Gujin Dan is a Chinese medicine formulation that stimulates cell proliferation and differentiation by controlling multiple genes involved in MC3T3-E1 cells

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 identi�ed 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 signi�cantly promoted the proliferation of MC3T3-E1 cells, as well as the synthesis and mineralisation of the extracellular matrix. GJD signi�cantly 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 signi�cantly 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.


Introduction
Osteoporosis (OP) is the most common disease of bone metabolism and is rooted as an imbalance in bone remodelling, where bone resorption is greater than bone formation.With the ageing population, the prevalence of osteoporosis and the incidence of fragility fractures in China has increased signi cantly over the past 30 years [1,2], with studies showing that bone loss (-2.5 < T-score < -1.0 SD) is as high as 44% and osteoporosis (T-score ≤ -2.5 SD) is as high as 37% in northeast China [3].Although many drugs and modalities are currently available for the treatment of OP and have achieved some success, the increased side effects caused by long-term medication can also greatly reduce patient compliance [4][5][6].Chinese medicine has a low-cost effect with fewer side effects, such as Xian-ling-gu-bao, which has also been used to treat osteoporosis with good results [7].
MC3T3-E1 is an osteoblast precursor cell line derived from the mouse parietal region, a useful model for osteoblast development-related experiments, representing immature osteoblasts.It has been shown that MC3T3-E1 is a continuous source of osteoblast regeneration that avoids the need for repeated preparation of primary osteoblasts and has a better balance and homogeneity than primary osteoblasts [8,9].Gujin Dan (GJD), developed by Professor Liu Bailing, is one of the typical Chinese medicine, which consists of 19 herbs acting synergistically, and has been used for decades in the treatment of cervical spondylosis, lumbar disc herniation, osteoarthritis and osteoporosis in the Department of Orthopaedics and Traumatology of Jilin Province Hospital of Traditional Chinese Medicin, China [10][11][12].Clinically, the formulation is usually in the form of an oral pill for the treatment of patients with osteoporosis.Although its effect is effective and available in our hospital for the treatment of osteoporotic diseases, the exact molecular mechanism of its e cacy in the treatment of osteoporosis remains to be elucidated.
In this experiment, GJD aqueous extract was prepared, and the main components of the aqueous extract were detected by high-performance liquid chromatography (HPLC).The effect of GJD on the differentiation and proliferation of MC3T3-E1 cells was investigated and con rmed.Transcriptome analysis was performed by RNA sequence analysis of MC3T3-E1 cells in response to GJD treatment, and differentially expressed genes were screened by bioinformatics analysis.These results provide ideas to unravel the effects of GJD on osteoblasts and osteoblast-associated bone diseases.

Preparation of GJD aqueous extracts
GJD was obtained from the A liated Hospital of Changchun University of Chinese Medicine (Changchun, China).The formulation of GJD is composed of 19 Chinese materia medica (CMM), namely Drynariae Rhizoma, Spatholobi Caulis, Epimedium, Radix Rehmanniae Praeparata, Paederia Scandens, Cynanchum paniculatum, Curcuma longa, Achyranthes Bidentata, Pericarpium Citri Reticulatae, Radix Paeoniae Alba, Cornu Cervi Degelatinatum, Liquorice, Eucommia ulmoides, Testudinis Carapax et Plustrum, Corydalis yanhusuo, Eupolyphaga Sinensis Walker, Zaocys Dhumnade, Coicis Semen, Cornus O cinalis.The CMM mixture of GJD was extracted by re ux with distilled water and then passed through a 0.45-μm hollow bre cartridge (GE Healthcare, USA).The ltrate was freeze-dried in a Heto PowerDry LL3000 freeze-dryer (Thermo, USA) and stored at -80°C.Before using the drug in the experiment, 0.1 g of GJD freeze-dried powder was added directly to 10 ml of culture medium and vortexed to dissolve, and left to stand for 15 min.After the drug and medium were completely dissolved, the dissolved mixture was ltered through a 0.22-μm hollow bre cartridge (GE Healthcare, USA) and added to the cells.

Culture of MC3T3-E1 osteoblast-like cells and induction of osteoblast differentiation
MC3T3-E1 cells were cultured in DMEM (Hyclone, China) containing 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% CO 2 .MC3T3-E1 cells were inoculated into 24-well cell culture plates at a density of 3 × 10 4 cells/well, and replace with fresh medium every 2-3 days.When the cells reached 80% fusion, the cell culture medium was discarded and add the osteogenic differentiation medium (DMEM supplemented with 10% FBS, 50 μg/mL Ascorbic acid, 10 mM β-Glycerophosphate) was treated with the optimal concentration of GJD for 7 days, replace osteogenic differentiation medium every 2-3 days, and these cells were then used for further studies.

Cell proliferation assay
MC3T3-E1 cell viability was assessed using the Cell Counting Kit-8 (CCK-8) (Sigma, USA) according to the manufacturer's protocol.Brie y, MC3T3-E1 cells (ATCC, USA) were inoculated at a density of 3 × 10 4 cells/well into 96-well cell culture plates (Thermo, USA) (100 μl) and incubated in a humidi ed incubator (Thermo, USA) containing 5% CO 2 at 37°C for 24 hours.The cell culture medium was then discarded and MC3T3-E1 cells were treated with different concentrations of GJD (0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/ml dissolved in the medium; 100 μl per well) and subsequently incubated for 24 hr.Followed by the addition of 10ul of CCK-8 reagent and the cells were incubated for 1 h.Absorbance was measured at 450 nm using an In nite 200 PRO ELISA (Life Sciences, USA).Cell proliferation rate was calculated as the percentage of cell viability after treatment with different concentrations of GJD, respectively.

Alkaline Phosphatase Staining and Activity Measurement
MC3T3-E1 cells were inoculated into 24-well cell culture plates at a density of 3 × 10 4 cells/well, and replace with fresh medium every 2-3 days.When the cells reached 80% fusion, the cell culture medium was discarded and add the osteogenic differentiation medium was treated with the optimal concentration of GJD for 7 days, and replace osteogenic differentiation medium every 2-3 days.After discarding the cell culture medium and rinsing the residue with PBS, the cells were xed in 4% paraformaldehyde at 4°C for 30 minutes, then rinsed again with PBS and stained with the BCIP/NBT Alkaline Phosphatase (ALP) staining kit (Beyotime, China) according to the manufacturer's protocols by mixing 33 μl of BCIP solution, 66 μl of NBT solution and 10.1 ml of BCIP/NBT working solution was mixed and added to the cells for 2 hours.Images were taken with a light microscope (Olympus, Japan) and a digital camera.MC3T3-E1 cells were treated as described above and proteins were extracted to determine the enzymatic activity of alkaline phosphatase.The alkaline phosphatase assay kit (Beyotime, China) was used accordingly with the manufacturer's protocol to measure alkaline phosphatase activity.In nite 200 PRO ELISA was used to measure the absorbance of alkaline phosphatase activity of 405 nm.

Alizarin Red S Staining and Calcium Assay
MC3T3-E1 cells were inoculated into 24-well cell culture plates at a density of 3 x 10 4 cells/well, and replace with fresh medium every 2-3 days.When the cells reached 80% fusion, the cell culture medium was discarded and add the osteogenic differentiation medium was treated with the optimal concentration of GJD for 7 days, replacing the osteogenic differentiation medium every 2-3 days.After discarding the medium and rinsing the residue with PBS, the cells were xed in a xative solution using the Osteoblast Mineralisation Nodule Staining Kit (Beyotime, China) according to the manufacturer's instructions for 30 minutes, rinsed three times with PBS, and stained with an appropriate amount of Alizarin Red S staining solution for 30 minutes at room temperature.Images were taken with a light microscope (Olympus, Japan) and a digital camera and quanti ed using a plate reader (Life science, USA) at an optical density (OD) of 405 nm [13].
2.7 RNA puri cation and Illumina sequencing MC3T3-E1 cells were inoculated into 6-well cell culture plates at a density of 10 x 10 5 cells/well and incubated for 24 h.MC3T3-E1 cells were treated with GJD for 24 h or left untreated.The cell culture medium was then discarded, and each well was thoroughly rinsed with cold PBS buffer.According to the Manufacturer's instruction, TRIzol (Invitrogen, USA) was used to isolate total RNA, and Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to assess the RNA integrity.TruSeq Stranded mRNA kit (Illumina, USA) was used to generate double-end mRNA libraries following the manufacturer's protocol.high-throughput sequencing of mRNA libraries was performed on the Illumina HiSeq 2500 platform (Illumina, USA).

RNA-Seq data analysis
After RNA-seq, clean reads are obtained by trimming the raw reads to remove low-quality reads and splice sequences.The dataset is stored in the NCBI Sequence Read Archive (SRA) database under accession number PRJNA780206.Clean reads were aligned to the mouse (Mus musculus) reference genome using HISAT [14].Gene expression levels were measured using the Fpkm algorithm [15].Non-redundant (NR) and Swiss-Prot protein databases were annotated with BLAST.DEGseq was used to analyse differentially expressed genes.Genes with a log2 fold change ≥ 1 or ≤ -1 and with a p-value ≤ 0.001 were considered to be differentially expressed.The False discovery rate FDR was generated using the Benjamini and Hochberg methods[16].

Quantitative real-time PCR (qRT-PCR) validation
qRT-PCR was used to verify the expression levels of differentially expressed genes.Brie y, total RNA was isolated using TRIzol (Invitrogen, USA) according to the manufacturer's protocol.cDNA was synthesised using the iScript cDNA Synthesis Kit (Bio-Rad, USA) and standard ampli cation conditions were applied using the SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, USA) on the CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) on the CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) to amplify standard ampli cation conditions.Gene expression levels were normalised to the mouse glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene as an internal reference gene and calculated using the 2 -ΔΔCT method [17].

Chemical Quality Control of GJD
The chemical quality control results for GJD are shown in Figure 1.The chemical compound compositions were the same as previously reported and included Paeoni orin, Pinoresinol diglucoside, Loganin, Liquiritin, Acteoside, Tetrahydropalmatine, Naringin, Icariin, Ammonium glycyrrhetate and Catalpol.

GJD promotes the proliferation of MC3T3-E1 cells in a dose-dependent manner
The proliferation of MC3T3-E1 cells due to the effects of GJD treated 24 hours measured by CCK-8 assay as shown in Figure 2. Cell viability was signi cantly increased in a dose-dependent manner in the GJD treatment compared to the untreated control (0 mg/ml).The GJD concentration of 3.2 mg/ml was chosen for subsequent experiments as treatment with 3.2 mg/ml had a signi cant effect on cell viability.

Alkaline phosphatase and Alizarin Red S Results
MC3T3-E1 cells cultured for 7 days after GJD treatment by osteogenic induction of differentiation were observed microscopically by ALP and ARS staining of cells compared to untreated cells.We observed that MC3T3-E1 cells after treatment by GJD could promote cell proliferation and differentiation.Furthermore, the results of quantitative analysis of ARS and ALP in each group were consistent with the microphotographic results (Figure 3-4).

Sequencing, genome mapping and functional annotation
After Illumina sequencing and data processing, 44,599,448 and 47,323,148 clean reads were obtained for MC3T3-E1 cells not treated with GJD (blank) and MC3T3-E1 cells treated with GJD, respectively, as shown in Table 1.The quality assessment indicated that the Q30 percentage was greater than 95% and the GC content percentage was approximately 51%.For the blank and GJD treated samples, 41,360,834 and 44,185,514 reads were compared to the mouse genome respectively.In total, 12,977 of 13,070 (blanks) and 13,282 of 13,383 (GJD) transcripts were annotated against the non-redundant (NR) NCBI Protein Data Bank and the Swiss-Prot database, respectively.

Comparative analysis of differentially expressed genes
The 362 genes that were identi ed via differential expression analysis were signi cantly differentially expressed between the GJD-treated and Blank groups (log 2-fold change ≥ 1 or ≤ -1 and p ≤ 0.001), including 346 up-regulated genes and 16 down-regulated genes (GJD vs. blank), as shown in Table 2.

Gene Ontology (GO) functional enrichment analysis of DEGs
Based on the functional enrichment analysis of DEGs, the identi ed DEGs were classi ed into the following GO categories: biological processes, cellular components, and molecular functions, as shown in Figure 5.The classi cation of biological processes shows that DGEs mainly include the regulation of the cellular process, metabolic process, single-organism process, and biological regulation.The cellular component also shows that the majority of DGEs are located in the cell, cell part and organelle regions.
For molecular function classi cation, the results show that DGEs are mainly associated with binding, catalytic activity, nucleic acid binding transcription factor activity and molecular function regulator.

GJD increases the expression levels of multiple genes that positively regulate the proliferation of MC3T3-E1 cells or osteoblasts
As GJD promotes the proliferation of MC3T3-E1 cells, we rst analysed differentially expressed genes that positively regulate the proliferation of MC3T3-E1 cells or osteoblasts.We identi ed 10 differentially expressed genes based on the results from RNA-seq analysis that directly promote cell proliferation, including Adamts1, Mcam, Cyr61, Fos, Cebpd, Fosl2, Sirt1, Nipbl, Sema3c and Kcnq1ot1.The expression levels of these genes were signi cantly increased in response to GJD treatment, as shown in Table 3.

GJD treatment increases the expression levels that inhibit osteoblast apoptosis to maintain cellular activity
We analysed differentially expressed genes involved in the inhibition of osteoblast apoptosis.Overall, the expression levels of the four genes were signi cantly altered in response to GJD treatment.These genes are normally altered when cells are in a proliferative state and play a key role in the inhibition of apoptosis, as shown in Table 4.

GJD treatment maintains cellular activity and proliferation by inhibiting osteoclast growth or differentiation
We went on to analyse differentially expressed genes involved in the inhibition of osteoclast growth and differentiation.In general, the signi cant upregulation of the expression levels of 10 genes was in response to GJD treatment.These genes are normally down-regulated when the cells are in a proliferative state and play a key role in inhibiting osteoclast proliferation, as shown in Table 5.

GJD increases the expression levels of several genes that promote differentiation of MC3T3-E1 cells and osteoblasts
Among the identi ed DEGs, we analysed differentially expressed genes involved in promoting cell differentiation.In response to the GJD treatment, the expression levels of 20 genes were signi cantly upregulated.These genes are normally up-regulated and Pdzrn3 is down-regulated when the cells are in a differentiated state.As shown in Table 6.

Validation of RNA-Seq data by qRT-PCR
To verify the accuracy of the RNA-seq results, eight differentially expressed genes (Adamts1, Mcan, Cebpd, Gadd45a, Bcl3, Nfkbiz, junB, Klf10 and Hspa1b) were selected and their expression pro les were veri ed using qRT-PCR.The speci c primers used in this experiment are listed in Table 8.
Relative ploidy changes for each gene were normalized to the internal reference gene Gapdh.qRT-PCR measurements of the expression levels of selected differentially expressed genes were consistent with the results of RNA-seq analysis, as shown in Figure 6.

Discussion
GJD is a Chinese medicinal preparation widely used for the treatment of osteoporotic diseases and has been used for decades in Jilin Provincial Hospital of Traditional Chinese Medicine.However, the precise molecular mechanisms behind the ability of GJD to treat these diseases remain to be elucidated.In this study, we investigated the effects of GJD on MC3T3-E1 cells using state-of-the-art RNA-seq technology.From the results of the CCK-8 assay, in a dose-dependent manner, the proliferation of MC3T3-E1 cells was signi cantly promoted by GJD.We then performed RNA-seq to further explore the mechanisms that are responsible for regulating cell proliferation in response to GJD treatment.
We rst analysed the differentially expressed genes involved in increased cell proliferation.The signi cant increase in the expression levels of several genes involved in promoting cell proliferation via GJD include Adamts1, Mcam, Cyr61, Fos, Cebpd, Fosl2, Nipbl, Sema3c and Sirt1.Adamts1 is a secreted multifunctional metalloproteinase and an early responder to parathyroid hormone (PTH) in osteoblasts.
Upregulation of Adamts1 promotes osteoblast growth and mineralisation and is an effective regulator of bone remodelling [18].Mcam gene expression is in response to "metaphyseal mesenchymal progenitors" (MMPs), which play a key role in osteoblast proliferation and differentiation.MSCs express marker genes [19].It is well known that bone morphogenetic proteins (BMPs), especially BMP-2, are key regulators of osteogenesis, and the literature demonstrates that CYR61 enhances BMP-2 mRNA and protein expression in a time-and dose-dependent manner to signi cantly increase proliferation and osteoblast differentiation in MC3T3-E1 cells and primary cultured osteoblasts, and is associated with multiple pathways, such as the ILK and ERK signalling pathway, and the canonical Wnt pathway [20].Fos (also known as the c-fos gene) is part of the AP-l (activator protein 1) transcription factor, which can increase MC3T3-E1 cell proliferation through JNK, ERK and p38 MAPK signalling pathways [21].Several studies have shown that Cebpd is associated with target genes for a variety of biological functions, including growth arrest, apoptosis, differentiation, stem cell self-renewal and tumour suppression, and that in osteoblasts and MC3T3-E1 cells, Cebpd upregulation promotes cell proliferation and differentiation and plays a key role in bone growth and bone remodelling [22].The literature reports that SIRT1, a regulator of bone mass, is closely related to bone metabolism and bone mass and that SIRT1 overexpression promoted proliferation, differentiation and prevented autophagic apoptosis of MC3T3-E1 cells and was able to inhibit osteoclast differentiation [23][24][25].Kcnq1ot1 is associated with a variety of tumourigenesis, and upregulation of Kcnq1ot1 promotes MC3T3-E1 cell proliferation and osteoblast differentiation [26].Our results show that GJD treatment signi cantly promotes osteoblast proliferation by upregulating the expression levels of functional genes involved in promoting cell proliferation.
In line with these results, we also found that the levels of several genes involved in the inhibition of osteoclast differentiation were signi cantly upregulated after GJD treatment, such as Bcl3, Bcl6, Nfkbiz, Sirt1, Clcf1, and Kcnq1ot1.It has been shown that B-cell chronic lymphatic leukaemia protein 3 (Bcl3) interacts with tumour necrosis factor receptor-associated factor 6 through its anchor protein repeat domain to attenuate RANKL-induced osteoclast bone resorption activity and inhibit osteoclast growth in bone marrow-derived macrophages in vitro [27].B cell lymphoma 6 (Bcl6) is a negative regulator of osteoclastogenesis and overexpression of Bcl6 effectively inhibits osteoclast differentiation in vitro [28].Nfkbiz is a key transcription factor involved in osteoclast differentiation, and upregulation of Nfkbiz can mediate impaired osteoclast differentiation [29].Galectin-3 (encoded by the LGALS3 gene) is a member of the β-galactoside-binding lectin family that play multiple roles in cell growth, differentiation and aggregation, interfering with RANLK-mediated signalling and has an intrinsic inhibitory effect on osteoclastogenesis, and overexpression of Lgals3 promotes osteogenic differentiation [30,31].Wisp1, a member of the CCN family that is found in mineralized tissues and is produced by osteoblasts and their precursors, down-regulation of Wisp1 promotes osteoclast differentiation, whereas up-regulation promotes osteoblast differentiation [32].
We further found that the expression levels of several genes involved in the inhibition of apoptosis were signi cantly altered after GJD treatment, such as Gadd45a, Birc3, Sirt1 and Hspa1b.Gadd45a is a target gene for the FoxO3a transcription factor, Gadd45a is also a DNA damage repair gene, and its upregulation eliminates pro-apoptotic genes in damaged or abnormal cells [33].Birc3 is part of a family of apoptosis-inhibiting proteins that effectively mediates resistance to cell death [34].Negative regulation of Hspa1b, an apoptosis-related factor, inhibits osteoblast apoptosis [35].Our results show that GJD treatment inhibits apoptosis through signi cant changes in the expression levels of functional genes involved in the inhibition of apoptosis.
As well, we analysed that the expression levels of most genes involved in MC3T3-E1 cells and osteoblasts to promote their differentiation and mineralisation were signi cantly upregulated after GJD treatment, such as Lgals3, Adamts1, Junb, Egr1, Klf10, Atf6, Wisp1, Malat1, Btg2, Sirt1, Sertad4, Zfyve16, Creb5, Snai2, Fblim1, Fam46a, Calcrl, Kcnq1ot1, Pdzrn3.Overexpression of the AP-1 transcription factor JunB induces osteoblast differentiation [36].Egr1 co-regulates osteoblast differentiation with several osteoblast-related genes and Egr1 overexpression contributes to osteoblast differentiation [37,38].Klf10 is a member of the Krüppel-like family of transcription factors, Klf10 upregulation effectively promotes osteoblast differentiation [39].Osteocalcin is a target gene of ATF6, ATF6 upregulation promotes osteoblast differentiation [40].Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is associated with a variety of human tumours and Malat1 upregulation mediates the regulation of osteoblast activity and pro-differentiation [41].Sertad4 is a newly identi ed gene associated with osteoporosis, and upregulation of Sertad4 promotes osteoblast differentiation [42].The Zinc nger fyve domain containing 16 (ZFYVE16, also known as Endo n) is involved in the BMP signalling pathway playing an important role in promoting osteoblast differentiation [43].Creb5 is relevant to the treatment of several cancer diseases, and studies of osteoblast differentiation have revealed that this Creb5 upregulation promotes osteoblast differentiation [44].Snail2 is a marker of malignancy in epithelial tumours, and upregulation of Snail2 was shown to promote increased osteoblast alkaline phosphatase activity and promote differentiation [45].Fblim1 is a key regulator of bone homeostasis and deletion of the Fblim1 gene results in increased differentiation of osteoclasts in vivo [46].FAM46A is a gene associated with skeletal dysplasia and bone mineralization and plays an important role in bone regulation, and FAM46A de ciency can lead to fracture susceptibility and long bone curvature [47].CALCRL (Calcitonin receptor-like receptor) is associated with disease and CALCRL upregulation promotes osteoblast differentiation [48].PDZRN3 is a member of the PDZ domain-containing RING nger family of proteins and plays an important role in the negative feedback control of osteoblast differentiation by inhibiting Wnt-β-catenin signalling [49].
Based on our RNA-seq, we also analysed the expression levels of osteoblast markers, including Atf4, Fn1, Usp7, Sox4, Col16a1, Spp1, Bmp1, Runx2, Bglap, Col12a1 and Alpl, which were slightly increased in response to GJD treatment.Based on our RNA-seq, we also analysed the expression levels of osteoblast markers, including Atf4, Fn1, Usp7, Sox4, Col16a1, Spp1, Bmp1, Runx2, Bglap, Col12a1 and Alpl, which were slightly increased in response to GJD treatment.Studies con rmed that SPP1, Fn1, Bglap, Col16a1, and Col12a1 are the main bone matrix proteins with associated genes [50][51][52][53].ATF4, SOX4, RUNX2 are key transcription factors associated with bone formation [54][55][56].It has been shown that ubiquitinspeci c proteinase 7 (USP7) is involved in the regulation of various biological functions and plays an important role in osteoblast differentiation [57].Bmp1 is a member of the bone morphogenetic protein family and plays an important role in bone remodelling [58].Alpl is a marker of osteoblasts and is mostly used in the important assessment of bone formation [59].
There are also genes in the RNA-Seq that are signi cantly upregulated or downregulated by drug interventions, which we have not included in the analysis because they are not relevant to our topic, so we have only analysed genes associated with osteoporosis disease.Other data may also be useful for subsequent studies of other diseases.
The GJD is based on the clinical experience of Professor Bailing Liu, a master of Chinese medicine, and is designed under the guidance of the basic theory of Chinese medicine, with the theory that "the kidney is the master of bone" and "treating the kidney also treats the bone".Among the nineteen CMMs in GJD, Bonesetter, Epimedium, Radix Rehmanniae Praeparata, Achyranthes bidentata, Eucommia ulmoides and Cornus o cinalis are the drugs that play the most important role in Chinese medicine formulation, the so-called monarchs and ministers, all these herbs are drugs that play a role in Chinese medicine in tonifying the kidneys and strengthening the bones.The so-called kidney is not simply limited to one organ but is a function of the Zang viscera system under the general theory of Chinese medicine.
The Qi in the Zang viscera kidney is Kidney Qi, which promotes and regulates the growth and development of the bones [60,61].Rhizoma Dynariae is one of the medicines in the Chinese Pharmacopoeia, mainly used for the treatment of bone-related diseases.Its main component is Flavonoids, which have been proven in numerous studies to have the effect of reducing bone loss and promoting bone formation [62].Epimedium has been shown to promote osteogenic differentiation and thus induce bone formation [63].Radix Rehmanniae Praeparata is one of the most commonly used herbs in Chinese medicine and has been shown in numerous experimental studies to have therapeutic effects on diseases such as osteoporosis through its many active ingredients [64].Achyranthes bidentata is used in many prescriptions in Chinese medicine, which shows its wide range of effects, and studies have shown that this herb has signi cant anti-osteoporosis effects [65].Eucommia ulmoides is a common herbal medicine used in Chinese medicine for the treatment of osteoporosis, which has the effect of regulating bone metabolism and is mostly used for the prevention and treatment of osteoporosis [66].
Studies of Cornus O cinalis extract were found to inhibit osteoclast differentiation and promote osteoblast differentiation [67].In addition, studies have shown that the Curcuminoid-enriched Turmeric extract in Curcuma Longa helps retain BMD in trend-model rats, as well as the microarchitectural structure and Trabecular connectivity of bone [68].Pericarpium Citri Reticulatae contains Hesperetin, according to the research, Hesperetin effectively suppressed Rankl-induced osteoclastogenesis, osteoclastic bone resorption and F-actin ring [69].Radix Paeoniae Alba containing Paeoni orin has been widely used in the treatment of osteoporosis.Paeoni orin treatment of osteoporosis can be divided into two general directions: inhibiting osteoclast activity [70] and improving osteoblast activity [71,72], It slows bone resorption by inhibiting osteoclast activity and promotes bone formation by increasing osteoblast activity.Corydalis Yanhusuo contains Tetrahydropalmatine, Experimental studies in vivo and in vitro have shown that Tetrahydropalmatine inhibits osteoclast formation by inhibiting NF-κB and MAPK pathways [73].Coicis Semen extract has been shown to promote osteoblast proliferation and effectively relieve osteoporosis in animals [73][74][75].
In conclusion, our results suggest that the various herbs and their active ingredients in GJD synergistically promote osteoblast proliferation and differentiation and also inhibit osteoclast differentiation and in ammation.

Conclusion
This study shows that GJD, a Chinese medicine formulation that has been used for decades to treat osteoporosis and other bone metabolic diseases, signi cantly promoted the viability and proliferation of MC3T3-E1 cells, kept the cells in a sustained proliferative state, promoted their differentiation and inhibited osteoclastogenesis and differentiation.These effects were achieved through the synergistic interaction of the active ingredients of the various herbs in GJD, in which increased functional gene expression levels involved in osteoblast proliferation and differentiation and decreased the expression levels of genes involved in osteoclastogenesis and differentiation.Treatment with GJD also reduced the expression levels of genes involved in cellular pro-apoptosis.Thus, this study signi cantly deepens the current understanding of the molecular effects of GJD on MC3T3-E1 cells.This study would also provide Tables

Figures
Figures

Figure 1 A
Figure 1

Figure 2 Effects
Figure 2

Table 1
Statistical sequencing results of Blank and GJD.

Table 3
The expression levels of multiple genes that positively regulate the proliferation of MC3T3-E1 cells or osteoblasts (Blank versus GJD).

Table 4
Positive regulation of differentially expressed genes that inhibit osteoblast apoptosis (Blank versus GJD).

Table 5
Differential gene expression that inhibits the growth or differentiation of osteoclasts (Blank versus GJD).

Table 7
The expression level of osteoblast markers (Blank versus GJD).

Table 8
Primer sequences used in qRT-PCR validation.