目的：骨血管生成与成骨密切相关，提示促血管生成分子与成骨细胞之间存在关联。血管生成前分子血管内皮生长因子（VEGF）及其受体Flt-1、Flk-1和成纤维细胞生长因子（FGF-2）在类风湿性关节炎（RA）发病机制中起着重要作用。白细胞介素（IL-35）在RA中被证明是一种抗炎细胞因子。然而，所涉及的机制还没有完全理解。本研究旨在探讨白介素-35是否对RA成骨细胞的血管生成及其相关的信号通路有影响。

方法：用成骨细胞株MC3T3E1细胞体外研究白介素-35对成骨细胞增殖、凋亡及促血管生成分子mRNA和蛋白的影响。采用细胞增殖试剂盒-8（CCK-8）法和流式细胞术检测白介素-35对细胞增殖和凋亡的影响。分别采用实时聚合酶链反应（PCR）和酶联免疫吸附试验（ELISA）检测促血管生成分子的表达及白介素-35、骨形成、血管生成之间的关系及相关的信号通路。

结果：白细胞介素-35在体外呈剂量依赖性促进成骨细胞增殖，并抑制其凋亡。白介素-35增加了成骨细胞对基底和肿瘤坏死因子α诱导的促血管生成分子的表达。白花丹素阻断Th17/IL-17信号通路可抑制IL-35在成骨细胞中的促血管生成作用。

结论：这些结果表明，IL-35通过促进成骨细胞增殖、抑制凋亡和通过Th17/IL-17相关信号通路上调促血管生成分子来促进骨形成和血管生成。我们的发现扩展了目前对RA骨形成和血管生成调控机制的理解。

附原文：

OBJECTIVES: Angiogenesis in bone and osteogenesis appear to be closely linked, suggesting the existence of molecular crosstalk between pro-angiogenic molecules and osteoblasts. The pro-angiogenic molecules vascular endothelial growth factor (VEGF) with its receptors Flt-1, Flk-1 and fibroblast growth factor (FGF)-2 play a crucial role in born formation, an early and critical event in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-35 is demonstrated to be an anti-inflammatory cytokine in RA. However, the mechanisms involved are not fully understood. This study aims to investigate whether IL-35 has an impact on angiogenesis in osteoblasts and its related signalling pathway in RA.

METHODS: The effects of IL-35 on osteoblasts proliferation, apoptosis and pro-angiogenic molecules mRNA and protein were examined using osteoblast-like MC3T3E1 cells in vitro. The effects of IL-35 on proliferation and apoptosis were examined using cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Pro-angiogenic molecules expression were assessed by real time PCR and ELISA, respectively. The signalling pathway between IL-35, bone formation, angiogenesis and signalling pathway was also investigated.

RESULTS: IL-35 promoted osteoblasts proliferation and inhibited apoptosis in a dose-dependent manner in vitro. IL-35 increased basal and TNF-alpha induced pro-angiogenic molecules expression by osteoblasts. Blocking the Th17/IL-17 signalling pathway with plumbagin inhibited the pro-angiogenic effects of IL-35 in osteoblasts.

CONCLUSIONS: These results suggested that IL-35 promotes bone formation and angiogenesis by fostering osteoblasts proliferation, inhibiting apoptosis and upregulating pro-angiogenic molecules through Th17/IL-17 related-signalling pathway. Our findings extend the current understanding of mechanisms modulating bone formation and angiogenesis in RA.

引自：Liu S, Li Y, Xia L, Shen H, Lu J. IL-35 prevent bone loss through promotion of bone formation and angiogenesis in rheumatoid arthritis. Clinical and experimental rheumatology 2019