Wenshen Tongluo Zhitong decoction regulates the bone fat differentiation balance of bone marrow mesenchymal stem cells through exosomal miR-342-3p

Wumiti·Taxi; Wang Lining; Li Muzhe; Sun Jie; Chen Shuangliu; Zhu Yihua; Zhou Shijie; Ma Yong; Guo Yang

doi:10.12307/2026.672

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.