Ginsenoside Rb2 exhibits therapeutic value for male osteoporosis in orchiectomy mice by suppressing osteoclastogenesis and modulating NF-κB/MAPK signaling pathways.

Osteoporosis (OP) is a systemic disorder characterized by decreased bone mass as well as deteriorated microarchitecture. Although OP in men is common, it has received much less attention than that in women. Ginseng, a famous traditional herb in Asia, is used to strengthen and repair bones by invigorating vital bioenergy and maintaining body homeostasis in dietary intake and clinical applications. However, there is currently no study investigating the impact of ginseng and its active compounds on male osteoporosis. In this study, RNA sequencing and bioinformatic analysis were conducted to reveal the influence of Ginsenoside-Rb2 on RAW264.7 cells and its underlying signaling pathways. The potential anti-osteoporosis effects of Rb2 as well as its molecular mechanisms were elucidated in RAW264.7 cells and BMMs by TRAP staining, F-actin belt staining, qRT-PCR and WB. Moreover, orchiectomy (ORX) was utilized to demonstrate the influence of Rb2 on bone mass loss in vivo by micro-CT scanning, and H&E, TRAP, and IHC staining. The results suggested that Rb2 suppressed osteoclastogenesis and mitigated bone loss in orchiectomy mice through NF-κB/MAPK signaling pathways. These findings indicate that ginseng as well as its active component Rb2 have potential therapeutic value in the management of osteoporosis in men.

Myricitrin promotes osteogenesis and prevents ovariectomy bone mass loss via the PI3K/AKT signalling pathway.

This study aimed to explore the effect of myricitrin on osteoblast differentiation in mice immortalised bone marrow mesenchymal stem cells (imBMSCs). Additionally, ovariectomy (OVX) mice were employed to examine the effect of myricitrin on bone trabecular loss in vivo. The effect of myricitrin on the proliferation of imBMSCs was evaluated using a cell counting kit-8 assay. Alizarin red staining, alkaline phosphatase staining were performed to elucidate osteogenesis. Furthermore, qRT-PCR and western blot determined the expression of osteo-specific genes and proteins. To screen for candidate targets, mRNA transcriptome genes were sequenced using bioinformatics analyses. Western blot and molecular docking analysis were used to examine target signalling markers. Moreover, rescue experiments were used to confirm the effect of myricitrin on the osteogenic differentiation of imBMSCs. OVX mice were also used to estimate the delay capability of myricitrin on bone trabecular loss in vivo using western blot, micro-CT, tartaric acid phosphatase (Trap) staining, haematoxylin and eosin staining, Masson staining and immunochemistry. In vitro, myricitrin significantly enhanced osteo-specific genes and protein expression and calcium deposition. Moreover, mRNA transcriptome gene sequencing and molecular docking analysis revealed that this enhancement was accompanied by an upregulation of the PI3K/AKT signalling pathway. Furthermore, copanlisib, a PI3K inhibitor, partially reversed the osteogenesis promotion induced by myricitrin. In vivo, western blot, micro-CT, hematoxylin and eosin staining, Masson staining, Trap staining and immunochemistry revealed that bone trabecular loss rate was significantly alleviated in the myricitrin low- and high-dose groups, with an increased expression of osteopontin, osteoprotegerin, p-PI3K and p-AKT compared to the OVX group. Myricitrin enhances imBMSC osteoblast differentiation and attenuate bone mass loss partly through the upregulation of the PI3K/AKT signalling pathway. Thus, myricitrin has therapeutic potential as an antiosteoporosis drug.

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.

Naringenin protects against iron overload-induced osteoarthritis by suppressing oxidative stress.

BACKGROUND: The traditional Chinese medicine Gusuibu, the rhizome of Rhizoma Drynariae, is used to treat rheumatism and fractures. Naringenin (NAR) is an active ingredient in Gusuibu and has significant anti-inflammatory and antioxidant effects. However, the role of naringenin in iron overload-induced osteoarthritis (IOOA) is unknown. HYPOTHESIS: NAR reduces cartilage damage in IOOA. METHODS: The effects of NAR on the viability of IOOA chondrocytes and the synthesis ability of type II collagen were evaluated using cell counting kit (CCK8) and toluidine blue assays. To determine the mechanism of action and characteristics of NAR, the intracellular iron ion content, apoptosis rate, and mitochondrial membrane potential (MMP) change, and malondialdehyde (MDA) levels, as well as the degree of reactive oxygen species (ROS) and lipid hydroperoxide (LPO) accumulation in the cells were detected in vitro and verified using western blotting and quantitative real-time PCR (qRT-PCR). To verify the role of NAR in vivo, IOOA mice were established using iron dextran and surgery-induced destabilised medial meniscus. Changes in the articular cartilage and subchondral bone were examined using Safranin O-fast Green staining (S-O), haematoxylin-eosin staining (H&E), and microcomputed tomography (µCT). RESULTS: In vitro, NAR attenuated the impairment of cell viability, apoptosis, and MMP caused by ferric ammonium citrate and interleukin-1ß co-culture, increased the levels of MDA, reduced the expression of matrix metallopeptidase (MMP)3, MMP13, and Bax, and restored the expression of type II collagen (Col II). NAR showed a slight iron accumulation-reducing effect. NAR alleviated the accumulation of ROS and LPO in IOOA chondrocytes and upregulated antioxidant genes nuclear factor E2-related factor 2 (NRF2) and haem oxygenase 1 (HO-1). When ML385, a specific NRF-2 inhibitor, was added, the protective effect of NAR was significantly inhibited. In vivo, NAR reduced synovitis and attenuated cartilage damage and subchondral bone proliferation in IOOA mice. CONCLUSIONS: NAR can reduce oxidative stress through the NRF2-HO-1 pathway, alleviate cartilage damage under iron overload, and has the potential to treat IOOA.

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.

Jintiange Capsule May Have a Positive Effect on Pain Relief and Functional Activity in Patients with Knee Osteoarthritis: A Meta-Analysis of Randomized Trials.

BACKGROUND: Knee osteoarthritis (KOA) occurs frequently in the elderly and causes pain, especially when they walk. Traditional Chinese medicine treatment is effective in releasing knee osteoarthritis. Jintiange (JTG) capsule is widely used in treating knee osteoarthritis, but its clinical effects such as pain relief are still unclear. This meta-analysis aims to evaluate the clinical results systematically and negative effects of JTG capsule in patients with knee osteoarthritis. METHODS: A meta-analysis of clinical randomized controlled trials (RCTs) on JTG capsule treatment was carried out in KOA patients. The search time was from the establishment of the database to May 2021. The database included PubMed, Cochrane Library, EMBASE, Web of Science database, Chinese Biomedical database (CBM), Chinese VIP information, China National Knowledge Infrastructure (CNKI), and WanFang database. The outcome indicators were extracted from the included literature and analyzed, and the risk of bias was assessed through Cochrane Handbook 5.0.1. RESULTS: Twenty-two articles analyzed in this study involved 1887 patients. JTG capsule used alone or used with other interventions affects total effective rate significantly (RR: 1.19; 95% Cl: 1.11, 1.29; P=0.045), VAS score (SMD: -0.74; 95% Cl: -0.90, -0.59; P ≤ 0.001), WOMAC score (SMD: -0.77; 95% Cl: -0.96, -0.59; P ≤ 0.001), and Lequesne score (SMD: -0.82; 95% Cl: -1.02, -0.61; P=0.010). CONCLUSION: Our current evidence indicated that JTG capsule may release the pain of KOA patients and improve their functional activity. However, considering the unsatisfactory quality of the included trials, more high-quality trials are needed to prove this issue.