【摘要】 背景：中药及其有效成分在调节外泌体miRNA功能方面的潜在作用为骨代谢疾病治疗开辟了新的途径。目的：探讨温肾通络止痛方通过调控脂肪间充质干细胞来源外泌体miR-342-3p改善骨髓间充质干细胞骨脂分化平衡的作用及机制。方法：分离培养1周龄SD大鼠脂肪间充质干细胞及骨髓间充质干细胞，温肾通络止痛方含药血清干预脂肪间充质干细胞后超速离心分离外泌体。外泌体与骨髓间充质干细胞共培养48 h后采用碱性磷酸酶染色、茜素红染色和油红O染色评估骨髓间充质干细胞的骨脂分化能力；利用高通量测序筛选差异miRNA，生信分析下游靶基因，用qPCR验证关键miRNA及靶基因表达。采用qPCR评估miR-342-3p及靶基因PDGFRα对骨髓间充质干细胞成骨、成脂分化基因表达的影响；Western blot法检测miR-342-3p及PDGFRα对下游磷脂酰肌醇3激酶/蛋白激酶B通路蛋白表达的影响。结果与结论：温肾通络止痛方显著促进脂肪间充质干细胞来源外泌体的分泌且更易被骨髓间充质干细胞摄取；测序结果显示温肾通络止痛方干预后的脂肪间充质干细胞来源外泌体中miR-342-3p表达上调最为明显，其通过靶向抑制PDGFRα促进骨髓间充质干细胞成骨分化(Runx2、Osterix mRNA表达上调)并抑制成脂分化(PPARγ、CEBPα mRNA表达下调)；miR-342-3p可促进磷脂酰肌醇3激酶、蛋白激酶B磷酸化蛋白表达，激活磷脂酰肌醇3激酶/蛋白激酶B信号通路，调控骨髓间充质干细胞骨脂分化平衡。以上结果表明温肾通络止痛方通过脂肪间充质干细胞外泌体miR-342-3p靶向PDGFRα/磷脂酰肌醇3激酶/蛋白激酶B轴调控骨髓间充质干细胞的骨脂分化平衡，为中药复方防治骨质疏松提供了分子机制依据。更多还原

【Abstract】 BACKGROUND: The potential role of traditional Chinese medicine and its active ingredients in regulating the function of exosomal miRNA has opened up a new way for the treatment of bone metabolic diseases. OBJECTIVE: To investigate the effect and mechanism of Wenshen Tongluo Zhitong decoction in improving bone fat differentiation balance in bone marrow mesenchymal stem cells by regulating adipocyte derived exosome miR-342-3p. METHODS: Adipose mesenchymal stem cells and bone marrow mesenchymal stem cells of 1-week-old SD rats were isolated and cultured. Adipose-derived mesenchymal stem cells were treated with serum containing Wenshen Tongluo Zhitong decoction, and then exosomes were extracted by high-speed centrifugation method. After exosomes were co-cultured with bone marrow mesenchymal stem cells for 48 hours, alkaline phosphatase staining, alizarin red staining, and oil red O staining were used to evaluate the bone-fat differentiation ability of bone marrow mesenchymal stem cells. High-throughput sequencing was used to screen differential miRNAs, and downstream target genes were analyzed by bioinformatics, and the expression of key miRNAs and target genes was verified by qPCR. qPCR was used to evaluate the effects of miR-342-3p and its target gene PDGFRα on the expression of osteogenic and adipogenic differentiation genes of bone marrow mesenchymal stem cells. Western blot assay was used to detect the effects of miR-342-3p and PDGFRα on the expression of downstream phosphoinositide 3-kinase/protein kinase B pathway proteins.RESULTS AND CONCLUSION: Wenshen Tongluo Zhitong decoction significantly promoted the secretion of exosomes derived from adipose mesenchymal stem cells and made them more easily taken up by bone marrow mesenchymal stem cells. Sequencing results showed that the expression of miR-342-3p in exosomes derived from adipose mesenchymal stem cells after Wenshen Tongluo Zhitong decoction intervention was the most obvious, which promoted the osteogenic differentiation of bone marrow mesenchymal stem cells(up-regulated Runx2 and Osterix mRNA expression) and inhibited adipogenic differentiation(down-regulated PPARγ and CEBPα mRNA expression) by targeting and inhibiting PDGFRα. miR-342-3p could promote the phosphorylation expression of phosphoinositide 3-kinase and protein kinase B, activate the phosphoinositide 3-kinase/protein kinase B signaling pathway, and regulate the balance of bone and fat differentiation of bone marrow mesenchymal stem cells. The above results indicate that Wenshen Tongluo Zhitong decoction regulates the balance of bone-fat differentiation of bone marrow mesenchymal stem cells by targeting the PDGFRα/phosphoinositide 3-kinase/protein kinase B axis through exosome miR-342-3p of adipose mesenchymal stem cells, which can provide a molecular mechanism basis for the prevention and treatment of osteoporosis with traditional Chinese medicine compound.更多还原