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Investigating the Molecular Control of Deer Antler Extract on Articular Cartilage Baojin Yao1, Zhenwei Zhou1, Mei Zhang2, Xiangyang Leng3* and Daqing Zhao1*

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Investigating the Molecular Control of Deer Antler Extract on Articular Cartilage Baojin Yao1, Zhenwei Zhou1, Mei Zhang2, Xiangyang Leng3* and Daqing Zhao1* Yao et al. Journal of Orthopaedic Surgery and Research (2021) 16:8 https://doi.org/10.1186/s13018-020-02148-w RESEARCH ARTICLE Open Access Investigating the molecular control of deer antler extract on articular cartilage Baojin Yao1, Zhenwei Zhou1, Mei Zhang2, Xiangyang Leng3* and Daqing Zhao1* Abstract 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. Keywords: Deer antler extract, Articular cartilage, RNA sequencing, Molecular mechanism, Therapeutic targets Background deer antler are water-soluble proteins that play potential Deer antler is considered as a precious traditional Chinese roles in bone formation and repair [2–6]. However, little medicinal material and has been widely used to reinforce is known regarding their effects on cartilage development kidney’s yang, nourish essence, and strengthen bone func- and growth. Deer antler is a fantastic mammalian append- tion [1]. The most prominent bioactive components in age with characteristics of rapid growth and annually re- generation [7–9]. The longitudinal growth of the antler * Correspondence: [email protected]; [email protected] was considered as a modified endochondral ossification 3The Affiliated Hospital of Changchun University of Chinese Medicine, process that is analogous to the long bone growth origi- Changchun 130117, China nated from mesenchymal condensation, chondrocytic dif- 1Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China ferentiation, and ossification [10]. During rapid growth Full list of author information is available at the end of the article stage, deer antler is capable to grow as rapidly as 2 cm per © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yao et al. Journal of Orthopaedic Surgery and Research (2021) 16:8 Page 2 of 12 day, which represents the fastest rate of cartilaginous tis- supernatant was further clarified by filtering through a sue growth in the mammal kingdom [11]. Hollow Fiber Membrane Filter Column (GE Healthcare, Antler growth is driven by the growth center that is lo- USA) and lyophilized with a Heto PowerDry LL3000 cated in the distal tip consisted of mesenchyme and cartil- Freeze Dryer prior to storage at − 80 °C (Thermo, USA). age tissues [12]. According to the application of deer antler in traditional Chinese medicine, the antler is divided into wax slice, powder slice, blood slice, and bone slice Experimental animals from distal tip to proximal base, and the wax slice is lo- Twenty male Sprague-Dawley (SD) rats (SPF grade, 7 cated in the distal tip of antler (growth center), which has weeks old) were obtained from the Changchun Yisi la- the highest percentage of bioactive components [13, 14]. boratory animal technology Co, Ltd. (Changchun, China) In our previous studies, we generated the deer antler ex- with production license number SCXK (Ji) 2016-0003. tract (DAE), and the proportion of protein was 70% ac- The rats were housed with a constant temperature of cording to the protein concentration assay [15]. 23 °C accompanied with a relative humidity of 50% in an We also performed a series of analyses to investigate the air-conditioned room and exposed to a 12/12-h (light/ effects of deer antler extract (DAE) on chondrocyte prolif- dark) cycle. All animal protocols were approved by the eration, differentiation, and apoptosis and demonstrated Institutional Animal Care and Use Committee of Chang- that freshly aqueous extracts from sika deer antlers at chun University of Chinese Medicine, and all experimen- rapid growth stage could enhance chondrocyte viability tal procedures were performed in accordance with and promote chondrocyte proliferation, but inhibit chon- corresponding standards and guidelines. drocyte differentiation, maturation, and apoptosis. Fur- thermore, our results suggested that DAE play potential Drug administration and cartilage collection roles in boosting the abilities of chondrocytes against oxi- All rats were randomly divided into Blank group and dative, inflammatory, and immune stresses [15, 16]. DAE group (10 rats per group) after 7-day adaptive feed- In the present study, we carried out state-of-the-art ing. The drug administration was carried out as previ- RNA sequencing (RNA-seq) experiment combined with ously described [5]. The rats in DAE group were orally quantitative real-time polymerase chain reaction (qRT- administrated with DAE at a dose of 0.2 g/kg per day for PCR) verification assay to explore the molecular control 3 weeks, and the rats in Blank group were fed with and therapeutic targets of DAE on articular cartilage. We drinking water. The administrated dose for DAE in rat demonstrated that DAE significantly increased the expres- experiment was calculated based on the body surface sion levels of functional genes involved in cartilage forma- area normalization method [17]. Articular cartilage from tion, growth, and repair and decreased the expression each rat was harvested in the early morning after 3 levels of susceptibility genes involved in the pathophysi- weeks of DAE administration. Briefly, all rats were eu- ology of osteoarthritis. Thus, DAE might serve as a candi- thanized with CO2 inhalation and cervical dislocation to date supplement for maintaining cartilage homeostasis assure death. Articular cartilage from either the Blank and preventing cartilage degeneration and inflammation. group or the DAE group was carefully removed from the These effects were possibly achieved by accelerating the underlying subchondral bone from the left knee joint expression of functional genes involved in chondrocyte with a scalpel blade accompanied with a stereo micro- commitment, survival, proliferation, and differentiation scope (Nikon, Japan) following Katagiri’s methods [18] and suppressing the expression of susceptibility genes in- and stored at − 80 °C for RNA extraction. volved in the pathophysiology of osteoarthritis. Methods RNA isolation and sequencing DAE preparation Cartilage from each group was pooled together and pul- The DAE used in the following experiments was the same verized into a powder in liquid nitrogen, respectively. as the ones that were prepared as previously described Total RNA was isolated from the cartilage samples with [15]. All experiments were approved by the Institutional the TRIzol reagent (Invitrogen, USA) according to the Animal Ethics Committee of Changchun University of manufacturer’s instructions. The quality of RNA was Chinese Medicine. Briefly, deer antlers in the rapid growth assessed using an Agilent 2100 Bioanalyzer (Agilent phase were obtained from three 4-year-old adult sika Technologies, USA). Illumina 2 × 150 paired-end mRNA
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