Ginwa Enterprise (Group) Inc.

Chronic inflammatory pain represents a significant global health burden, seriously affecting the patient's quality of life. Jin-Tian-Ge Capsules (JTG), a substitute for natural tiger bone, has been approved in China for the treatment of osteoporosis, osteoarthritis and rheumatoid arthritis. Clinical observations show that JTG can mitigate chronic pain associated with the above bone-related diseases. However, the potential mechanism underlying the anti-inflammatory and analgesic effects of JTG remains unclear.

To clarify the effect of JTG on relief of inflammatory pain and its potential mechanism.

The anti-inflammatory effect of JTG was evaluated using LPS-stimulated RAW264.7 cells. An inflammatory pain mouse model was developed using complete Freund's adjuvant (CFA), and used to validate the effect of JTG. Paw withdrawal latency (PWL) was determined using thermal hyperalgesia test to characterize the pain threshold of mice. Metabolite alterations caused by JTG serum metabolomics in CFA mice were determined to explore the potential mechanism in relief of inflammatory pain.

JTG effectively decreased the levels of inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), nitric oxide, IL-6, IL-1β, TNF-α and prostaglandin E2, and suppressed the phosphorylation of JNK, p38, ERK, and p65, as well as nuclear transport of p65 in LPS-induced RAW264.7 cells. In vivo, JTG significantly increased PWL and alleviated inflammatory pain by promoting the expression of β-endorphin and μ-opioid receptor in hypothalamic tissues. In addition, JTG inhibited paw swelling, paw skin tissue disorders, the expression of TNF-α and COX-2, and the phosphorylation of JNK, p38, ERK, and p65 in CFA-induced mouse paw skin tissues, thereby alleviating inflammation and pain. Metabolomics results suggest that JTG may participate in modulating glycerophgspholipid (GPL), arginine and arachidonic acid metabolisms, and also inhibit the expression of the key enzymes of GPL metabolism including phosphoethanolamine cytidylyltransferase 2 (PCYT2), phosphocholine cytidylyltransferase A (PCYT1A), and phospholipase A2G6 (PLA2G6) in skin tissues of CFA-induced inflammatory mice. In addition, JTG suppressed the expression of transient receptor potential vanilloid 1 (TRPV1) and protein kinase C (PKC) in hypothalamic tissues of CFA-induced inflammatory mice, thereby alleviating inflammation and pain.

JTG attenuated inflammatory pain through modulation of GPL/PKC/TRPV1 pathway.

Tiger bone, which had long been used in traditional Chinese medicine, had the action of removing wind and alleviating pain, strengthening the sinews and bones, and often used to treat bone impediment, and atrophic debility of bones in TCM clinical practice. As a substitute of natural bone tiger, artificial tiger bone Jintiange (JTG), has been approved by the State Food and Drug Administration of China for relief the symptom of osteoporosis, such as lumbago and back pain, lassitude in loin and legs, flaccidity and weakness legs, and walk with difficulty based on TCM theory. JTG has similar chemical profile to natural tiger bone, and contains mineral substance, peptides and proteins, and has been shown to protect bone loss in ovariectomized mice and exert the regulatory effects on osteoblast and osteoclast activities. But how the peptides and proteins in JTG modulate bone formation remains unclear.

To investigate the stimulating effects of JTG proteins on osteogenesis and explore the possible underlying mechanisms.

JTG proteins were prepared from JTG Capsules by extracting calcium, phosphorus and other inorganic elements using SEP-PaktC18 desalting column. MC3T3-E1 cells were treated with JTG proteins to evaluate their effects and explore the underlying mechanisms. Osteoblast proliferation was detected by CCK-8 method. ALP activity was detected using a relevant assay kit, and bone mineralized nodules were stained with alizarin red-Tris-HCl solution. Cell apoptosis was analyzed by flow cytometry. Autophagy was observed by MDC staining, and autophagosomes were observed by TEM. Nuclear translocations of LC3 and CHOP were detected by immunofluorescence and observed under a laser confocal microscope. The expression of key proteins related to osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways was analyzed by Western Blot analysis.

JTG proteins improved osteogenesis as evidenced by the alteration of proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, inhibited their apoptosis, and enhanced autophagosome formation and autophagy. They also regulated the expression of key proteins of PI3K/AKT and ER stress pathways. In addition, PI3K/AKT and ER stress pathway inhibitors could reverse the regulatory effects of JTG proteins on osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways.

JTG proteins increased the osteogenesis and inhibited osteoblast apoptosis by enhancing autophagy via PI3K/AKT and ER stress signaling pathways.

Tiger bone, which had been one of the most famous traditional Chinese medicine for 2000 years, was originate from the skeleton of Panthera tigris L., and had the actions of anti-inflammatory, analgesic, immune-regulatory and promoting healing of bone fracture, and was used for the treatment of osteoporosis and rheumatoid arthritis. Jin-Tian-Ge (JTG), the artificial tiger bone powder, were prepared from skeletons of several farmed animals to substitute the natural tiger bone, and has been used for the treatment of osteoporosis in clinical practice. However, the characteristic and mechanism of action of JTG for the therapy of osteoporosis need to be further evidenced by using modern pharmacological methods. The aim of this work is to investigate the bone-protective effects of JTG, and explore the possible underlying mechanism.

Ovariectomy (OVX) rats were orally administrated JTG or estradiol valerate (EV) for 12 weeks. We investigated the pharmacodynamic effects of JTG on anti-bone loss in OVX rats, and also investigated the role of JTG in promoting osteogenesis and inhibiting osteoclast differentiation.

JTG increased the bone mineral density (BMD), improved the bone microarchitecture and biomechanical properties in ovariectomized rast, whereas reversed the bone high turnover in OVX rats as evidenced by serum biochemical markers in OVX rats. JTG increased osteogenic differentiation of BMSCs in vitro, and up-regulated the expression of the key proteins of BMP and Wnt/β-catenin pathways. JTG also inhibited the osteoclastogenesis of BMM as evidenced by the alteration of the TRAP activity, F-actin construction and the expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, Cathepsin K (Ctsk) and matrix metallopeptidase 9 (MMP9) of OCs induced with RANKL and LPS, reduced the expression and phosphorylation of NF-κB in OCs.

JTG prevented bone loss in OVX rats and increased osteogenic differentiation of BMSCs through regulation of the BMP and Wnt/β-catenin pathway, inhibited osteoclastogenesis by suppressing the NF-κB pathway, suggesting that JTG had the potentials for prevention and treatment of osteoporosis by modulating formation and differentiation of osteoblast and osteoclast.