Orcinol gentiobioside inhibits RANKL-induced osteoclastogenesis by promoting apoptosis and suppressing autophagy via the JNK1 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoporosis (OP) is a metabolic disorder characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Curculigo orchioides Gaertn has a long history of application in traditional Chinese and Indian medicine for treating OP. Orcinol gentiobioside (OGB) is a principal active constituent derived from Curculigo orchioides Gaertn and has been shown to have anti-OP activity. However, the therapeutic efficacy and mechanism of OGB in modulating osteoclastic bone resorption remain undefined. AIM OF THE STUDY: To evaluate the effect of OGB on the formation, differentiation and function of osteoclasts derived from bone marrow macrophages (BMMs), and further elucidate the underlying action mechanism of OGB in OP. MATERIALS AND METHODS: Osteoclasts derived from BMMs were utilized to evaluate the effect of OGB on osteoclast formation, differentiation and bone resorption. Tartrate-resistant acid phosphatase (TRAP) staining and activity assays were conducted to denote the activity of osteoclasts. Osteoclast-related genes and proteins were determined by RT-PCR and Western blotting assays. The formation of the F-actin ring was observed by confocal laser microscopy, and bone resorption pits were observed by inverted microscopy. The target of OGB in osteoclasts was predicted by using molecular docking and further verified by Cellular Thermal Shift Assay (CETSA) and reversal effects of the target activator. The apoptosis of osteoclasts was analyzed by flow cytometry, and autophagic flux in osteoclasts was determined by confocal laser microscopy. RESULTS: OGB inhibited osteoclast formation and differentiation, osteoclast-related genes and proteins expression, F-actin ring formation, and bone resorption activity. Molecular docking and CETSA analysis demonstrated that OGB exhibited good affinity for c-Jun N-terminal Kinase 1 (JNK1). In addition, OGB induced apoptosis and inhibited autophagy in osteoclasts, and the JNK agonist anisomycin reversed the increase in apoptosis and inhibition of autophagy induced by OGB in osteoclasts. CONCLUSION: OGB inhibited osteoclastogenesis by promoting apoptosis and diminishing autophagy via JNK1 signaling.

Jintiange proteins promote osteogenesis and inhibit apoptosis of osteoblasts by enhancing autophagy via PI3K/AKT and ER stress pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM: To investigate the stimulating effects of JTG proteins on osteogenesis and explore the possible underlying mechanisms. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: JTG proteins increased the osteogenesis and inhibited osteoblast apoptosis by enhancing autophagy via PI3K/AKT and ER stress signaling pathways.

[Research progress on anti-inflammatory effects of plant-derived cannabinoid type 2 receptor modulators].

Excessive and persistent inflammatory responses are a potential pathological condition that can lead to diseases of various systems, including nervous, respiratory, digestive, circulatory, and endocrine systems. Cannabinoid type 2 receptor(CB2R) belongs to the G protein-coupled receptor family and is widely distributed in immune cells, peripheral tissues, and the central nervous system. It plays a role in inflammatory responses under various pathological conditions. The down-regulation of CB2R activity is an important marker of inflammation and and CB2R modulators have been shown to have anti-inflammatory effects. This study explored the relationship between CB2R and inflammatory responses, delved into its regulatory mechanisms in inflammatory diseases, and summarized the research progress on CB2R modulators from plants other than cannabis, including plant extracts and monomeric compounds, in exerting anti-inflammatory effects. The aim is to provide new insights into the prevention and treatment of inflammatory diseases.

Monotropein Protects against Inflammatory Bone Loss and Suppresses Osteoclast Formation and Bone Resorption by Inhibiting NFATc1 via NF-κB and Akt/GSK-3ß Pathway.

Monotropein (Mon) is a kind of iridoid glycoside plant secondary metabolite primarily present in some edible and medicinal plants. The aim of this study was to investigate the effect of Mon on lipopolysaccharide (LPS)-induced inflammatory bone loss in mice and osteoclasts (OCs) derived from bone marrow-derived macrophages (BMMs), and explore the mechanisms underlying the effect of Mon on LPS-induced osteoclastogenesis. It was found that Mon markedly attenuated deterioration of the bone micro-architecture, enhanced tissue mineral content (TMC) and bone volume/total volume (BV/TV), reduced structure model index (SMI) and trabecular separation/spacing (Tb.Sp) in the bone tissue and decreased the activities of tartrate resistant acid phosphatase-5b (TRACP-5b), receptor activator NF-κB (RANK), and receptor activator NF-κB ligand (RANKL) as well as the serum levels of interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) in LPS-treated mice. In addition, Mon treatment reduced the number of TRAP positive OCs in the bone tissue of LPS-treated mice and also exerted a stronger inhibitory effect on formation, differentiation, and F-actin ring construction of OCs derived from BMMs. Mon significantly inhibited the expression of the nuclear factor of activated T-cells c1 (NFATc1) and the immediate early gene (C-Fos) and nuclear translocation of NFATc1 in LPS-treated OCs, thereby inhibiting the expression of matrix metalloproteinase-9 (MMP-9), cathepsin K (CtsK), and TRAP. Mon significantly inhibited the expression of TRAF6, phosphorylation of P65, and degradation of IKBα, thus inhibiting the activation of NF-κB pathway in LPS-induced inflammatory mice and OCs derived from BMMs, and also inhibited LPS-induced phosphorylation of protein kinase B (Akt) and Glycogen synthase kinase 3ß (GSK-3ß) in OCs derived from BMMs. In conclusion, these results suggested that Mon could effectively inhibit osteoclastogenesis both in vitro and in vivo and therefore may prove to be potential option for prevention and treatment of osteoclastic bone resorption-related diseases.

Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects.

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects-including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism-without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors-ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.

[Research progress on natural cannabinoid receptor type 2( CB2R)regulators and their effects on bone metabolism].

Cannabinoid receptor type 2( CB2 R),a member of the G protein-coupled receptor( GPCR) superfamily,has a variety of biological activities,such as regulating pain response,resisting inflammation and fibrosis,and mediating bone metabolism. Some CB2 R regulators exhibit a good regulatory effect on bone metabolism. Cannabinoids in Cannabis sativa can cause psychoactive effects despite various pharmacological actions they exerted by targeting CB2 R. Therefore,it is of great significance to discover CB2 R regulators in non-Cannabis plants for finding new lead compounds without psychoactive effects and elucidating the action mechanism of plant drugs. The present study clarifies the discovery,structure,and physiological functions of CB2 R,especially its regulatory effects on bone metabolism,summarized CB2 R regulators extracted from non-Cannabis plants,and systematically analyzes the regulatory effects of CB2 R regulators on bone metabolism in animals,osteoblasts,and osteoclasts,to provide a scientific basis for the discovery of new CB2 R regulators and the development of anti-osteoporotic drugs.

Enterolactone has stronger effects than enterodiol on ovarian cancer.

BACKGROUND: Ovarian cancer is one of the three leading gynecological malignancies, characterized by insidious growth, highly frequent metastasis, and quick development of drug resistance. As a result, this disease has low 5-year survival rates. Estrogen receptor inhibitors were commonly used for the treatment, but only 7% to 18% of patients respond to anti-estrogen therapies. Therefore, more effective therapies to inhibit estrogen-related tumors are urgently needed. Recently, phytoestrogens, such as lignans with estrogen-like biological activities, have attracted attention for their potential effects in the prevention or treatment of estrogen-related diseases. Enterodiol (END) and enterolactone (ENL) are mammalian lignans, which can reduce the risk of various cancers. However, the effects of END and ENL on ovarian cancer are not adequately documented. METHODS: We used in vitro assays on the ES-2 cell line to evaluate the inhibiting effects of END and ENL on ovarian cancer cell proliferation, invasion and migration ability and in vivo xenograft experiments on nude mice to validate the anticancer effects of END and ENL. RESULTS: The in vitro assays demonstrated that high-dose END and ENL could obviously inhibit ovarian malignant properties, including cancerous proliferation, invasion, and metastasis. Compared to END, ENL behaved in a better time-dose dependent manner on the cancer cells. The in vivo experiments showed that END (1 mg/kg), ENL (1 mg/kg) and ENL (0.1 mg/kg) suppressed tumor markedly, and there were statistically significant differences between the experimental and control groups in tumor weight and volume. Compared to END, which have serious side effects to the animals at high concentration such as 1 mg/kg, ENL had higher anticancer activities and less side effects in the animals than END at the same concentrations, so it would be a better candidate for drug development. CONCLUSION: END and ENL both have potent inhibitory effects on ovarian cancer but ENL possesses a more effective anti-cancer capability and less side effects than END. Findings in this work provide novel insights into ovarian cancer therapeutics with phytoestrogens and encourage their clinical applications.