Rhizoma drynariae total flavonoids combined with calcium carbonate ameliorates bone loss in experimentally induced Osteoporosis in rats via the regulation of Wnt3a/β-catenin pathway

Background Rhizoma drynariae, a traditional Chinese herb, is commonly used in treatment of bone healing in osteoporotic fractures. However, whether the Rhizoma drynariae total flavonoids (RDTF) can promote the absorption of calcium and enhance the bone formation is unclear. The aim of the present study was to investigate the preventive effects of RDTF combined with calcium carbonate (CaCO3) on estrogen deficiency-induced bone loss. Methods Three-month-old Sprague–Dawley rats were ovariectomized (OVX) and then treated with CaCO3, RDTF, and their admixtures for ten weeks, respectively. The bone trabecular microstructure, bone histopathological examination, and serum biomarkers of bone formation and resorption were determined in the rat femur tissue. The contents of osteoprotegerin (OPG), receptor activator of the NF-κB (RANK), and its ligand (RANKL) in marrow were analyzed by ELISA, and the protein expressions of Wnt3a, β-catenin, and phosphorylated β-catenin (p-β-catenin) were analyzed by Western blot. Statistical analysis was conducted by using one-way analysis of variance (ANOVA) followed by LSD post hoc analysis or independent samples t test using the scientific statistic software SPSS version 20.0 Results RDTF combined with CaCO3 could promote osteosis and ameliorate bone loss to improve the repair of cracked bone trabeculae of OVX rats. Furthermore, RDTF combined with CaCO3 also could prevent OVX-induced decrease in collagen fibers in the femoral tissue of ovariectomized rats and promote the regeneration of new bone or cartilage tissue, while CaCO3 supplementation promoted the increase in bone mineral content. Nevertheless, there was no difference in the expression of Wnt3a, β-catenin and p-β-catenin between osteopenic rats and RDTF treated rats, but RDTF combined with CaCO3 could activate the Wnt3a/β-catenin pathway. Conclusions RDTF combined with CaCO3 could ameliorate estrogen deficiency-induced bone loss via the regulation of Wnt3a/β-catenin pathway.

Prediction of Rhizoma Drynariae Targets in the Treatment of Osteoarthritis Based on Network Pharmacology and Experimental Verification

Rhizoma Drynariae has been widely used for the treatment of osteoarthritis (OA), but its potential targets and molecular mechanisms remain to be further explored. Targets of Rhizoma Drynariae and OA were predicted by relevant databases, and a protein-protein interaction (PPI) network was constructed to identify key targets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to obtain related pathways and then select significant pathways associated with OA. The OA chondrocyte model was established by inflammatory factor-induced SW1353 chondrocytes, and molecular docking was conducted to verify the above theoretical prediction. The results showed that a total of 86 Rhizoma Drynariae-OA interaction targets were identified, among which IL-6 and AKT1 were the key targets in the PPI network. Luteolin was the most critical component of Rhizoma Drynariae. KEGG results indicated that the effects of Rhizoma Drynariae on OA are associated with the PI3K/AKT, TNF, IL-17, apoptosis, and HIF-1 signaling pathway. The PI3K/AKT pathway can activate the downstream NF-κB pathway and further regulate the transcription and expression of downstream IL-6, IL-17, HIF-1α, Bax, and TNF, suggesting that the PI3K/AKT/NF-κB pathway is the critical pathway in the treatment of OA with Rhizoma Drynariae. Active components of Rhizoma Drynariae and key proteins of the PI3K/AKT/NF-κB signaling pathway were subjected to molecular docking, whose results showed that luteolin and IKK-α played a critical role. In vitro experiments indicated that both aqueous extracts of Rhizoma Drynariae (AERD) and luteolin inhibited the expression of IL-6 and HIF-1α and suppressed the activation of PI3K/AKT/NF-κB, IL-17, and TNF pathways. The measurement of mitochondrial membrane potential (Δψm) indicated that AERD and luteolin can decrease the LPS-induced early apoptotic cells. Luteolin had a more prominent inhibitory effect than AERD in the abovementioned in vitro experiments. In conclusion, the therapeutic mechanism of Rhizoma Drynariae against OA may be closely related to the inhibition of the PI3K/AKT/NF-κB pathway and downstream pathways, and luteolin plays a vital role in the treatment.

Rhizoma Drynariae protects against Canaglifloxacin induced bone loss

Osteoporosis is a condition marked by a loss of bone mass and degradation of the bone microstructure, both of which lead to increased fragility and consequent fragility fractures, particularly in the elderly. Rhizoma Drynariae (DRE) is one of the most often used herbal remedies for osteoporosis therapy. Transdermal drug administration is a well- established new method for drug delivery that offers numerous benefits over conventional routes. Wistar albino rats were split into five groups of six rats each: vehicle control, diabeticgroup, DRE group, Canagliflozin (CGF), and CGF + DRE group. Each medication was given by gastric gavage once a day for 35 days. The drug canagliflozin appears to raise the risk of fractur. When compared to the control group, DRE treatment increased bone strength at the femoral diaphysis in osteoporotic fractures in rats by increasing ultimate load and stiffness. The goal of this study is to investigate the anti-osteoporosis effects of DRE in diabetic rats co-treated with CGF. Blood glucose levels and bone mineral density (BMD) were measured. According to the data, DRE produced a significant increase in bone amount. DRE may help prevent and cure diabetic osteoporosis by increasing bone mineral density, according to one study. Keywords: Rhizoma Drynariae Diabetic osteoporosis, Streptozotocin induced diabetes, Canagliflozin

Rhizoma Drynariae promotes the Osteogenic differentiation of Bone Mesenchymal Stem Cells by activating the Wnt/β-catenin Signaling Pathway

Background: Rhizoma Drynariae (RD), a traditional Chinese medicine with pleiotropic biological activities, exerts a protective effect against age-related osteoporosis. Osteoporosis is a serious clinical problem and characterized by the deterioration in bone volume and strength, mainly due to the dysfunction of bone marrow stromal cells (BMSCs). However, it remains unclear whether RD-containing serum regulates the osteogenic differentiation of BMSCs via Wnt/β-catenin signaling pathway. Methods: The effects of RD-containing serum on the osteogenesis of BMSCs were detected via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), western blotting, alkaline phosphatase (ALP) activity assay and alizarin red staining. Using Dickkopf-related protein-1 (DKK1), an inhibitor of the Wnt/β-catenin signaling pathway, we examined whether RD-containing serum regulates osteoblast differentiation of BMSCs via the Wnt/b-catenin signalling pathway. Results: The results showed that RD-containing serum promoted the BMSC proliferation and ALP activities, as well as up-regulated the expression of osteogenic markers and Wnt/β-catenin pathway-related genes, i.e., runt-related transcription factor 2, Sp7, osteocalcin, β-catenin and Wnt3a, in BMSCs. Moreover, we found that RD-containing serum activated the Wnt/β-catenin pathway. Adding DKK1 to the RD-containing serum could decrease the promotion of osteogenic differentiation of RD-containing serum on BMSCs. Conclusions: Collectively, RD-containing serum could promote the osteogenic differentiation of BMSCs, and the potential mechanism may involve regulation of Wnt/β-catenin signaling.

Total Flavonoids of Rhizoma Drynariae Promotes Differentiation of Osteoblasts and Growth of Bone Graft in Induced Membrane Partly by Activating Wnt/β-Catenin Signaling Pathway

Total flavonoids of Rhizoma drynariae (TFRD), a Chinese medicine, is widely used in the treatment of fracture, bone defect, osteoporosis and other orthopedic diseases, and has achieved good effects. Purpose of this trial was to explore efficacy of TFRD on bone graft’s mineralization and osteoblasts’ differentiation in Masquelet induced membrane technique in rats. Forty male Sprague-Dawley rats were randomly divided into high dose group (H-TFRD), middle dose group (M-TFRD), low dose group (L-TFRD) and control group (control). The critical size bone defect model of rats was established with 10 rats in each group. Polymethyl methacrylate (PMMA) spacer was implanted into the defect of right femur in rats. After the formation of the induced membrane, autogenous bone was implanted into the induced membrane. After 12 weeks of bone graft, bone tissues in the area of bone graft were examined by X-ray, Micro-CT, hematoxylin-eosin (HE) and Masson trichrome staining to evaluate the growth of the bone graft. The β-catenin, c-myc, COL1A1, BMP-2 and OPN in bone graft were quantitatively analyzed by Western blot and Immunohistostaining. Osteoblasts were cultured in the medium containing TFRD. Cell Counting Kit-8 (CCK-8) method, Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, Western blot, RT-PCR and other methods were used to detect the effects of TFRD on the proliferation of osteoblasts and the regulation of Wnt/β-catenin signaling pathway. In vivo experiments showed that the growth and mineralization of bone graft in TFRD group was better. Moreover, the expression of Wnt/β-catenin and osteogenesis-related proteins in bone tissue of TFRD group was more than that in other groups. In vitro experiments indicated that osteoblasts proliferated faster, activity of ALP was higher, number of mineralized nodules and proteins related to osteogenesis were more in TFRD group. But blocking Wnt/β-catenin signaling pathway could limit these effects. Therefore, TFRD could promote mineralization of bone graft and differentiation of osteoblasts in a dose-dependent manner during growing period of the bone graft of induced membrane technique, which is partly related to the activation of Wnt/β-catenin signaling pathway.

Total Flavonoids of Rhizoma Drynariae Restore the MMP/TIMP Balance in Models of Osteoarthritis by Inhibiting the Activation of the NF- κ B and PI3K/AKT Pathways

Total flavonoids of Rhizoma Drynariae (TFRD) have been shown to have beneficial effects on osteoarthritis (OA) clinically, but the mechanisms have not been elucidated. In this study, we investigated the effect of TFRD on articular cartilage in an OA rat model established by the Hulth method and in SW1353 chondrocytes induced by the proinflammatory factor interleukin-1β (IL-1β). The results showed that TFRD could alleviate the pathological changes in knee cartilage in OA model rats. In vivo, the qPCR analysis indicated that the mRNA levels of matrix metalloproteinases, MMP-1, MMP-3, and MMP-13, were decreased, while tissue inhibitor of matrix metalloproteinases- (TIMP-) 4 was increased in cartilage, and these changes could be partially prevented by TFRD. In vitro experiments showed that IL-1β could significantly increase the expression of MMP-1, MMP-3, and MMP-13 and decrease the expression of TIMP-4 in SW1353 cells at the mRNA and protein levels. TFRD could increase the expression of MMP-3 and MMP-13 and decrease the expression of TIMP-4. Transfection of siRNA and addition of pathway inhibitors were used to clarify that inhibition of NF- κ B and PI3K/AKT pathway decreased MMP-1, MMP-3, and MMP-13 and increased TIMP-4 expression. We also found that in IL-1β-induced SW1353 cells, TFRD pretreatment had a modest inhibitory effect on p-AKT (Ser473) and reversed the increase of nuclear factor kappa-B (NF- κ B ) p65 in nuclear fraction and the decrease of inhibitor of NF- κ B I κ B - α in the cytosolic fraction. Further immunofluorescence confirmed that TFRD can inhibit IL-1β-induced NF- κ B p65 translocation to the nucleus to some extent. In conclusion, TFRD showed chondroprotective effects by restoring the MMP/TIMP balance in OA models by suppressing the activation of the NF- κ B and PI3K/AKT pathways.