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Antlers are the sole mammalian organs capable of continuous regeneration. This distinctive feature has evolved into various biomedical models. Research on mechanisms of antler growth, development, and ossification provides valuable insights for limb regeneration, cartilage-related diseases, and cancer mechanisms. Here, ribonucleic acid sequencing (RNA-seq) and four-dimensional data-independent acquisition (4D DIA) technologies were employed to examine gene and protein expression differences among four tissue layers of the Chinese milu deer antler: reserve mesenchyme (RM), precartilage (PC), transition zone (TZ), cartilage (CA). Overall, 4611 differentially expressed genes (DEGs) and 2388 differentially expressed proteins (DEPs) were identified in the transcriptome and proteome, respectively. Among the 828 DEGs common to both omics approaches, genes from the collagen, integrin, and solute carrier families, and signaling molecules were emphasized for their roles in the regulation of antler growth, development, and ossification. Bioinformatics analysis revealed that in addition to being regulated by vascular and nerve regeneration pathways, antler growth and development are significantly influenced by numerous cancer-related signaling pathways. This indicates that antler growth mechanisms may be similar to those of cancer cell proliferation and development. This study lays a foundation for future research on the mechanisms underlying the rapid growth and ossification of antlers.

Osteoporosis, a metabolic disorder, remains challenging to treat due to limited understanding of its underlying mechanism. The annual cycle of "cyclic physiological osteoporosis (CPO)" and its full reversal in male deer represents a unique natural model for studying this condition. Deer antlers, weighing up to 25 kg/pair, derive over 60% of their mineral contents from deer skeleton during mineralization. Based on the literature, we propose to divide CPO and its reversal into two phases: Phase I (approximately 115 days): from hard antler casting to the end of antler linear growth, marked by simultaneous robust antler ossification and CPO development; and Phase II (up to 165 days): from end of Phase I to the onset of antler skin shedding, characterized by complete antler mineralization and CPO reversal. This review analyzes the paradoxical occurrence of robust antler ossification and skeleton CPO within the same endocrine microenvironment during phase I; total antler mineralization and full reversal of deer skeleton CPO in phase II. Furthermore, we will discuss potential insights for osteoporosis treatment using deer materials from the period of Phase II. Our goal is to identify novel substances and therapies that could be applied in clinical setting to effectively treat osteoporosis., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage., Methods: A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry., Results: ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco's modified eagle medium) groups ( p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs ( p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1)., Conclusions: ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Background: Sika deer (Cervus nippon) holds significance among cervids, with three genomes recently published. However, these genomes still contain hundreds of gaps and display significant discrepancies in continuity and accuracy. This poses challenges to functional genomics research and the selection of an appropriate reference genome. Thus, obtaining a high-quality reference genome is imperative to delve into functional genomics effectively., Findings: Here we report a high-quality consensus genome of male sika deer. All 34 chromosomes are assembled into single-contig pseudomolecules without any gaps, which is the most complete assembly. The genome size is 2.7G with 23,284 protein-coding genes. Comparative genomics analysis found that the genomes of sika deer and red deer are highly conserved, an approximately 2.4G collinear regions with up to 99% sequence similarity. Meanwhile, we observed the fusion of red deer's Chr23 and Chr4 during evolution, forming sika deer's Chr1. Additionally, we identified 607 transcription factors (TFs) that are involved in the regulation of antler development, including RUNX2, SOX6, SOX8, SOX9, PAX8, SIX2, SIX4, SIX6, SPI1, NFAC1, KLHL8, ZN710, JDP2, and TWST2, based on this consensus reference genome., Conclusions: Our results indicated that we acquired a high-quality consensus reference genome. That provided valuable resources for understanding functional genomics. In addition, discovered the genetic basis of sika-red hybrid fertility and identified 607 significant TFs that impact antler development., (© 2024. The Author(s).)

Objective: Velvet antler polypeptide (VAP) has been shown to play important roles in the immune and nervous systems. The purpose of this study was to investigate the protective effects of VAP on cerebral ischemic injury with the involvement of NF-κB signaling pathway in vitro., Materials and Methods: PC-12 cells stimulated by oxygen-glucose deprivation/reperfusion (OGD/R) was used to mimic cerebral ischemic injury in vitro. The levels of ROS, SOD, and intracellular concentrations of Ca2+ were measured by the relevant kits. Meanwhile, the expressions of inflammatory cytokines (IL-6, IL-1β, and TNF-α) were determined by ELISA kit assay. In addition, MTT, EdU, and flow cytometry assays were used to measure the cell proliferation and apoptosis. Besides which, the related proteins of NF-κB signaling pathway were measured by western blotting assay., Results: VAP alleviated cerebral ischemic injury by reducing OGD/R-induced oxidative stress, inflammation, and apoptosis in PC-12 cells in a time dependent manner. Mechanistically, VAP inhibited the levels of p-p65 and p-IkB-α in a time dependent manner, which was induced by OGD/R operation. Moreover, NF-κB agonist diprovocim overturned the suppression effects of VAP on OGD/R-induced oxidative stress, inflammation, and apoptosis in PC-12 cells., Conclusions: The results demonstrate that VAP may alleviate cerebral ischemic injury by suppressing the activation of NF-κB signaling pathway., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Hepatic stellate cell (HSC) activation is a crucial step in the development of liver fibrosis. Previous studies have shown that antler stem cells (AnSCs) inhibited HSC activation, suggesting that this may be achieved through secreting or releasing peptides. This study aimed to investigate whether AnSC-derived peptides (AnSC-P) could reduce liver fibrosis. The results showed that AnSC-P effectively reduced liver fibrosis in rats. Furthermore, we found that thymosin β10 (Tβ-10) was rich in AnSC-P, which may be the main component of AnSC-P contributing to the reduction in liver fibrosis. A further study showed that Tβ-10 reduced liver fibrosis in rats, with a reduction in HYP and MDA levels in the liver tissues, a decrease in the serum levels of ALP, ALT, AST, and TBIL and an increase in TP and ALB. Moreover, Tβ-10 decreased the expression levels of the genes related to the TGF-β/SMAD signaling pathway in vivo. In addition, Tβ-10 also inhibited TGF-β1-induced HSC activation and decreased the expression levels of the TGF-β/SMAD signaling pathway-related genes in HSCs in vitro. In conclusion, antler Tβ-10 is a potential drug candidate for the treatment of liver fibrosis, the effect of which may be achieved via inhibition of the TGFβ/SMAD signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lactobacillus curvatus HY7602 fermented antler (FA) ameliorates sarcopenia and improves exercise performance by increasing muscle mass, muscle fiber regeneration, and mitochondrial biogenesis; however, its anti-fatigue and antioxidant effects have not been studied. Therefore, this study aimed to investigate the anti-fatigue and antioxidant effects and mechanisms of FA. C2C12 and HepG2 cells were stimulated with 1 mM of hydrogen peroxide (H 2 O 2 ) to induce oxidative stress, followed by treatment with FA. Additionally, 44-week-old C57BL/6J mice were orally administered FA for 4 weeks. FA treatment (5-100 μg/mL) significantly attenuated H 2 O 2 -induced cytotoxicity and reactive oxygen species (ROS) production in both cell lines in a dose-dependent manner. In vivo experiments showed that FA treatment significantly increased the mobility time of mice in the forced swimming test and significantly downregulated the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), and lactate. Notably, FA treatment significantly upregulated the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione/oxidized glutathione ratio (GSH/GSSG) and increased the mRNA expression of antioxidant genes ( SOD1 , SOD2 , CAT , GPx1 , GPx2 , and GSR ) in the liver. Conclusively, FA is a potentially useful functional food ingredient for improving fatigue through its antioxidant effects.

Background: Type 1 diabetes mellitus (T1D) represents a severe threat to human health. Persistent hyperglycemia and dyslipidemia can lead to damaged liver function, while effective interventions for these complications are currently lacking. Deer antler stem cells (AnSCs), a novel type of adult stem cells, significantly reduced liver injury, which was speculated to be achieved through the paracrine pathway., Methods: In this study, AnSC-conditioned medium (AnSC-CM) was used to treat C57BL/6 mice with T1D symptoms induced by streptozotocin (STZ). The therapeutic effects of AnSC-CM on T1D were evaluated, and the underlying mechanism was investigated., Results: It was shown that AnSC-CM alleviated the T1D symptom: decreased body weight, increased blood glucose levels and islet lesions, and reduced insulin secretion. Moreover, AnSC-CM treatment improved liver function and mitigated liver injury in T1D mice. Impressively, the therapeutic effects of AnSC-CM on T1D were better than those of bone marrow mesenchymal stem cell-CM (BMSC-CM). The mechanistic study revealed that AnSC-CM significantly downregulated the NF-κB signaling pathway in both pancreatic and liver tissues., Conclusions: Therapeutic effects of AnSC-CM on STZ-induced T1D and liver injury may be achieved through targeting the NF-κB signaling pathway., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Deer antlers are a bony organ solely able to acquired distinct unique attributes during evolution and all these attributes are against thus far known natural rules. One of them is as the fastest animal growing tissue (2 cm/day), they are remarkably cancer-free, despite high cell division rate. Although tumor-like nodules on the long-lived castrate antlers in some deer species do occur, but they are truly benign in nature. In this review, we tried to find the answer to this seemingly contradictory phenomenon based on the currently available information and give insights into possible clinic application. The antler growth center is located in its tip; the most intensive dividing cells are resident in the inner layer of reserve mesenchyme (RM), and these cells are more adopted to osteosarcoma rather than to normal bone tissues in gene expression profiles but acquire their energy mainly through aerobic oxidative phosphorylation pathway. To counteract propensity of neoplastic transformation, antlers evolved highly efficient apoptosis exactly in the RM, unparalleled by any known tissues; and annual wholesale cast to jettison the corps. Besides, some strong cancer suppressive genes including p53 cofactor genes and p53 regulator genes are highly positively selected by deer, which would have certainly contributed to curb tumorigenesis. Thus far, antler extracts and RM cells/exosomes have been tried on different cancer models either in vitro or in vivo, and all achieved positive results. These positive experimental results together with the anecdotal folklore that regular consumption of velvet antler is living with cancer-free would encourage us to test antlers in clinic settings., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Aim: We isolated a novel polypeptide PNP1 from velvet antler and investigated the role of PNP1 in ischemia reperfusion and its associated mechanism., Methods: We built the ischemia reperfusion mouse model by the middle cerebral artery occlusion (MCAO) approach. Thereafter, PNP-1 was injected via the tail vein, and neurological function was scored. Meanwhile, the tissue injury level was detected through hematoxylin & eosin (HE) and immunohistochemical (IHC) staining, inflammatory factor levels were determined with enzyme-linked immunosorbent assay (ELISA), while protein levels through Western blotting. In addition, vascular endothelial cells were used to construct the oxygen-glucose deprivation (OGD) injury model in vitro, so as to detect the intervention effect of PNP1 on endothelial injury. Additionally, microglial cells were utilized to construct the inflammatory injury model to examine the impact of PNP1 on the polarization of microglial cells., Results: PNP1 suppressed hypoxic cerebral injury in MCAO mice, decreased the tissue inflammatory factors, promoted tissue angiogenesis, and reduced the ischemic penumbra area. Experimental results in vitro demonstrated that, PNP1 suppressed vascular endothelial cell injury, and inhibited microglial M1 polarization as well as inflammatory response., Conclusion: Velvet antler polypeptide PNP1 isolated in this study has the anti-ischemic cerebral injury effect, and its mechanism is associated with suppressing vascular endothelial cell injury and microglial cell inflammatory response., Competing Interests: Declaration of competing interest No competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram-negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

MicroRNAs (miRNAs) play a crucial role in maintaining the balance between the rapid growth and suppression of tumorigenesis during antler regeneration. This study investigated the role of a novel miRNA, PC-3p-2869 (miR-PC-2869), in antler growth and its therapeutic potential in human osteosarcoma and chondrosarcoma. Stem-loop RT-qPCR showed that miR-PC-2869 was expressed extensively in diverse layers of antler tissues. Overexpression of miR-PC-2869 suppressed the proliferation and migration of antler cartilage cells. Similarly, heterologous expression of miR-PC-2869 reduced the proliferation, colony formation, and migration of osteosarcoma cell line MG63 and U2OS and chondrosarcoma cell line SW1353. Moreover, 18 functional target genes of miR-PC-2869 in humans were identified based on the screening of the reporter library. Among them, 15 target genes, including CDK8, EEF1A1, and NTN1, possess conserved miR-PC-2869-binding sites between humans and red deer ( Cervus elaphus ). In line with this, miR-PC-2869 overexpression decreased the expression levels of CDK8, EEF1A1, and NTN1 in MG63, SW1353, and antler cartilage cells. As expected, the knockdown of CDK8, EEF1A1, or NTN1 inhibited the proliferation and migration of MG63, SW1353, and antler cartilage cells, demonstrating similar suppressive effects as miR-PC-2869 overexpression. Furthermore, we observed that CDK8, EEF1A1, and NTN1 mediated the regulation of c-myc and cyclin D1 by miR-PC-2869 in MG63, SW1353, and antler cartilage cells. Overall, our work uncovered the cellular functions and underlying molecular mechanism of antler-derived miR-PC-2869, highlighting its potential as a therapeutic candidate for bone cancer.

The antler is the unique mammalian organ found to be able to regenerate completely and periodically after loss, and the continuous proliferation and differentiation of mesenchymal cells and chondrocytes together complete the regeneration of the antler. Circular non-coding RNAs (circRNAs) are considered to be important non-coding RNAs that regulate body development and growth. However, there are no reports on circRNAs regulating the antler regeneration process. In this study, full-transcriptome high-throughput sequencing was performed on sika deer antler interstitial and cartilage tissues, and the sequencing results were verified and analyzed. The competing endogenous RNA (ceRNA) network related to antler growth and regeneration was further constructed, and the differentially expressed circRNA2829 was screened out from the network to study its effect on chondrocyte proliferation and differentiation. The results indicated that circRNA2829 promoted cell proliferation and increased the level of intracellular ALP. The analysis of RT-qPCR and Western blot demonstrated that the mRNA and protein expression levels of genes involved in differentiation rose. These data revealed that circRNAs play a crucial regulatory role in deer antler regeneration and development. CircRNA2829 might regulate the antler regeneration process through miR-4286-R+1/FOXO4.

A significant variety of cell growth factors are involved in the regulation of antler growth, and the fast proliferation and differentiation of various tissue cells occur during the yearly regeneration of deer antlers. The unique development process of velvet antlers has potential application value in many fields of biomedical research. Among them, the nature of cartilage tissue and the rapid growth and development process make deer antler a model for studying cartilage tissue development or rapid repair of damage. However, the molecular mechanisms underlying the rapid growth of antlers are still not well studied. MicroRNAs are ubiquitous in animals and have a wide range of biological functions. In this study, we used high-throughput sequencing technology to analyze the miRNA expression patterns of antler growth centers at three distinct growth phases, 30, 60, and 90 days following the abscission of the antler base, in order to determine the regulatory function of miRNA on the rapid growth of antlers. Then, we identified the miRNAs that were differentially expressed at various growth stages and annotated the functions of their target genes. The results showed that 4319, 4640, and 4520 miRNAs were found in antler growth centers during the three growth periods. To further identify the essential miRNAs that could regulate fast antler development, five differentially expressed miRNAs (DEMs) were screened, and the functions of their target genes were annotated. The results of KEGG pathway annotation revealed that the target genes of the five DEMs were significantly annotated to the "Wnt signaling pathway", "PI3K-Akt signaling pathway", "MAPK signaling pathway", and "TGF-β signaling pathway", which were associated with the rapid growth of velvet antlers. Therefore, the five chosen miRNAs, particularly ppy-miR-1, mmu-miR-200b-3p, and novel miR-94, may play crucial roles in rapid antler growth in summer.

Velvet antler is a traditional Chinese medicine with various pharmacological values, which is an important raw material for traditional Chinese medicinal wine. Nevertheless, the chemical compositions and bioactivities of velvet antler residue used for making medicinal wine are rarely reported, leading to a waste of resources. In this study, a velvet antler protein (VA-pro) was extracted from velvet antler residue by simulating the gastrointestinal digestion, and its composition, structural characteristics and in vivo anti-tumor activities were determined and investigated. VA-pro possessed high purity with a relatively low molecular weight as 22.589 kDa under HPLC, one- and two-dimensional electrophoresis, and it contained high contents of Pro, Gly, Glu and Ala. Besides, the secondary structure of VA-pro was dominated by β-turn and β-sheet, and VA-pro possessed similar protein sequence, isoelectric point and amino acid compositions to hypothetical protein G4228&#95;020061. The in vivo results substantiated that VA-pro could improve the body weights and immune organ indices, increase the expressions of sera cytokines and regulate the distributions of T and B lymphocytes subsets in peripheral blood of S180 tumor-bearing mice. Furthermore, VA-pro could effectively inhibit solid S180 tumors growth by inducing S phase cell cycle arrest mediated through mitochondria. To summarize, our study provided theoretical support that VA-pro had the potential to be used as an immunopotentiator in immunocompromised or cancer-bearing hosts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Antlers have been widely studied due to their unique physiological characteristics, such as rapid growth, periodic shedding and regeneration. However, little is known about how antler growth is regulated by long non-coding RNA (lncRNA). The aim of the present study was to identify the lncRNAs expression profile and explore the function of lncRNAs during the antler growth. Herein, RNA-sequencing technology (RNA-seq) was performed on the three growth periods (early developmental period: EP, middle developmental period: MP, later developmental period: LP) of male sika deer ( Cervus nippon ) antler, 16 differentially expressed lncRNAs (DE lncRNAs) and 11 DE lncRNAs were identified in EP vs MP and MP vs LP related to cell proliferation and cell differentiation, respectively. Finally, lncRNAs-mRNAs co-expression networks were constructed based on the identified DE lncRNAs and their potential trans-target genes. The result reveals that lncRNAs may play diverse roles in different periods of antler growth. It provides a novel perspective for revealing the molecular mechanism of antler growth.

Long non-coding RNA play an importantr role in the differentiation of chondrocytes. This study aims to explore the role of long non-coding RNA in the transcriptional regulation of Notch4. In previous studies, it has been found that Notch signal can be used as the downstream of TGF-β signal to affect the proliferation and differentiation of deer antler chondrocytes, but the specific mechanism remains unclear. Here we found that lncRNA27785.1 was involved in the regulation of TGF-β/ Smad3 signal and Notch4 gene. The overexpression lncRNA27785.1 can negatively regulate the expression of Notch4 to inhibit cell proliferation and differentiation, while interference with lncRNA27785.1 can promote the expression of Notch4 gene to promote the proliferation and differentiation of chondrocytes. Subsequently, through luciferase experiment and CHIP experiment, we found that lncRNA27785.1 is regulated by Smad3 transcription, and Smad3 inhibited the expression of lncRNA27785.1. In addition, activated TGF-β signaling can reduce the inhibitory effect of lncRNA27785.1 on Notch4 signaling. In summary, we found that lncRNA27785.1 and TGF-β/Smad3 play an important role in Notch4 signaling. Our findings provided evidence to explain how TGF-β signaling regulate the Notch signaling pathway to influence chondrocyte proliferation and differentiation by a specific lncRNA27785.1., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The demand for low-salt foods is increasing due to their health benefits. Umami is known to enhance salty, and a large amount of umami components have been identified in edible fungi. 5'-nucleotides and umami amino acids from nine species of edible fungi were quantified. The equal umami concentration (EUC) in nine edible fungi was within the range of 37.7-1317.72 g MSG/100 g, and umami intensity as determined by electronic tongue and sensory evaluation was within the range of 11.22-13.53 and 2.85-5.55, respectively. Antler fungus had the highest umami intensity. Umami amino acids and nucleotides could increase salty intensity of NaCl at medium and high concentrations. The enzymatic hydrolysate of Antler fungus at higher concentrations could more effectively enhance salty taste of NaCl at lower concentration. This synergistic effect between umami and salty indicates that Antler fungus can potentially be used as an ingredient in low-salt foods., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Although peroxiredoxin 2 (PRDX2) plays a vital role in relieving oxidative stress, its physiological function in cartilage development remains almost unknown. In this study, we found that the expression of PRDX2 significantly increased in the chondrocytes compared with pre-chondrocytes. PRDX2 knockdown significantly decreased the expression of extracellular matrix (ECM) protein (Col2a and Aggrecan), which led to blocked cartilage formation. Moreover, PRDX2 knockdown also inhibited the expression of connective tissue growth factor (CTGF). CTGF is an important growth factor that regulates synthesis of ECM proteins. We explored the possible regulatory mechanism by which PRDX2 regulated the expression of CTGF. Our results demonstrated that PRDX2 knockdown downregulated the expression of CTGF by inhibiting Wnt5a/Yes-associated protein 1 (YAP1) pathway. In addition, PRDX2 knockdown promoted the expression of interleukin 6 (IL-6), indicating PRDX2 expression had an anti-inflammatory function during antler growth. Mechanistically, PRDX2 knockdown promoted cartilage matrix degradation by activating the IL-6-mediated Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) signaling pathway. These results reveal that PRDX2 is a potential regulator that promotes cartilage extracellular matrix synthesis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint deformity and disability. Deer velvet antler (DA), a traditional Chinese medicine, has been used to treat various types of arthritis for several thousands of years, but the underlying mechanisms are unknown. Herein, we investigated the anti-arthritic and anti-inflammatory effects of DA in vitro and in vivo . The ethyl acetate layer of DA ethanol extract (DA-EE-EA) was used to treat tumor necrosis factor (TNF)-[Formula: see text]-stimulated fibroblast-like synoviocyte MH7A cells, collagen-induced arthritis DBA/1 mice, and SKG mice with zymosan-induced arthritis. DA-EE-EA reduced nitric oxide production, prostaglandin E2 levels, and levels of pro-inflammatory cytokines including interleukin (IL)-1[Formula: see text], IL-6, and IL-8 in MH7A cells. DA-EE-EA also downregulated the phosphorylation of mitogen-activated protein kinase p38 and c-Jun N-terminal kinase and the translocation of nuclear factor kappa B p65. Intraperitoneal injection of DA-EE-EA for 3 weeks substantially reduced clinical arthritis scores in vivo models. Pathohistological images of the hind paws showed that DA-EE-EA reduced immune cell infiltration, synovial hyperplasia, and cartilage damage. The levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha, IL-1[Formula: see text], IL-6, IL-8, IL-17A, and interferon-gamma, decreased in the hind paw homogenates of DA-EE-EA-treated mice. We also identified several potential components, such as hexadecanamide, oleamide, erucamide, and lysophosphatidylcholines, that might contribute to the anti-inflammatory effects of DA-EE-EA. In conclusion, DA-EE-EA has the potential to treat RA by regulating inflammatory responses. However, the individual components of DA-EE-EA and the underlying anti-inflammatory mechanisms need further investigation in future studies.

Antler is a special bone tissue that has the ability to regenerate completely periodically. It is the fastest growing bone in the animal kingdom. Antler provides a valuable research model for bone growth and mineralization. Antler grows longitudinally by endochondral ossification with their growth center located in its tip. Many scholars have carried out detailed studies on morphology and gene expression of antler tip. However, few scholars have analyzed the protein expression patterns of antler tip at different development stages. This study used label-free proteomics approach to analyze the protein expression dynamics of the antler tip in six developmental periods (15, 25, 45, 65, 100 and 130 days after the previous antler cast) and costal cartilage. In result, 2052 proteins were confidently quantified, including 1937 antler proteins and 1044 costal cartilage proteins. Moreover, 913 antler core proteins and 132 antler-special proteins were obtained. Besides, the stages special proteins and differentially expressed proteins (DEPs) in different development stages were analyzed. A total of 875 DEPs were determined by one-way AVOVA. It is found that the growth period (15, 25, 45 and 65 days) showed more up-regulated protein including several chondrogenesis-associated proteins (collagen types II, collagen types XI, HAPLN1, PAPSS1 and PAPSS2). In ossification stages, the up-regulated proteins related with lysosome (CTSD, CTSB, MMP9, CAII) indicated that the antler has higher bone remodeling activity. Given the up-regulated expression of immune-related molecules (S100A7, CATHL7, LTF, AZU1, ELANE and MPO), we speculate that the local immune system may contribute to the ossification of antler tip. In conclusion, proteomics technology was used to deeply analyze the protein expression patterns of antler at different development stages. This provides a strong support for the research on the molecular regulation mechanism of rapid growth and ossification of velvet antler.

Ethnopharmacological Relevance: Hard antler extract (HAE) is a traditional Chinese medicine and has potent antitumor, antioxidative, anti-inflammatory, and immunomodulatory activities. Previous studies have demonstrated that HAE can inhibit human prostate cancer metastasis and murine breast cancer proliferation. However, the effect of HAE on human breast cancer cells has not been clarified., Aim of the Study: To investigate the effects and underlying mechanism of HAE on self-renewal of stem-like cells and spontaneous and transforming growth factor (TGF)-β1-enhanced wound healing, invasion and epithelial-mesenchymal transition (EMT) in breast cancer cells., Methods: HAE was prepared from sika deer by sequential enzymatic digestions and the active compounds were determined by HPLC. The effects of HAE on the viability, mammosphere formation, wound healing and invasion of MDA-MB-231 and SK-BR3 cells were determined. The impact of HAE treatment on spontaneous and TGF-β1-promoted EMT and the nuclear factor (NF)-κB signaling in breast cancer cells was examined by quantitative RT-PCR and western blotting., Results: Treatment with HAE at varying concentrations did not change the viability of breast cancer cells. However, HAE at 0.25 or 0.5 mg/mL significantly reduced the number and size of formed mammospheres, and inhibited spontaneous and TGF-β1-enhanced wound healing, invasion and EMT in MDA-MB-231 and SK-BR3 cells in a dose-dependent manner. TGF-β1 treatment significantly decreased IκBα expression and increased NF-kBp65 phosphorylation in breast cancer cells, indicating that TGF-β1 enhanced NF-κB signaling. In contrast, HAE treatment attenuated the spontaneous and TGF-β1-enhanced NF-κB signaling in breast cancer cells., Conclusion: Our data indicated that HAE inhibited the self-renewal of stem-like cells and spontaneous and TGF-β1-enhanced wound healing, invasion and EMT in breast cancer cells by attenuating the NF-κB signaling in vitro., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The desert hedgehog (Dhh) is crucial for spermatogenesis and Leydig cell differentiation, but little is known regarding its physiological function in cartilage. In this study, Dhh mRNA was abundant in antler chondrocytes, where it advanced cell proliferation concomitant with accelerated transition from the G1 to the S phase and induced elevation of the hypertrophic chondrocyte markers, Col X and Runx2. Silencing of Ptch1 resulted in appreciable Smo accumulation and enhanced rDhh stimulation of Smo, whose impediment by cyclopamine obscured the proliferative function of Dhh and alleviated its guidance of chondrocyte differentiation. Further analysis evidenced the noteworthy positive action of Smo in the bridging between Dhh and Gli transcription factors. Obstruction of Gli1 by GANT58 caused the failed stimulation of Col X and Runx2 by rDhh. Analogously, siRNA against Gli1-3 hindered chondrocyte differentiation in the context of rDhh. Simultaneously, Gli transcription factors mediated the regulation of Dhh on Foxa1, Foxa2, and Foxa3, whose knockdown impaired chondrocyte differentiation. Attenuation of Foxa antagonized the augmentation of Col X and Runx2 generated by rDhh. Collectively, Dhh signaling through its target Foxa appears to induce antler chondrocyte proliferation and differentiation., (© 2020 Wiley Periodicals, Inc.)

Deer antlers are the only mammalian organs capable of complete renewal. Antler renewal is a stem cell-based [antler stem cells (ASCs)] process. Maintenance and activation of the ASCs require them to be located in a specialized microenvironment (niche), and to interact with the cells resident in the niche. Based on previous experiments we found that niche of the ASCs is provided by the closely associated enveloping skin, which currently was known includes dermal papilla cells (DPCs) and epidermal cells. Antler generation/regeneration are triggered by the interactions between ASCs and the niche. In the present study, we established an in vitro co-culture system in which ASCs and DPCs, were cultured together to mimic the in vivo state. A MLEFF strategy was adopted to identify the interactive molecules from the co-culture system. In total, 128 molecules were identified and over 60% belonged to exosomes. Important biological processes that were activated by these molecules included osteoblast differentiation, angiogenesis, and the PI3K-AKT signaling pathway. In so doing, we have significantly simplified the process for identifying interactive molecules, which may be the key signals for triggering antler formation/renewal. Further study of these molecules will help us to gain insights into the mechanism of mammalian organ regeneration.

Velvet antler (VA) is crucial and precious nourishment in China and some countries in Southeast Asia and has excellent anti-fatigue effect. The incidence of fatigue syndrome has increased gradually. VA can be a potential source of anti-fatigue products. Therefore, we investigated the anti-fatigue activity of different sections (upper, middle, and basal section) of VA from different species (red deer and sika deer) via loading swimming test in mice. Furthermore, nucleosides are one kind of active components in VA which could considerably reduce fatigue in mice. In order to explore whether the nucleosides are correlated with anti-fatigue effect, the contents of eight nucleosides (uracil, cytidine, hypoxanthine, xanthine, thymine, inosine, guanosine, and adenosine) were determined simultaneously using high-performance liquid chromatography. The results indicated that the swimming time in mice was increased from basal to upper section, which was consistent with the change trend of the total contents of eight nucleosides of VA. Therefore, we speculated that the contents of nucleosides in VA may affect its anti-fatigue effect. Furthermore, the contents of nucleosides were also used as a reference for evaluating the quality of different parts of VA obtained from red and sika deer., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

Velvet antlers (VA) have been used as medicines and nutraceuticals for over 2000 years. Meanwhile, deer antlers are the only mammalian organs that can fully regenerate after annual shedding. The antler formation and regeneration rely on the stem cells resident in antlerogenic periosteum (AP), transplantation of which can induce ectopic antler formation. Here, a comprehensive quantitative proteomic analysis of antlerogenic periosteal cells (AP cells), compared with the adjacent facial periosteal cells (FP cells), was carried out, from both extracellular and intracellular perspectives. In this study, the stable isotope labeling by amino acids in cell culture (SILAC) was applied to ensure the precision of quantification. Then, the protein equalization strategy and reverse-phase liquid chromatography (RPLC) separation in high pH were utilized to improve the depth of proteome profiling. Proteomics analysis of the conditioned media (CM) from AP and FP cells showed that significantly over-expressed extracellular proteins in AP cells were involved in cell proliferation, angiogenesis and neurogenesis. Combining the extracellular and intracellular proteomes, we found several potential secreted proteins might regulate antler formation and regeneration, such as SFRP4 and LUM. These results provide new insight into the underlying mechanism of antler formation and regeneration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acetaminophen (APAP)-induced acute kidney injury (AKI) has turned into a typical reason for clinic obtained renal disappointment. In any case, the advancement of prophylaxis procedures and endorsed treatments for APAP-AKI is restricted. Sika deer antler protein (SDAPR) has a protective effect on drug-induced AKI. The reason for this investigation was to clarify the material premise and atomic instrument of the defensive impact of SDAPR on APAP-induced AKI. We conducted column chromatography on SDAPR extracted with Sephadex G-100 and obtained proteins of 2 different components named sika deer antler protein 1 (SDAP1) and sika deer antler protein 2 (SDAP2), respectively. MTT assay and xCELLigence Real-Time Cell Analysis showed that SDAPR, SDAP1 and SDAP2 had protective effects on APAP-induced cytotoxicity in Human kidney tubular epithelial (HK-2) cells. Therefore, we conducted proteomic analysis on SDAPR, SDAP1 and SDAP2. What's more, we inspected them viability of avoiding renal damage in APAP mice and HK-2 cells model. Contrasted and saline, SDAPR, SDAP1 and SDAP2 pretreatment portion conditionally fundamentally constricted heights in kidney damage Markers and the histological changes of renal cylindrical wounds, diminished the quantity of apoptosis-positive rounded cells, initiated NF-E2 p45-related factor 2 (Nrf2), inhibition Forkhead box O 1 transcription factors (FoxO1) and brought down the degrees of renal oxidative stress and apoptosis prompted by APAP. The above security of SDAPR, SDAP1 and SDAP2 was nullified by the Phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) in HK-2 cells. In this manner, our outcomes exhibit that, SDAPR, SDAP1 and SDAP2 against APAP-induced oxidative stress and apoptosis by initiating Nrf2 and restraint FoxO1 through PI3K/Akt signaling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antlers are bony appendages of deer that undergo periodic regeneration from the top of permanent outgrowths (the pedicles) of the frontal bones. Of the "less familiar" bone types whose study was advocated by John Currey to gain a better understanding of structure-function relationships of mineralized tissues and organs, antlers were of special interest to him. The present review summarizes our current knowledge about the evolution, development, structure, mineralization, and biomechanics of antlers and how their formation is affected by environmental factors like nutrition. Furthermore, the potential role of antlers as a model in bone biology and several fields of biomedicine as well as their use as a monitoring tool in environmental studies are discussed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Backgroud: Reindeer is the only deer species that both male and female produce antlers, which provides a particularly interesting case in studying the differences between antlers of the two sexes. Alpha 3(VI) Collagen Gene (COL6A3), forms a microfibrillar network associated with the structural integrity and biomechanical properties, has been found to be one of the differentially expressed genes in antler mesenchyme of female and male reindeer., Objective and Methods: The promoter sequence of reindeer COL6A3 gene was obtained using the cloning technology and analyzed by the bioinformatics methods. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the COL6A3 promoter in reindeer antler mesenchyme. Real-time quantitative PCR was used to detect COL6A3 expression in the antler mesenchyme of female and male reindeer., Results: Sequence analysis revealed that the reindeer COL6A3 partial promoter sequence was 983 bp including the possible promoter region at + 105 bp to + 155 bp. Homology and phylogenetic analysis indicated that the COL6A3 promoter of reindeer had the closest genetic distance with Bos taurus, Capra hircus and Ovis aries. BSP results indicated that the methylation level of COL6A3 promoter in the female reindeer antler mesenchyme was significantly higher than in the male. Correlating with increased methylation status, we also found that COL6A3 mRNA expression in female reindeer antler mesenchyme was significantly lower than in the male., Conclusion: The higher methylation level of the COL6A3 gene in female reindeer antler mesenchyme coincides with decreased COL6A3 mRNA expression, thereby affecting the transposon silencing mechanism and possibly contributing to apparent differences of antlers in female and male reindeer.

Ruminants are the only extant mammalian group possessing bony (osseous) headgear. We obtained 221 transcriptomes from bovids and cervids and sequenced three genomes representing the only two pecoran lineages that convergently lack headgear. Comparative analyses reveal that bovid horns and cervid antlers share similar gene expression profiles and a common cellular basis developed from neural crest stem cells. The rapid regenerative properties of antler tissue involve exploitation of oncogenetic pathways, and at the same time some tumor suppressor genes are under strong selection in deer. These results provide insights into the evolutionary origin of ruminant headgear as well as mammalian organ regeneration and oncogenesis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

Glycosaminoglycans (GAG) from the velvet antlers of Sika deer (Cervus nippon) at the different growing stages (Fukurozuno, Anshi, and Santajo) of bred and wild deer were isolated and their concentrations and sulfation patterns were analyzed. GAG were digested with chondroitinase ABC, ACI, heparinase-I and -III, and keratanase-II into the corresponding repeating disaccharides of chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan, heparan sulfate (HS), and keratan sulfate. Cartilaginous tissues contained CS-DS at high concentrations with an almost equal ratio of 4- and 6-sulfates, while 4-sulfate-type CS-DS predominantly occupied ossified tissues, but at low concentrations. High O- and N-sulfation degrees of HS correspond to high ossification. Dynamic quantitative changes in CS-DS and compositional changes in CS-DS and HS were closely associated with the mineralization of deer antlers.

Osteocalcin is a major non-collagenous component of the bone extracellular matrix and is considered to be an indicative factor of osteoblast differentiation. In the present study, we detected osteocalcin expression in different antler areas and growth phases by immunohisto- chemistry. Osteocalcin was highly expressed in all areas during the mineralization period and in mesenchymal cell and chondrocyte areas during the rapid growth period. The nucleotide sequence of the osteocalcin gene in sika deer antler was determined. The open reading frame was 303 bp encoding a protein of 100 amino acids. The estimated molecular mass of osteocalcin was 10.38 kDa and the theoretical isoelectric point was 5.37. The osteocalcin gene with a 6× His-tag at the C-terminus was cloned into the pGEX-4T1 vector and expressed in Escherichia coli under optimal conditions. The recombinant soluble protein fused with GST was purified with Ni-NTA resin. The purified osteocalcin protein exhibited a significant increase in HA adhesion and promoted antler chondrocyte proliferation. Osteocalcin is an important factor in regulating the rapid growth and differentiation of deer antlers., (Copyright© by the Polish Academy of Sciences.)

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.

Deer antlers are unusual mammalian organs that can fully regenerate after annual shedding. Stem cells resident in the pedicle periosteum (PPCs) provide the main cell source for antler regeneration. Central to various cellular processes are plasma membrane proteins, but the expression of these proteins has not been well documented in antler regeneration. In the present study, plasma membrane proteins of PPCs and facial periosteal cells (FPCs) were analyzed using label-free liquid chromatography⁻mass spetrometry (LC⁻MS/MS). A total of 1739 proteins were identified. Of these proteins, 53 were found solely in the PPCs, 100 solely in the FPCs, and 1576 co-existed in both PPCs and FPCs; and 39 were significantly up-regulated in PPCs and 49 up-regulated in FPCs. In total, 226 gene ontology (GO) terms were significantly enriched from the differentially expressed proteins (DEPs). Five clusters of biological processes from these GO terms comprised responses to external stimuli, signal transduction, membrane transport, regulation of tissue regeneration, and protein modification processes. Further studies are required to demonstrate the relevancy of these DEPs in antler stem cell biology and antler regeneration.

Background: Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration., Methods: To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies., Results: Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10., Conclusion: Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.

Velvet antler (VA), the unossified antler from members of the family Cervidae, has been used in traditional Chinese medicines and health foods for over 2000 years in enhancement of kidney function and treatment or prevention of cardiovascular, immunological and gynaecological disease. The aim of this study was to investigate the anti-inflammatory effect of velvet antler water extracts from Formosan sambar deer (Rusa unicolor swinhoei, SVAE) and red deer (Cervus elaphus, RVAE). Results indicated that both SVAE and RVAE significantly reduced the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) productions in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells at concentrations above 200 μg mL -1 . SVAE seems to demonstrate a better anti-inflammatory effect than that of RVAE in vitro. Both SVAE and RAVE also enhanced the anti-inflammatory cytokine IL-10 production in LPS-stimulated RAW 264.7 cells. The results of MTT assay indicated that SVAE and RVAE did not exhibit any cytotoxicity in LPS-stimulated RAW 264.7 cells. Two-dimensional (2D) gel electrophoresis analysis revealed that the levels of 6 specific proteins were different between these two velvet antlers samples. Furthermore, the storage period was the major factor affecting the anti-inflammatory activity of SAVE. In this study, we demonstrated the difference of anti-inflammatory effect and the protein profile between SVAE and RVAE. SVAE showed better anti-inflammatory potential than RVAE. In the future, the anti-inflammatory active components and their related mechanisms should be further investigated., (Copyright © 2018. Published by Elsevier B.V.)

Understanding the role of the PI3K/AKT pathway in regulating basic antler stem cell parameters and angiogenesis may provide an insight into the mechanisms underlying mammalian appendage development. The present study took multiple approaches in vitro to investigate the effects of the PI3K/AKT pathway on antler stem cells. By addition of LY294002, proliferation rate of the antlerogenic periosteum (AP) cells was decreased significantly ( p <0.01), while the proliferation rate of the pedicle periosteum (PP) cells decreased to a lesser extent; the cytoskeleton of the AP cells was essentially collapsed; and the PP cells significantly shrunken. By addition of LY294002 or KU-0063794, formation of networking tubular structures from HUVECs in the AP or PP cell conditioned medium was significantly inhibited; whereas, expression level of VEGF-B mRNA in the AP or PP cells was decreased by the former, and increased by the latter significantly. Therefore, the results suggest that the PI3K/AKT pathway is involved in proliferation and differentiation of the AP and the PP cells, and plays a more important role in the former than in the latter.

Hypoxic-ischemic encephalopathy (HIE) is a complex condition which is associated with high mortality and morbidity. However, few promising treatments for HIE exist. In the present study, the central objective was to identify the therapeutic effect of pilose antler polypeptides (PAP) on HIE in rats. Sprague-Dawley (SD) rats (14 days old) were used and divided into three groups, including control group, hypoxic-ischemia (HI) group and PAP group. After 21 days of treatment, locomotor activity was improved in PAP-treated rats, brain atrophy was decreased and cerebral edema was mitigated to some extent. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis indicated that PAP administration decreased the expressions of inflammatory cytokines and apoptosis genes in hippocampus compared with HI group. Furthermore, the mRNA expressions of genes related to neurotrophic factors were significantly increased in the hippocampus. In addition, the expressions of oxidative stress markers were all down-regulated after PAP administration. Moreover, PAP up-regulated both the mRNA and protein levels of SDF1 and CXCR4, which may activate the SDF1/CXCR4 axis to moderate brain injury. These results suggest that PAP may be potentially used in the treatment of HIE.

Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well-known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4, and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1., (© 2017 Wiley Periodicals, Inc.)

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.

Background/aims: Sika deer (Cervus nippon Temminck) antler is traditional animal medicine of renal protection in East Asia. This study measured the effect of sika deer antler protein (SDAPR) on gentamicin (GM)-induced cytotoxicity in HEK293 cells, and investigated the effect of SDAPR against GM-induced nephrotoxicity in mice., Methods: HEK293 cells viability and oxidative stress were measured in HEK293 cells while flow cytometry was used for apoptosis analysis. The acute kidney injury biomarkers, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin c (Cys-C), were repeatedly measured by ELISA assay. ICR male mice were randomly assigned six groups: Control, GM with vehicle, single SDAPR, GM with SDAPR at three concentrations 50, 100, 200 mg/kg/d, p.o., 10 d. GM was injected for 8 consecutive days (100 mg/kg/d, i.p.). Renal function, oxidative stress and levels of inflammatory factors were measured in vivo. Renal tissues were stained with H&E to observe pathological changes., Results: Pretreatment with SDAPR (0.5-4.0 mg/mL) significantly improved cell viability. Treatment with SDAPR could reduce KIM-1, NGAL and Cys-C activity. SDAPR could improve antioxidant defense and attenuated apoptosis on HEK293 cells. SDAPR also ameliorated GM-induced histopathologic changes, and decreased blood urea nitrogen (BUN) and serum creatinine (Cr). Additionally, SDAPR significantly regulated oxidative stress marker and interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) inflammatory cytokines., Conclusion: These results show that SDAPR could be an effective dietary supplement to relieve GM-induced nephrotoxicity by improved antioxidase activity, suppressed inflammation, and inhibited apoptosis in vitro and vivo., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Antlers are the cranial appendages of deer that regenerate each year. This renewal provides a model to explore molecules involved in mammalian organ regeneration. The cellular distributions of the brain-derived neurotrophic factor (BDNF) and the isoforms of its cognate receptor Trk tyrosine kinase receptor (TrkB) were localized by immunohistochemistry in sections of growing red deer antler. BDNF and TrkB full length were widely expressed in the integument, perichondrium, periosteum and bone. The truncated isoform receptor was particularly evidenced in integument and vascular inner dermis, but very light reaction was observed in cartilage and bone, both at the site of endochondral and intramembranous ossification. These observations were also assessed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred in chondroprogenitor cells; however the full-length TrkB receptor was down regulated in osteocartilaginous compartments, in which the truncated isoform was up regulated. The truncated isoform is a dominant-negative receptor that inhibits the full length receptor signalling, even if the truncated isoform not only has this function. This study establishes the presence of BDNF and its receptor in the different cellular compartments of growing antler. Their transcripts assessed by RT-PCR indicate a local synthesis of these molecules that may contribute to the modulation of antler growth, acting as autocrine and/or paracrine factors independently of nerve supply. Among the plethora of other molecular signals and growth factors affecting the antler growth, the local production of BDNF and its cognate receptor could be of interest in understanding their role in antler renewal and to delineate the different involvement of the receptor isoforms., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Lu Rong, velvet antler (VA), is a traditional Chinese medicine, which is used as a food supplement and therapeutic drug in China, Japan, Russia, New Zealand and Southeast Asia. The regenerative characteristics of VA have resulted in great research interest, particularly regarding the fields of organ grafting and stem cell differentiation. Various VA proteomic studies verified that proteins act as the primary bioactive components of VA. The present study aimed to investigate if VA proteins (VA‑pro) influence endothelial progenitor cell (EPC) viability. Various methods have previously been used to investigate VA‑pro, including freeze‑drying technology, ultrasonic wave methods, high performance liquid chromatography‑mass spectrometry, EPCs extraction and culture. Results demonstrated that VA‑pro promoted EPCs proliferation and migration, particularly at a concentration of 1 mg/ml. Furthermore, VA‑pro increased the activation level of Notch1 intracellular domain and Hes1, and the level of phosphorylated‑Akt and phosphorylated‑mechanistic target of rapamycin. VA‑pro may therefore affect EPC viability via regulation of the Notch and Akt signaling pathways. The present study revealed the effects and potential molecular mechanism of VA‑pro on EPCs, and suggested an association between VA regeneration characteristics and the optimization of EPC viability. These findings may contribute to EPC transplantation research and aid in providing a novel treatment method for vascular diseases in the future.

Proteins are the most prominent bioactive component in deer antler velvet. The aim of the present study was to track the fate of protein of antler velvet by protein digestomics. The peptide profile identified by LC-MS/MS and the in vitro bioactivity of antler velvet aqueous extract (AAE) were investigated in simulated gastrointestinal digestion. A total of 23, 387 and 417 peptides in AAE, gastric and pancreatic digests were identified using LC-MS/MS, respectively. Collagens, the predominant proteins, released 34 peptides in gastric digests and 146 peptides in pancreatic digests. The gastric and pancreatic digests presented dipeptidyl peptidase IV (DPP-IV) and prolyl endopeptidase (PEP) inhibition activities. Four peptides from digests were proved to be DPP-IV and PEP inhibitory peptides. The results showed that the peptides released from antler velvet protein contributed to the bioactivity of antler velvet during digestion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Every spring, deer cast their old antlers and initiate a regeneration process, which yields a new set of antlers of up to 1m in length. Over the course of three months, branches of the trigeminal nerve, originating from the frontal skull, innervate velvet, a modified skin that covers the regenerating antler. The rate of growth of these axons reaches up to 2cm per day making them the fastest regenerating axons in adult mammals. Here, we aim to identify the factors secreted by velvet that promote such high speed axon growth. Our experiments with cultures of adult rat trigeminal neurons demonstrate that conditioned medium harvested from velvet organotypic cultures has greater axon growth-promoting properties than a medium conditioned by normal skin. The axon growth-promoting effects of velvet act synergistically with the extracellular matrix (ECM) protein laminin, a component of the basal lamina present in the deer antler. Our proteomic analyses identified several axon growth promoters in the velvet-conditioned medium (VCM), including soluble proteins such as nerve growth factor (NGF) and apolipoprotein A-1, as well as matrix extracellular proteins, such as periostin and SPARC. Additional in vitro analyses allowed us to determine that a synergic relationship between periostin and NGF may contribute to neurite growth-promoting effects of velvet secretome. A combinatorial approach using these factors may promote regeneration at high speeds in patients with peripheral neuropathies., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

As the only known organ that can completely regenerate in mammals, deer antler is of real significance in the field of regenerative medicine. Recent studies have shown that the regenerative capacity of the antlers comes from the pedicle periosteum and the cells resident in the periosteum possess the attributes of stem cells. Currently, the molecular mechanism of antler regeneration remains unclear. In the present study, we compared the potentiated and dormant antler stem cells using isobaric tags for the relative and absolute quantification (iTRAQ) labeling of the peptides, coupled with two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the proteome profiles. Proteins were identified by searching against the NCBI nr database and our own Cervine transcriptome database, and bioinformatics analysis was conducted to identify the differentially expressed proteins. Based on this searching strategy, we identified 169 differentially expressed proteins in total, consisting of 70 up- and 99 down-regulated in the potentiated vs. dormant antler stem cells. Reliability of the iTRAQ was confirmed via quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of selected genes. We identified transduction pathways through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, such as HIF-1 and PI3K-AKT signaling pathways that play important roles in regulating the regeneration of antlers. In summary, the initiation stage of antler regeneration, a process from dormant to potentiated states in antler stem cells, is regulated by multiple proteins and complicated signal networks., Competing Interests: The authors declare no conflict of interest.

MicroRNAs (miRNAs) can effectively regulate gene expression at the post-transcriptional level and play a critical role in tissue growth, development and regeneration. Our previous studies showed that antler regeneration is a stem cell-based process and antler stem cells reside in the periosteum of a pedicle, the permanent bony protuberance, from which antler regeneration takes place. Antlers are the only mammalian organ that can fully regenerate and hence provide a unique opportunity to identify miRNAs that are involved in organ regeneration. In the present study, we used next generation sequencing technology sequenced miRNAs of the stem cells derived from either the potentiated or the dormant pedicle periosteum. A population of both conserved and 20 deer-specific miRNAs was identified. These conserved miRNAs were derived from 453 homologous hairpin precursors across 88 animal species, and were further grouped into 167 miRNA families. Among them, the miR-296 is embryonic stem cell-specific. The potentiation process resulted in the significant regulation (>±2 Fold, q value <0.05) of conserved miRNAs; 8 miRNA transcripts were down- and 6 up-regulated. Several GO biology processes and the Wnt, MAPK and TGF-beta signaling pathways were found to be up-regulated as part of antlerogenic stem cell potentiation process. This research has identified miRNAs that are associated either with the dormant or the potentiated antler stem cells and identified some target miRNAs for further research into their role played in mammalian organ regeneration.