SPI1 Regulates RARA to Alleviate Articular Cartilage Degeneration in Osteoarthritis Mice
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摘要:
目的 探讨Spi-1原癌基因（Spi-1 proto-oncogene，SPI1）通过调控视黄酸受体α（retinoic acid receptor alpha，RARA）表达对骨关节炎（osteoarthritis，OA）小鼠软骨的作用并阐明具体调控机制。 方法 IL-1β诱导人软骨细胞构建OA细胞模型，并在细胞中转染pcDNA-NC、pcDNA-SPI1和pcDNA-RARA，通过Western blot检测SPI1、RARA、MMP3、ADAMTS5、MMP13和COL2A1蛋白，ELISA检测TNF-α和IL-6水平，流式细胞术检测细胞凋亡率。内侧半月板失稳术（destabilized medial meniscus，DMM）法诱导OA小鼠模型，并注射过表达SPI1的腺相关病毒，通过苏木精-伊红染色、番红固绿染色、免疫组织化学染色、Western blot和ELISA实验评估SPI1表达对OA小鼠软骨的作用。通过双荧光素酶报告基因实验、染色质免疫沉淀和Western blot实验验证SPI1与RARA的作用关系。 结果 SPI1和RARA在IL-1β诱导的软骨细胞和OA小鼠中表达降低（P < 0.001），过表达SPI1或过表达RARA可抑制IL-1β诱导的MMP3、ADAMTS5、MMP13表达（P < 0.001）、细胞凋亡（P < 0.001）和细胞因子TNF-α和IL-6水平（P < 0.001），促进COL2A1表达（P < 0.001）。同时过表达SPI1和RARA可进一步增加过表达RARA对IL-1β诱导软骨细胞的作用（P < 0.001）。机制研究表明，SPI1可作为转录因子与RARA启动子结合并促进RARA转录。动物研究表明，过表达SPI1可缓解DMM诱导的小鼠关节软骨损伤，抑制炎症反应（P < 0.001）。 结论 过表达SPI1通过促进RARA表达，减轻软骨细胞损伤从而缓解小鼠OA。 Abstract:Objective To investigate the effect of the Spi-1 proto-oncogene （SPI1） on cartilage in osteoarthritis （OA） mice by regulating the expression of retinoic acid receptor alpha （RARA）, and to elucidate the underlying regulatory mechanism. Methods An OA cell model was established by inducing human chondrocytes with IL-1β, and the cells were transfected with pcDNA-NC, pcDNA-SPI1, and pcDNA-RARA. Western blot was used to detect the protein levels of SPI1, RARA, MMP3, ADAMTS5, MMP13, and COL2A1; ELISA was used to measure TNF-α and IL-6 levels; and flow cytometry was used to determine the apoptosis rate. An OA mouse model was induced by destabilization of the medial meniscus （DMM）, followed by injection of adeno-associated virus overexpressing SPI1. The effect of SPI1 expression on the cartilage of OA mice was evaluated by hematoxylin-eosin staining, safranin O-fast green staining, immunohistochemical staining, Western blot, and ELISA. The regulatory relationship between SPI1 and RARA was verified by dual-luciferase reporter assay, chromatin immunoprecipitation, and Western blot. Results The expression of SPI1 and RARA was decreased in IL-1β-induced chondrocytes and in OA mice （P < 0.001）. Overexpression of SPI1 or RARA inhibited IL-1β-induced expression of MMP3, ADAMTS5, and MMP13 （P < 0.001）, apoptosis （P < 0.001）, and levels of the cytokines TNF-α and IL-6 （P < 0.001）, while promoting COL2A1 expression （P < 0.001）. Co-overexpression of SPI1 and RARA further enhanced the effects of RARA overexpression on IL-1β-induced chondrocytes （P < 0.001）. Mechanistic studies revealed that SPI1, acting as a transcription factor, bound to the RARA promoter and promoted RARA transcription. Animal studies demonstrated that overexpression of SPI1 alleviated DMM-induced articular cartilage damage and suppressed the inflammatory responsein mice （P < 0.001） . Conclusion Overexpression of SPI1 attenuates chondrocyte damage by promoting RARA expression, thereby ameliorating OA in mice. -
Key words:
- Osteoarthritis /
- Cartilage degeneration /
- SPI1 /
- RARA /
- Cytokines
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图 1 SPI1过表达缓解IL-1β诱导的软骨细胞损伤（$\bar x \pm s $，n = 3）
A：RT-qPCR检测不同浓度IL-1β处理对细胞中SPI1 mRNA表达的影响；B～C：Western blot检测不同浓度IL-1β处理对细胞中SPI1蛋白表达的影响；D～E：转染pcDNA-SPI1对SPI1蛋白表达的影响；F～J：过表达SPI1对细胞中的MMP3、ADAMTS5、MMP13和COL2A1蛋白表达的作用；K～L：ELISA检测细胞上清液中TNF-α和IL-6浓度；M～N：Annexin V-FITC/PI试剂盒检测软骨细胞凋亡率；与NC组比较，**P < 0.01，***P < 0.001；与IL-1β+pcDNA-NC组比较，ΔΔΔP < 0.001。
Figure 1. Overexpression of SPI1 alleviates IL-1β-induced chondrocyte injury （$\bar x \pm s $，n = 3）
图 2 SPI1调控RARA转录（$\bar x \pm s $，n = 3）
A：JASPAR数据库预测与RARA结合的SPI1碱基序列；B：Western blot检测软骨细胞中RARA蛋白表达；与NC组比较，***P < 0.001；与IL-1β+pcDNA-NC组比较，ΔΔΔP < 0.001；C：双荧光素酶报告基因实验检测荧光素酶活性；与pcDNA-NC组比较，***P < 0.001；D：ChIP-qPCR实验检测SPI1与RARA启动子结合情况；与IgG组相比，***P < 0.001。
Figure 2. SPI1 regulates RARA transcription （$\bar x \pm s $，n = 3）
图 3 SPI1通过RARA调节IL-1β诱导的软骨细胞损伤（$\bar x \pm s $，n = 3）
A～C：Western blot检测转染pcDNA-RARA和pcDNA-SPI1对RARA和SPI1蛋白表达的影响；与NC组比较，***P < 0.001；与IL-1β+pcDNA-NC组比较，ΔΔΔP < 0.001；与IL-1β+pcDNA-RARA组比较，▲▲▲P < 0.001；D～H：Western blot检测过表达RARA和SPI1对MMP3、ADAMTS5、MMP13和COL2A1蛋白表达的作用；I～J：ELISA试剂盒检测过表达RARA和SPI1对促炎细胞因子TNF-α和IL-6的作用；K～L：Annexin V-FITC/PI试剂盒检测过表达RARA和SPI1对细胞凋亡的影响；与IL-1β+pcDNA-NC组比较，**P < 0.01，***P < 0.001；与IL-1β+pcDNA-RARA组比较，ΔΔP < 0.01，ΔΔΔP < 0.001。
Figure 3. SPI1 modulates IL-1β-induced chondrocyte injury via RARA （$\bar x \pm s $，n = 3）
图 4 SPI1过表达缓解OA小鼠软骨退变（$\bar x \pm s $，n = 6）
A～B：小鼠关节软骨HE染色及OARSI评分；C：关节软骨番红固绿染色；D～G：IHC染色检测关节软骨中MMP3和COL2A1阳性细胞比例及统计结果；与sham组比较，***P < 0.001；与DMM+AAV-NC组比较，ΔΔΔP < 0.001。
Figure 4. Overexpression of SPI1 alleviates cartilage degeneration in OA mice （$\bar x \pm s $，n = 6）
图 5 过表达SPI1通过上调RARA缓解OA小鼠软骨退变（$\bar x \pm s $，n = 6）
A～E：Western blot检测小鼠关节软骨中的MMP3、ADAMTS5、MMP13和COL2A1蛋白表达及定量分析；F～H：Western blot检测关节软骨中的SPI1和RARA蛋白表达及定量分析；I～J：ELISA检测关节软骨中的TNF-α和IL-6水平；与sham组比较，***P < 0.001；与DMM+AAV-NC组比较，ΔΔΔP < 0.001。
Figure 5. Overexpression of SPI1 alleviates cartilage degeneration in OA mice by upregulating RARA （$\bar x \pm s $，n = 6）
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[1] Yue L, Berman J. What is osteoarthritis?[J]. JAMA, 2022, 327(13): 1300. [2] DeMoya C D, Joenathan A, Lawson T B, et al. Advances in viscosupplementation and tribosupplementation for early-stage osteoarthritis therapy[J]. Nat Rev Rheumatol, 2024, 20(7): 432-451. doi: 10.1038/s41584-024-01125-5 [3] Li G Z, Ji R J, Xu C P, et al. On patient quality of life: Impacts of knee osteoarthritis on pain, anxiety, depression, fatigue and sleep disorders[J]. Nurs Open, 2025, 12(7): e70264. doi: 10.1002/nop2.70264 [4] Geng N, Fan M, Kuang B, et al. 10-hydroxy-2-decenoic acid prevents osteoarthritis by targeting aspartyl β hydroxylase and inhibiting chondrocyte senescence in male mice preclinically[J]. Nat Commun, 2024, 15(1): 7712. doi: 10.1038/s41467-024-51746-3 [5] Fang Y, Chen W, Li Z, et al. The role of a key transcription factor PU. 1 in autoimmune diseases[J]. Front Immunol, 2022, 13: 1001201. doi: 10.3389/fimmu.2022.1001201 [6] Li Q, Zhang L, You W, et al. PRDM1/BLIMP1 induces cancer immune evasion by modulating the USP22-SPI1-PD-L1 axis in hepatocellular carcinoma cells[J]. Nat Commun, 2022, 13(1): 7677. doi: 10.1038/s41467-022-35469-x [7] Wang C, Wang Y, Gu Y, et al. SPI1 facilitates microfracture-mediated cartilage regeneration in the elderly by enhancing bone marrow stromal cells ctemness[J]. J Tissue Eng, 2025, 16: 20417314241311073. [8] Wang C, Zeng Z, Wang T, et al. Unraveling the spatial and signaling dynamics and splicing kinetics of immune infiltration in osteoarthritis synovium[J]. Front Immunol, 2025, 16: 1521038. doi: 10.3389/fimmu.2025.1521038 [9] Stegen S, Rinaldi G, Loopmans S, et al. Glutamine metabolism controls chondrocyte identity and function[J]. Dev Cell, 2020, 53(5): 530-544. e8. [10] Kodandapani R, Pio F, Ni C Z, et al. A new pattern for helix-turn-helix recognition revealed by the PU. 1 ETS-domain-DNA complex[J]. Nature, 1996, 380(6573): 456-460. doi: 10.1038/380456a0 [11] Xu Y, Liu X, Chen H, et al. Spi1 aggravates neuropathic pain by modulating Clec7a-mediated neuroinflammation and microglial phagocytosis[J]. J Headache Pain, 2025, 26(1): 158. doi: 10.1186/s10194-025-02089-x [12] Fang D, Chen J, Yang S, et al. Silencing of SPI1/OLAH axis inhibits pancreatic acinar cell oxidative stress, inflammation and apoptosis to alleviate hyperlipidemic acute pancreatitis[J]. Pancreas, 2025, 54(10): e887-e895. doi: 10.1097/MPA.0000000000002533 [13] Du J, An Z. SPI1 affects Aβ1–42-induced microglia-mediated neuroinflammation by transcriptionally regulating FOSL2 expression[J]. J Neuroimmunol, 2025, 406: 578650. doi: 10.1016/j.jneuroim.2025.578650 [14] Wang C, Zeng Z, Wang T, et al. Unraveling the spatial and signaling dynamics and splicing kinetics of immune infiltration in osteoarthritis synovium[J]. Front Immunol, 2025, 16: 1521038. doi: 10.3389/fimmu.2025.1521038 [15] Zu X, Chen S, Li Z, et al. SPI1 activates mitochondrial unfolded response signaling to inhibit chondrocyte senescence and relieves osteoarthritis[J]. Bone Res, 2025, 13(1): 47. doi: 10.1038/s41413-025-00421-4 [16] Yan S, Lu T, Yang H, et al. Decreased histone H3K9 dimethylation in synergy with DNA demethylation of Spi-1 binding site contributes to ADAMTS-5 expression in articular cartilage of osteoarthritis mice[J]. J Cell Physiol, 2024, 239(12): e31444. doi: 10.1002/jcp.31444 [17] Gudas L J. Synthetic retinoids beyond cancer therapy[J]. Annu Rev Pharmacol Toxicol, 2022, 62: 155-175. doi: 10.1146/annurev-pharmtox-052120-104428 [18] Lou J, Zhang Y, Tao J, et al. Infection-related pathway activation and vulnerability to arsenic trioxide in acute promyelocytic leukemia with TTMV: : RARA[J]. Leukemia, 2025, 39(2): 490-494. doi: 10.1038/s41375-024-02481-7 [19] Chang H Y, Koh V C Y, Md Nasir N D, et al. MED12, TERT and RARA in fibroepithelial tumours of the breast[J]. J Clin Pathol, 2020, 73(1): 51-56. doi: 10.1136/jclinpath-2019-206208 [20] Testa U, Pelosi E. Function of PML-RARA in Acute Promyelocytic Leukemia[J]. Adv Exp Med Biol, 2024: 321-339. [21] Cassim Bawa F N, Gopoju R, Xu Y, et al. Retinoic acid receptor alpha (RARα) in macrophages protects from diet-induced atherosclerosis in mice[J]. Cells, 2022, 11(20): 3186. doi: 10.3390/cells11203186 [22] DiKun K M, Tang X H, Fu L, et al. Retinoic acid receptor α activity in proximal tubules prevents kidney injury and fibrosis[J]. Proc Natl Acad Sci U S A, 2024, 121(7): e2311803121. doi: 10.1073/pnas.2311803121 [23] Yi P, Xu X, Yao J, et al. Analysis of mRNA expression and DNA methylation datasets according to the genomic distribution of CpG sites in osteoarthritis[J]. Front Genet, 2021, 12: 618803. doi: 10.3389/fgene.2021.618803 -
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