Guo Fang, Song Fuxing, Chen Zhenjiang, Niu Na, Sun Lina, Yan Min, Liu Min
Department of Pediatrics, Jinan City People's Hospital, No. 001, Xuehu Street, Changshao North Road, Laiwu District, Jinan, 271100, China.
J Inflamm (Lond). 2025 Jun 19;22(1):23. doi: 10.1186/s12950-025-00450-0.
Infantile pneumonia (IP) is a significant cause of morbidity and mortality in young children. Mesenchymal stem cells (MSCs) have emerged as potential therapeutic agents in pneumonia due to their immunomodulatory properties. The study analyzed the role of MSCs from bone marrow in IP and the underlying mechanism.
Human embryonic lung fibroblasts (WI-38) were stimulated using lipopolysaccharide (LPS) to mimic an IP cell model. This study employed flow cytometry to analyze the expression of hematopoietic markers and marker proteins on MSCs. The differentiation potential of MSCs was assessed through microscopy, oil red O staining, and alkaline phosphatase (ALP) assays. The localization of exosomes in WI-38 cells was observed using the cell membrane green fluorescent probe DIO. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry assays were used to analyze the expression of mRNA or protein. Cell viability, proliferation, and apoptosis were evaluated using Cell counting kit-8, 5-Ethynyl-2'-deoxyuridine, and flow cytometry assays, respectively. Enzyme-linked immunosorbent assays were conducted to measure cytokine levels. A mouse model of pneumonia was utilized to assess the therapeutic potential of MSC-derived exosomes on lung injury. Co-immunoprecipitation (Co-IP) assay was performed to study the interaction between Cbl proto-oncogene B (CBLB) and mitogen-activated protein kinase 14 (MAPK14).
MSC-derived exosomes could be transferred into LPS-induced WI-38 cells, where they mitigated the inhibitory effects of LPS on CBLB mRNA expression. These exosomes improved WI-38 cell proliferation, reduced apoptosis, and decreased the production of pro-inflammatory cytokines including IL-6, IL-1β, and TNF-α by regulating CBLB after LPS treatment. Moreover, in a mouse model, MSC-derived exosomes protected against LPS-induced lung injury, whereas the effect was reversed after treatment with the exosomes isolated from CBLB-deficient MSCs. In addition, CBLB was found to destabilize MAPK14 protein expression in WI-38 cells. Further, overexpression of CBLB ameliorated LPS-induced inhibitory effect on cell proliferation and promoting effects on cell apoptosis and inflammation in WI-38 cells by regulating MAPK14.
MSC-derived exosomal CBLB has therapeutic potential in ameliorating the progression of IP probably by ubiquitinating MAPK14, which could lead to novel clinical interventions for treating this condition.
小儿肺炎（IP）是幼儿发病和死亡的重要原因。间充质干细胞（MSCs）因其免疫调节特性已成为肺炎潜在的治疗药物。本研究分析了骨髓间充质干细胞在小儿肺炎中的作用及其潜在机制。
使用脂多糖（LPS）刺激人胚肺成纤维细胞（WI-38）以模拟小儿肺炎细胞模型。本研究采用流式细胞术分析间充质干细胞上造血标志物和标志物蛋白的表达。通过显微镜检查、油红O染色和碱性磷酸酶（ALP）测定评估间充质干细胞的分化潜能。使用细胞膜绿色荧光探针DIO观察外泌体在WI-38细胞中的定位。采用定量逆转录聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法和免疫组织化学测定法分析mRNA或蛋白质的表达。分别使用细胞计数试剂盒-8、5-乙炔基-2'-脱氧尿苷和流式细胞术测定法评估细胞活力、增殖和凋亡。进行酶联免疫吸附测定以测量细胞因子水平。利用肺炎小鼠模型评估间充质干细胞来源的外泌体对肺损伤的治疗潜力。进行免疫共沉淀（Co-IP）测定以研究Cbl原癌基因B（CBLB）与丝裂原活化蛋白激酶14（MAPK14）之间的相互作用。
间充质干细胞来源的外泌体可以转移到LPS诱导的WI-38细胞中，减轻LPS对CBLB mRNA表达的抑制作用。这些外泌体通过在LPS处理后调节CBLB来改善WI-38细胞增殖、减少凋亡并降低促炎细胞因子（包括IL-6、IL-1β和TNF-α）的产生。此外，在小鼠模型中，间充质干细胞来源的外泌体可预防LPS诱导的肺损伤，而用从CBLB缺陷型间充质干细胞中分离的外泌体处理后，这种作用会逆转。此外，发现CBLB可使WI-38细胞中MAPK14蛋白表达不稳定。此外，CBLB的过表达通过调节MAPK14改善了LPS诱导的对WI-38细胞增殖的抑制作用以及对细胞凋亡和炎症的促进作用。
间充质干细胞来源的外泌体CBLB可能通过泛素化MAPK14在改善小儿肺炎进展方面具有治疗潜力，这可能为治疗这种疾病带来新的临床干预措施。
