10-Hydroxy-2-decenoic acid facilitates osteogenic differentiation via the enhancement of autophagy and antioxidant capacity

doi:10.12307/2026.455

Abstract

Abstract:

BACKGROUND: 10-Hydroxy-2-decenoic acid (10-HDA) exhibits potent anti-inflammatory, antioxidant, and immunomodulatory effects, but its role in regulating bone metabolism remains unclear.
OBJECTIVE: To investigate the regulatory effects and underlying mechanisms of 10-HDA on bone remodeling.
METHODS: (1) Bone marrow-derived mesenchymal stem cells were isolated from rats and treated with different concentrations of 10-HDA (0, 0.5, 1, 2, and 4 mmol/L). Cytoskeletal staining was used to assess cell morphology, while live/dead staining and the cell counting kit-8 assay were employed to evaluate cell viability and proliferation. (2) Bone marrow-derived mesenchymal stem cells were isolated from rats and treated with different concentrations of 10-HDA (0, 0.5, 1, and 2 mmol/L). After osteogenic induction, alkaline phosphatase staining and alizarin red staining were conducted to assess osteogenic differentiation. The expression of osteogenesis-related proteins was analyzed by western blot and immunofluorescence staining. (3) Mouse bone marrow mononuclear cells were induced to differentiate into macrophages by adding osteoclast-inducing differentiation medium containing different concentrations of 10-HDA (0, 0.5, 1, and
2 mmol/L). Tartrate-resistant acid phosphatase staining and F-actin staining were used to evaluate osteoclast formation. (4) Rat bone marrow mesenchymal stem cells were starved for 6 hours before conventional culture and grouped as follows: control group (no intervention), 10-HDA group, 10-HDA + AS1842856 group, 10-HDA + EX-527 group. 10-Hydroxy-2-decenoic acid was administered at a concentration of 0.5 mmol/L. AS1842856 was a forkhead box transcription factor O1 inhibitor, and EX-527 was a silent information regulator 1 inhibitor. Furthermore, activation of the silent information regulator 1/forkhead box transcription factor O1 signaling pathway and expression of autophagy- and antioxidant-related proteins were assessed by western blot and immunofluorescence. (5) Rat bone marrow mesenchymal stem cells were cultured in five groups: control group, H₂O₂ group, and H₂O₂ + 10-HDA group. The intervention concentration of 10-HDA was 0.5 mmol/L. Corresponding drug interventions were administered 24 hours after H₂O₂ exposure. After osteogenic induction, alkaline phosphatase staining and alizarin red staining were conducted. (6) Rat bone marrow mesenchymal stem cells were cultured in five groups: control group, H₂O₂ group, H₂O₂+10-HDA group, H₂O₂+10-HDA+AS1842856 group, and H₂O₂+10-HDA+EX-527 group. The intervention concentration of 10-HDA was 0.5 mmol/L. Corresponding drug interventions were administered 24 hours after H₂O₂ exposure. Western blot analysis was performed to detect the expression of silent information regulator 1/forkhead box transcription factor O1 signaling pathway and antioxidant-related proteins. TUNEL staining and β-galactosidase staining were performed to evaluate apoptosis and senescence.
RESULTS AND CONCLUSION: (1) At the concentration of 0.5, 1, and 2 mmol/L, 10-HDA promoted the proliferation of bone marrow-derived mesenchymal stem cells. Therefore, these three intervention concentrations were selected for subsequent experiments. (2) 10-HDA (0.5 mmol/L) significantly enhanced the osteogenic differentiation and mineralization of bone marrow-derived mesenchymal stem cells and increased the expression of osteogenesis-related proteins, as evidenced by alkaline phosphatase staining, alizarin red staining, western blot and immunofluorescence staining. (3) Tartrate-resistant acid phosphatase staining and F-actin staining revealed that 0.5 mmol/L 10-HDA significantly inhibited osteoclast formation. (4) Western blot and immunofluorescence staining revealed that 10-HDA activated the silent information regulator 1/forkhead box transcription factor O1 signaling pathway to promote deacetylation and nuclear translocation of forkhead box transcription factor O1, thereby upregulating autophagy-related proteins and antioxidant enzymes. (5) Alkaline phosphatase and alizarin red staining revealed that 10-HDA promotes osteogenic differentiation and mineralization in rat bone marrow-derived mesenchymal stem cells under oxidative stress conditions. (6) Western blot analysis revealed that under oxidative stress conditions, 10-HDA enhanced the antioxidant capacity of rat bone marrow mesenchymal stem cells by activating the silent information regulator 1/forkhead box transcription factor O1 signaling pathway. TUNEL and β-galactosidase staining revealed that under oxidative stress conditions, 10-HDA reduced apoptosis and senescence in rat bone marrow mesenchymal stem cells by activating the silent information regulator 1/forkhead box transcription factor O1 signaling pathway. To conclude, 10-HDA enhances autophagy and antioxidant capacity by regulating the silent information regulator 1/forkhead box transcription factor O1 signaling pathway, thereby promoting osteogenic differentiation.
Key words: osteoporosis; bone marrow-derived mesenchymal stem cells; 10-hydroxy-2-decenoic acid; osteogenic differentiation; osteoclasts; silent information regulator 1; autophagy; antioxidant

Key words: osteoporosis, bone marrow-derived mesenchymal stem cells, 10-hydroxy-2-decenoic acid, osteogenic differentiation, osteoclasts, silent information regulator 1, autophagy, antioxidant

CLC Number:

Hu Jie, He Hui, Ma Fengyu, Shen Xiaotian, Yuan Zhangqin, Liang Ting, Han Fengxuan. 10-Hydroxy-2-decenoic acid facilitates osteogenic differentiation via the enhancement of autophagy and antioxidant capacity[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(25): 6433-6445.

Figures/Tables 8

2.1 不同浓度10-HDA对大鼠骨髓间充质干细胞活性和骨架的影响如图1A所示，低浓度(0.5，1，2 mmol/L)10-HDA组的大鼠骨髓间充质干细胞铺展良好，F-肌动蛋白结构完整且分布有序；在4 mmol/L 10-HDA处理后，部分细胞明显出现收缩，骨架结构紊乱，提示该浓度可能具有一定的细胞毒性作用。活死染色结果显示，4 mmol/L 10-HDA组红色荧光死细胞较多，0.5，1，2 mmol/L 10-HDA组红色荧光死细胞较少，如图1B，C所示，表明0.5-2 mmol/L 10-HDA具有良好的细胞相容性。CCK-8检测结果显示，培养第3，5天，0.5，1，2 mmol/L 10-HDA可促进大鼠骨髓间充质干细胞增殖，而4 mmol/L 10-HDA抑制大鼠骨髓间充质干细胞的增殖，见图1D。综上所述，0.5，1，2 mmol/L 10-HDA具有良好的细胞相容性，可促进大鼠骨髓间充质干细胞的增殖。10-HDA作为一种不饱和脂肪酸衍生物，在高浓度下可能插入细胞膜或线粒体膜中，影响膜的流动性与完整性、破坏细胞器功能，尤其是线粒体膜电位，从而激活细胞凋亡通路[32]，因此，后续实验选择0.5，1，2 mmol/L作为10-HDA适宜处理浓度，以兼顾细胞活性与实验安全性。 2.2 不同浓度10-HDA对大鼠骨髓间充质干细胞成骨分化的影响为评估10-HDA的促成骨潜能，采用碱性磷酸酶染色和茜素红染色分别检测它对成骨早期活性和晚期基质矿化的影响。如图2A，B所示，对照组表现出较低的碱性磷酸酶活性和较弱的矿化结节形成，提示在未诱导条件下未启动成骨分化；相比之下，诱导组在成骨诱导培养基处理后碱性磷酸酶染色和钙沉积增强，证实成骨过程被成功激活。0.5，1 mmol/L 10-HDA组碱性磷酸酶活性高于诱导组(P < 0.05)，2 mmol/L 10-HDA组的碱性磷酸酶活性与诱导组相似，见图2C，表明低浓度10-HDA可促进成早期阶段的骨分化。与碱性磷酸酶染色结果一致，茜素红染色显示，0.5，1 mmol/L 10-HDA组钙化结节数量多于诱导组(P < 0.05)，如图2D所示，提示低浓度10-HDA对成骨晚期分化及钙沉积具有促进作用。综上所述，10-HDA在适当浓度下能显著促进成骨分化，增强成骨早期活性和晚期矿化能力。 2.3 不同浓度10-HDA对大鼠骨髓间充质干细胞成骨相关蛋白表达的影响为进一步探究10-HDA的促成骨作用，采用Western blot检测关键成骨标志蛋白表达，其中，Ⅰ型胶原是骨组织细胞外基质的主要成分，主要参与成骨早期的基质合成；RUNX2是成骨细胞谱系分化所必需的关键转录因子；碱性磷酸酶是成骨活性的早期标志物，而骨桥蛋白和骨钙素则在基质成熟和矿化过程中发挥重要作用。如图2E，F所示，0.5 mmol/L 10-HDA处理显著上调了成骨标志蛋白表达，然而，随着10-HDA浓度升高(1，2 mmol/L)，这一促进作用逐渐减弱。免疫荧光染色显示，与诱导组相比，0.5 mmol/L 10-HDA显著增强了Ⅰ型胶原和RUNX2蛋白表达，见图3，表明基质合成增加、成骨分化能力增强，进一步证实0.5 mmol/L 10-HDA可通过上调关键调控蛋白和结构蛋白促进早期成骨分化。 2.4 不同浓度10-HDA对破骨细胞生成的影响抗酒石酸酸性磷酸酶是成熟破骨细胞表达的标志性酶，而F-actin环是破骨细胞形成骨吸收腔的关键结构。抗酒石酸酸性磷酸酶与细胞骨架染色显示，0.5 mmol/L 10-HDA处理后，抗酒石酸酸性磷酸酶阳性的多核细胞数量显著减少，并且F-actin环结构出现破坏或缺失，但当10-HDA浓度增加至1，2 mmol/L 时该抑制作用并未进一步增强，如图4所示，提示10-HDA的抗破骨分化作用中可能存在一个浓度阈值。综上所述，10-HDA不仅能促进成骨细胞介导的骨形成，还能有效抑制破骨细胞生成，从而有助于构建有利于骨再生的微环境。"

达、p-FOXO1/FOXO1比值升高(P < 0.05)，自噬相关蛋白微管相关蛋白1轻链3与Beclin-1表达升高(P < 0.05)，P62蛋白表达降低(P < 0.05)，提示自噬活性增强；成骨相关蛋白Ⅰ型胶原、β-连环蛋白、RUNX2、骨桥蛋白表达升高(P < 0.05)，见图5A-D。与0.5 mmol/L 10-HDA组比较，0.5 mmol/L 10-HDA+AS1842856组、0.5 mmol/L 10-HDA+EX-527组SIRT1、微管相关蛋白1轻链3、Beclin-1、Ⅰ型胶原、β-连环蛋白、RUNX2、骨桥蛋白表达及p-FOXO1/FOXO1比值均降低(P < 0.05)，P62蛋白表达升高(P < 0.05)，见图5A-D。表明使用FOXO1抑制剂AS1842856处理后0.5 mmol/L 10-HDA的上述作用被部分逆转，提示自噬在10-HDA的成骨机制中起关键作用；使用SIRT1特异性抑制剂EX-527处理后，SIRT1蛋白表达被有效抑制，FOXO1激活程度及Beclin-1表达下降，P62出现积累；与此同时，10-HDA诱导的成骨标志物水平显著降低，说明SIRT1的抑制削弱了10-HDA增强自噬和促成骨的作用。为进一步探究10-HDA是否促进FOXO1的激活，进行了免疫荧光染色以观察FOXO1的亚细胞定位。FOXO1进入细胞核是它转录活化的关键标志，只有转位至细胞核后才能调控参与自噬和成骨分化的下游基因表达[36]。如图5E，F所示，对照组中FOXO1主要分布于胞质中，激活水平较低；0.5 mmol/L 10-HDA处理后，细胞核中FOXO1荧光显著增强，提示FOXO1转录活性提高；联合FOXO1抑制剂AS1842856处理后FOXO1核定位部分减弱，而联合SIRT1抑制剂EX-527后FOXO1核定位显著减少。这些结果表明，10-HDA通过激活SIRT1促进FOXO1核转位，进而可能增强自噬及成骨基因的转录活性。 2.6 10-HDA在氧化应激微环境中对骨髓间充质干细胞的保护作用及机制探究通过H2O2诱导骨髓间充质干细胞进入氧化应激状态，采用碱性磷酸酶和茜素红染色评估成骨分化，如图6A-C所示。H2O2处理显著抑制了骨髓间充质干细胞的成骨分化，表现为碱性磷酸酶染色强度和面积的显著下降以及钙结节形成减少；0.5 mmol/L 10-HDA处理显著恢复了骨髓间充质干细胞的成骨能力，表明0.5 mmol/L 10-HDA在氧化应激条件下具有保护作用并可促进成骨。为探究SIRT1/FOXO1信号通路的功能，研究通过Western blot检测该通路相关蛋白及抗氧化蛋白超氧化物歧化酶2和过氧化氢酶的表达水平，如图6D，E所示。H2O2处理后，大鼠骨髓间充质干细胞内SIRT1蛋白表达显著下调，p-FOXO1/FOXO1比值降低，超氧化物歧化酶2和过氧化氢酶蛋白表达亦有所减少；0.5 mmol/L 10-HDA处理后可显著恢复上述蛋白表达，尤其是超氧化物歧化酶2蛋白表达明显上调；相反，在与AS1842856或EX-527联合处理的情况下，10-HDA的保护作用明显减弱。这些结果提示，10-HDA可能通过激活SIRT1/FOXO1信号通路来增强"

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