Quercetin protects against iron overload-induced osteoporosis through activating the Nrf2/HO-1 pathway.

AIMS: Eucommia is the tree bark of Eucommia japonica, family Eucommiaceae. In traditional Chinese medicine, Eucommia is often used to treat osteoporosis. Quercetin (QUE), a major flavonoid extract of Eucommia japonica, has been reported to have anti-osteoporosis effects. However, there are no studies reporting the mechanism of QUE in the treatment of iron overload-induced osteoporosis. This study set out to investigate the therapeutic effects of QUE against iron overload-induced bone loss and its potential molecular mechanisms. MATERIALS AND METHODS: In vitro, MC3T3-E1 cells were used to study the effects of QUE on osteogenic differentiation, anti-apoptosis and anti-oxidative stress damage in an iron overload environment (FAC 200 µM). In vivo, we constructed an iron overload mouse model by injecting iron dextrose intraperitoneally and assessed the osteoprotective effects of QUE by Micro-CT and histological analysis. KEY FINDINGS: In vitro, we found that QUE increased the ALP activity of MC3T3-E1 cells in iron overload environment, promoted the formation of bone mineralized nodules and upregulated the expression of Runx2 and Osterix. In addition, QUE was able to reduce FAC-induced apoptosis and ROS production, down-regulated the expression of Caspase3 and Bax, and up-regulated the expression of Bcl-2. In further studies, we found that QUE activated the Nrf2/HO-1 signaling pathway and attenuated FAC-induced oxidative stress damage. The results of the in vivo study showed that QUE was able to reduce iron deposition induced by iron dextrose and attenuate bone loss. SIGNIFICANCE: Our results suggested that QUE protects against iron overload-induced osteoporosis by activating the Nrf2/HO-1 signaling pathway.

Biochanin A protects against iron overload associated knee osteoarthritis via regulating iron levels and NRF2/System xc-/GPX4 axis.

BACKGROUND: Iron homeostasis plays a positive role in articular cartilage health. Excessive iron or iron overload can induce oxidative stress damage in chondrocytes and ferroptosis cell death, advancing knee osteoarthritis (KOA). However, up to date, few effective agents treat iron overload-induced KOA (IOKOA). Chinese herbal medicine (CHM) provides abundant resources for drug selection to manage bone metabolic conditions, including osteoporosis. Biochanin A (BCA) is a novel bioactive multifunctional natural compound isolated from Huangqi, which has protective effects on bone loss. Nevertheless, the function and mechanism of BCA in treating IOKOA are still elusive. PURPOSE: This study seeks to uncover the potential therapeutic targets and mechanisms of BCA in the management of KOA with iron accumulation. METHODS: Iron dextrin (500 mg/kg) was intraperitoneally injected into mice to establish the iron overloaded mice model. OA was induced through surgery, and the progression was evaluated eight weeks following surgery. OA severity was evaluated with micro-CT and Safranin-O/Fast green staining in vivo. Iron deposition in the knee joint and synovium was assessed using Perl's Prussian blue staining. Ferric ammonium citrate (FAC) was then administered to primary chondrocytes to evaluate iron regulators mediated iron homeostasis. Toluidine blue staining was utilized to identify chondrocytes in vitro. The vitality of the cells was assessed using the CCK-8 test. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. The intracellular iron level was detected utilizing the calcein-AM test. Reactive oxygen species (ROS), lipid-ROS, and mitochondrial membrane potentiality were reflected via fluorescence density. Utilizing RT-qPCR and western blotting, the expression level was determined. RESULTS: Micro-CT and histological staining of knee joints showed greater cartilage degradation and higher iron buildup detected in iron-overloaded mice. BCA can reduce iron deposition and the severity of KOA. Toluidine blue staining and the CCK-8 assay indicated that BCA could rescue chondrocytes killed by iron. Cell apoptosis rates were increased due to iron overload but improved by BCA. Further, the intracellular content of iron, ROS, and lipid-ROS was increased with ferric ammonium citrate (FAC) treatment but restored after treatment with different concentrations of BCA. JC-1 staining revealed that BCA could reduce mitochondrial damage induced by iron overload. CONCLUSION: Iron overload was shown to promote chondrocyte ferroptosis in vivo and in vitro. Moreover, iron overload suppressed the expression of collagen II and induced MMP expression by catalyzing ROS generation with mitochondrial dysfunction. Our results showed that BCA could directly reduce intracellular iron concentration by inhibiting TfR1 and promoting FPN but also target the Nrf2/system xc-/GPX4 signaling pathway to scavenge free radicals and prevent lipid peroxidation. The results of this research indicate that BCA regulates iron homeostasis during the progression of osteoarthritis, which can open a new field of treatment for KOA.

Bioinformatics analysis combined with experimental validation to explore the mechanism of XianLing GuBao capsule against osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: XianLing GuBao Capsule (XLGB) is often used to treat osteoarthritis (OA), osteoporosis, fractures, and other musculoskeleton disorders. However, the molecular mechanism of XLGB for treating OA is still unclear. AIM OF THE STUDY: This study set out to uncover the molecular mechanism underlying the treatment of osteoarthritis with XLGB. MATERIALS AND METHODS: Disease genes were obtained from CTD, DisGeNET, and GeneCards databases, and XLGB drug targets were obtained from ETCM and target genes predicted by XLGB metabolic components reported in the literature. Then we used the Venn diagram viewer to extract disease and drug intersection genes as potential therapeutic genes for Protein-protein interaction (PPI), GO terminology, and KEGG pathway analysis. Subsequently, we performed qRT-PCR, Western blot and histological analysis to validate the therapeutic effect of XLGB against OA and its molecular mechanism. RESULTS: A total of 1039 OA genes and 949 XLGB target genes were collected, and finally 188 potential therapeutic target genes were obtained. PPI network analysis indicated that the main target genes for XLGB to treat OA include Akt1, Mapk3, Il-6, Il-1ß, Ptgs2, Mmp9, etc. The results of KEGG and GO enrichment analysis suggested that XLGB may treat OA by anti-inflammatory and reducing extracellular matrix degradation. In vitro, XLGB down-regulated the expressions of Mmp3, Mmp9, Mmp12, Mmp13, Cox-2, Il-6, increased the expression of Collagen II and Sox9. Mechanistically, XLGB inhibits the activation of PI3K/AKT/NF-κB and MAPK pathways. Moreover, the results of animal experiments indicated that XLGB reduced cartilage destruction, bone resorption, and synovitis in osteoarthritic rats. CONCLUSIONS: XLGB has a protective effect against OA by suppressing PI3K/AKT/NF-κB and MAPK signaling. Our study provides a theoretical basis for XLGB in the treatment of osteoarthritis.

Gegen Qinlian Decoction ameliorates type 2 diabetes osteoporosis via IGFBP3/MAPK/NFATc1 signaling pathway based on cytokine antibody array.

BACKGROUND: Osteoporosis affects more than half the patients with type 2 diabetes mellitus (T2DM). Up to data, there is no effective clinical practice in managing type 2 diabetes osteoporosis (T2DOP) because of its complex pathogenesis. Gegen Qinlian Decoction (GQD) has been used for the long-term management of T2DM. However, the underlying mechanism of GQD in the treatment of T2DOP remains unknown. PURPOSE: To reveal the role of GQD in T2DOP and its potential therapeutic targets in the management of T2DOP. STUDY DESIGN: The effect of GQD on T2DOP was observed in db/db mice in four groups: model group, GQD low-dose group (GQD-L), GQD high-dose group (GQD-H), and metformin (positive control) group. C57BL/6J mice were used as the negative control group. METHODS: Quantitative phytochemical analysis of GQD was performed using high-performance liquid chromatography (HPLC). Micro-CT and hematoxylin-eosin (H&E) staining were used to evaluate bone histomorphometry. To screen for candidate targets of GQD, a cytokine antibody array was used, followed by bioinformatics analysis. Quantitative real-time PCR (qRT-PCR) and western blotting (WB) were used to determine expression levels. RESULTS: The major active components of GQD were confirmed by HPLC. Micro-CT and H&E staining showed that bone mass was significantly increased in the GQD-H group compared with the model group. Antibody arrays revealed that the expression of insulin-like growth factor binding protein 3 (IGFBP3) was elevated in the GQD-H group. The MAPK pathway was identified using bioinformatics analysis. Additionally, the levels of osteoclastogenesis-related genes, including cathepsin K (Ctsk), acid phosphatase 5 (Acp5), matrix metallopeptidase 9 (Mmp9), and ATPase H+ transporting V0 subunit D2 (Atp6v0d2) were significantly decreased in the GQD-H group. Compared with the model group, high-dosage GQD inhibited phosphorylation of extracellular signal-regulated kinases (ERKs) and P38 mitogen-activated protein kinase (MAPK) and the expression of c-Fos and nuclear factor of activated T cells 1 (NFATc1). CONCLUSION: GQD plays a protective role in T2DOP by upregulating IGFBP3 expression and downregulating the IGFBP3/MAPK/NFATc1 signaling pathway. IGFBP3 in serum may also be a novel biomarker in the treatment of T2DOP. Our current findings not only expand the application of GQD, but also provide a theoretical basis and guidance for T2DOP.

12-Deoxyphorbol 13-acetate inhibits RANKL-induced osteoclastogenesis via the attenuation of MAPK signaling and NFATc1 activation.

Osteoporosis, characterized by bone loss and microstructure damage, occurs when osteoclast activity outstrips osteoblast activity. Natural compounds with inhibitory effect on osteoclast differentiation and function have been evidenced to protect from osteoporosis. After multiple compounds screening, 12-deoxyphorbol 13-acetate (DPA) was found to decline RANKL-induced osteoclastogenesis dose-dependently by attenuating activities of NFATc1 and c-Fos, followed by decreasing the level of osteoclast function-associated genes and proteins including Acp5, V-ATPase-d2 and CTSK. Mechanistically, we found that DPA suppressing RANKL-induced downstream signaling pathways, including MAPK signaling pathway and calcium oscillations. Furthermore, the in vivo efficacy of DPA was further confirmed in an OVX-induced osteoporosis mice model. Collectively, the results in our presentation reveal that DPA might be a promising compound to manage osteoporosis.

Study on the potential active components and molecular mechanism of Xiao Huoluo Pills in the treatment of cartilage degeneration of knee osteoarthritis based on bioinformatics analysis and molecular docking technology.

BACKGROUND: Knee osteoarthritis is a common joint degenerative disease. Xiao Huoluo Pills (XHLP) has been used to treat degenerative diseases such as osteoarthritis and hyperosteogeny. However, XHLP's specific effective ingredients and mechanism of action against osteoarthritis have not been explored. Therefore, bioinformatics technology and molecular docking technology are employed in this study to explore the molecular basis and mechanism of XHLP in the treatment of knee osteoarthritis. METHODS: Public databases (TCMSP, Batman-TCM, HERB, DrugBank, and UniProt) are used to find the effective active components and corresponding target proteins of XHLP (screening conditions: OB > 30%, DL ≥ 0.18). Differentially expressed genes related to cartilage lesions of knee osteoarthritis are obtained based on the GEO database (screening conditions: adjust P value < 0.01, |log2 FC|≥1.0). The Venn package in R language and the BisoGenet plug-in in Cytoscape are adopted to predict the potential molecules of XHLP in the treatment of knee osteoarthritis. The XHLP-active component-target interaction network and the XHLP-knee osteoarthritis-target protein core network are constructed using Cytoscape software. Besides, GO/KEGG enrichment analysis on core genes is performed using the Bioconductor package and clusterProfiler package in the R language to explain the biological functions and signal pathways of the core proteins. Finally, molecular docking is performed through software such as Vina, LeDock, Discovery Studio 2016, PyMOL, AutoDockTools 1.5.6, so as to verify the binding ability between the active components of the drug and the core target protein. RESULTS: XHLP has been screened out of 71 potentially effective active compounds for the treatment of OA, mainly including quercetin, Stigmasterol, beta-sitosterol, Izoteolin, and ellagic acid. Knee osteoarthritis cartilage lesion sequencing data (GSE114007) was screened out of 1672 differentially expressed genes, including 913 upregulated genes and 759 downregulated genes, displayed as heat maps and volcano maps. Besides, 33 core target proteins are calculated by Venn data package in R and BisoGenet plug-in in Cytoscape. The enrichment analysis on these target genes revealed that the core target genes are mainly involved in biological processes such as response to oxygen levels, mechanical stimulus, vitamin, drug, and regulation of smooth muscle cell proliferation. These core target genes are involved in signaling pathways related to cartilage degeneration of knee osteoarthritis such as TNF signaling pathway and PI3K-Akt signaling pathway. Finally, the molecular docking verification demonstrates that some active components of the drug have good molecular docking and binding ability with the core target protein, further confirming that XHLP has the effect of inhibiting cartilage degeneration in knee osteoarthritis. CONCLUSIONS: In this study, based on the research foundation of bioinformatics and molecular docking technology, the active components and core target molecules of XHLP for the treatment of cartilage degeneration of knee osteoarthritis are screened out, and the potential mechanism of XHLP inhibiting cartilage degeneration of knee osteoarthritis is deeply explored. The results provide theoretical basis and new treatment plan for XHLP in the treatment of knee osteoarthritis.

Sanhuang Jiangtang tablet protects type 2 diabetes osteoporosis via AKT-GSK3ß-NFATc1 signaling pathway by integrating bioinformatics analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Sanhuang Jiangtang tablet (SHJTT), has been widely used to treat type 2 diabetes mellitus (T2DM). However, the potential and mechanism of SHJTT in treating type 2 diabetes osteoporosis (T2DOP) has not been reported. AIM OF THE STUDY: The aim of this work was to investigate the role and the underlying molecular mechanism of SHJTT in managing type 2 diabetes osteoporosis. MATERIALS AND METHODS: The target genes of each component consisting of SHJTT were obtained by searching the ETCM database. The target genes of osteoporosis and diabetes were individually acquired by analyzing the DisGeNET and OMIM disease databases. Then the potential therapeutic genes were obtained from the intersection of the herbal medicine targets and the disease targets which were imported into the R and STRING platform for the analysis of GO terms, KEGG pathways and PPI network. The key modules of PPI network were constructed by Cytoscape software. Finally, leptin receptor deficiency (db/db) mice were confirmed as an animal model of type 2 diabetic osteoporosis (T2DOP) through phenotype assessment and the key genes of SHJTT against T2DOP were validated by quantitative real-time PCR (qRT-PCR). RESULTS: A total of 786 target genes of SHJTT were obtained from ETCM. Simultaneously, a total of 3906 osteoporosis and type 2 diabetes associated targets were acquired from DisGeNET and OMIM databases. Then, 97 common targets were found by overlapping them. On the basis of the GO and KEGG enrichment analysis and PPI network, we found that the related pathway of SHJTT in type 2 diabetes osteoporosis was AKT-GSK3ß-NFATc1 pathway which is tightly associated with osteoclast differentiation. The expression of key genes including Akt1, Mapk3, Gsk3ß, Mmp9, Nfkb1 were significantly down-regulated by SHJTT in T2DOP mice (p < 0.05). CONCLUSIONS: SHJTT had a protective effect on T2DOP via regulating AKT-GSK3ß-NFATc1 signaling pathway. This study might provide a theoretical basis for the application of SHJTT for the treatment of type 2 diabetic osteoporosis.

Effectiveness and safety of warm needle acupuncture on insomnia: Protocol for a systematic review and meta-analysis.

BACKGROUND: Warm needle acupuncture (WNA) combines acupuncture and moxibustion, which is an integral part of the acupuncture therapy. Insomnia is a common sleep disorder, which affects sub-healthy people and patients with chronic disease. The clinical practice indicates that WNA has a therapeutic effect on insomnia. Here we will provide a protocol to explore the effectiveness and safety of WNA for insomnia. METHODS: We will search the randomized controlled trails (RCT) literatures of WNA for insomnia in 9 electronic databases, including 5 English databases [PubMed, Web of Science, EMBASE, the Cochrane Central Register of Controlled Trials (Cochrane Library), and WHO International Clinical Trials Registry Platform (TCTRP)] and 4 Chinese databases [Chinese National Knowledge Infrastructure (CNKI), Chinese VIP Information, Wanfang Database, and Chinese Biomedical Literature Database (CBM)]. Sleep quality value of the patient will be considered as the primary outcome and the secondary outcome will include biochemical, indicators total scores on the insomnia severity index, quality of life, adverse events caused by WNA, and changes of symptom in Traditional Chinese Medicine. The selection of the studies will be performed by EndnoteX7 software. All analyses will be conducted by using RevMan software V5.3. RESULT: This study will provide a rational synthesis of current evidences for warm needle acupuncture on insomnia. CONCLUSION: The conclusion of this study will provide evidence to judge the effectiveness and safety of WNA on insomnia. REGISTRATION: PROS-PERO CRD42018112645.