Atractylodes lancea volatile oils target ADAR2‐miR‐181a‐5p signaling to mesenchymal stem cell chondrogenic differentiation

Atractylodeslancea Rhizoma (Rhizoma atractylodis [RA]) has long been recommended for the treatment of arthritis in traditional Chinese medicine, but its mechanism of action is still unclear. RA contains a large amount of Atractylodes lancea volatile oils (Atr). In this study, we investigated whether Atr can promote mesenchymal stem cells (MSCs) chondrogenic differentiation. The Atr were extracted from RA by steam distillation method, and the effect of Atr on MSCs was detected by the CCK8 assay. The optimal concentration of Atr for MSCs cultivation was 3 μg/ml. The differentially expressed miR-181a-5p was screened by miRNA microarray assay, and its mimics and inhibitors were transfected into MSCs. It was found that the inhibitor of miR-181a-5p could upregulate cartilage-specific genes such as SOX9, COL2A1, and ACAN. Meanwhile, we also found that the expression of gene editing enzyme ADAR2 was significantly increased in the chondrogenic differentiation of MSCs induced by Atr, and the bases of precursor sequence of miR-181a-5p were changed from A to G. After ADAR2 deletion, the expression of cartilage-specific genes was significantly down-regulated and the precursor sequence bases of miR-181a-5p were not changed. Bioinformatics analysis revealed that the predicted target gene of miR-181a-5p was yingyang1 (YY1), and the targeting relationship was verified by dual-luciferase reporter assay. After deleting YY1, the expression of cartilage-specific genes was significantly down-regulated. In conclusion, our study demonstrated that Atr can promote chondrogenic differentiation of MSC through regulation of the ADAR2-miR-181a-5p signaling pathway. This may provide a new insight into the possible mechanism of traditional Chinese medicine (Atr) in treating inflammatory joint diseases.中药苍术(atractylodlancea Rhizoma, RA)在中医临床研究中发现其对关节软骨类疾病具有疗效，但它的作用机制尚不清楚。苍术的主要活性成分是苍术挥发油(Atractylodes lancea volatile oils)，我们的研究发现苍术挥发油能促进间充质干细胞(MSC)成软骨细胞分化。本研究采用水蒸气蒸馏法提取苍术挥发油，通过CCK8实验检测苍术挥发油对MSC的毒性作用，以3μg/ml为最佳作用浓度并用于后续MSC的诱导培养。通过miRNA芯片筛选出差异表达的miR-181a-5p，将其模拟物及抑制物转染至MSC内，发现miR-181a-5p的抑制物可以上调软骨基因SOX9、COL2A1及ACAN。同时我们发现，在苍术挥发油诱导MSC成软骨细胞分化中，基因编辑酶ADAR2的表达显著上升，miR-181a-5p的前体序列碱基发生了A-G的改变；删失ADAR2酶后，软骨基因的表达显著下调，miR-181a-5p的前体序列碱基没有改变。而后使用生物信息学网站预测miR-181a-5p的靶基因是Yingyang1 (YY1)，通过构建双荧光素酶报告基因验证靶向关系；删失YY1后，软骨基因的表达显著下调。综上所述，苍术挥发是通过ADAR2- miR-181a-5p信号通路进而促进MSC成软骨细胞分化，本研究可为中药苍术治疗关节软骨类疾病的机制研究提供新的见解。.