M-CSF Mediates Macrophage Polarization to Regulate Chondrocyte Function in Osteoarthritis via the CSF1R/NF-κB Signaling Pathway
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摘要:
目的 探讨巨噬细胞集落刺激因子（macrophage colony-stimulating factor，M-CSF）通过调控集落刺激因子1受体（colony stimulating factor 1 receptor，CSF1R）介导骨关节炎（osteoarthritis，OA）中巨噬细胞极化对软骨细胞功能的影响，并阐明其具体作用机制。 方法 采用脂多糖（lipopolysaccharide，LPS）和干扰素-γ（interferon-gamma，IFN-γ）诱导小鼠巨噬细胞RAW 264.7和原代分离的小鼠骨髓来源巨噬细胞（bone marrow-derived macrophages，BMDM）建立OA细胞模型，在RAW 264.7细胞中分别转染sh-NC和sh-M-CSF，并使用CSF1R抑制剂PLX3397进行处理。通过Western blot、免疫荧光和酶联免疫吸附实验（enzyme linked immunosorbent assay，ELISA）检测M-CSF和CSF1R对RAW 264.7细胞极化的影响。利用Transwell共培养系统将小鼠软骨细胞与RAW 264.7或BMDM细胞共培养，利用细胞计数试剂盒、Annexin V-FITC/PI凋亡检测试剂盒、ELISA、Western blot和免疫荧光分析RAW 264.7细胞极化对软骨细胞功能的影响。 结果 经LPS和IFN-γ处理后，RAW 264.7和BMDM细胞发生M1极化并伴随促炎细胞因子分泌增加（P < 0.01），M-CSF和CSF1R表达上调（P < 0.001）。与诱导后的RAW 264.7或BMDM细胞共培养的软骨细胞活性降低（P < 0.001）、细胞凋亡率升高（P < 0.001）、促炎细胞因子水平上升（P < 0.001）。敲低M-CSF或使用PLX3397处理抑制RAW 264.7细胞的M1极化（P < 0.05）以及NF-κB信号通路激活（P < 0.05），并减轻其对软骨细胞的损伤作用（P < 0.001）。敲低M-CSF同时经PLX3397处理组进一步抑制M1极化（P < 0.01），促进软骨细胞的功能恢复（P < 0.001）。 结论 敲低M-CSF通过抑制CSF1R表达和NF-κB通路激活，减弱LPS和IFN-γ诱导的RAW 264.7细胞M1极化，从而缓解软骨细胞损伤。 Abstract:Objective To investigate the effects of macrophage colony-stimulating factor （M-CSF） on chondrocyte function through macrophage polarization mediated by colony-stimulating factor 1 receptor （CSF1R） in osteoarthritis （OA）, and to elucidate the underlying mechanism. Methods Lipopolysaccharide （LPS） and interferon-gamma （IFN-γ） were used to induce mouse RAW 264.7 macrophages and primary mouse bone marrow-derived macrophages （BMDMs） to establish an OA cell model. RAW 264.7 cells were transfected with sh-NC or sh-M-CSF and treated with the CSF1R inhibitor PLX3397. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assay （ELISA） were used to assess the effects of M-CSF and CSF1R on RAW 264.7 macrophage polarization. Mouse chondrocytes were co-cultured with RAW 264.7 cells or BMDMs using a Transwell co-culture system. Cell Counting Kit, Annexin V-FITC/PI apoptosis assays, ELISA, Western blotting, and immunofluorescence were used to analyze the effects of RAW 264.7 cell polarization on chondrocyte function. Results Following LPS and IFN-γ treatment , RAW 264.7 and BMDM cells underwent M1 polarization, accompanied by increased secretion of pro-inflammatory cytokines （P < 0.01） and upregulated M-CSF and CSF1R expression （P < 0.001）. Chondrocyte viability decreased （P < 0.001）, apoptosis increased （P < 0.001）, and pro-inflammatory cytokine levels rose （P < 0.001） after co-culture with induced RAW 264.7 cells or BMDMs. M-CSF knockdown or PLX3397 treatment inhibited M1 polarization （P < 0.05） and NF-κB pathway activation （P < 0.05） in RAW 264.7 cells and alleviated their damaging effects on chondrocytes （P < 0.001）. Combined M-CSF knockdown and PLX3397 treatment further inhibited M1 polarization （P < 0.01） and promoted functional recovery of chondrocytes （P < 0.001）. Conclusion M-CSF knockdown attenuates LPS- and IFN-γ-induced M1 polarization of RAW 264.7 cells by inhibiting CSF1R expression and NF-κB pathway activation, thereby alleviating chondrocyte injury. -
Key words:
- Osteoarthritis /
- Macrophage /
- M-CSF /
- Chondrocyte /
- CSF1R
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图 1 LPS和IFN-γ诱导RAW 264.7细胞M1极化并上调M-CSF（$\bar x \pm s $，n = 3）
A：细胞培养上清液中TNF-α水平；B：细胞培养上清液中IL-6水平；C：细胞培养上清液中IL-10水平；D：细胞培养上清液中TGF-β水平；E～I：Western blot检测RAW 264.7细胞中INOS、CD86、ARG1和CD206蛋白条带及相对表达分析；J～L：ARG1和INOS的IF染色代表性图像及平均荧光强度统计分析；M～O：Western blot检测M-CSF和CSF1R蛋白条带及相对表达分析；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 1. LPS and IFN-γ induce M1 polarization and upregulate M-CSF in RAW 264.7 cells （$\bar x \pm s $，n = 3）
图 2 LPS和IFN-γ诱导BMDM的M1极化并上调M-CSF（$\bar x \pm s $，n = 3）
A：细胞培养上清液中TNF-α水平；B：细胞培养上清液中IL-6水平；C：细胞培养上清液中IL-10水平；D：细胞培养上清液中TGF-β水平；E～I：Western blot检测BMDM细胞中INOS、CD86、ARG1和CD206蛋白条带及相对表达分析；J～L：BMDM中ARG1和INOS的IF染色代表性图像及平均荧光强度统计分析；M～O：Western blot检测M-CSF和CSF1R蛋白条带及相对表达分析；**P < 0.01；***P < 0.001。
Figure 2. LPS and IFN-γ induce M1 polarization and upregulate M-CSF in BMDM （$\bar x \pm s $，n = 3）
图 3 RAW 264.7细胞M1极化抑制软骨细胞活性促进凋亡（$\bar x \pm s $，n = 3）
A：CCK-8检测软骨细胞活力；B～C：Annexin V-FITC/PI细胞凋亡试剂盒检测软骨细胞凋亡率及分析结果；D～G：Western blot检测凋亡相关蛋白Bcl-2、Bax和cleaved caspase 3蛋白条带及相对表达分析；***P < 0.001。
Figure 3. M1-polarized RAW 264.7 cells inhibit chondrocyte viability and promote apoptosis （$\bar x \pm s $，n = 3）
图 4 BMDM细胞M1极化抑制软骨细胞活性促进凋亡（$\bar x \pm s $，n = 3）
A：CCK-8检测与BMDM细胞共培养的软骨细胞活力；B～C：Annexin V-FITC/PI细胞凋亡试剂盒检测软骨细胞凋亡率及统计分析；D～G：Western blot检测凋亡相关蛋白Bcl-2、Bax和cleaved caspase 3蛋白条带及相对表达分析；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 4. M1 polarization of BMDMs inhibits chondrocyte viability and promotes apoptosis （$\bar x \pm s $，n = 3）
图 5 RAW 264.7细胞M1极化促进软骨细胞变性和炎症反应（$\bar x \pm s $，n = 3）
A～E：Western blot检测软骨细胞中MMP3、ADAMTS5、MMP13和COL2A1蛋白条带及相对表达分析；F～G：COL2A1的IF染色代表性图像及平均荧光分析；H～I：ELISA试剂盒检测软骨细胞培养上清液中TNF-α和IL-6水平；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 5. M1 polarization of RAW 264.7 cells promotes chondrocyte degeneration and inflammatory responses （$\bar x \pm s $，n = 3）
图 6 BMDM细胞M1极化促进软骨细胞变性和炎症反应（$\bar x \pm s $，n = 3）
A～E：Western blot检测与BMDM细胞共培养的软骨细胞中MMP3、ADAMTS5、MMP13和COL2A1蛋白条带和相对表达分析；F～G：COL2A1的IF染色代表性图像和平均荧光强度分析；H～I：ELISA试剂盒检测软骨细胞培养上清液中TNF-α和IL-6浓度；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 6. M1 polarization of BMDM cells promotes chondrocyte degeneration and inflammatory responses （$\bar x \pm s $，n = 3）
图 7 敲低M-CSF通过下调CSF1R抑制RAW 264.7细胞M1极化（$\bar x \pm s $，n = 3）
A～C：Western blot检测敲低M-CSF和PLX3397处理后M-CSF和CSF1R蛋白条带和相对表达分析；D～H：Western blot检测RAW 264.7细胞极化标志物INOS、CD86、ARG1和CD206蛋白条带和相对表达分析；I～K：ARG1和INOS免疫荧光染色代表性图像和平均荧光强度分析；L～O：ELISA检测炎性细胞因子TNF-α、IL-6、IL-10和TGF-β浓度；P～R：Western blot检测P65、p-P65、IKKβ、p-IKKβ蛋白条带和p-P65、p-IKKβ相对表达分析；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 7. Knockdown of M-CSF inhibits M1 polarization in RAW 264.7 cells by downregulating CSF1R （$\bar x \pm s $，n = 3）
图 8 敲低M-CSF通过下调CSF1R促进软骨细胞增殖并抑制凋亡（$\bar x \pm s $，n = 3）
A～B：RAW 264.7细胞中敲低M-CSF和PLX3397处理对软骨细胞凋亡的影响及凋亡率分析；C：CCK-8检测软骨细胞活力；D～G：与敲低M-CSF和PLX3397处理的RAW 264.7细胞共培养软骨细胞中Bcl-2、Bax和cleaved caspase3蛋白条带及相对表达分析；***P < 0.001。
Figure 8. Knockdown of M-CSF promotes chondrocyte proliferation and inhibits apoptosis by downregulating CSF1R （$\bar x \pm s $，n = 3）
图 9 敲低M-CSF通过下调CSF1R缓解软骨细胞变性（$\bar x \pm s $，n = 3）
A～E：Western blot检测与敲低M-CSF和PLX3397处理的RAW 264.7细胞共培养软骨细胞中MMP3、ADAMTS5、MMP13和COL2A1蛋白条带及相对表达分析；F～G：IF检测COL2A1代表性图像及平均荧光强度分析；H～I：ELISA试剂盒检测软骨细胞培养上清液中TNF-α和IL-6水平；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 9. M-CSF knockdown alleviates chondrocyte degeneration by downregulating CSF1R （$\bar x \pm s $，n = 3）
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