for ARDS development in septic patients. LPS-induced AM-derived Exos may promote ferroptosis of PMVEC cells by transporting miR-155, and this mechanism may be related to suppression of Nrf2 expression. -
Key words:
- Sepsis /
- Alveolar macrophages /
- Exosome /
- Pulmonary microvascular endothelial cells /
- Ferroptosis /
- miR-155
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图 1 Exos的鉴定
A：TEM观察Exos（比例尺=100 nm）；B：Western blot检测Calnexin、CD9、CD63和TSG101蛋白表达；C：通过纳米粒子追踪分析进行Exos定量。
Figure 1. Identification of exosomes
图 2 ARDS组和非ARDS组AM来源的Exos中炎症相关miRNA的差异表达
*P < 0.05。
Figure 2. Differential expression of inflammation-related miRNAs in alveolar macrophage-derived exosomes between ARDS group and non-ARDS group
图 3 LPS诱导的AM来源的Exos中miR-155表达
A：在用LPS预处理（LPS-Exos）或不用LPS预处理（NC-Exos）的Raw 264.7细胞来源的Exos中miR-155的表达；B：PMVEC细胞摄取PKH67标记的Exos（×100，比例尺=100 μm）；C：在共孵育LPS-Exos或NC-Exos后，PMVEC细胞中miR-155的表达；**P < 0.01；****P < 0.0001。
Figure 3. Expression of miR-155 in alveolar macrophage-derived exosomes induced by LPS
图 4 AM来源的Exos促进PMVEC细胞铁死亡
A：PMVEC细胞在与LPS-Exos或NC-Exos共孵育后的生存活力；与Con组相比，***P < 0.001；与NC-Exos组相比，###P < 0.001；B～D：与LPS-Exos或NC-Exos共孵育后PMVEC细胞中Fe2+、SOD、ROS水平变化；E：免疫印迹检测PMVEC细胞中GPX4和Nrf2蛋白表达；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 4. Alveolar macrophage-derived exosomes promote ferroptosis of pulmonary microvascular endothelial cells
图 5 LPS诱导的AM来源Exos通过miR-155/Nrf2信号通路促进PMVEC细胞铁死亡
A～B：转染miR-155 Inhibitor或Inhibitor-NC后，检测miR-155在Raw 264.7细胞和Raw 264.7细胞来源Exos中的表达；C：与经miR-155 Inhibitor预处理的LPS-Exos共孵育后，使用CCK-8测定法检测PMVEC细胞的细胞活力；与Con组相比，*P < 0.05；***P < 0.001；与Inhibitor-NC+LPS-Exos组相比，###P < 0.001；D～F：PMVEC细胞中Fe2+、SOD、ROS水平变化；G：免疫印迹检测PMVEC细胞中GPX4和Nrf2蛋白表达；H：双荧光素酶活性分析miR-155和Nrf2的关系；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 5. LPS-induced alveolar macrophage-derived exosomes promote ferroptosis of pulmonary microvascular endothelial cells via the miR-155/Nrf2 signaling pathway
表 1 用于qPCR的引物
Table 1. Primers used for quantitative real-time PCR
引物 序列 （5′-3′） miR-15a F: GCCGAGTAGCAGCACATAAT R: CTCAACTGGTGTCGTGGA miR-15b F: GCCGAGTAGCAGCACATCAT R: CTCAACTGGTGTCGTGGA miR-21 F: GCCGAGTAGCTTATCAGACT R: CTCAACTGGTGTCGTGGA miR-27b F: GCCGAGTTCACAGTGGCTA R: CTCAACTGGTGTCGTGGA miR-93 F: TCGGCAGGCAAAGTGCTGTTCGT R: CTCAACTGGTGTCGTGGA miR-125a F: TCGGCAGGTCCCTGAGACCCTTTA R: CTCAACTGGTGTCGTGGA miR-146a F: GCCGAGTGAGAACTGAATTC R: CTCAACTGGTGTCGTGGA miR-155 F: GCCGAGTTAATGCTAATTGTG R: CTCAACTGGTGTCGTGGA U6 F: CTCGCTTCGGCAGCACA R: AACGCTTCACGAATTTGCGT 
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表 2 ARDS和非ARDS患者组中受试者的基本特征[（$\bar x \pm s $），M（M25，M75），n（%）]
Table 2. Baseline characteristics of subjects in ARDS and non-ARDS groups [（$\bar x \pm s $），M（M25，M75），n（%）]
特征 ARDS组（n = 21） 非ARDS组（n = 85） z/t/χ2 P 年龄（岁） 66.75 ±16.47 74.19 ± 11.05 2.479 0.017* 男性 11 （52.4） 50 （58.8） 0.286 0.593 APACHE II评分 28.55 ± 8.30 26.36 ± 8.60 1.113 0.269 体质量指数（kg/m2） 24.75 ± 4.65 22.87 ± 5.15 1.536 0.128 查尔森指数 2.70 ± 1.50 2.35 ± 1.60 0.883 0.379 心血管疾病 6 （28.6） 26 （30.6） 0.033 0.857 高血压 15 （71.4） 49 （57.6） 1.337 0.248 慢性阻塞性肺病 2 （9.5） 16 （18.8） 1.033 0.309 哮喘 1 （4.8） 4 （4.7） <0.001 0.991 肺结核 1 （4.8） 3 （3.5） 0.070 0.791 癌症 3 （14.3） 12 （14.1） <0.001 0.984 糖尿病 13 （61.9） 42 （49.4） 1.053 0.305 卒中 5 （23.8） 19 （22.4） 0.020 0.886 慢性肾病 8 （38.1） 20 （23.5） 1.838 0.175 SOFA评分（分） 12.09 ± 4.4 8.80 ± 3.15 3.936 0.001* 白细胞（109/L） 16.70（10.95，25.05） 15.30（10.25，20.10） 1.411 0.161 单核细胞（109/L） 6.00（3.00，8.35） 3.40（2.00，6.00） 1.642 0.104 淋巴细胞（109/L） 10.00（3.00，13.25） 7.40（4.00，14.25） 0.095 0.924 C反应蛋白（mg/L） 232.40（51.05，345.70） 138.60（46.60，242.65） 1.259 0.208 降钙素原（ng/mL） 4.71（0.59，41.34） 2.80（0.62，14.24） 0.694 0.487 乳酸（mmol/L） 26.65（19.38，67.70） 19.90（14.70，35.40） 2.167 0.030* *P < 0.05。
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