Shi Qiyun, Tian Taotao, Li Yanfeng
Medical Center of Cervical and Lumbar Pain (Tui Na Therapy), Luoyang Orthopedic-Traumatological Hospital Of Henan Province (Henan Provincial Orthopedic Hospital), Luoyang, China.
Medical Center of Microsurgery, Luoyang Orthopedic-Traumatological Hospital Of Henan Province (Henan Provincial Orthopedic Hospital), Zhengzhou, China.
Front Immunol. 2025 May 9;16:1491928. doi: 10.3389/fimmu.2025.1491928. eCollection 2025.
To establish a cross-tissue bioinformatics model for identifying conserved key genes and immune infiltration mechanisms in ischemia-reperfusion injury (IRI) with experimental validation in limb IRI, including pharmacological targeting of the WNT5A/PLC pathway.
Transcriptomic data from CTGF-stimulated cardiac myocytes (GSE36073) were analyzed as a surrogate for limb IRI due to shared pathological mechanisms. Random forest, LASSO regression, algorithms identified feature genes, validated in a rat limb IRI model using RT-qPCR, and histology. Pharmacological inhibition (WNT5A inhibitor Box5, PLC inhibitor U-73122) was performed to assess pathway involvement. Immune cell infiltration patterns were analyzed via CIBERSORT.
From 169 differentially expressed genes (116 upregulated, 53 downregulated), machine learning identified four key genes (WNT5A, PLCG, ITPR1, CAMK2A), significantly upregulated in experimental limb IRI (P<0.01). Pharmacological inhibition confirmed their functional roles: Box5 and U-73122 treatment reduced expression of WNT5A and PLC versus IRI controls (P<0.05), showing IRI-induced muscle fiber disruption, edema, and inflammation. Immune analysis revealed myeloid polarization shifts (increased M1, decreased M2 macrophages; P<0.05). WNT5A correlated negatively with memory immune cells, while PLCG, ITPR1, and CAMK2A correlated with lymphocyte subpopulations.
We identified a conserved molecular signature across cardiac and skeletal muscle IRI, with WNT5A/PLC pathway components as mechanistically validated therapeutic targets. Our cross-tissue bioinformatic approach, reinforced by pharmacological and histological evidence, provides a novel framework for IRI analysis when direct patient data are unavailable. Combined targeting of macrophage polarization and cellular activation the WNT5A/PLC axis may offer synergistic therapeutic potential.
建立一种跨组织生物信息学模型，用于识别缺血再灌注损伤（IRI）中保守的关键基因和免疫浸润机制，并在肢体IRI中进行实验验证，包括对WNT5A/PLC通路的药物靶向作用。
由于病理机制相同，将CTGF刺激的心肌细胞转录组数据（GSE36073）作为肢体IRI的替代数据进行分析。采用随机森林、LASSO回归算法识别特征基因，并在大鼠肢体IRI模型中通过RT-qPCR和组织学进行验证。进行药物抑制（WNT5A抑制剂Box5、PLC抑制剂U-73122）以评估通路参与情况。通过CIBERSORT分析免疫细胞浸润模式。
从169个差异表达基因（116个上调，53个下调）中，机器学习识别出四个关键基因（WNT5A、PLCG、ITPR1、CAMK2A），在实验性肢体IRI中显著上调（P<0.01）。药物抑制证实了它们的功能作用：与IRI对照组相比，Box5和U-73122处理降低了WNT5A和PLC的表达（P<0.05），显示IRI诱导的肌纤维破坏、水肿和炎症。免疫分析显示髓系极化转变（M1增加，M2巨噬细胞减少；P<0.05）。WNT5A与记忆免疫细胞呈负相关，而PLCG、ITPR1和CAMK2A与淋巴细胞亚群相关。
我们在心脏和骨骼肌IRI中识别出一个保守的分子特征，WNT5A/PLC通路成分作为经机制验证的治疗靶点。我们的跨组织生物信息学方法，得到了药理学和组织学证据的支持，为在缺乏直接患者数据时进行IRI分析提供了一个新框架。联合靶向巨噬细胞极化和细胞活化的WNT5A/PLC轴可能具有协同治疗潜力。
