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目的 本实验基于Wnt/β-连环蛋白（Wnt/β-catenin）通路探讨胰岛素样生长因子结合蛋白-4（IGFBP-4）通过抑制滑膜巨噬细胞M1型极化改善骨关节炎（OA）滑膜炎症。方法 C57bl/6雄性小鼠膝关节腔内注射碘乙酸钠构建骨关节炎模型，关节腔内注射IGFBP-4过表达/敲减腺病毒载体进行干预。微型计算机断层扫描技术（Micro-CT）观察膝关节软骨下骨变化；HE染色观察膝关节滑膜病理形态；ELISA检测小鼠血清肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）水平；免疫荧光染色观察滑膜巨噬细胞M1型极化；实时荧光定量PCR和Western blot法检测滑膜IGFBP-4、β-catenin、环氧化酶2（COX2）、诱导型一氧化氮合酶（iNOS）和CD86表达。脂多糖（LPS）诱导RAW264.7细胞炎症模型，利用IGFBP-4蛋白和Wnt/β-catenin通路激动剂SKL2001进行干预。CCK-8法检测细胞活性；流式细胞术检测M1型极化；ELISA检测细胞培养上清TNF-α、IL-6水平；Western blot法检测细胞Wnt/β-catenin通路相关蛋白表达。结果 OA组小鼠软骨下骨表面缺损，IGFBP-4过表达组骨表面缺损减轻，IGFBP-4敲减组缺损加重。OA组滑膜组织增生增厚明显，IGFBP-4过表达组滑膜组织增生增厚较少，IGFBP-4敲减组滑膜组织增生增厚程度严重。OA组小鼠滑膜组织F4/80和iNOS共定位表达高于Sham组；与OA组相比，IGFBP-4过表达组F4/80和iNOS共定位表达降低，IGFBP-4敲减组F4/80和iNOS共定位表达升高。OA组小鼠血清TNF-α、IL-1β水平以及滑膜中β-catenin、COX2、iNOS和CD86表达高于Sham组；与OA组相比，IGFBP-4过表达组TNF-α、IL-1β水平以及β-catenin、COX2、iNOS和CD86表达明显降低，IGFBP-4敲减组上述因子表达明显升高。与Control组相比，LPS组细胞β-catenin、COX-2表达明显升高，M1型极化增多，TNF-α和IL-6水平升高；LPS联合IGFBP-4组细胞β-catenin、COX2表达，M1型极化以及TNF-α和IL-6水平低于LPS组、LPS和IGFBP-4联合SKL2001组；与LPS和IGFBP-4联合SKL2001组相比，LPS联合SKL2001组细胞β-catenin、COX2表达，M1型极化及TNF-α和IL-6水平升高。结论 IGFBP-4能显著抑制巨噬细胞M1型极化减轻滑膜炎症，其机制可能与抑制Wnt/β-catenin信号通路有关。
Abstract：Objective To investigate the mechanism by which insulin-like growth factor-binding protein-4 (IGFBP-4) ameliorates synovial inflammation in osteoarthritis (OA) by inhibiting M1-type polarization of synovial macrophages via the Wnt/β-catenin signaling pathway. Methods The OA model was established in male C57bl/6 mice by intra-articular injection of sodium iodoacetate. Adenoviral vectors carrying IGFBP-4 overexpression or knockdown sequences were injected into the knee joint cavity for in vivo intervention. Micro-computed tomography (Micro-CT) was used to observe the subchondral bone changes in the knee joint. Hematoxylin-eosin (HE) staining was used to observe the pathological morphology of knee synovial tissues. ELISA was applied to detect serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in mice. Immunofluorescence staining was used to evaluate M1 polarization level of the synovium macrophages. Quantitative real-time PCR and Western blot were used to measure the expression of IGFBP-4， β-catenin， cyclooxygenase-2 (COX-2)， inducible nitric oxide synthase (iNOS)， and CD86 in synovial tissue. In vitro， a RAW264.7 macrophage inflammatory model was induced by lipopolysaccharide (LPS)， and cells were treated with recombinant IGFBP-4 protein and the specific Wnt/β-catenin pathway agonist SKL2001. Cell viability was measured by CCK?8 assay. Flow cytometry was used to quantify M1 macrophage polarization. ELISA was used to measure levels of TNF-α and IL-6 in cell culture supernatants， and Western blot was performed to analyze the expression of key proteins involved in the Wnt/β-catenin signaling pathway. Results Micro-CT showed that the subchondral bone surface exhibited defects in OA mice， which were alleviated by IGFBP-4 overexpression and aggravated by IGFBP-4 knockdown. Pathological staining revealed obvious synovial hyperplasia and thickening in OA mice， which was attenuated by IGFBP-4 overexpression and exacerbated by IGFBP-4 knockdown. Immunofluorescence results demonstrated that the co-localization of F4/80 and iNOS in synovial tissues was significantly higher in the OA group than in the sham group， and this upregulation was suppressed by IGFBP-4 overexpression and enhanced by IGFBP-4 knockdown. Consistently， serum levels of TNF-α and IL-1β， as well as the protein expression of β-catenin， COX2， iNOS and CD86 in synovial tissues， were remarkably elevated in the OA group versus the sham group. IGFBP-4 overexpression significantly downregulated the levels of these inflammatory factors and pathway-related proteins， whereas IGFBP-4 knockdown exerted the opposite effects. In vitro experiments verified that LPS stimulation significantly upregulated β-catenin and COX2 expression， promoted M1 macrophage polarization， and increased the secretion of TNF-α and IL-6. IGFBP-4 treatment effectively inhibited LPS-induced inflammatory responses and Wnt/β-catenin pathway activation. Notably， the protective effects of IGFBP-4 were partially abrogated by SKL2001-mediated Wnt/β-catenin pathway activation. Conclusion IGFBP-4 can effectively inhibit M1 polarization of synovial macrophages and alleviates synovial inflammation， and its protective mechanism is closely associated with the negative regulation of the Wnt/β-catenin signaling pathway.
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基本信息:
DOI：10.13423/j.cnki.cjcmi.010177
中图分类号:R686.7
引用信息:
[1]乔玉雪,赵雪梅,王欣笛,等.IGFBP-4通过Wnt/β-catenin通路抑制巨噬细胞M1型极化改善滑膜炎症[J].细胞与分子免疫学杂志().DOI:10.13423/j.cnki.cjcmi.010177.
基金信息:
蒙药研发国家地方联合工程研究中心开放基金项目(MDK2021023)
2026-07-10
2026-07-10
2026-07-10