Therapeutic potential of a prominent dihydroxyflavanone pinocembrin for osteolytic bone disease: In vitro and in vivo evidence.

Background/objective: As the pivotal cellular mediators of bone resorption and pathological bone remodeling, osteoclasts have emerged as a prominent target for anti-resorptive interventions. Pinocembrin (PIN), a predominant flavonoid found in damiana, honey, fingerroot, and propolis, has been recognized for its potential therapeutic effects in osteolysis. The purpose of our project is to investigate the potential of PIN to prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms. Methods: The study commenced by employing protein-ligand molecular docking to ascertain the specific interaction between PIN and nuclear factor-κB (NF-κB) ligand (RANKL). Subsequently, PIN was introduced to bone marrow macrophages (BMMs) under the stimulation of RANKL. The impact of PIN on osteoclastic activity was assessed through the utilization of a positive TRAcP staining kit and a hydroxyapatite resorption assay. Furthermore, the study investigated the generation of reactive oxygen species (ROS) in osteoclasts induced by RANKL using H2DCFDA. To delve deeper into the underlying mechanisms, molecular cascades triggered by RANKL, including NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were explored using Luciferase gene report, western blot analysis, and quantitative real-time polymerase chain reaction. Moreover, an estrogen-deficient osteoporosis murine model was established to evaluate the therapeutic effects of PIN in vivo. Results: In this study, we elucidated the profound inhibitory effects of PIN on osteoclastogenesis and bone resorption, achieved through repression of NF-κB and NFATc1-mediated signaling pathways. Notably, PIN also exhibited potent anti-oxidative properties by mitigating RANKL-induced ROS generation and augmenting activities of ROS-scavenging enzymes, ultimately leading to a reduction in intracellular ROS levels. Moreover, PIN effectively abrogated the expression of osteoclast-specific marker genes (Acp5, Cathepsin K, Atp6v0d2, Nfatc1, c-fos, and Mmp9), further underscoring its inhibitory impact on osteoclast differentiation and function. Additionally, employing an in vivo mouse model, we demonstrated that PIN effectively prevented osteoclast-induced bone loss resultant from estrogen deficiency. Conclusion: Our findings highlight the potent inhibitory effects of PIN on osteoclastogenesis, bone resorption, and RANKL-induced signaling pathways, thereby establishing PIN as a promising therapeutic candidate for the prevention and management of osteolytic bone diseases. The translational potential of this article: PIN serves as a promising therapeutic agent for the prevention and management of osteolytic bone diseases and holds promise for future clinical applications in addressing conditions characterized by excessive bone resorption. PIN is a natural compound found in various sources, including damiana, honey, fingerroot, and propolis. Its widespread availability and potential for therapeutic use make it an attractive candidate for further investigation and development as a clinical intervention.

Prediction of musculoskeletal pain after the first intravenous zoledronic acid injection in patients with primary osteoporosis: development and evaluation of a new nomogram.

OBJECTIVE: To construct a new prediction nomogram to predict the risk of musculoskeletal pain in patients with primary osteoporosis who receive zoledronic acid intravenously for the first time. METHOD: Clinical data of 368 patients with primary osteoporosis who received the first intravenous injection of zoledronic acid in our hospital from December 2019 to December 2022 were studied. Patients were divided into a musculoskeletal pain group (n = 258) and a non-musculoskeletal pain group (n = 110) based on the presence or absence of musculoskeletal pain 3 days after injection. Statistically significant predictors were screened by logistic regression analysis and the minimum absolute contraction and selection operator (LASSO) to construct a nomogram. The nomogram was evaluated by the receiver operating characteristic (ROC) curve, the calibration curve, the C-index, and the decision curve analysis (DCA) and verified in a validation cohort. RESULTS: The independent predictors of the nomogram were age, serum 25-hydroxyvitamin D, NSAIDs, prior Vitamin D intake, and BMI. The area under the ROC curve (AUC) was 0.980 (95% CI, 0.915-0.987), showing excellent predictive performance. The nomogram c index was 0.980, and the nomogram c index for internal verification remained high at 0.979. Moreover, calibration curves show that the nomogram has good consistency. Finally, the DCA showed that the net benefit of the nomogram was 0.20-0.49. CONCLUSION: Musculoskeletal pain is a common symptom of APR in OP patients treated with intravenous zoledronic acid. Risk factors for musculoskeletal pain after zoledronic acid injection in OP patients were: non-use of NSAIDs, youth (<80 years old), serum 25 (OH) D<30ng /mL, no prior intake of vitamin D, BMI<24 kg /m2. A nomogram constructed from the above predictors can be used to predict musculoskeletal pain after the first zoledronic acid injection.

Huang-Lian-Jie-Du Decoction Attenuates Atherosclerosis and Increases Plaque Stability in High-Fat Diet-Induced ApoE-/- Mice by Inhibiting M1 Macrophage Polarization and Promoting M2 Macrophage Polarization.

Macrophage polarization plays a vital impact in triggering atherosclerosis (AS) progression and regression. Huang-Lian-Jie-Du Decoction (HLJDD), a famous traditional Chinese decoction, displays notable anti-inflammatory and lipid-lowering effects in different animal models. However, its effects and mechanisms on AS have not been clearly defined. We determined whether HLJDD attenuated atherosclerosis and plaques vulnerability by regulating macrophage polarization in ApoE-/- mice induced by high-fat diet (HFD). Furthermore, we investigated the effects of HLJDD on macrophage polarization in oxidized low-density lipoprotein (ox-LDL) induced RAW264.7 cells. For in vivo assay, compared with the model group, HLJDD ameliorated lipid metabolism, with significantly decreased levels of serum triglyceride, total cholesterol (CHOL), and lipid density lipoprotein. HLJDD suppressed serum tumor necrosis factor α (TNF-α) and IL-1ß levels with increased serum IL-10 level, and inhibited mRNA level of NLRP3 inflammasome in carotid tissues. HLJDD enhanced carotid lesion stability by decreasing macrophage infiltration together with increased expression of collagen fibers and α-SMA. Moreover, HLJDD inhibited M1 macrophage polarization, which decreased the expression and mRNA levels of M1 markers [inducible nitric oxide synthase (iNOS) and CD86]. HLJDD enhanced alternatively activated macrophage (M2) activation, which increased the expression and mRNA levels of M2 markers (Arg-1 and CD163). For in vitro assay, HLJDD inhibited foam cell formation in RAW264.7 macrophages disturbed by ox-LDL. Besides, groups with ox-LDL plus HLJDD drug had a lower expression of CD86 and mRNA levels of iNOS, CD86, and IL-1ß, but higher expression of CD163 and mRNA levels of Arg-1, CD163, and IL-10 than ox-LDL group. Collectively, our results revealed that HLJDD alleviated atherosclerosis and promoted plaque stability by suppressing M1 polarization and enhancing M2 polarization.

Integrating Network Pharmacology and Experimental Validation to Decipher the Mechanism of Action of Huanglian Jiedu Decoction in Treating Atherosclerosis.

BACKGROUND: This study used network pharmacology, molecular docking and experimental validation to assess the effects of Huanglian Jiedu Decoction (HLJDD) on atherosclerosis (AS). METHODS: The components and targets of HLJDD were analyzed using the Traditional Chinese Medicine Systems Pharmacology database, and information on the genes associated with AS was retrieved from the GeneCards and OMIM platforms. Protein-protein interactions were analyzed using the STRING platform. A component-target-disease network was constructed using Cytoscape. GO and KEGG analyses were performed to identify molecular biological processes and signaling pathways, and the predictions were verified experimentally. Molecular docking was conducted with ChemOffice software, PyMOL software and Vina to verify the correlation of targets and compounds. RESULTS: HLJDD contained 31 active compounds, with quercetin, kaempferol, moupinamide and 5-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone as the core compounds. The most important biotargets of HLJDD in AS were ICAM-1, CD31 and RAM-11. The molecular docking results showed that the molecular docking interaction energy between the 3 key targets and the 4 high-degree components were much less than -5 kJ∙mol-1. The experimental validation results showed that HLJDD might treat AS mainly by reducing TC, TG and LDL-C and increasing HDL-C, upregulating CD31 expression, reducing ICAM-1 and RAM-11 expression, and downregulating inflammatory factors, including CRP, IL-6 and TNF-α. These results support the network pharmacology data and demonstrate that HLJDD affects the expression of core genes and alters the leukocyte transendothelial migration signaling pathway. CONCLUSION: Based on network pharmacology and experimental validation, our study indicated that HLJDD exerted anti-AS effect through upregulating CD31 expression and reducing the expression of ICAM-1 and RAM-11. HLJDD may be a potential therapeutic drug to the prevention of AS.

Total flavonoids of rhizoma drynariae ameliorates bone formation and mineralization in BMP-Smad signaling pathway induced large tibial defect rats.

Osteogenesis and angiogenesis acts as an essential role in repairing large tibial defects (LTDs). Total flavonoids of rhizoma drynariae (TFRD), a traditional Chinese medicinal herb, is reported to show anabolic effects on fracture healing. However, whether TFRD could improve the bone formation and angiogenesis in LTDs remains unknown. The purpose of this study was to evaluate the effect of TFRD on bone formation and angiogenesis in LTDs in distraction osteogenesis (DO). Using a previously established fracture model, LTD rats was established with circular external fixator (CEF). All rats then randomly divided into TFRD low dosage group (with DO), TFRD medium dosage group (with DO), TFRD high dosage group (with DO), model group (with DO) and blank group (without DO). Twelve weeks after treatment, according to X-ray and Micro-CT, TFRD groups (especially in medium dosage group) can significantly promote the formation of a large number of epiphyses and improve new bone mineralization compared with model group, and the results of HE and Masson staining and in vitro ALP level of BMSC also demonstrated the formation of bone matrix and mineralization in the TFRD groups. Also, angiographic imaging suggested that total flavonoids of TFRD was able to promote angiogenesis in the defect area. Consistently, TFRD significantly increased the levels of BMP-2, SMAD1, SMAD4, RUNX-2, OSX and VEGF in LTD rats based on ELISA and Real-Time PCR. In addition, we found that ALP activity of TFRD medium dosage group reached a peak after 10 days of induction through BMSC cell culture in vitro experiment. TFRD promoted bone formation in LTD through activation of BMP-Smad signaling pathway, which provides a promising new strategy for repairing bone defects in DO surgeries.