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Background. As in philosophy of traditional Chinese medicine (TCM), the theory of “kidney governing bones” has been demonstrated by a series of scientific studies. Furthermore, many groups including ours have explored the molecular mechanisms related to bone development, growth, and regeneration using modern biology technologies, such as RNA sequencing (RNA-Seq) and isobaric tags for relative and absolute quantification (ITRAQ), and have demonstrated that the underlying molecular mechanisms were highly consistent with the “kidney governing bones” theory. Objective. Kidney-yang deficiency (YD), as a pathological condition, has a passive effect on the skeleton growth; more specifically, it is a state of skeletal metabolic disorder. However, the exact molecular mechanisms related to the “kidney governing bones” theory under the control of multiple organs and systems are still unknown. Methods. In this study, we performed RNA-Seq analysis to investigate and compare the gene expression patterns of six types of tissue (bone, cartilage, kidney, testicle, thyroid gland, and adrenal gland) from YD rats and normal rats and analyzed the interaction effects controlled by multiple functional genes and signaling pathways between those tissues. Results. Our results showed that, in the state of YD, the functions of bone and cartilage were inhibited. Furthermore, multiple organs involving the reproductive, endocrine, and urinary systems were also investigated, and our results showed that YD could cause dysfunctions of these systems by downregulating multiple functional genes and signaling pathways that positively regulate the homeostasis of these tissues. Conclusion. We ensure that “kidney governing bones” was not a simple change in a single gene but the changes in complex biological networks caused by functional changes in multiple genes. This also coincides with the holistic view of TCM, which holds that the human body itself is an organic whole and the functional activities of each organ coordinate with each other.

Background Chondrocyte proliferation and differentiation play pivotal roles in regulating cartilage formation, endochondral bone formation, and repair. Cartilage damage and underdevelopment may cause severe joint diseases. Various transcription factors regulate cartilage development. Nuclear factor 1 B (Nfib) is a transcription factor that plays a regulatory role in various organs. However, the effect and mechanism of Nfib on the proliferation and differentiation of chondrocytes in cartilage are still largely unknown. Methods and results In the present study, we investigated the gene expression patterns in primary chondrocytes with Nfib overexpression or silencing by RNA sequencing (RNA-seq) technology. The results showed that Nfib overexpression significantly up-regulated genes that are related to chondrocyte proliferation and extracellular matrix (ECM) synthesis and significantly down-regulated genes related to chondrocyte differentiation and ECM degradation. However, with Nfib silencing, the genes involved in promoting chondrocyte differentiation were significantly up-regulated, whereas those involved in promoting chondrocyte proliferation were significantly down-regulated. Furthermore, quantitative real-time PCR (qRT-PCR), western blot, alcian blue staining and immunofluorescence staining assays further confirmed that Nfib potentially promotes chondrocyte proliferation and extracellular synthesis but inhibits differentiation. Conclusions The molecular mechanism of Nfib in promoting chondrocyte proliferation and inhibiting differentiation was probably achieved by stimulating Sox9 and its downstream genes. Thus, this study adds new insights regarding the underlying molecular mechanism of transcriptional regulation in cartilage.

Deer velvet antlers are the young horns of male deer that are not ossified and densely overgrown. Velvet antler and its preparations have been widely used in the treatment of postmenopausal osteoporosis (PMOP) in recent years, although its mechanism of action in the human body remains unclear. To screen the effective ingredients and targets of velvet antler in the treatment of PMOP using network pharmacology and to explore the potential mechanisms of velvet antler action in such treatments, we screened the active ingredients and targets of velvet antler in the BATMAN-TCM database. We also screened the relevant targets of PMOP in the GeneCards and OMIM databases and then compared the targets at the intersection of both velvet antler and PMOP. We used Cytoscape 3.7.2 software to construct a network diagram of “disease-drug-components-targets” and a protein-protein interaction (PPI) network through the STRING database and screened out the core targets; the R language was then used to analyze the shared targets between antler and PMOP for GO-enrichment analysis and KEGG pathway-annotation analysis. Furthermore, we used the professional software Maestro 11.1 to verify the predictive analysis based on network pharmacology. Hematoxylin-eosin (H&E) staining and micro-CT were used to observe the changes in trabecular bone tissue, further confirming the results of network pharmacological analysis. The potentially effective components of velvet antler principally include 17β-E2, adenosine triphosphate, and oestrone. These components act on key target genes such as AKT1, IL6, MAPK3, TP53, EGFR, SRC, and TNF and regulate the PI3K/Akt-signaling and MAPK-signaling pathways. These molecules participate in a series of processes such as cellular differentiation, apoptosis, metabolism, and inflammation and can ultimately be used to treat PMOP; they reflect the overall regulation, network regulation, and protein interactions.

Background Keratinocytes and fibroblasts represent the major cell types in the epidermis and dermis of the skin and play a significant role in maintenance of skin homeostasis. However, the biological characteristics of keratinocytes and fibroblasts remain to be elucidated. The purpose of this study was to compare the gene expression pattern between keratinocytes and fibroblasts and to explore novel biomarker genes so as to provide potential therapeutic targets for skin-related diseases such as burns, wounds, and aging. Methods Skin keratinocytes and fibroblasts were isolated from newborn mice. To fully understand the heterogeneity of gene expression between keratinocytes and fibroblasts, differentially expressed genes (DEGs) between the two cell types were detected by RNA-seq technology. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the known genes of keratinocytes and fibroblasts and verify the RNA-seq results. Results Transcriptomic data showed a total of 4309 DEGs (fold-change > 1.5 and q-value

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.

Clinical treatment of Osteoarthritis (OA) remains a challenge due to the poor self-regeneration ability of cartilage. Deer antler is the only cartilage tissue that can completely regenerate each year. Insulin-like growth factor 1 (IGF-1) is one of the major active components in the deer antler that participate in regulating the rapid regeneration of deer antler cartilage. This has led us to speculate that deer IGF-1 might potentially become a candidate drug for reducing damage and inflammation of OA. Thus, we aimed to explore the underlying mechanism of deer IGF-1 in chondrocyte proliferation, differentiation, and inflammation response. Deer, mouse, and human IGF-1 amino acid sequences and protein structures were aligned using CLUSTAL and PSIPRED. The underlying molecular mechanism of deer IGF-1 on primary chondrocytes was investigated by RNA-sequencing (RNA-seq) technology combined with various experiments. Cytokine interleukin-1β (IL-1β) was used to induce the inflammation response of primary chondrocytes. We found that deer IGF-1 was more similar to human IGF-1 than mouse IGF-1. qRT-PCR and immunofluorescence assay indicated that deer IGF-1 had stronger effects than mouse IGF-1. We also found that the deer IGF-1 enhanced the expression of cell proliferation, differentiation, and extracellular matrix (ECM)-related genes, but decreased the expression of ECM-degrading genes. Deer IGF-1 also attenuated the IL-1β-induced inflammatory and ECM degradation in chondrocytes. This study provides insight into the molecular mechanisms of deer IGF-1 on primary chondrocyte viability and presents a candidate for combatting inflammatory responses in OA development.

Eucommia leaves are dry leaves of Eucommia ulmoides which have long been considered as a functional health food for the treatment of hypertension, hypercholesterolemia, fatty liver, and osteoporosis. With the recent development of Chinese medicine, Eucommia leaves are widely used for tonifying the kidneys and strengthening bone. However, the specific molecular mechanism of Eucommia leaves for strengthening bone remains largely unknown. Osteoblasts are the main functional cells of bone formation; thus, it is essential to study the effect of Eucommia leaves on osteoblasts to better understand their mechanism of action. In the present study, we prepared an aqueous extract of Eucommia leaves (ELAE) and determined its content by high-performance liquid chromatography (HPLC). The effects of ELAE on MC3T3-E1 cells were investigated by CCK-8 assay, alkaline phosphatase (ALP), and Alizarin red S staining assays, combined with RNA sequencing (RNA-seq) and qRT-PCR validation. We demonstrated that ELAE had a significant promoting effect on the proliferation of MC3T3-E1 cells and significantly enhanced extracellular matrix synthesis and mineralization, which were achieved by regulating various functional genes and related signaling pathways. ELAE significantly increased the expression level of genes promoting cell proliferation, such as Rpl10a, Adnp, Pex1, Inpp4a, Frat2, and Pcdhga1, and reduced the expression level of genes inhibiting cell proliferation, such as Npm1, Eif3e, Cbx3, Psmc6, Fgf7, Fxr1, Ddx3x, Mbnl1, and Cdc27. In addition, ELAE increased the expression level of gene markers in osteoblasts, such as Col5a2, Ubap2l, Dkk3, Foxm1, Col16a1, Col12a1, Usp7, Col4a6, Runx2, Sox4, and Bmp4. Taken together, our results suggest that ELAE could promote osteoblast proliferation, differentiation, and mineralization and prevent osteoblast apoptosis. These findings not only increase our understanding of ELAE on the regulation of bone development but also provide a possible strategy to further study the prevention and treatment of osteogenic related diseases by ELAE.

Background Delayed union of most tibial fractures due to their special anatomical structures.So an effective animal model is very important to study the mechanism and method of fracture healing.However, due to the small tibia of mice, the operation is difficult, and the surgical model requires high surgical skills. The construction of the fixation model of intramedullary nail for this fracture has improved and simplified the traditional fixation model of intramedullary nail, which not only achieves the purpose of constructing the fracture model, but also makes it more simple and effective.Therefore, the aim of the current study was to develop a new mouse model to study fracture healing of tibia. Methods We chose a combination between an open osteotomy and intramedullary stabilization. The 22G needle was inserted into the fracture end in a closed manner by using an open approach for osteotomy at the middle and lower 1/3 level of the tibia.Fractured tibia were analyzed using microcomputed tomography and histology at days 7,14,21and 28after surgery. All animals displayed normal limb loading and a physio-logical gait pattern within the first three days after fracture. No animals were lost due to surgery or anesthesia. Results X-ray confirmed that the fracture types obtained by the fracture modeling method were transverse fractures. X-ray, Micro-CT, immunohistochemistry, histological staining and Real-time PCR showed that the fracture healing of mice was typical endochondral ossification, with high repeatability. Conclusion The mouse tibial fracture model established by intramedullary nailing is safe, rapid and simple. Its fracture healing is a typical intrachondral ossification with high repeatability, which can be better used for the study of molecular mechanism and clinical transformation of fracture healing and bone metabolism.

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.

Deer antlers are unique cranial appendages capable of regeneration and rapid growth. In addition, deer antlers have been widely used in traditional Chinese medicine to promote the function of the kidneys, reproductive system, bones and nervous system. It has been shown that water-soluble substances are the major bioactive components within the deer antlers. In this study, we prepared aqueous extracts from deer antlers during a rapid growth stage. We investigated the effects of antler extracts on primary chondrocytes by analyzing their protein expression patterns using isobaric tags for relative and absolute quantitation technology. We demonstrated that antler extracts promote chondrocyte proliferation and prevent chondrocyte differentiation and apoptosis by controlling multiple cellular processes involved in genomic stability, epigenetic alterations, ribosome biogenesis, protein synthesis and cytoskeletal reorganization. Antler extracts significantly increased the expression levels of proliferation markers Mki67 and Stmn1 and differentiation inhibitor Acp5 as well as cellular apoptosis inhibitors Ndufa4l2 and Rcn1. Thus, this study has greatly expanded our current knowledge of the molecular effects of antler extracts on chondrocytes. It has also shed new light on possible strategies to prevent damage to and to treat cartilage and its related diseases by using aqueous extracts from growing Sika deer antlers.

Cartilage is a resilient and smooth connective tissue that is found throughout the body. Among the three major types of cartilage, namely hyaline cartilage, elastic cartilage, and fibrocartilage, hyaline cartilage is the most widespread type of cartilage predominantly located in the joint surfaces (articular cartilage, AC). It remains a huge challenge for orthopedic surgeons to deal with AC damage since it has limited capacity for self-repair. Xiphoid cartilage (XC) is a vestigial cartilage located in the distal end of the sternum. XC-derived chondrocytes exhibit strong chondrogenic differentiation capacity. Thus, XC could become a potential donor site of chondrocytes for cartilage repair and regeneration. However, the underlying gene expression patterns between AC and XC are still largely unknown. In the present study, we used state-of-the-art RNA-seq technology combined with validation method to investigate the gene expression patterns between AC and XC, and identified a series of differentially expressed genes (DEGs) involved in chondrocyte commitment and differentiation including growth factors, transcription factors, and extracellular matrices. We demonstrated that the majority of significantly up-regulated DEGs (XC vs. AC) in XC were involved in regulating cartilage regeneration and repair, whereas the majority of significantly up-regulated DEGs (XC vs. AC) in AC were involved in regulating chondrocyte differentiation and maturation. This study has increased our knowledge of transcriptional networks in hyaline cartilage and elastic cartilage. It also supports the use of XC-derived chondrocytes as a potential cell resource for cartilage regeneration and repair.

Background Deer Sinew serves as a medicinal food, and has been used for treating skeletal diseases, especially bone diseases in a long history. Thus, it could become an alternative option for the prevention and therapeutic remedy of bone-related diseases. In our previous study, we established an optimal extraction process of the enzymatic hydrolysates from Chinese Sika deer sinews (DSEH), and we demonstrated that DSEH significantly promoted the proliferation of MC3T3-E1 cells (an osteoblast-like cell line) with a certain dose-effect relationship. However, the precise molecular mechanism of deer sinew in regulating bone strength is still largely unknown. The aim of this study was to explore the underlying molecular mechanism of DSEH on MC3T3-E1 cells proliferation and extracellular matrix synthesis. Methods Preparation and quality control were performed as previously described. The effect of DSEH at different administrated concentrations on cell proliferation was measured using both CCK-8 and MTT assays, and the capacity of DSEH on extracellular matrix synthesis was detected by Alizarin red staining and quantification. The gene expression pattern change of MC3T3-E1 cells under the treatment of DSEH was investigated by RNA-seq analysis accompanied with validation methods. Results We demonstrated that DSEH promoted MC3T3-E1 cell proliferation and extracellular matrix synthesis by regulating multiple functional genes. DSEH significantly increased the expression levels of genes that promoted cell proliferation such as Gstp1, Timp1, Serpine1, Cyr61, Crlf1, Thbs1, Ctgf, P4ha2, Sod3 and Nqo1. However, DSEH significantly decreased the expression levels of genes that inhibited cell proliferation such as Mt1, Cdc20, Gas1, Nrp2, Cmtm3, Dlk2, Sema3a, Rbm25 and Hspb6. Furthermore, DSEH mildly increased the expression levels of osteoblast gene markers. Conclusions Our findings suggest that DSEH facilitate MC3T3-E1 cell proliferation and extracellular matrix synthesis to consolidate bone formation and stability, but prevent MC3T3-E1 cells from oxidative stress-induced damage, apoptosis and further differentiation. These findings deepened the current understanding of DSEH on regulating bone development, and provided theoretical support for the discovery of optional prevention and treatment for bone-related diseases.

Background Deer antler is a zoological exception due to its fantastic characteristics, including amazing growth rate and repeatable regeneration. Deer antler has been used as a key ingredient in traditional Chinese medicine relating to kidney and bone health for centuries. The aim of this study was to dissect the molecular regulation of deer antler extract (DAE) on xiphoid cartilage (XC). Methods The DAE used in this experiment was same as the one that was prepared as previously described. The specific pathogen-free (SPF) grade Sprague-Dawley (SD) rats were randomly divided into blank group (n =10) and DAE group (n =10) after 1-week adaptive feeding. The DAE used in this experiment was same as the one that was prepared as previously described. The rats in DAE group were fed with DAE for 3 weeks at a dose of 0.2 g/kg per day according to the body surface area normalization method, and the rats in blank group were fed with drinking water. Total RNA was extracted from XC located in the most distal edge of the sternum. Illumina RNA sequencing (RNA-seq) in combination with quantitative real-time polymerase chain reaction (qRT-PCR) validation assay was carried out to dissect the molecular regulation of DAE on XC. Results We demonstrated that DAE significantly increased the expression levels of DEGs involved in cartilage growth and regeneration, but decreased the expression levels of DEGs involved in inflammation, and mildly increased the expression levels of DEGs involved in chondrogenesis and chondrocyte proliferation. Conclusions Our findings suggest that DAE might serve as a complementary therapeutic regent for cartilage growth and regeneration to treat cartilage degenerative disease, such as osteoarthritis.

In-stent restenosis (ISR) is the main factor affecting the outcome of percutaneous coronary intervention (PCI), and its main pathological feature is neointimal hyperplasia. Huotan Jiedu Tongluo decoction (HTJDTLD) is an effective traditional Chinese medicine (TCM) prescription for the treatment of vascular stenosis diseases. However, the precise anti-ISR mechanism of HTJDTLD remains unclear. Here, we investigated whether HTJDTLD can inhibit the excessive activation of endoplasmic reticulum stress (ERS) and reduce the level of autophagy factors through regulating the PERK-eIF2α-ATF4 pathway, thereby inhibiting the proliferation of the intima of blood vessels damaged by balloon injury (BI) and preventing the occurrence of ISR. In this study, a 2F Fogarty balloon was used to establish a common carotid artery (CCA) BI model in male Sprague-Dawley rats. Then, HTJDTLD (16.33 g/kg/d) or atorvastatin (1.19 mg/kg/d) was administered by gavage. Four weeks later, hematoxylin-eosin (HE) and Masson staining of the injured CCA were performed to observe the histological changes in the CCA. Immunohistochemistry (IHC) was used to assess the proliferation and dedifferentiation of vascular smooth muscle cells (VSMCs) in the CCA. Western blotting and RT-PCR were used to measure the expression of ERS- and autophagy-related proteins and mRNAs in the CCA. The results indicated that HTJDTLD significantly alleviated BI-induced carotid artery intimal hyperplasia and fibrosis and reduced the neointimal area (NIA) and NIA/medial area (MA) ratio. In addition, HTJDTLD inhibited the proliferation and dedifferentiation of VSMCs, reduced the expression of proliferating cell nuclear antigen (PCNA), and increased the smooth-muscle-α-actin- (SMα-actin-) positive area. HTJDTLD also significantly reduced the expression of the ERS-related factors: GRP78, p-PERK/PERK, p-eIF2α/eIF2α, ATF4, and CHOP. In addition, the expression of the autophagy-related factors, Beclin1, LC3B, and ATG12, was significantly decreased. In addition, in vitro experiments showed that HTJDTLD inhibited the above-mentioned ERS signal molecules in human umbilical vein endothelial cells (HUVEC) and rat aortic smooth muscle cells (A7R5) induced by tunicamycin (TM) and played a crucial role in protecting cells from damage. HTJDTLD may be a very promising drug for the treatment of ISR.

Background Deer antler is considered as a precious traditional Chinese medicinal material and has been widely used to reinforce kidney’s yang, nourish essence, and strengthen bone function. The most prominent bioactive components in deer antler are water-soluble proteins that play potential roles in bone formation and repair. The aim of this study was to explore the molecular control and therapeutic targets of deer antler extract (DAE) on articular cartilage. Methods DAE was prepared as previously described. All rats were randomly divided into Blank group and DAE group (10 rats per group) after 7-day adaptive feeding. The rats in DAE group were orally administrated with DAE at a dose of 0.2 g/kg per day for 3 weeks, and the rats in Blank group were fed with drinking water. Total RNA was isolated from the articular cartilage of knee joints. RNA sequencing (RNA-seq) experiment combined with quantitative real-time polymerase chain reaction (qRT-PCR) verification assay was carried out to explore the molecular control and therapeutic targets of DAE on articular cartilage. Results We demonstrated that DAE significantly increased the expression levels of functional genes involved in cartilage formation, growth, and repair and decreased the expression levels of susceptibility genes involved in the pathophysiology of osteoarthritis. Conclusions DAE might serve as a candidate supplement for maintaining cartilage homeostasis and preventing cartilage degeneration and inflammation. These effects were possibly achieved by accelerating the expression of functional genes involved in chondrocyte commitment, survival, proliferation, and differentiation and suppressing the expression of susceptibility genes involved in the pathophysiology of osteoarthritis. Thus, our findings will contribute towards deepening the knowledge about the molecular control and therapeutic targets of DAE on the treatment of cartilage-related diseases.

Additional file 1: Table S1.

Xianling Gubao Capsule (XGC), a kind of capsule preparation of Chinese herbal officially approved for sale by the National Medical Products Administration (NMPA), has the effect of tonifying kidney and strengthening bones. Although the impact of XGC in treating bone diseases has been widely studied, the effect of XGC in kidney injury is unknown yet. The kidney injury model is established by intraperitoneal injection with cadmium chloride (CdCl2). Before model establishment, each XGC group was pregavaged with XGC for 10 d. After 10 d, CdCl2 was injected intraperitoneally into the model group and each XGC group, each XGC group continued to be gavaged with XGC for 4 weeks, and the control group was gavaged with equal doses of distilled water once daily. The level of serum urea nitrogen (BUN) and serum creatinine (Cr) is evaluated by kit. The effect of XGC on protecting kidney injury in mice with kidney injury is analyzed by histopathology (HE stain), immunohistochemistry (IHC), and real-time fluorescence quantitative PCR (RT-qPCR). The results show that CdCl2 significantly increases the level BUN and Cr in serum and results in remarkable pathological changes in the nephron, including tubule edema, congestion, and necrosis. While oral administration of XGC can significantly decrease BUN and Cr in serum and prevent and protect the kidney from the above injuries. In addition, the protein expression of p-mTOR was remarkably reduced, and the ratio of LC3II/LC3I protein and mRNA was significantly increased in mice with oral administration of XGC. Our findings suggest that XGC can prevent and protect kidney injury by improving the state of renal tubular hyperemia and necrosis and reduce the level of BUN and Cr in cadmium poisoning mice.

Background: Deer antler is considered as a precious traditional Chinese medicinal material, and has been widely used to reinforce kidney’s yang, nourish essence and strengthen bone function. The most prominent bioactive components in deer antler are water-soluble proteins that play potential roles in bone formation and repair. The aim of this study was to explore the molecular control and therapeutic targets of deer antler extract (DAE) on articular cartilage.Methods: DAE was prepared as previously described. All rats were randomly divided into Blank group and DAE group (10 rats per group) after 7-day adaptive feeding. The rats in DAE group were orally administrated with DAE at a dose of 0.2 g/kg per day for 3 weeks and the rats in Blank group were fed with drinking water. Total RNA was isolated from the articular cartilage of knee joints. RNA sequencing (RNA-seq) experiment combined with quantitative real-time polymerase chain reaction (qRT-PCR) verification assay were carried out to explore the molecular control and therapeutic targets of DAE on articular cartilage.Results: We demonstrated that DAE significantly increased the expression levels of functional genes involved in cartilage formation, growth and repair, and decreased the expression levels of susceptibility genes involved in the pathophysiology of osteoarthritis. Conclusions: DAE might serve as a candidate supplement for maintaining cartilage homeostasis, and preventing cartilage degeneration and inflammation. These effects were possibly achieved by accelerating the expression of functional genes involved in chondrocyte commitment, survival, proliferation and differentiation, and suppressing the expression of susceptibility genes involved in the pathophysiology of osteoarthritis. Thus, our findings will contribute towards deepening the knowledge about the molecular control and therapeutic targets of DAE on the treatment of cartilage related diseases.

Background Deer antler is considered as a precious traditional Chinese medicinal material, and has been widely used to reinforce kidney’s yang, nourish essence and strengthen bone function. The most prominent bioactive components in deer antler are water-soluble proteins that play potential roles in bone formation and repair. The aim of this study was to explore the molecular control and therapeutic targets of deer antler extract (DAE) on articular cartilage. Methods DAE was prepared as previously described. All rats were randomly divided into Blank group and DAE group (10 rats per group) after 7-day adaptive feeding. The rats in DAE group were orally administrated with DAE at a dose of 0.2 g/kg per day for 3 weeks and the rats in Blank group were fed with drinking water. Total RNA was isolated from the articular cartilage of knee joints. RNA sequencing (RNA-seq) experiment combined with quantitative real-time polymerase chain reaction (qRT-PCR) verification assay were carried out to explore the molecular control and therapeutic targets of DAE on articular cartilage. Results We demonstrated that DAE played a potential role in promoting cartilage formation, growth and repair, and inhibiting cartilage degeneration and inflammation. These effects were possibly achieved by accelerating the expression of functional genes involved in chondrocyte commitment, survival, proliferation and differentiation, and suppressing the expression of susceptibility genes involved in the pathophysiology of osteoarthritis. Conclusions DAE might serve as a chondroprotective agent for treating cartilage degeneration and inflammation by boosting the ability of cartilage growth and repair. Thus, our findings will contribute towards deepening the knowledge about the molecular control and therapeutic targets of DAE on the treatment of osteoarthritis.

Background Deer antlers have become a valuable model for biomedical research due to the capacities of regeneration and rapid growth. However, the molecular mechanism of rapid antler growth remains to be elucidated. The aim of the present study was to compare and explore the molecular control exerted by the main beam and brow tine during rapid antler growth. Methods The main beams and brow tines of sika deer antlers were collected from Chinese sika deer (Cervus nippon) at the rapid growth stage. Comparative transcriptome analysis was conducted using RNA-Seq technology. Differential expression was assessed using the DEGseq package. Functional Gene Ontology (GO) enrichment analysis was accomplished using a rigorous algorithm according to the GO Term Finder tool, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was accomplished with the R function phyper, followed by the hypergeometric test and Bonferroni correction. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to verify the RNA levels for differentially expressed mRNAs. Results The expression levels of 16 differentially expressed genes (DEGs) involved in chondrogenesis and cartilage development were identified as significantly upregulated in the main beams, including transcription factor SOX-9 (Sox9), collagen alpha-1(II) chain (Col2a1), aggrecan core protein (Acan), etc. However, the expression levels of 17 DEGs involved in endochondral ossification and bone formation were identified as significantly upregulated in the brow tines, including collagen alpha-1(X) chain (Col10a1), osteopontin (Spp1) and bone sialoprotein 2 (Ibsp), etc. Conclusion These results suggest that the antler main beam has stronger growth capacity involved in chondrogenesis and cartilage development compared to the brow tine during rapid antler growth, which is mainly achieved through regulation of Sox9 and its target genes, whereas the antler brow tine has stronger capacities of endochondral bone formation and resorption compared to the main beam during rapid antler growth, which is mainly achieved through the genes involved in regulating osteoblast and osteoclast activities. Thus, the current research has deeply expanded our understanding of the intrinsic molecular regulation displayed by the main beam and brow tine during rapid antler growth.

Background Guzhi Zengsheng Zhitongwan (GZZSZTW) is an effective Chinese medicinal formulation for the treatment of osteoarthritis (OA) designed according to the “kidney governing bone” theory, which has been widely used as a golden guide for treating bone and cartilage diseases in traditional Chinese medicine. The aim of this study was to explore the molecular mechanism underlying its effects on the bone and kidney. Methods Preparation and quality control were performed as previously described. Since GZZSZTW is orally administered in the form of pills prepared in boiled water, the Chinese materia medica (CMM) mixture of this formula was extracted with distilled water by a reflux method and was then filtered through a 0.45-μm Hollow Fiber Cartridge (GE Healthcare, USA). The filtrate was freeze-dried by a Heto PowerDry LL3000 Freeze Dryer (Thermo, USA) and stored at − 80 °C. The effects of GZZSZTW on gene expression and regulation of both kidney and bone tissues were investigated using a state-of-the-art RNA-seq technology. Results We demonstrated that GZZSZTW could enhance kidney function and suppress bone formation and resorption by modulating the activities of osteoblast and osteoclast, and might subsequently contribute to the inhibition of osteophyte formation during the process of OA. These effects might be achieved by the synergistic interactions of various herbs and their active components in GZZSZTW, which increased the expression levels of functional genes participating in kidney function, regulation, and repair, and then decreased the expression levels of genes involved in bone formation and resorption. Thus, our findings were consistent with the “kidney governing bone” theory, which has been widely used as a guide in clinical practice for thousands of years. Conclusions This study has deepened the current knowledge about the molecular effects of GZZSZTW on bone and kidney regulation. Furthermore, this study might be able to provide possible strategies to further prevent and treat joint diseases by using traditional Chinese medicinal formulations following the “kidney governing bone” theory.

Additional file 2: Table S2. List of the top 30 highly expressed DEGs in the main beams (main beams vs. brow tines)

Additional file 2: Table S2 Statistical analysis of differentially expressed genes in bone and kidney (GZZSZTW vs. Blank)

Additional file 3: Table S3 Primer sequences for qRT-PCR validation

Additional file 4: Table S4. Gene-specific primers used for qRT-PCR verification

Additional file 1: Table S1. Statistics for the sequencing and assembly results

Additional file 3: Table S3. List of the top 30 highly expressed DEGs in the brow tines (main beams vs. brow tines)

Additional file 1: Table S1. Statistical summary of sequencing and read assembly

Deer antlers are unique mammalian appendages that grow faster than any other known organ. During the rapid growth stage at 60 days after casting the previous antler, the maximal antler growth rate is up to or even more than 2 cm/day. Antler growth is driven by the growth center located in the antler tip. The growth center consists of several tissue layers from distal to proximal, including the skin, mesenchyme, precartilage, and cartilage. To analyze the gene expression patterns of the antler growth center in a tissue-differential manner and explore the molecular mechanism responsible for rapid antler growth, we used an RNA-Seq method to analyze gene expression patterns in different tissues of the Sika deer antler growth center during the rapid growth stage. We demonstrated considerable diversity in the expression levels of functional genes among different tissues within the antler growth center. These tissue-differentially expressed genes included transcription factors, growth factors, and extracellular matrix proteins. We identified a series of genes that contribute to chondrogenesis from mesenchymal cell condensation to chondrocyte differentiation. The genes identified in the antler growth center at the rapid growth stage provide valuable insight into the physiological mechanisms underlying rapid antler growth.

Deer antlers are amazing natural appendages that grow faster than any other known mammalian bone. Antler growth occurs at the tip and is initially cartilage, which is later replaced by bone tissue. However, little is known regarding the precise role of cooperation between cell lineages and functional genes in regulating antler growth, and molecular mechanisms responsible for rapid growth remain elusive. In this study, we use an RNA-Seq approach to elucidate the full spectrum of cell lineages, functional genes, and their cooperative interactions during antler growth. We identify Sox9 as a pivotal transcription factor during chondrogenesis and skeletal development expressed in the chondrocyte lineage from the multipotent mesenchymal precursor stage through most subsequent cell differentiation stages with a particularly strong activity in proliferating and prehypertrophic chondrocytes. Furthermore, we analyze the miRNA expression patterns at initial growth stage and rapid growth stage and identify several miRNAs that involve in regulating antler chondrogenesis and rapid growth. Among these miRNAs, miR-140 plays pivotal role during antler growth by targeting Sox9 and vice versa.

Chondrocytes are the sole cell type present within cartilage, and play pivotal roles in controlling the formation and composition of health cartilage. Chondrocytes maintain cartilage homeostasis through proliferating, differentiating and synthesizing different types of extracellular matrices. Thus, the coordinated proliferation and differentiation of chondrocytes are essential for cartilage growth, repair and the conversion from cartilage to bone during the processes of bone formation and fracture healing. Runx3, a transcription factor that belongs to the Runx family, is significantly upregulated at the onset of cartilage mineralization and regulates both early and late markers of chondrocyte maturation. Therefore, Runx3 may serve as an accelerator of chondrocyte differentiation and maturation. However, the underlying molecular mechanism of Runx3 in regulating chondrocyte proliferation and differentiation remains largely to be elucidated. In the present study, we used state-of-the-art RNA-seq technology combined with validation methods to investigate the effect of Runx3 overexpression or silencing on primary chondrocyte proliferation and differentiation, and demonstrated that Runx3 overexpression possibly inhibited chondrocyte proliferation but accelerated differentiation, whereas Runx3 silencing possibly promoted chondrocyte proliferation but suppressed differentiation. Furthermore, Runx3 overexpression possibly decreased the expression levels of Sox9 and its downstream genes via Sox9 cartilage-specific enhancers, and vice versa for Runx3 silencing.

Background Guzhi Zengsheng Zhitongwan (GZZSZTW) is an effective formula of traditional Chinese herbal medicine and has been widely applied in the treatment of joint diseases for many years. The aim of this study was to dissect the molecular targets and signaling pathways of Guzhi Zengsheng Zhitongwan based on the analysis of serum proteomics. Methods The Chinese herbs of GZZSZTW were immersed in 5 l distilled water and boiled with reflux extraction method. The extract was filtered, concentrated and freeze-dried. The chemical profile of GZZSZTW extract was determined by high-performance lipid chromatography (HPLC). The 7-week old Sprague-Dawley (SD) rats in GZZSZTW groups were received oral administration at doses of 0.8, 1.05, and 1.3 g/kg per day and the rats in blank group were fed with drinking water. Serum samples were collected from the jugular veins. Primary chondrocyte viability was evaluated by CCK-8 assay. A full spectrum of the molecular targets and signaling pathways of GZZSZTW were investigated by isobaric tags for relative and absolute quantitation (iTRAQ) analysis and a systematic bioinformatics analysis accompanied with parallel reaction monitoring (PRM) and siRNA validation. Results GZZSZTW regulated a series of functional proteins and signaling pathways responsible for cartilage development, growth and repair. Functional classification analysis indicated that these proteins were mainly involved in the process of cell surface dynamics. Pathway analysis mapped these proteins into several signalling pathways involved in chondrogenesis, chondrocyte proliferation and differentiation, and cartilage repair, including hippo signaling pathway, cGMP-PKG signaling pathway, cell cycle and calcium signaling pathway. Protein–protein interaction analysis and siRNA knockdown assay identified an interaction network consisting of TGFB1, RHO GTPases, ILK, FLNA, LYN, DHX15, PKM, RAB15, RAB1B and GIPC1. Conclusions Our results suggest that the effects of GZZSZTW on treating joint diseases might be achieved through the TGFB1/RHO interaction network coupled with other proteins and signaling pathways responsible for cartilage development, growth and repair. Therefore, the present study has greatly expanded our knowledge and provided scientific support for the underlying therapeutic mechanisms of GZZSZTW on treating joint diseases. It also provided possible alternative strategies for the prevention and treatment for joint diseases by using traditional Chinese herbal formulas.

Traditional Chinese medicine has been proven to be effective in treating human diseases according to a long-term observation for more than 2000 years. However, the precise molecular mechanisms of a majority of the medications are still largely unknown. Deer antler has been clinically used as an effective animal medication in traditional Chinese medicine for many centuries. Previous studies have demonstrated that antler extracts play crucial roles in promoting bone and cartilage development, growth and repair. However, the underlying molecular mechanism remains to be elucidated. In the present study, we applied isobaric tags for relative and absolute quantitation (iTRAQ) technology and a systematic bioinformatics analysis accompanied with validation method to obtain a full spectrum of the serum protein profiles under deer antler extract treatment. We identified a complex interaction network formed by the positive regulation of Tropomyosins (Tpm1, 2 and 4), WD repeat-containing protein 1 (Wdr1), Alpha-actinin-1 (Actn1) and Destrin (Dstn) and the negative regulation of Alpha-2-macroglobulin (A2m), Serine protease inhibitor A3 N (Serpina3n) and Apolipoproteins (Apoh and Apof), which coordinately interact with multiple proteins and signaling pathways. Our results suggest that the therapeutic effects of deer antler extract on treating bone diseases might achieved though the regulation of bone formation and remodeling by controlling a series of serum proteins and signaling pathways that were essential for osteoblast and osteoclast activities. Thus, this study has greatly deepened the current knowledge about the molecular mechanism of therapeutic effects of deer antler extract on bone diseases such as osteoporosis.

Antler growth is a unique event compared to other growth and development processes in mammals. Antlers grow extremely fast during the rapid growth stage when growth rate peaks at 2 cm per day. Antler growth is driven by a specific endochondral ossification process in the growth center that is in the distal region of the antler tip. In this study, we used state-of-art RNA-seq technology to analyze the expression profiles of mRNAs and miRNAs during antler growth. Our results indicated that the expression levels of multiple genes involved in chondrogenesis and endochondral ossification, including Fn1, Sox9, Col2a1, Acan, Col9a1, Col11a1, Hapln1, Wwp2, Fgfr3, Comp, Sp7 and Ihh, were significantly increased at the rapid growth stage. Our results also indicated that there were multiple differentially expressed miRNAs interacting with differentially expressed genes with opposite expression patterns. Furthermore, some of the miRNAs, including miR-3072-5p, miR-1600, miR-34-5p, miR-6889-5p and miR-6729-5p, simultaneously interacted with and controlled multiple genes involved in the process of chondrogenesis and endochondral ossification. Therefore, we established a miRNA-mRNA regulatory network by identifying miRNAs and their target genes that were differentially expressed in the antler growth centers by comparing the rapid growth stage and the initial growth stage.

Deer antlers are amazing appendages with the fastest growth rate among mammalian organs. Antler growth is driven by the growth center through a modified endochondral ossification process. Thus, identification of signaling pathways functioning in antler growth center would help us to uncover the underlying molecular mechanism of rapid antler growth. Furthermore, exploring and dissecting the molecular mechanism that regulates antler growth is extremely important and helpful for identifying methods to enhance long bone growth and treat cartilage- and bone-related diseases. In this study, we build a comprehensive intercellular signaling network in antler growth centers from both the slow growth stage and rapid growth stage using a state-of-art RNA-Seq approach. This network includes differentially expressed genes that regulate the activation of multiple signaling pathways, including the regulation of actin cytoskeleton, calcium signaling, and adherens junction. These signaling pathways coordinately control multiple biological processes, including chondrocyte proliferation and differentiation, matrix homeostasis, mechanobiology, and aging processes, during antler growth in a comprehensive and efficient manner. Therefore, our study provides novel insights into the molecular mechanisms regulating antler growth and provides valuable and powerful insight for medical research on therapeutic strategies targeting skeletal disorders and related cartilage and bone diseases.

SOX4, together with SOX11 and SOX12, forms group C of SRY-related (SOX) transcription factors. They play key roles, often in redundancy, in multiple developmental pathways, including neurogenesis and skeletogenesis. De novo SOX11 heterozygous mutations have been shown to cause intellectual disability, growth deficiency, and dysmorphic features compatible with mild Coffin-Siris syndrome. Using trio-based exome sequencing, we here identify de novo SOX4 heterozygous missense variants in four children who share developmental delay, intellectual disability, and mild facial and digital morphological abnormalities. SOX4 is highly expressed in areas of active neurogenesis in human fetuses, and sox4 knockdown in Xenopus embryos diminishes brain and whole-body size. The SOX4 variants cluster in the highly conserved, SOX family-specific HMG domain, but each alters a different residue. In silico tools predict that each variant affects a distinct structural feature of this DNA-binding domain, and functional assays demonstrate that these SOX4 proteins carrying these variants are unable to bind DNA in vitro and transactivate SOX reporter genes in cultured cells. These variants are not found in the gnomAD database of individuals with presumably normal development, but 12 other SOX4 HMG-domain missense variants are recorded and all demonstrate partial to full activity in the reporter assay. Taken together, these findings point to specific SOX4 HMG-domain missense variants as the cause of a characteristic human neurodevelopmental disorder associated with mild facial and digital dysmorphism.

Chinese materia medica (CMM) are essential components of traditional Chinese medicine, and Chinese medicinal formulas consisting of 2 or more types of CMM are widely used. These formulations have played a pivotal role in health protection and disease control for thousands of years. Guzhi Zengsheng Zhitongwan (GZZSZTW), which represents one of the Chinese medicinal formulations, has been used for several decades to treat joint diseases. However, the exact molecular mechanism underlying its efficacy in treating osteoarthritis remains to be elucidated. In the present study, we investigated the effects of GZZSZTW on primary chondrocytes. We demonstrated that GZZSZTW significantly promoted chondrocyte viability, maintained chondrocytes in a continuous proliferative state, and prevented their further differentiation. These effects were achieved by the synergistic interactions of various herbs and their active components in GZZSZTW through an increase in the expression levels of functional genes participating in chondrocyte commitment and proliferation and a decrease in the expression levels of genes involved in chondrocyte differentiation. GZZSZTW treatment also decreased the expression levels of genes that inhibited chondrocyte proliferation. Thus, this study has greatly deepened the current knowledge about the molecular effects of GZZSZTW on chondrocytes. It has also shed new light on possible strategies to further prevent and treat cartilage-related diseases by using traditional Chinese medicinal formulations.

The Sika deer antler is well known for its unique ability to regenerate repeatedly and grow rapidly. Furthermore, it is a precious traditional Chinese medicine and has been widely used for more than 20 centuries. The major bioactive components within the antlers are water-soluble proteins, polypeptides, and free amino acids. Many studies have shown that water-soluble antler extracts play pivotal roles in wound healing, immune system modulation, anti-oxidation, and anti-inflammation. However, the exact effects on chondrocytes are still largely unknown. In this study, we prepared fresh, aqueous extracts from growing deer antlers in a rapid growth stage. We isolated the chondrocytes from neonatal mouse rib cartilage and investigated the effects of antler extracts on chondrocyte viability. We also used the RNA-Seq method to analyze the gene expression pattern under antler extract treatment. We demonstrated that fresh extracts from Sika deer antlers in a rapid growth stage significantly promoted chondrocyte viability and kept chondrocytes proliferating continuously, while blocking maturation and further differentiation. Additionally, our results indicated that antler extracts might serve as a potent anti-oxidant, anti-inflammatory agent, and immune modulator to boost the abilities of chondrocytes against oxidative, inflammatory, and immune stresses. Thus, this study has greatly deepened our current knowledge of the molecular control of antler extracts on chondrocytes. It has also shed light on possible new strategies to further prevent and treat diseases of cartilage and other related diseases.