Background: With the development of Traditional Chinese medicine (TCM) in recent years, the use of TCM in the treatment of osteoporosis has received much attention and research. Gujin Dan (GJD) is one of the representative Chinese medicine formulations that work synergistically with 19 herbs and has been used for decades to treat cervical spondylosis, lumbar disc herniation, osteoarthritis and osteoporosis. However, the exact molecular mechanism by which GJD is used to strengthen bones in the treatment of osteoporosis remains largely unknown. Methods: In this study, an aqueous extract of GJD was prepared and its components were identified by high-performance liquid chromatography (HPLC). The effect of GJD aqueous extract on MC3T3-E1 cells was determined by Cell Counting Kit-8 (CCK-8) assay, alkaline phosphatase (ALP), and alizarin red S staining (ARS), combined with RNA sequencing (RNA-seq) and qRT-PCR. Results: Our study showed that GJD significantly promoted the proliferation of MC3T3-E1 cells, as well as the synthesis and mineralisation of the extracellular matrix. GJD significantly increased the expression levels of genes that promote cell proliferation such as Adamts1, Mcam, Cyr61, Fos, Cebpd, Fosl2, Sirt1, Nipbl, Sema3c and Kcnq1ot1, up-regulated genes that inhibit apoptosis such as Gadd45a, Birc3, up-regulated genes that inhibit osteoclastogenesis such as Bcl6, Nfkbiz, Clcf1, Bcl3, Lgals3, Wisp1, Dusp1 and Fblim1, up-regulated genes that promote MC3T3-E1 cell differentiation such as Junb, Egr1, Klf10, Atf6, Malat1, Btg2, Sertad4, Zfyve16, Tet2, Creb5, Snai2, Fam46a, Calcrl and Pdzrn3. In addition, GJD mildly upregulated the expression levels of gene markers such as Atf4, Fn1, Usp7, Sox4, Col16a1, Spp1, Bmp1, Runx2, Bglap, Col12a1, and Alpl in osteoblasts. Conclusions: Our results show that GJD promotes the differentiation and proliferation of MC3T3-E1 cells, inhibits osteoclast formation, and prevents osteoblast apoptosis. The present study significantly improves the current understanding of the molecular effects of GJD on MC3T3-E1 cells. This study also provides a new strategy for the further use of Chinese medicinal preparations against bone metabolism-related diseases.
Background: The development of Chinese medicine has been practised in China over a long period of time, and China has long used single medicines in various forms of decoction to treat illnesses, and later learned to combine several medicines to form formulas to enhance the effects of the medicines. The use of Chinese herbal medicines and formulas has played a pivotal role in the prevention and treatment of diseases in China since ancient times. The application of Chinese herbal preparations in the field of osteoporosis treatment has received widespread attention, and Gujin Dan(GJD) is one of the representative herbal formulas, however, the exact minute mechanism of its treatment of osteoporosis remains to be elucidated.Methods: In the study, we prepared an aqueous extract of GJD and measured the effect of different administration concentrations of GJD on cell proliferation by CCK-8 assay, and the effect of GJD on cell differentiation ability by Alizarin Red S Staining, Alkaline Phosphatase Staining and quantitative assay. Changes in gene expression patterns of MC3T3-E1 cells under GJD treatment were investigated by RNA-seq analysis and validation methods.Results: We demonstrate that GJD promotes the proliferation and differentiation of Mc3t3-e1 cells through the regulation of multiple functional genes. This was mainly achieved by regulating the expression levels of four categories of genes that promote the proliferation of Mc3t3-e1 cells or osteoblasts, inhibit apoptosis and autophagy, inhibit osteoclast formation and differentiation, and promote osteoblast differentiation. In addition, GJD slightly increased the expression levels of gene markers in osteoblasts. Conclusions: Our findings suggest that GJD promotes proliferation and differentiation of MC3T3-E1 cells and inhibits osteoclastogenesis and differentiation, as well as apoptosis and autophagy, through the synergistic interaction of various herbs and their active components in GJD. This study has significantly improved the current understanding of the molecular effects of GJD on MC3T3-E1 cells. This study also provides new ideas for possible strategies to further prevent and treat bone metabolism-related diseases using traditional Chinese medicinal preparations.
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