Total flavonoids of Rhizoma drynariae promote angiogenesis and osteogenesis in bone defects.

Bone defects are difficult to heal, which conveys a heavy burden to patients' lives and their economy. The total flavonoids of Rhizoma drynariae (TFRD) can promote the osteogenesis of distraction osteogenesis. However, the dose effect is not clear, the treatment period is short, and the quality of bone formation is poor. In our study, we observed the long-term effects and dose effects of TFRD on bone defects, verified the main ingredients of TFRD in combination with network pharmacology for the first time, explored its potential mechanism, and verified these findings. We found that TFRD management for 12 weeks regulated osteogenesis and angiogenesis in rats with 4-mm tibial bone defects through the PI3K/AKT/HIF-1α/VEGF signaling pathway, especially at high doses (135 mg kg-1  d-1 ). The vascularization effect of TFRD in promoting human umbilical vein endothelial cells was inhibited by PI3K inhibitors. These results provide a reference for the clinical application of TFRD.

Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF­BB/VEGF/RUNX2/OSX signaling axis.

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney­tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co­immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow­derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet­derived growth factor (PDGF)­BB, VEGF, runt­related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription­quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF­BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF­BB. The protein and mRNA levels of PDGF­BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF­BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic­osteogenic coupling to regenerate bone defects during DO via the PDGF­BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Comparative physiological and transcriptomic analyses illuminate common mechanisms by which silicon alleviates cadmium and arsenic toxicity in rice seedlings.

The inessential heavy metal/loids cadmium (Cd) and arsenic (As), which often co-occur in polluted paddy soils, are toxic to rice. Silicon (Si) treatment is known to reduce Cd and As toxicity in rice plants. To better understand the shared mechanisms by which Si alleviates Cd and As stress, rice seedlings were hydroponically exposed to Cd or As, then treated with Si. The addition of Si significantly ameliorated the inhibitory effects of Cd and As on rice seedling growth. Si supplementation decreased Cd and As translocation from roots to shoots, and significantly reduced Cd- and As-induced reactive oxygen species generation in rice seedlings. Transcriptomics analyses were conducted to elucidate molecular mechanisms underlying the Si-mediated response to Cd or As stress in rice. The expression patterns of the differentially expressed genes in Cd- or As-stressed rice roots with and without Si application were compared. The transcriptomes of the Cd- and As-stressed rice roots were similarly and profoundly reshaped by Si application, suggesting that Si may play a fundamental, active role in plant defense against heavy metal/loid stresses by modulating whole genome expression. We also identified two novel genes, Os01g0524500 and Os06g0514800, encoding a myeloblastosis (MYB) transcription factor and a thionin, respectively, which may be candidate targets for Si to alleviate Cd and As stress in rice, as well as for the generation of Cd- and/or As-resistant plants. This study provides valuable resources for further clarification of the shared molecular mechanisms underlying the Si-mediated alleviation of Cd and As toxicity in rice.

Total Flavonoids of Rhizoma Drynariae Enhances Angiogenic-Osteogenic Coupling During Distraction Osteogenesis by Promoting Type H Vessel Formation Through PDGF-BB/PDGFR-ß Instead of HIF-1α/ VEGF Axis.

Background: Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney-tonifying traditional Chinese medicine Rhizoma Rrynariae, has been proved to be effective in treating osteoporosis, bone fractures and defects. However, pharmacological effects of TFRD on type H vessels, angiogenic-osteogenic coupling in distraction osteogenesis (DO) and the mechanism remain unclear. This study aims at investigating whether type H vessels exist in the DO model, effects of TFRD on angiogenic-osteogenic coupling and further elucidating the underlying mechanism. Methods: Rats models of DO and bone fracture (FR) were established, and then were separately divided into TFRD and control subgroups. Imageological and histological analyses were performed to assess bone and vessel formation. Immunofluorescent staining of CD31 and endomucin (Emcn) was conducted to determine type H vessel formation. Matrigel tube formation, ALP and Alizarin Red S staining assays were performed to test the effects of TFRD on angiogenesis or osteogenesis of endothelial precursor cells (EPCs) or bone marrow-derived mesenchymal stem cells (BMSCs). Additionally, expression levels of HIF-1α, VEGF, PDGF-BB, RUNX2 and OSX were determined by ELISA, qPCR or western blot, respectively. Results: The in vivo results indicated more formed type H vessels in DO groups than in FR groups and TFRD obviously increased the abundance of type H vessels. Moreover, groups with higher abundance of type H vessels showed better angiogenesis and osteogenesis outcomes. Further in vitro experiments showed that TFRD significantly promoted while blocking PDGF-BB remarkably suppressed the angiogenic activity of EPCs under stress conditions. The levels of p-AKT and p-ERK1/2, downstream mediators of the PDGF-BB pathway, were up-regulated by TFRD but blocked by function blocking anti-PDGF-BB antibody. In contrast, the activated AKT and ERK1/2 and corresponding tube formation were not affected by the HIF-1α inhibitor. Besides, blocking PDGF-BB inhibited the osteogenic differentiation of the stretched BMSCs, but TFRD enhanced the osteogenic activity of BMSCs and ameliorated the inhibition, with more calcium nodes, higher ALP activity and mRNA and protein levels of RUNX2 and OSX. Conclusion: Type H vessels exist in the DO model and TFRD enhances angiogenic-osteogenic coupling during DO by promoting type H vessel formation via PDGF-BB/PDGFR-ß instead of HIF-1α/VEGF axis.