GJD is a Chinese medicinal preparation widely used for the treatment of osteoporotic diseases and has been used for decades in Jilin Provincial Hospital of Traditional Chinese Medicine. However, the precise molecular mechanisms behind the ability of GJD to treat these diseases remain to be elucidated. In this study, we investigated the effects of GJD on MC3T3-E1 cells using state-of-the-art RNA-seq technology. From the results of the CCK-8 assay, in a dose-dependent manner, the proliferation of MC3T3-E1 cells was significantly promoted by GJD. We then performed RNA-seq to further explore the mechanisms that are responsible for regulating cell proliferation in response to GJD treatment.
We first analysed the differentially expressed genes involved in increased cell proliferation. The significant increase in the expression levels of several genes involved in promoting cell proliferation via GJD include Adamts1, Mcam, Cyr61, Fos, Cebpd, Fosl2, Nipbl, Sema3c and Sirt1. Adamts1 is a secreted multifunctional metalloproteinase and an early responder to parathyroid hormone (PTH) in osteoblasts. Upregulation of Adamts1 promotes osteoblast growth and mineralisation and is an effective regulator of bone remodelling [18]. Mcam gene expression is in response to "metaphyseal mesenchymal progenitors" (MMPs), which play a key role in osteoblast proliferation and differentiation. MSCs express marker genes [19]. It is well known that bone morphogenetic proteins (BMPs), especially BMP-2, are key regulators of osteogenesis, and the literature demonstrates that CYR61 enhances BMP-2 mRNA and protein expression in a time- and dose-dependent manner to significantly increase proliferation and osteoblast differentiation in MC3T3-E1 cells and primary cultured osteoblasts, and is associated with multiple pathways, such as the ILK and ERK signalling pathway, and the canonical Wnt pathway [20]. Fos (also known as the c-fos gene) is part of the AP-l (activator protein 1) transcription factor, which can increase MC3T3-E1 cell proliferation through JNK, ERK and p38 MAPK signalling pathways [21]. Several studies have shown that Cebpd is associated with target genes for a variety of biological functions, including growth arrest, apoptosis, differentiation, stem cell self-renewal and tumour suppression, and that in osteoblasts and MC3T3-E1 cells, Cebpd upregulation promotes cell proliferation and differentiation and plays a key role in bone growth and bone remodelling [22]. The literature reports that SIRT1, a regulator of bone mass, is closely related to bone metabolism and bone mass and that SIRT1 overexpression promoted proliferation, differentiation and prevented autophagic apoptosis of MC3T3-E1 cells and was able to inhibit osteoclast differentiation [23-25]. Kcnq1ot1 is associated with a variety of tumourigenesis, and upregulation of Kcnq1ot1 promotes MC3T3-E1 cell proliferation and osteoblast differentiation [26]. Our results show that GJD treatment significantly promotes osteoblast proliferation by upregulating the expression levels of functional genes involved in promoting cell proliferation.
In line with these results, we also found that the levels of several genes involved in the inhibition of osteoclast differentiation were significantly upregulated after GJD treatment, such as Bcl3, Bcl6, Nfkbiz, Sirt1, Clcf1, and Kcnq1ot1. It has been shown that B-cell chronic lymphatic leukaemia protein 3 (Bcl3) interacts with tumour necrosis factor receptor-associated factor 6 through its anchor protein repeat domain to attenuate RANKL-induced osteoclast bone resorption activity and inhibit osteoclast growth in bone marrow-derived macrophages in vitro [27]. B cell lymphoma 6 (Bcl6) is a negative regulator of osteoclastogenesis and overexpression of Bcl6 effectively inhibits osteoclast differentiation in vitro [28]. Nfkbiz is a key transcription factor involved in osteoclast differentiation, and upregulation of Nfkbiz can mediate impaired osteoclast differentiation [29]. Galectin-3 (encoded by the LGALS3 gene) is a member of the β-galactoside-binding lectin family that play multiple roles in cell growth, differentiation and aggregation, interfering with RANLK-mediated signalling and has an intrinsic inhibitory effect on osteoclastogenesis, and overexpression of Lgals3 promotes osteogenic differentiation [30,31]. Wisp1, a member of the CCN family that is found in mineralized tissues and is produced by osteoblasts and their precursors, down-regulation of Wisp1 promotes osteoclast differentiation, whereas up-regulation promotes osteoblast differentiation [32].
We further found that the expression levels of several genes involved in the inhibition of apoptosis were significantly altered after GJD treatment, such as Gadd45a, Birc3, Sirt1 and Hspa1b. Gadd45a is a target gene for the FoxO3a transcription factor, Gadd45a is also a DNA damage repair gene, and its upregulation eliminates pro-apoptotic genes in damaged or abnormal cells [33]. Birc3 is part of a family of apoptosis-inhibiting proteins that effectively mediates resistance to cell death [34]. Negative regulation of Hspa1b, an apoptosis-related factor, inhibits osteoblast apoptosis [35]. Our results show that GJD treatment inhibits apoptosis through significant changes in the expression levels of functional genes involved in the inhibition of apoptosis.
As well, we analysed that the expression levels of most genes involved in MC3T3-E1 cells and osteoblasts to promote their differentiation and mineralisation were significantly upregulated after GJD treatment, such as Lgals3, Adamts1, Junb, Egr1, Klf10, Atf6, Wisp1, Malat1, Btg2, Sirt1, Sertad4, Zfyve16, Creb5, Snai2, Fblim1, Fam46a, Calcrl, Kcnq1ot1, Pdzrn3. Overexpression of the AP-1 transcription factor JunB induces osteoblast differentiation [36]. Egr1 co-regulates osteoblast differentiation with several osteoblast-related genes and Egr1 overexpression contributes to osteoblast differentiation [37,38]. Klf10 is a member of the Krüppel-like family of transcription factors, Klf10 upregulation effectively promotes osteoblast differentiation [39]. Osteocalcin is a target gene of ATF6, ATF6 upregulation promotes osteoblast differentiation [40]. Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is associated with a variety of human tumours and Malat1 upregulation mediates the regulation of osteoblast activity and pro-differentiation [41]. Sertad4 is a newly identified gene associated with osteoporosis, and upregulation of Sertad4 promotes osteoblast differentiation [42]. The Zinc finger fyve domain containing 16 (ZFYVE16, also known as Endofin) is involved in the BMP signalling pathway playing an important role in promoting osteoblast differentiation [43]. Creb5 is relevant to the treatment of several cancer diseases, and studies of osteoblast differentiation have revealed that this Creb5 upregulation promotes osteoblast differentiation [44]. Snail2 is a marker of malignancy in epithelial tumours, and upregulation of Snail2 was shown to promote increased osteoblast alkaline phosphatase activity and promote differentiation [45]. Fblim1 is a key regulator of bone homeostasis and deletion of the Fblim1 gene results in increased differentiation of osteoclasts in vivo [46]. FAM46A is a gene associated with skeletal dysplasia and bone mineralization and plays an important role in bone regulation, and FAM46A deficiency can lead to fracture susceptibility and long bone curvature [47]. CALCRL (Calcitonin receptor-like receptor) is associated with disease and CALCRL upregulation promotes osteoblast differentiation [48]. PDZRN3 is a member of the PDZ domain-containing RING finger family of proteins and plays an important role in the negative feedback control of osteoblast differentiation by inhibiting Wnt-β-catenin signalling [49].
Based on our RNA-seq, we also analysed the expression levels of osteoblast markers, including Atf4, Fn1, Usp7, Sox4, Col16a1, Spp1, Bmp1, Runx2, Bglap, Col12a1 and Alpl, which were slightly increased in response to GJD treatment. Based on our RNA-seq, we also analysed the expression levels of osteoblast markers, including Atf4, Fn1, Usp7, Sox4, Col16a1, Spp1, Bmp1, Runx2, Bglap, Col12a1 and Alpl, which were slightly increased in response to GJD treatment. Studies confirmed that SPP1, Fn1, Bglap, Col16a1, and Col12a1 are the main bone matrix proteins with associated genes [50-53]. ATF4, SOX4, RUNX2 are key transcription factors associated with bone formation [54-56]. It has been shown that ubiquitin-specific proteinase 7 (USP7) is involved in the regulation of various biological functions and plays an important role in osteoblast differentiation [57]. Bmp1 is a member of the bone morphogenetic protein family and plays an important role in bone remodelling [58]. Alpl is a marker of osteoblasts and is mostly used in the important assessment of bone formation [59].
There are also genes in the RNA-Seq that are significantly upregulated or downregulated by drug interventions, which we have not included in the analysis because they are not relevant to our topic, so we have only analysed genes associated with osteoporosis disease. Other data may also be useful for subsequent studies of other diseases.
The GJD is based on the clinical experience of Professor Bailing Liu, a master of Chinese medicine, and is designed under the guidance of the basic theory of Chinese medicine, with the theory that "the kidney is the master of bone" and "treating the kidney also treats the bone". Among the nineteen CMMs in GJD, Bonesetter, Epimedium, Radix Rehmanniae Praeparata, Achyranthes bidentata, Eucommia ulmoides and Cornus officinalis are the drugs that play the most important role in Chinese medicine formulation, the so-called monarchs and ministers, all these herbs are drugs that play a role in Chinese medicine in tonifying the kidneys and strengthening the bones. The so-called kidney is not simply limited to one organ but is a function of the Zang viscera system under the general theory of Chinese medicine. The Qi in the Zang viscera kidney is Kidney Qi, which promotes and regulates the growth and development of the bones [60,61]. Rhizoma Dynariae is one of the medicines in the Chinese Pharmacopoeia, mainly used for the treatment of bone-related diseases. Its main component is Flavonoids, which have been proven in numerous studies to have the effect of reducing bone loss and promoting bone formation [62]. Epimedium has been shown to promote osteogenic differentiation and thus induce bone formation [63]. Radix Rehmanniae Praeparata is one of the most commonly used herbs in Chinese medicine and has been shown in numerous experimental studies to have therapeutic effects on diseases such as osteoporosis through its many active ingredients [64]. Achyranthes bidentata is used in many prescriptions in Chinese medicine, which shows its wide range of effects, and studies have shown that this herb has significant anti-osteoporosis effects [65]. Eucommia ulmoides is a common herbal medicine used in Chinese medicine for the treatment of osteoporosis, which has the effect of regulating bone metabolism and is mostly used for the prevention and treatment of osteoporosis [66]. Studies of Cornus Officinalis extract were found to inhibit osteoclast differentiation and promote osteoblast differentiation [67]. In addition, studies have shown that the Curcuminoid-enriched Turmeric extract in Curcuma Longa helps retain BMD in trend-model rats, as well as the microarchitectural structure and Trabecular connectivity of bone [68]. Pericarpium Citri Reticulatae contains Hesperetin, according to the research, Hesperetin effectively suppressed Rankl-induced osteoclastogenesis, osteoclastic bone resorption and F-actin ring [69]. Radix Paeoniae Alba containing Paeoniflorin has been widely used in the treatment of osteoporosis. Paeoniflorin treatment of osteoporosis can be divided into two general directions: inhibiting osteoclast activity [70] and improving osteoblast activity [71,72], It slows bone resorption by inhibiting osteoclast activity and promotes bone formation by increasing osteoblast activity. Corydalis Yanhusuo contains Tetrahydropalmatine, Experimental studies in vivo and in vitro have shown that Tetrahydropalmatine inhibits osteoclast formation by inhibiting NF-κB and MAPK pathways [73]. Coicis Semen extract has been shown to promote osteoblast proliferation and effectively relieve osteoporosis in animals [73-75].
In conclusion, our results suggest that the various herbs and their active ingredients in GJD synergistically promote osteoblast proliferation and differentiation and also inhibit osteoclast differentiation and inflammation.