Investigating the molecular control of deer antler extract on articular cartilage

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.

day, which represents the fastest rate of cartilaginous tissue growth in the mammal kingdom [11].
Antler growth is driven by the growth center that is located in the distal tip consisted of mesenchyme and cartilage 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 from distal tip to proximal base, and the wax slice is located in the distal tip of antler (growth center), which has the highest percentage of bioactive components [13,14]. In our previous studies, we generated the deer antler extract (DAE), and the proportion of protein was 70% according to the protein concentration assay [15].
We also performed a series of analyses to investigate the effects of deer antler extract (DAE) on chondrocyte proliferation, differentiation, and apoptosis and demonstrated that freshly aqueous extracts from sika deer antlers at rapid growth stage could enhance chondrocyte viability and promote chondrocyte proliferation, but inhibit chondrocyte differentiation, maturation, and apoptosis. Furthermore, our results suggested that DAE play potential roles in boosting the abilities of chondrocytes against oxidative, inflammatory, and immune stresses [15,16].
In the present study, we carried out state-of-the-art RNA sequencing (RNA-seq) experiment combined with quantitative real-time polymerase chain reaction (qRT-PCR) verification assay to explore the molecular control and therapeutic targets of DAE on articular cartilage. 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. Thus, 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.

DAE preparation
The DAE used in the following experiments was the same as the ones that were prepared as previously described [15]. All experiments were approved by the Institutional Animal Ethics Committee of Changchun University of Chinese Medicine. Briefly, deer antlers in the rapid growth phase were obtained from three 4-year-old adult sika deers. The antlers were chopped into small pieces and thoroughly washed with ice water. The clean antler pieces were completely homogenized with a Tissue Homogenizer (Voshin, China) and centrifuged with an Eppendorf 5804R Refrigerated Centrifuge (Eppendorf, Germany). The supernatant was further clarified by filtering through a Hollow Fiber Membrane Filter Column (GE Healthcare, USA) and lyophilized with a Heto PowerDry LL3000 Freeze Dryer prior to storage at − 80°C (Thermo, USA).

Experimental animals
Twenty male Sprague-Dawley (SD) rats (SPF grade, 7 weeks old) were obtained from the Changchun Yisi laboratory animal technology Co, Ltd. (Changchun, China) with production license number SCXK (Ji) 2016-0003. The rats were housed with a constant temperature of 23°C accompanied with a relative humidity of 50% in an air-conditioned room and exposed to a 12/12-h (light/ dark) cycle. All animal protocols were approved by the Institutional Animal Care and Use Committee of Changchun University of Chinese Medicine, and all experimental procedures were performed in accordance with corresponding standards and guidelines.

Drug administration and cartilage collection
All rats were randomly divided into Blank group and DAE group (10 rats per group) after 7-day adaptive feeding. The drug administration was carried out as previously described [5]. 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. The administrated dose for DAE in rat experiment was calculated based on the body surface area normalization method [17]. Articular cartilage from each rat was harvested in the early morning after 3 weeks of DAE administration. Briefly, all rats were euthanized with CO 2 inhalation and cervical dislocation to assure death. Articular cartilage from either the Blank group or the DAE group was carefully removed from the underlying subchondral bone from the left knee joint with a scalpel blade accompanied with a stereo microscope (Nikon, Japan) following Katagiri's methods [18] and stored at − 80°C for RNA extraction.

RNA isolation and sequencing
Cartilage from each group was pooled together and pulverized into a powder in liquid nitrogen, respectively. Total RNA was isolated from the cartilage samples with the TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The quality of RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Illumina 2 × 150 paired-end mRNA libraries were prepared with the TruSeq Stranded mRNA kit (Illumina, USA) according to the manufacturer's instructions. Transcriptome sequencing was carried out by RNA-seq method on an Illumina HiSeq 2500 platform (Illumina, USA).

RNA-seq data analysis
After RNA-seq, raw reads in FASTQ format were first processed by perl scripts. High-quality clean reads were generated by removing the low-quality reads and adapter sequences. The clean reads from each sample were aligned to the rat (Rattus norvegicus) reference genome via HISAT software [19]. Gene expression levels were evaluated by calculating the relative transcript abundance using the FPKM algorithm [20]. Genes with an FPKM smaller than 0.2 were considered not expressed and removed [21]. The transcripts were annotated using the BLAST program against the National Center for Biotechnology Information (NCBI) non-redundant (NR) and Swiss-Prot protein databases [22]. Differentially expressed genes (DEGs) between the Blank group and DAE group were identified using the DEGseq R package [23]. Transcripts with a log 2 fold change ≥ 1 or ≤ − 1 and with a p value ≤ 0.001 were defined as DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was analyzed using an R function phyper, and the hypergeometric test and Bonferroni correction were carried out for multiple testing corrections. GO terms or pathways with an adjusted p value (Q value) less than 0.05 were recognized to be significantly enriched [24].

Verification of RNA-seq data by qRT-PCR
To verify the RNA-seq data, qRT-PCR was carried out to detect the expression levels of DEGs identified by RNAseq analysis. Briefly, total RNA was isolated using TRIzol reagent (Invitrogen, USA) following the manufacturer's instructions. cDNA synthesis was performed using an iScript cDNA Synthesis Kit (Bio-Rad, USA) and amplified using a SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, USA) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) under standard amplification conditions. Gene expression levels were calculated according to the 2 −ΔΔCT method by normalizing the rat glyceraldehyde 3-phosphate dehydrogenase gene (Gapdh) [25].

Statistic summary of RNA-seq data
The messenger RNAs (mRNAs) of articular cartilage from rats with or without the treatment of DAE were sequenced using Illumina paired-end sequencing technology. The raw reads were uploaded in the NCBI Sequence Read Archive (SRA) database under BioProject accession number PRJNA642711. As shown in Table 1, 39,698,758 (Blank group) and 40,202,822 (DAE group) clean reads were obtained after trimming away the lowquality and adapter sequences, respectively, and the quality control results showed that the Q30 value was greater than 94%, and the GC content was around 51%. Therefore, the sequencing results were accurate and reliable for further analysis. By performing data mapping and annotation, 37,619,438 (Blank group) and 38,032, 508 (DAE group) reads were mapped to the rat (Rattus norvegicus) genome; subsequently, 12,821 out of 15,664 (Blank group) and 12,902 out of 15,733 (DAE group) transcripts were obtained by annotating against the nonredundant (NR) NCBI protein database and Swiss-Prot database, respectively.

DEGs identification, GO and KEGG enrichment analysis
By comparing the Blank group and DAE group, 308 genes were identified as DEGs under the established criteria (log 2 fold change ≥ 1 or ≤ − 1 and p ≤ 0.001), including 208 upregulated genes (log 2 fold change ≥ 1 and p ≤ 0.001) and 100 downregulated genes (log 2 fold change ≤ − 1 and p ≤ 0.001), as shown in Table 2. GO enrichment analyses were carried out to gain insight into the biological functions of DEGs under DAE treatment, as shown in Fig. 1. Under the category of cellular component, the significantly enriched GO terms were mainly classified into extracellular region, extracellular region part, extracellular space, extracellular matrix, and proteinaceous extracellular matrix; under the category of molecular function, the significantly enriched GO terms were mainly classified into protein binding, transporter activity, substrate-specific transporter activity, protein heterodimerization activity, and tetrapyrrole binding; under the category of biological process, the significantly enriched GO terms were mainly classified into singlemulticellular organism process, developmental process, single-organism developmental process, anatomical structure development, and multicellular organism development. KEGG pathway enrichment analyses were carried out to further explore the possible functional pathways of DEGs under DAE treatment, as shown in   Fig. 2; the significant enriched pathways were predominantly mapped to thyroid hormone signaling pathway, protein digestion and absorption, PI3K-AKT signaling pathway, nitrogen metabolism, ECM-receptor interaction, and cell adhesion molecules (CAMs).

Gene expression levels of DEGs validated by qRT-PCR
The expression levels of a series of DEGs were validated by qRT-PCR assay, including 6 significantly upregulated genes and 6 significantly downregulated genes. The specific gene primers for qRT-PCR were listed in Table 5.
The relative fold change of each gene was normalized to the internal reference gene Gapdh. The expression levels of the selected DEGs validated by qRT-PCR exhibited similar expression patterns as those of the RNA-seq analysis, as shown in Fig. 3.

Discussion
For many years, RNA-seq has become an essential tool for studying the dynamic and complex characteristics of the transcriptome, especially for the mRNA molecules, which encode proteins following the central dogma of molecular biology [26,27]. Meanwhile, many scientists in the research field of Chinese medicine have paid more and more attention to explore the molecular control of Chinese herbs and formulations by taking advantage of RNA-seq technology [28,29]. Our previous studies have shown that DAE play potential roles in regulating chondrocyte proliferation and differentiation [15,16]. Therefore, in the present study, we further investigated the effects of DAE on articular cartilage using a state-of-theart RNA-seq technology accompanied with validation method to obtain the precise molecular mechanism of DAE on cartilage homeostasis. In total, 308 DEGs were identified, including 208 upregulated genes and 100 downregulated genes by comparing DAE-treated group with Blank group (DAE vs. Blank). According to the GO enrichment analysis, the significantly enriched GO terms were predominantly involved in extracellular matrix synthesis, binding activity, and developmental process. Those functional gene groups play pivotal roles in regulating cartilage homeostasis [30][31][32]. Based on the KEGG enrichment analysis, the significantly enriched pathways were predominantly involved in thyroid hormone signaling pathway, protein digestion and absorption, PI3K-AKT signaling pathway, nitrogen metabolism, ECM-receptor interaction, and cell adhesion molecules (CAMs). Among those enriched signaling pathways, thyroid hormone signaling pathway, PI3K-AKT signaling pathway, ECM-receptor interaction, and cell adhesion molecules (CAMs) have been considered to play crucial roles in articular cartilage maintenance and osteoarthritis pathogenesis [33][34][35][36]. Thus, these results suggest that DAE play potential role in regulating articular cartilage homeostasis by controling multiple functional group genes and signaling pathways.
Among the significantly upregulated DEGs under DAE treatment, 36 genes that participate in cartilage formation, growth, and repair were identified. For instance, Hapln1, Col9a1, Comp, Cnmd, Matn3, Col27a1, Matn1, and Dpt are extracellular matrixes that play key roles in regulating chondrocyte metabolism and functions via cell-matrix interaction [37][38][39][40][41]. Scrg1 is a stimulator of chondrogenesis that has a potential role in tissue engineering of articular cartilage [42]. Tfrc, also known as CD71, is a transferrin receptor that is essential for cartilage maturation during embryonic development [43]. Vegfa, a member of the Vegf growth factor family, is a key component to support chondrocyte survival [44]. Ca2, Ca12, and Ca9 are family members of carbonic anhydrases, which are important for cartilage homeostasis. All of them are significantly expressed in the articular chondrocytes, and Ca2 is mainly localized in the proliferating chondrocytes [45]. Rsad2, also known as viperin, is highly expressed in the middle zone of articular cartilage [46]. Tal1 is a basic helix-loop-helix transcription factor in the articular chondrocytes and serves as a crucial regulator during chondrocyte maturation [47].
Csgalnact1 is a glycosyltransferase that is necessary for the biosynthesis of chondroitin sulfate proteoglycans in cartilage, particularly in the proliferating chondrocytes [48]. Chrdl1, a secreted glycoprotein, is considered to be a juvenile chondrocyte-specific factor that stimulates stem cell growth [49]. Thbs3 is mainly expressed in the proliferating chondrocytes and inhibits cartilage clarification [50]. Cd55 is a complement decay-accelerating factor in the cartilage that plays pivotal role in protecting chondrocytes from possible damage [51]. Nog serves as an inhibitor of bone morphogenetic proteins (BMPs) and prevents cartilage degeneration and osteoarthritis development by inhibiting Il1β and Bmp2 expression [52]. Esf1 is an essential nucleolar protein that is required for cartilage formation [53]. Arntl, also known as Bmal1, plays crucial role in controling cartilage homeostasis through modulating TGF-β signaling [54]. Clcn3 is a highly expressed channel protein in chondrocytes during cartilage development and plays a key role in cell volume regulation [55]. Wisp3 is a multi-domain protein that maintains cartilage integrity and prevents chondrocyte hypertrophy [56]. Mug1 serves as an inhibitor of proteolytic enzyme that prevents the degradation of cartilage extracellular matrix [57]. In addition, Cdc25b, Mki67, Bub1, Ccne2, Mphosph8, G2e3, Dyrk3, and Cep70 are considered to be essential genes involved in cell proliferation [58][59][60][61][62][63][64]. Thus, these results suggest that DAE might serve as a candidate supplement for maintaining cartilage homeostasis. Among the significantly downregulated DEGs under DAE treatment, 31 genes involved in osteoarthritis susceptibility were identified. For instance, Col1a1 and Col4a1 are collagen fibers that were associated with the progression of osteoarthritis [65]. S100a4, a member of the S100 protein family, is involved in cartilage degradation of osteoarthritis pathophysiology [66]. Fabp4 is a fatty acid-binding protein that serves as a biomarker for knee osteoarthritis [67]. Creb3l1 is a transcription factor significantly upregulated at the early stage of osteoarthritis [68]. Ccl9, also called macrophage inflammatory protein-1 gamma (MIP-1γ), is a small cytokine belonging to the CC chemokine family that was shown to be highly expressed during osteoarthritis progression [69]. Rbp7, a family member of the cellular retinol-binding proteins, is significantly upregulated in osteoarthritic chondrocytes [70]. Notch3 is a family member of Notch receptors, and genetic deletion of Notch3 or the blockade of Notch3 signaling prevents joint damage and attenuates inflammation of inflammatory arthritis [71].
In addition to the above findings, we also compared the expression levels of genes that are well known to characterize hyaline cartilage, such as Sox9, Sox5, Sox6, Wwp2, Acan, Col2a1, Col9a1, Col11a1, Hapln1, Comp, Matn1, Ptch1, Fgfr3, Runx2, and Runx3. As shown in Table S1, the expression levels of a majority of these genes were slightly upregulated under the DAE treatment. However, the expression level of Sox9 was slightly downregulated, which indicates that DAE might regulate articular chondrocytes through other transcription factors and related signaling pathways.
Nevertheless, there are still some limitations in the present study. First, this study lacks pathological analysis of animal model by performing the histological, behavioral, and biochemical evaluations; further experiments in the future still need to be carried out to investigate the effects of DAE on osteoarthritis model to get insight into their exact roles in regulating cartilage repair and regeneration. Second, this study included only a small sample size, although it is very difficult to obtain enough qualified articular cartilage from rats, which largely limits the sample size. However, more sample sizes still need to be included in future studies, and it is better to have at least 3 to 5 independent tissue samples be independently assessed for statistical analysis of a molecular outcome.

Conclusion
In summary, the present study demonstrated that 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 cartilage formation, growth, and repair 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. The asterisk *, **, and *** indicate significant differences using the Student t test with p value < 0.05, < 0.01, and < 0.001, respectively. Gene expression levels for individual genes are presented as the ratio of (fold change) the DAE group to the Blank group