| 周渝洲,肖权洲,陈特,等.TNFAIP3通过NF-κB通路介导巨噬细胞极化影响强直性脊柱炎.骨科,2025,16(2): 144-151. |
| TNFAIP3通过NF-κB通路介导巨噬细胞极化影响强直性脊柱炎 |
| TNFAIP3 influences ankylosing spondylitis by mediating macrophage polarization through the NF-κB pathway |
| 投稿时间：2024-07-23 |
| DOI：10.3969/j.issn.1674-8573.2025.02.007 |
| 中文关键词: 肿瘤坏死因子-α诱导蛋白3 强直性脊柱炎 巨噬细胞 过表达 核因子-κB通路 |
| 英文关键词: TNFAIP3 Ankylosing spondylitis Macrophages Overexpression NF-κB pathway |
| 基金项目:湖南省卫生健康委科研计划项目（202104071114） |
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| 中文摘要: |
| 目的 探究过表达肿瘤坏死因子-α诱导蛋白3（TNFAIP3）是否会抑制核因子-κB（NF-κB）通路影响巨噬细胞极化状态，从而调控强直性脊柱炎（AS）的炎症反应，并初步阐明相关的分子机制。方法 采用1,3-β-D葡聚糖注射BALB/c小鼠构建AS模型，为模型组。AS小鼠注射空载体为AS+oe-NC组，注射TNFAIP3质粒为AS+oe-TNFAIP3组。通过实时荧光定量聚合酶链反应（qPCR）和蛋白印迹法（Western blot）分别检测三组小鼠脊柱组织中TNFAIP3的mRNA和蛋白表达水平，验证过表达效率；苏木精-伊红（HE）染色评估脊柱组织的病理变化。免疫组化检测脊柱周围组织中炎症因子［白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）］的表达水平。通过荧光染色标记CD11b-FITC/CD86（M1型巨噬细胞）和CD11b-FITC/CD206（M2型巨噬细胞），评估巨噬细胞极化状态。Western blot检测NF-κB通路关键蛋白IκB激酶α（I-kappa-B kinase alpha/β，IKKα/β）、核因子κB亚基p65（Nuclear factor kappa-B subunit p65，p65）、磷酸化核因子κB亚基p65（Phosphorylated nuclear factor kappa-B subunit p65，p-p65）的表达水平。体外实验中，采用LPS和IL-4分别诱导RAW264.7巨噬细胞向M1型和M2型极化，分析TNFAIP3过表达对巨噬细胞极化的影响。结果 过表达TNFAIP3显著提高了TNFAIP3的mRNA和蛋白表达水平（P＜0.01），缓解了脊柱组织的病变。与模型组和AS+oe-NC组比较，AS+oe-TN-AIP3组脊柱周围组织中IL-1β、IL-6和TNF-α的表达量显著降低（P＜0.001）。巨噬细胞极化分析显示，AS+oe-TNFAIP3组M1型巨噬细胞（CD11b+CD86+）比例显著减少（P＜0.001），而M2型巨噬细胞（CD11b+CD206+）比例显著增加（P＜0.001）。Western blot结果显示，过表达TNFAIP3显著抑制NF-κB通路关键蛋白（IKKα/β、p65、p-p65）的激活（P＜0.01）。体外实验进一步证实，过表达TNFAIP3促进IL-4诱导的M2型极化（P＜0.001），同时抑制LPS诱导的M1型极化（P＜0.001）。结论 过表达TNFAIP3通过抑制NF-κB通路调控巨噬细胞极化，促进M2型极化并抑制M1型极化，从而缓解AS的炎症反应。本研究初步揭示了TNFAIP3在AS中的抗炎作用及其分子机制。 |
| 英文摘要: |
| Objective To investigate whether overexpression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3) suppresses the NF-κB pathway to regulate macrophage polarization, thereby modulating inflammatory responses in ankylosing spondylitis (AS), and to preliminarily elucidate the underlying molecular mechanisms. Methods An AS mouse model was constructed by injecting BALB/c mice with 1,3-β-D-glucan, forming the model group. AS mice were injected with empty vector as AS+oe NC group, and TNFAIP3 plasmid as AS+oe TNFAIP3 group. The overexpression efficiency of TNFAIP3 was validated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting to detect mRNA and protein expression levels in the spinal tissues of three groups of mice, respectively. Pathological changes in spinal tissues were assessed using hematoxylin-eosin (HE) staining. Immunohistochemistry was performed to evaluate the expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in peri-spinal tissues. Macrophage polarization status was analyzed via fluorescence staining for CD11b-FITC/CD86 (M1-type macrophages) and CD11b-FITC/CD206 (M2-type macrophages). Western blotting was used to detect the expression of key NF-κB pathway proteins, including I-kappa-B kinase alpha/beta (IKKα/β), nuclear factor kappa-B subunit p65 (p65), and phosphorylated p65 (p-p65). In vitro, RAW264.7 macrophages were polarized into M1- and M2-type using LPS and IL-4, respectively, to analyze the effects of TNFAIP3 overexpression on macrophage polarization. Results Overexpression of TNFAIP3 significantly increased TNFAIP3 mRNA and protein levels (P＜0.01), and alleviated the tissue lesions of the spinal tissues. Compared to the model group and AS+oe-NC groups, the AS+oe-TNFAIP3 group exhibited markedly reduced expression of IL-1β, IL-6, and TNF-α in peri-spinal tissues (P＜0.001). Macrophage polarization analysis revealed that the proportion of M1-type macrophages (CD11b+CD86+) was significantly decreased (P＜0.001), while that of M2-type macrophages (CD11b+CD206+) increased (P＜0.001) in the AS+oe-TNFAIP3 group. Western blotting demonstrated that TNFAIP3 overexpression significantly inhibited the activation of NF-κB pathway proteins (IKKα/β, p65, and p-p65) (P＜0.01). In vitro experiments further confirmed that TNFAIP3 overexpression promoted IL-4-induced M2 polarization (P＜0.001) and suppressed LPS-induced M1 polarization (P＜0.001). Conclusion Overexpression of TNFAIP3 alleviates AS inflammation by suppressing the NF-κB pathway to regulate macrophage polarization, promoting M2 polarization and inhibiting M1 polarization. Our study preliminarily reveals the anti-inflammatory role of TNFAIP3 in AS and its molecular mechanisms. |
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