We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon‐derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism. The self‐renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK‐8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real‐time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia‐induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self‐renewal ability and multi‐differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence. Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self‐renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α‐EGR1 axis in this process.

中文翻译：

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阿魏酸通过上调低氧引起的早期生长反应1来改善人肌腱干细胞的自我更新和分化。

我们旨在研究阿魏酸（FA）在体外对人肌腱干细胞（hTSCs）干性的潜在有益作用，并阐明其潜在的分子机制。hTSC的自我更新能力通过集落形成来评估，细胞增殖通过CCK-8试剂盒来确定。分别通过油红O，茜素红和阿尔辛蓝染色确定脂肪形成，成骨和软骨形成。通过实时PCR测量PPARγ，Col2A1，Acan，Runx2，HIF1α和EGR1的相对mRNA水平。Western blot检测HIF1α和EGR1蛋白水平。通过ChIP测定法分析了HIF1α与EGR1启动子的直接结合。低氧诱导的EGR1表达通过荧光素酶报告基因检测得到了验证。我们证明了FA治疗可以改善hTSC的自我更新能力和多分化潜力。FA诱导缺氧，进而通过与其缺氧反应元件共有序列直接关联而上调EGR1表达。此外，我们表明，HIF1α和EGR1都是FA对hTSC自我更新和分化的增强作用所必需的。因此，我们表征了FA对hTSC茎干的有益作用，并强调了HIF1α-EGR1轴在此过程中的关键作用。