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 [27, 28]. 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 [29, 30]. 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-the-art 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 [31-33]. Based on the KEGG enrichment analysis, the significant 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 [34-37]. 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 matrix that play key role in regulating chondrocyte metabolism and functions via cell-matrix interaction [38-42]. Scrg1 is a stimulator of chondrogenesis that has a potential role in tissue engineering of articular cartilage [43]. Tfrc, also known as CD71, is a transferrin receptor that is essential for cartilage maturation during embryonic development [44]. Vegfa, a member of the Vegf growth factor family, is a key component to support chondrocyte survival [45]. 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 [46]. Rsad2, also known as viperin, is highly expressed in the middle zone of articular cartilage [47]. Tal1 is a basic helix-loop-helix transcription factor in the articular chondrocytes, and serves as a crucial regulator during chondrocyte maturation [48].
Csgalnact1 is a glycosyltransferase that is necessary for the biosynthesis of chondroitin sulfate proteoglycans in cartilage, particularly in the proliferating chondrocytes [49]. Chrdl1, a secreted glycoprotein, is considered to be a juvenile chondrocyte-specific factor that stimulates stem cell growth [50]. Thbs3 is mainly expressed in the proliferating chondrocytes, and inhibits cartilage clarification [51]. Cd55 is a complement decay-accelerating factor in cartilage that play pivotal role in protecting chondrocytes from possible damage [52]. Nog serves as an inhibitor of bone morphogenetic proteins (BMPs), and prevents cartilage degeneration and osteoarthritis development by inhibiting Il1β and Bmp2 expression [53]. Esf1 is an essential nucleolar protein that is required for cartilage formation [54]. Arntl, also known as Bmal1, plays crucial role in controling cartilage homeostasis through modulating TGF-β signaling [55]. Clcn3 is a highly expressed channel protein in chondrocytes during cartilage development, and plays a key role in cell volume regulation [56]. Wisp3 is a multi-domain protein that maintains cartilage integrity and prevents chondrocyte hypertrophy [57]. Mug1 serves as an inhibitor of proteolytic enzyme that prevents the degradation of cartilage extracellular matrix [58]. In addition, Cdc25b, Mki67, Bub1, Ccne2, Mphosph8, G2e3, Dyrk3 and Cep70 are considered to be essential genes involved in cell proliferation [59-65]. 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 [66]. S100a4, a member of the S100 protein family, is involved in cartilage degradation of osteoarthritis pathophysiology [67]. Fabp4 is a fatty acid-binding protein that serves as a biomarker for knee osteoarthritis [68]. Creb3l1 is a transcription factor significantly upregulated at the early stage of osteoarthritis [69]. 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 [70]. Rbp7, a family member of the cellular retinol-binding proteins, is significantly upregulated in osteoarthritic chondrocytes [71]. 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 [72].
Cxcl16 (a chemokine ligand), Cd248 (a transmembrane glycoprotein), Ptx3 (an inflammation induced gene), Fcrl2 (a subtype of Fc receptor-like molecules), Tgfb1i1 (a transforming growth factor beta 1 induced gene), Nostrin (a nitric oxide synthase traffic inducer), Vwf (an adhesive and multimeric glycoprotein), Tnxb (a member of the tenascin family), Ahr (a ligand-activated transcription factor), Ifit1 (an interferon-induced protein), Il2rb (an interleukin-2 receptor subunit) and Clec9a (a C-type lectin) are involved in the inflammation associated with arthritis pathology [73-84]. Ramp3 is a receptor activity-modifying protein that is highly expressed during joint inflammation [85]. Cyp1b1 (a member of the cytochrome P450 enzyme family), Pcsk1n (an inhibitor of prohormone convertase 1), Dlx3 (a family member of homeobox proteins), Nkd2 (a regulator of inflammatory response), Mmp19 (a subtype of matrix metalloproteinases), Htra4 (a subtype of serine proteases), Aplnr (a G protein-coupled receptor of apelin), Tubb2b (a beta isoform of tubulin), Arg1 (a cytosolic manganese-dependent enzyme) and Clic5 (a chloride intracellular channel protein) are reported to be highly expressed under osteoarthritic condition [86-95]. Thus, these results suggest that DAE might serve as a candidate supplement for preventing cartilage degeneration and inflammation.
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.