Orcinol glucoside targeted p38 as an agonist to promote osteogenesis and protect glucocorticoid-induced osteoporosis.

BACKGROUND: Glucocorticoids (GC)-induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis, which leads to an increased risk of fracture in patients. The inhibition of the osteoblast effect is one of the main pathological characteristics of GIOP, but without effective drugs on treatment. PURPOSE: The aim of this study was to investigate the potential effects of orcinol glucoside (OG) on osteoblast cells and GIOP mice, as well as the mechanism of the underlying molecular target protein of OG both in vitro osteoblast cell and in vivo GIOP mice model. METHODS: GIOP mice were used to determine the effect of OG on bone density and bone formation. Then, a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS) method was used to identify the target of OG. Surface plasmon resonance (SPR), enzyme activity assay, molecular docking, and molecular dynamics were used to detect the affinity, activity, and binding site between OG and its target, respectively. Finally, the anti-osteoporosis effect of OG through the target signal pathway was investigated in vitro osteoblast cell and in vivo GIOP mice model. RESULTS: OG treatment increased bone mineral density (BMD) in GIOP mice and effectively promoted osteoblast proliferation, osteogenic differentiation, and mineralization in vitro. The CETSA-MS result showed that the target of OG acting on the osteoblast is the p38 protein. SPR, molecular docking assay and enzyme activity assay showed that OG could direct bind to the p38 protein and is a p38 agonist. The cellular study found that OG could promote p38 phosphorylation and upregulate the proteins expression of its downstream osteogenic (Runx2, Osx, Collagen Ⅰ, Dlx5). Meanwhile, it could also inhibit the nuclear transport of GR by increasing the phosphorylation site at GR226 in osteoblast cell. In vivo GIOP mice experiment further confirmed that OG could prevent bone loss in the GIOP mice model through promoting p38 activity as well as its downstream proteins expression and activity. CONCLUSIONS: This study has established that OG could promote osteoblast activity and revise the bone loss in GIOP mice by direct binding to the p38 protein and is a p38 agonist to improve its downstream signaling, which has great potential in GIOP treatment for targeting p38. This is the first report to identify OG anti-osteoporosis targets using a label-free strategy (CETSA-MS).

The protective effect of orcinol glucoside on dexamethasone induced osteoblast cell damage through inhibition of glucocorticoid receptor nucleus translocation / 中国药理学通报

Aim To investigate the protective effect of orcinol glucoside on dexamethasone(DEX)-induced osteoblast injury and its mechanism. Methods Primary osteoblasts were extracted from calvaria of neonatal mice and cultured in medium with DEX(1 μmol•L

Mechanism of Orcinol Glucoside in Treatment of Osteoporosis： An Exploration Based on Network Pharmacology and in Vitro Validation / 中国实验方剂学杂志

ObjectiveTo predict the therapeutic targets and related signaling pathways of orcinol glucoside （OG） in the treatment of osteoporosis by network pharmacology， and further clarify its mechanisms based on molecular docking and in vitro cell model. MethodThe pharmacological targets of OG were obtained from Similarity ensemble approach （SEA） and SwissTargetPrediction， and the targets related to osteoporosis from DisGeNET and GeneCards. The cross-analysis was conducted to screen the common targets between OG and osteoporosis. STRING was used to construct the protein-protein interaction （PPI） network， followed by topology analysis using CytoNCA plug-in of Cytoscape 3.7.2 to screen out the core targets. The obtained common targets were subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） analysis by g：Profiler. AutoDock Vina was utilized for molecular docking， and the in vitro cell experiments were then carried out for verifying the mechanism of OG in treating osteoporosis. ResultA total of 73 targets related to OG and osteoporosis were harvested，among which 14 were proved to be key targets by topological analysis. GO and KEGG functional enrichment analysis yielded 259 cell biological processes， mainly involving organonitrogen compound metabolic process， cell population proliferation， protein metabolic process， regulation of response to stress， and response to chemicals. Its mechanism of action might be related to advanced glycation end-product （AGE）-AGE receptor （RAGE） signaling pathway， interleukin-17 （IL-17） signaling pathway， and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. Molecular docking indicated that the binding energies of OG to Cyclin D1 （CCND1） and cyclin-dependent kinase 4 （CDK4） were the lowest and similar. The results of flow cytometry showed that compared with the normal group， OG group exhibited decreased proportion of cells in G0/G1 phase （P<0.01） and decreased proportion of cells in S phase （P<0.01）. As demonstrated by Western blot， compared with the normal group， OG up-regulated the protein expression levels of Cyclin D1 and CDK4 （P<0.05， P<0.01）. ConclusionOG alleviates osteoporosis via multiple targets and multiple pathways. It may exert the therapeutic effects by increasing Cyclin D1 and CDK4 protein expression to change cell cycle and promote cell proliferation.

Orcinol glucoside improves the depressive-like behaviors of perimenopausal depression mice through modulating activity of hypothalamic-pituitary-adrenal/ovary axis and activating BDNF- TrkB-CREB signaling pathway.

Orcinol Glucoside (OG), a phenolic glucoside isolated from C. orchioides, showed the antidepressant-like effect on chronic unpredictable mild stress (CUMS)-induced rats previously. This study was designed to determine whether OG could improve the depressive-like symptoms of perimenopausal depression (PMD) and the possible mechanisms involved. This research was performed on a PMD mice model established by a two-steps method of ovariectomy (OVX) followed CUMS. OG treatment effectively improved the depressive-like behaviors of OVX-CUMS mice, as indicated by increased sucrose intake in sucrose preference test (SPT), reduced immobility time in forced swimming test (FST), and tail suspension test (TST), lower frequency of grooming and defecation, increased actions of rearing, and prolonged duration in the center in open field test (OFT). OG treatment alleviated the OVX-CUMS induced dysfunction of hypothalamic-pituitary-ovarian (HPO) axis by increased serum estradiol (E2) and decreased ovarian hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH) in serum. Meanwhile, OG reversed the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis as evidenced by decreased CORT and ACTH in serum, reduced as well as the mRNA and protein expression of corticotropin-releasing hormone (CRH) in hypothalamus and hippocampus. Moreover, OG up-regulated the protein expression of BDNF, TrkB, and phosphorylation level of CREB and ERK1/2 in hippocampus. These findings demonstrated that OG improves depressive behaviors of OVX-CUMS mice by modulating of HPO/HPA axis dysfunction, and activating BDNF-TrkB-CREB signaling pathway.

Orcinol glucoside facilitates the shift of MSC fate to osteoblast and prevents adipogenesis via Wnt/ß-catenin signaling pathway.

BACKGROUND: During osteoporosis, bone mesenchymal stem cells (BMSCs) lineage commitment shifts to adipocytes, causing fat accumulation and bone loss in the skeleton. Seeking drugs that could reverse the adipocyte fate determination of BMSCs is critical for osteoporosis therapy. As a traditional Chinese medicine, Rhizoma Curculiginis (Xianmao) has been used to treat bone diseases and promote bone healing, while the effective constituent of it and the underlying mechanisms are unknown. OBJECTIVES: The aim of this study is to unveil the role of orcinol glucoside (OG), one constituent of Rhizoma Curculiginis, in osteoporosis and BMSCs lineage commitment and to explore the underlying mechanisms. METHODS: Micro-CT and three-point bending test were performed to determine the effect of OG on bone structure and strength. qT-PCR and Western blot were performed to determine the expression of osteogenic or adipogenic differentiation markers in BMSCs. Mineralization in differentiated BMSCs was assessed by Alizarin Red staining, and lipid accumulation in the cells was evaluated by Oil Red O staining. All measurements were performed at least three times. RESULTS: OG prevented bone loss by stimulating bone formation and attenuating fat formation in bone. In vitro, OG promoted osteoblastic differentiation and inhibited adipogenic differentiation of BMSCs. Inhibition of Wnt/ß-catenin by ICG-001 significantly reversed the effect of OG on osteogenic and adipogenic differentiation of BMSCs. CONCLUSION: Our study demonstrated the role of OG in alleviating bone loss and fat accumulation in osteoporotic bone, therefore bringing a new therapeutic means to the treatment of osteoporosis.

Integrated pathological cell fishing and network pharmacology approach to investigate main active components of Er-Xian decotion for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear. AIM OF THE STUDY: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects. MATERIALS AND METHODS: H2O2-induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H2O2-induced osteoblasts. RESULTS: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H2O2-induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy. CONCLUSIONS: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application.

Orcinol glucoside produces antidepressant effects by blocking the behavioural and neuronal deficits caused by chronic stress.

This study focused on the antidepressant potential of orcinol glucoside (OG) and its possible mechanisms of action. We established a depressed rat model using 3 consecutive weeks of chronic unpredictable mild stress (CUMS). The antidepressant-like effect of OG was revealed using the sucrose preference test, the open field test, the forced swimming test (FST), and the tail suspension test (TST). The activity of the hypothalamic-pituitary-adrenal (HPA) axis was evaluated by detecting the serum corticosterone (CORT) concentrations and mRNA expression of corticotrophin-releasing hormone (CRH) in the hypothalamus. The protein expression levels of brain-derived neurotrophic factor (BDNF) and total phosphorylated-ERK1/2 were detected by western blot. The results showed that OG treatment (1.5, 3, or 6mg/kg) alleviated the depression-like behaviour of rats under CUMS, as indicated by the increased sucrose preference and the decreased immobility in both the FST and TST, although the rearing frequency in the open field test increased only in the group that received the lowest dose (1.5mg/kg OG). Rats that received OG treatment exhibited reduced serum CORT levels and CRH mRNA expression in the hypothalamus, suggesting that the hyperactivity of the HPA axis in CUMS rats was reversed by OG treatment. Moreover, OG treatment upregulated the protein levels of BDNF and phosphorylated-ERK1/2 in the hippocampus, even above control levels. Our findings suggest that OG improved depressive behaviour in CUMS rats by downregulating HPA axis hyperactivity and increasing BDNF expression and ERK1/2 phosphorylation in the hippocampus.