Effects of cobimetinib versus combination of dasatinib and quercetin on stress-induced chondrocyte senescence

doi:10.12307/2026.432

Abstract

Abstract: BACKGROUND: Osteoarthritis is closely related to aging and characterized by degeneration of articular cartilage, subchondral bone sclerosis, and low-grade inflammatory responses. Aging and injury are significant triggers for inflammatory factors that mediate joint pathological changes. Cobimetinib, a MEK1 inhibitor, has an unclear effect on the inflammatory senescence of chondrocytes.
OBJECTIVE: To compare the effects of cobimetinib and the classical senolytic drug combination of dasatinib and quercetin (D+Q) in suppressing inflammation-induced chondrocyte senescence, and to explore their potential therapeutic value in osteoarthritis.
METHODS: Primary chondrocytes were isolated and cultured. Cell viability was assessed by cell counting kit-8 assay. Cells were divided into control, model, D+Q, and cobimetinib groups. Except for the control group, inflammatory senescence chondrocyte models were induced by interleukin-1β in the other three groups. The cobimetinib and D+Q groups were treated with cobimetinib and D+Q, respectively. Senescent phenotypes were evaluated using SA-β-Gal staining. The activation status of the MEK-ERK1/2 pathway, expression levels of senescence markers (P16, P21, P53) and senescence-associated secretory phenotype factors (such as inducible nitric oxide synthase, cyclooxygenase-2, chemokine 3, interleukin-6) were analyzed by real-time quantitative PCR, western blot, and immunofluorescence. Additionally, changes in molecules related to extracellular matrix synthesis and degradation (COL2A1, matrix metalloproteinase-13, and matrix metalloproteinase-3) were assessed.
RESULTS AND CONCLUSION: Both cobimetinib and D+Q could effectively inhibit the activation of the MEK-ERK1/2 pathway, ameliorate the senescent phenotypes of chondrocytes, as evidenced by downregulation of P16, P21, and P53, attenuation of senescence-associated secretory phenotype factor levels, restoration of COL2A1 expression, and suppression of matrix metalloproteinase-13 and matrix metalloproteinase-3. Overall, cobimetinib effectively alleviates interleukin-1β-induced inflammatory senescence in chondrocytes by inhibiting the MEK-ERK1/2 pathway. Compared with D+Q, it exhibits comparable anti-senescent effects while demonstrating greater potential in promoting matrix synthesis and inhibiting degradation.

Key words: osteoarthritis, cellular senescence, chondrocytes, cobimetinib, dasatinib, quercetin, senescence-associated secretory phenotype, MEK-ERK1/2 signaling pathway

CLC Number:

Zhao Minjun, , Wu Xubo, , Yin Jianli, , Ge Yangshuo, Ding Jiaying, Huang Chunmeng, Meng Tingting, Wang Xuezong, Liu Zhenfeng, Ding Daofang. Effects of cobimetinib versus combination of dasatinib and quercetin on stress-induced chondrocyte senescence[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(35): 9151-9158.

Figures/Tables 4

2.1 考比替尼对软骨细胞增殖活性的影响通过检测细胞存活率来确定考比替尼的最佳干预浓度。实验结果显示，软骨细胞存活率随考比替尼作用浓度的升高(0.01-5 μmol/L)而逐渐降低，见图1A。在相同浓度条件下，细胞存活率随着干预时间的延长而下降。当干预时间达到24 h且浓度为0.1 μmol/L时细胞存活率接近90%。而药物浓度为0.5 μmol/L时，软骨细胞存活率显著低于0 μmol/L组的80%，见图1A。基于上述结果，该研究选择0.1 μmol/L作为考比替尼干预的理想浓度。 2.2 软骨细胞形态观察结果原代SD大鼠软骨细胞培养2 d后，约70%细胞贴壁，细胞形态为类似圆形或椭圆形。白细胞介素1β处理24 h后，形态变得不规则，失去正常的圆形或椭圆形特征，呈现扁平状或伸展状，体积增大且出现纤维样改变。D+Q组处理后，细胞面积减小且趋向正常形态改变，生长速度明显放缓。考比替尼组细胞纤维样形态减少，细胞恢复卵圆形，且边界清晰，见图1B。 2.3 考比替尼对MEK-ERK1/2信号通路的调控作用为了验证考比替尼对MEK-ERK1/2信号通路的调控作用及与D+Q组的对比，采用Western Blot检测了白细胞介素1β刺激后软骨细胞中MEK1、p-MEK1、ERK1/2及p-ERK1/2蛋白的表达情况。结果显示，与对照组相比，白细胞介素1β刺激显著上调了p-MEK1和p-ERK1/2的表达水平，而MEK和ERK总蛋白的表达无明显变化，提示白细胞介素1β可激活MEK-ERK1/2信号通路。在干预组中，发现无论是考比替尼单独处理，还是D+Q组处理，均能明显抑制p-MEK1和p-ERK1/2的表达，见图1C-E。上述结果表明，考比替尼或D+Q联合处理均可有效抑制白细胞介素1β诱导的MEK-ERK1/2信号通路激活，提示它们在缓解炎症性软骨细胞衰老中的潜在机制可能与该通路的负向调控相关。 2.4 考比替尼与D+Q对白细胞介素1β诱导的软骨细胞衰老的缓解作用对比衰老相关β-半乳糖苷酶是衰老细胞最广泛使用的生物标志物之一[29]。两组药物分别与白细胞介素1β共同作用于"

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