Total flavonoids of Rhizoma drynariae improves tendon-bone healing for anterior cruciate ligament reconstruction in mice and promotes the osteogenic differentiation of bone mesenchymal stem cells by the ERR1/2-Gga1-TGF-ß/MAPK pathway.

BACKGROUND: Total flavonoids of Rhizoma drynariae (TFRD) is broadly used in the treatment of orthopedic diseases. Nevertheless, the effects and underlying mechanism of TFRD on tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) remain unclear. METHODS: The ACLR mouse model was established. Hematoxylin and Eosin (HE) staining was used for histological analysis of tendon-bone healing. Western blot was utilized to detect the levels of osteogenic related factors (ALP, OCN, RUNX2). The viability and alkaline phosphatase (ALP) activity of bone mesenchymal stem cells (BMSCs) were determined by Cell Counting Kit-8 (CCK-8) and ALP assays. The interaction of estrogen related receptor alpha (ESRRA), estrogen related receptor beta (ESRRB), and golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (Gga1) was detected by luciferase reporter assays. The levels of important proteins on the TGF-ß/MAPK pathway were measured by western blot. RESULTS: TFRD improved tendon-bone healing, restored biomechanics of ACLR mice and activated the TGF-ß/MAPK pathway. TFRD treatment also enhanced the viability and osteogenic differentiation of BMSCs in vitro. Then, we demonstrated that TFRD targeted ESRRA and ESRRB to transcriptionally activate Gga1 expression. Knockdown of ESRRA, ESRRB, or Gga1 suppressed the viability and osteogenic differentiation of TFRD-induced BMSCs, which was revealed to be restored by Gga1 overexpression. The overexpression of ESRRA, ESRRB, or Gga1 was demonstrated to promote the BMSC viability and osteogenic differentiation. TGF-ß1 treatment can reverse the impact of Gga1 inhibition on osteogenic differentiation in TFRD-induced BMSCs. CONCLUSION: TFRD improves tendon-bone healing in ACLR mouse models and facilitates the osteogenic differentiation of BMSCs through the ERR1/2-Gga1-TGF-ß/MAPK pathway, which might deepen our understanding of the underlying mechanism of TFRD in tendon-bone healing.

Enhancing cranial defect repair in rats: investigating the effect of combining Total Flavonoids from Rhizoma Drynariae with calcium phosphate/collagen scaffolds.

OBJECTIVE: To investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats. METHODS: The subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin-eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN). RESULTS: Upon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups. CONCLUSIONS: The synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.

Metabolite profiling of Drynariae Rhizoma using 1H NMR and HPLC coupled with multivariate statistical analysis.

Drynariae Rhizoma has been used to treat bone diseases and kidney deficiency in traditional medicine. Recently its aqueous extract was reported to enhance memory function. Although the Japanese standards for non-Pharmacopoeial crude drugs 2022 prescribed Drynaria roosii as the botanical origin, some counterfeits and both raw and stir-fired crude drugs are available in markets. To distinguish Drynariae Rhizoma derived from D. roosii appropriately from others and verify the validity of uses of stir-fried ones, 1H NMR-based metabolite profiling coupled with HPLC were performed. Raw samples derived from D. roosii contained naringin (1), neoeriocitrin (2), 5,7-dihydroxychromone-7-O-neohesperidoside (3), caffeic acid 4-O-ß-D-glucoside (4), protocatechuic acid (5), trans-p-coumaric acid 4-O-ß-D-glucoside (6), and kaempferol 3-O-α-L-rhamnoside 7-O-ß-D-glucoside (8). Stir-fried samples were characterized by presence of 5-hydroxymethyl-2-furaldehyde (13), and were divided into two types; one possessing similar composition to raw samples (Type I) and another without above components except 5 (Type II). Quantitative analyses using qHNMR and HPLC, followed by principal component analysis demonstrated that the raw samples had higher contents of 1 (0.93-9.86 mg/g), 2 (0.74-7.59 mg/g), 3 (0.05-2.48 mg/g), 4 (0.27-2.51 mg/g), 6 (0.14-1.26 mg/g), and 8 (0.04-0.52 mg/g), and Type II had a higher content of 5 (0.84-1.32 mg/g). The counterfeit samples derived from Araiostegia divaricata var. formosana were characterized by higher content of ( -)-epicatechin 3-O-ß-D-allopyranoside (10) (1.44-11.49 mg/g) without 1 and 2. These results suggested that Drynariae Rhizoma samples derived from other botanical origins and Type II stir-fried samples cannot substitute for D. roosii rhizome.

A rapid approach to capture the potential bioactive compounds from Rhizoma Drynariae, utilizing disease-associated mutation in calcium sensing receptor to alter the binding affinity for agonists.

Rhizoma Drynariae (RD) was used clinically to treat osteoporosis in China due to stimulating bone formation and inhibiting bone resorption, however, the bioactive constituents with the dual effect on bone are still unknown exactly. Disease-causing mutations in calcium sensing receptor (CaSR) can alter parathyroid hormone secretion and affect Ca2+ release from bone and Ca2+ reabsorption from kidney, which gives an indication that CaSR is a potential target for developing therapeutics to manage osteoporosis. Herein, a chromatographic approach was established, by immobilizing the mutant CaSR onto the surface of silica gels as stationary phase in a one-step procedure and then adding the different amino acids into mobile phase as competitors, for exploring the binding features of the known agonists and further screening ligands from RD. The mutant CaSR-coated column was prepared rapidly without the complicated purification and separation of the receptor, which had the large capacity of 13.1 mg CaSR /g silica gels and kept a good stability and specificity for at least 35 days. The CaSR mutation can weaken the binding affinities for three agonists, and the largest decreases occurred on the mutational site Thr151Met for neomycin, on the two sites of Asn118Lys and Glu191Lys for gentamicin-C, and on the site Phe612Ser for kanamycin, which gained new insights into their structure-function relationship. The potential bioactive compounds from RD were screened using the mutant CaSR-coated column and were recognized as coumaric acid 4-O-ß-D-glucopyranoside, caffeic acid, and naringin using UPLC-MS. Among them, naringin targeting CaSR gives a possible explanation that RD could manage osteoporosis. These results indicated that, such a rapid and simple method, utilizing disease-associated mutation in CaSR to alter the binding affinity for agonists, can be applied in capturing the potential bioactive compounds efficiently from complex matrices like herb medicines.

Network Pharmacology-Based Dissection of the Mechanism of Drynariae Rhizoma for Low Back Pain.

Objective: To explain the potential mechanisms of Drynariae Rhizoma (DR) in the treatment of low back pain (LBP). Design: Network pharmacology was used to reveal the potential mechanisms including collecting the active ingredients of DR, analyzing the common gene targets of LBP and DR, constructing protein-protein interaction (PPI) network, collecting protein classification, performing Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and verifying significant gene targets. Results: 234 different gene targets and 18 active compounds altogether were obtained. AKT1, VEGFA, and HIF1A were deemed to be major gene targets based on the degree values. According to GO analysis, steroid metabolic process involved 42 (18.10%) potential therapeutic LBP targets, neuronal cell body involved 24 (10.30%) potential therapeutic LBP targets, and protein serine/threonine kinase activity involved 28 (12.02%) potential therapeutic LBP targets in biological process (BP), cellular component (CC), and molecular function (MF), respectively. According to KEGG and pathway interaction analyses, the PI3K-Akt signaling pathway involved 34 (15.89%) potential therapeutic LBP targets, and PI3K-Akt signaling pathway played a significant role in the treatment of LBP. The mRNA expression levels of AKT1 and HIF1A were upregulated in healthy nucleus pulposus (NP) tissue than in degenerative NP tissue. In contrast, the mRNA expression level of VEGFA was downregulated in healthy NP tissue than in degenerative NP tissue. Conclusions: In this study, we identified a potential relationship between LBP and DR in this work, as well as a synergistic mechanism of DR in the treatment of LBP, which serves as a benchmark for further in vivo and in vitro research.

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.

Study on the Protective Effect and Mechanism of the Rhizoma Drynariae-Epimedium Formula on Osteoarthritis in Rats.

Purpose: The aim of the study was to study the protective effect of the Rhizoma Drynariae-Epimedium formula on osteoarthritis in rats and to explore its mechanism. Methods: Fifty SD rats were randomly divided into 5 groups, namely, the control group, model group, Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group, with 10 rats in each group. Knee arthritis models were established by injecting papain solution (10% papain + 0.03 mol/L L-cysteine mixture) into the knee joint cavity of SD rats on the 0th, 3rd, and 6th days of the experiment, respectively. The model group, Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group were given modeling treatment, while the control group was not given modeling treatment. The Rhizoma Drynariae group, Epimedium group, and the Rhizoma Drynariae-Epimedium group were, respectively, given corresponding solvent gavage treatment. Both the model group and the control group were given an equal volume of normal saline. Once a day, a total of 4 w were administered. The general conditions of the rats were observed and recorded, and the knee joint width and the knee joint swelling degree of the affected side were measured and compared. HE staining and Safranin O-fast green staining were used to compare the structural changes of cartilage. The concentrations of inflammatory factors IL-1ß, IL-6, and TNF-α in the joint cavity lavage fluid were determined by using ELISA. The expression of key proteins of the MAPK signaling pathway (p38, p-p38, ERK, p-ERK, JNK, and p-JNK) in joint synovial tissue was determined by western blotting. Results: After modeling, except for the normal activities of the SD rats in the control group, the rest of the groups showed lack of energy and a slight limp in the knee joints. The SD rats in the model group, Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group had local swelling of the knee joint, and the knee joint width was greater than those in the control group (p < 0.05). Compared with the model group, the knee joint swelling of SD rats in the Rhizoma Drynariae group, the Epimedium group, and the Rhizoma Drynariae-Epimedium group was significantly reduced. The knee joint swelling degree of SD rats in the Rhizoma Drynariae-Epimedium group was significantly lower than that in the Rhizoma Drynariae and Epimedium groups. HE staining and Safranin O-fast green staining showed that the cartilage structure of SD rats was severely damaged and eroded, and the subchondral bone mass was reduced. Compared with the model group, the damage of cartilage tissue in the Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group was less severe. In the Rhizoma Drynariae-Epimedium group, cartilage tissue structure damage and erosion were lighter than those of the Rhizoma Drynariae group and the Epimedium group. The concentrations of inflammatory factors IL-1ß, IL-6, and TNF-α in the articular cavity lavage fluid of SD rats in the model group, Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group were higher than those in the control group. Compared with the model group, the concentrations of IL-1ß, IL-6, and TNF-α in the joint cavity lavage fluid of the Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group were significantly decreased. In the Rhizoma Drynariae-Epimedium group, IL-1ß, IL-6, and TNF-α concentrations were lower than those of the Rhizoma Drynariae and Epimedium groups. Compared with the control group, the expression levels of p-p38, p-ERK, and p-JNK proteins in the model group, Rhizoma Drynariae group, Epimedium group, and Rhizoma Drynariae-Epimedium group were significantly increased. The expression levels of p-ERK, p-p38 and p-JNK in the Drynariae group, Epimedium group, and Drynariae-Epimedium group were significantly lower than those in the model group. The expression levels of p-ERK, p-p38, and p-JNK in the Rhizoma Drynariae-Epimedium group were significantly lower than those in the Rhizoma Drynariae and Epimedium groups. Conclusion: The Rhizoma Drynariae-Epimedium formula can play a protective role in the process of osteoarthritis by inhibiting the phosphorylation levels of p38, ERK, and JNK-related proteins in the cartilage tissue MAPK signaling pathway, reducing the inflammatory response.

Drynaria fortunei improves lipid profiles of elderly patients with postmenopausal osteoporosis via regulation of Notch1-NLRP3 inflammasome-mediated inflammation.

BACKGROUND: Dyslipidemia is a common comorbidity in elderly patients with postmenopausal osteoporosis (PMOP). Drynaria fortunei (Rhizoma drynariae) is well-known in traditional Chinese medicine for its ability to improve bone mineral density (BMD). However, whether and how Drynaria fortunei improves plasma lipid profiles in elderly PMOP patients remains unclear. METHODS: Eighty elderly female patients with concurrent PMOP and hyperlipemia were randomly assigned to Drynaria fortunei 2(n = 40) or control (n = 40) groups. The clinical efficacies of Drynaria fortunei were evaluated. At 0, 3-, 6-, 9-, and 12-month of follow-up, plasma levels of IL-1ß, IL-18, TNF-α, IL-6, IL-8, and IL-10 were measured using ELISA, whereas PBMC levels of NLRP3, ASC, caspase-1, NF-κB, SIRT1, and Notch1 were measured using RT-qPCR. PBMC isolated from PMOP patients were cultured and treated with Drynaria fortunei to determine its influence on NLRP3 inflammasome and associated cytokines. RESULTS: Drynaria fortunei effectively improved patients' BMD and lipid profiles. IL-1ß, IL-18, TNF-α, IL-6, IL-8 levels, as well as inflammasome-molecules of NLRP3, ASC, caspase-1, and NF-κB increased over time in the control group, but were significantly attenuated with Drynaria fortunei administration. In vitro, Drynaria fortunei suppressed NLRP3 inflammasome and associated cytokines by increasing SIRT1 or decreasing Notch1. Drynaria fortunei had inhibitory effects on NLRP3 inflammasome and Notch1 even when SIRT1 expression was suppressed. CONCLUSIONS: Drynaria fortunei has been demonstrated to significantly improve lipid profiles for elderly PMOP patients. Drynaria fortunei may down-regulate Notch1 independently of SIRT1 to suppress NLRP3 inflammasome-mediated inflammation, thus improving plasma lipid profile.

Desiccation and rehydration dynamics in the epiphytic resurrection fern Pleopeltis polypodioides.

The epiphytic resurrection-or desiccation-tolerant (DT)-fern Pleopeltis polypodioides can survive extreme desiccation and recover physiological activity within hours of rehydration. Yet, how epiphytic DT ferns coordinate between deterioration and recovery of their hydraulic and photosynthetic systems remains poorly understood. We examined the functional status of the leaf vascular system, chlorophyll fluorescence, and photosynthetic rate during desiccation and rehydration of P. polypodioides. Xylem tracheids in the stipe embolized within 3-4 h during dehydration. When the leaf and rhizome received water, tracheids refilled after ∼24 h, which occurred along with dramatic structural changes in the stele. Photosynthetic rate and chlorophyll fluorescence recovered to predesiccation values within 12 h of rehydration, regardless of whether fronds were connected to their rhizome. Our data show that the epiphytic DT fern P. polypodioides can utilize foliar water uptake to rehydrate the leaf mesophyll and recover photosynthesis despite a broken hydraulic connection to the rhizome.

Serial Exhaustive Extraction Revealed Antimicrobial and Antioxidant Properties of Platycerium stemaria (Beauv) Desv.

Microbial infections are increasing worldwide, and the widespread emergence of antibiotic-resistant pathogens poses a severe threat to public health. Medicinal plants are well-known sources of bioactive ingredients. This study was designed to determine the antimicrobial and antioxidant activities of extracts from Platycerium stemaria. The serial exhaustive extraction method using a solvent of increasing polarity from nonpolar (hexane) to polar (water) was designed to prepare crude extracts; liquid-liquid partition was used to fractionate of active extracts. The extracts and fractions were screened for antimicrobial activity on bacteria and yeasts using the microdilution method. The antioxidant activity was done using DPPH and FRAP assays. Out of the sixteen extracts screened, four (PsHex, PsH2O(H), PsMeOH(EA), and PsMeOH) exhibited potency with minimal inhibitory concentration (MIC) values ranging from 31.25 to 500 µg/mL. Out of the four extracts, two, including PsMeOH and PsMeOH(EA), exhibited DPPH radical scavenging activity with the antiradical power of 8.94 × 10-5 and 47.96 × 10-5, respectively, and ferric reducing antioxidant power values ranging from 0.34 to 61.53 µg equivalent Vit C/g of extract. The phytochemical screening of the promising crude extracts revealed flavonoids, glycosides, phenols, tannins, terpenoids, saponins, and anthraquinones. This study reports the antimicrobial and antioxidant activities of P. stemaria for the first time. The results showed that the serial exhaustive extraction approach used in this study allowed capturing the antimicrobial and antioxidant metabolites beyond the single extraction, indicating the need for a rigorous choice of an appropriate solvent and method for extracting P. stemaria. Further investigation is needed to characterize the active ingredients present in the promising extracts.

Network Pharmacology-Based Pharmacological Mechanism of the Chinese Medicine Rhizoma drynariae Against Osteoporosis.

d_abstr_R Rhizoma drynariae is the main traditional Chinese medicine used for the treatment of osteoporosis, but its anti-osteoporotic targeting mechanism has not been fully elucidated due to the complexity of its active ingredients. In this study, the pharmacological mechanism of action of Rhizoma drynariae against osteoporosis was studied by integrating pharmacological concepts. The pharmacokinetic characteristics of selected major active constituents of Rhizoma drynariae and the SMILES structural similarity were used to predict related targets. A literature search was conducted to identify known osteoporosis treatment targets, which were then combined with the predicted targets to construct the direct or indirect target interaction network map of Rhizoma drynariae against osteoporosis. Finally, data on the key targets of the interactions, ranked according to relevant node parameters obtained through pathway enrichment analysis and screening of key targets and active ingredients of Rhizoma drynariae, were used to perform molecular docking simulation. We screened 16 active ingredients of Rhizoma drynariae, and 7 key targets with direct or indirect effects with a high frequency were obtained. These main pathways were found to play important roles in the PI3k-akt signaling pathway, osteoclast differentiation, Wnt signaling pathway, and estrogen signaling pathway. Molecular docking showed that most active ingredients of Rhizoma drynariae had strong binding efficiency with key targets. Based on network pharmacology, we conclude that Rhizoma drynariae plays an anti-osteoporotic role by directly or indirectly targeting multiple major signaling pathways and influencing the proliferation and differentiation of multiple types of cells.

Subcellular distribution of cadmium in a novel potential aquatic hyperaccumulator - Microsorum pteropus.

Microsorum pteropus is a novel potential Cd (cadmium) aquatic hyperaccumulator. In the present study, hydroponic experiments were conducted to assess the accumulation and subcellular distribution of Cd in the root, stem and leaf of M. pteropus. SEM (scanning electron microscopy) - EDX (energy dispersive X-ray fluorescence spectrometer) and TEM (transmission electron microscopy) were used to observe the ultrastructure of different tissues under 500 µM Cd exposure. After exposure to 500 µM Cd for 7 days, the root, stem and leaf of M. pteropus can accumulate to be > 400 mg/kg Cd in dry mass with no significant influence on the growth. In the root and leaf of M. pteropus, the Cd was more likely to store in the cell wall fraction. However, Cd in the stem was mainly stored in both the cell wall fraction and the cytoplasm fraction. Under SEM observation and EDX detection, 1) Cd was found to be sequestrated in the epidermis or chelated in the root cells, 2) no significant deposit spots were observed in the stem, 3) Cd was found in the trichome of the leaf, and the sporangium was not damaged. TEM observations revealed 1) possible Cd precipitations in the root cell and 2) no significant ultrastructure variation in the stem, and 3) the chloroplast retained its structure and was not affected by the Cd. M. pteropus showed great capacity for Cd accumulation without influencing growth. In addition, the ultrastructure of all the tissues was not damaged by the Cd. M. pteropus showed a great potential in phytoremediation in heavy metal polluted water solutions, and may provide new directions for the study of resistance mechanisms of aquatic hyperaccumulators.

Effects of Rhizoma Drynariae Cataplasm on Fracture Healing in a Rat Model of Osteoporosis.

BACKGROUND Osteoporosis is an increasingly prevalent disease characterized by decreased bone mass and deterioration of the bone microstructure, which contribute to increased fragility and subsequent fragility fractures, especially in elderly individuals. Rhizoma Drynariae (DRE) is among the most frequently used herbal medicines for the treatment of osteoporosis. Transdermal delivery is a proven novel pathway for drug treatment and has several advantages over traditional drug delivery routes. MATERIAL AND METHODS Female Sprague-Dawley osteoporotic fracture model rats were divided into 3 groups: the control group, the DRE (90 mg/kg/day) group and the DRE cataplasm (containing 30 mg DRE, administered at right femur site daily) group. At 3 and 6 weeks after operation, we performed x-ray, histological, and biomechanical analyses, and evaluated bone marrow density of the femur. RESULTS Treatment with DRE increased callus formation and bone union compared with the control group. Moreover, DRE enhanced bone strength at the femoral diaphysis in the osteoporotic fractures in rats by increasing the ultimate load and stiffness compared with the control group. Furthermore, DRE restored the trabecular bone mineral density in the femur compared with the control group. DRE cataplasm application further enhanced the therapeutic effects against osteoporotic fracture in this rat model. CONCLUSIONS DRE cataplasm application might be useful against osteoporotic fracture.

Isolation and functional characterization of two Caffeoyl Coenzyme A 3-O-methyltransferases from the fern species Polypodiodes amoena.

Caffeoyl Coenzyme A 3-O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a hydroxyl moiety. CCoAOMTs are important for the synthesis of lignin, which provides much of the rigidity required by tracheophytes to enable the long distance transport of water. So far, no CCoAOMTs has been characterized from the ancient tracheophytes ferns. Here, two genes, each encoding a CCoAOMT (and hence denoted PaCCoAOMT1 and PaCCoAOMT2), were isolated from the fern species Polypodiodes amoena. Sequence comparisons confirmed that the product of each gene resembled enzymes known to be associated with lignin synthesis in higher plants. When either of the genes was heterologously expressed in E. coli, the resulting recombinant protein was able to methylate caffeoyl CoA, along with a number of phenylpropanoids, flavones and flavonols containing two vicinal hydroxyl groups. Their in vitro conversion rate when presented with either caffeoyl CoA or certain flavonoids as substrate was comparable with that of the Medicago sativa MsCCoAOMT. Their constitutive expression in Arabidopsis thaliana boosted the plants' lignin content, but did not affect that of methylated flavonols, indicating that both PaCCoAOMTs contributed to lignin synthesis and that neither was able to methylate flavonols in planta. The transient expression of a PaCCoAOMT-GFP fusion gene in tobacco demonstrated that in planta, PaCCoAOMTs are likely directed to the cytoplasm.

Cadmium accumulation capacity and resistance strategies of a cadmium-hypertolerant fern - Microsorum fortunei.

Microsorum fortunei (M. fortunei), a close relative to the cadmium (Cd) hyperaccumulator Microsorum pteropus, is an epiphytic Polypodiaceae fern with strong antioxidant activity. The Cd-accumulation capacities and Cd-resistance mechanisms of M. fortunei were analyzed in this study by measuring metal contents (Cd, Fe, Mg, Ca, Zn, Mn, K and Na) and chlorophyll fluorescence parameters (Fv/Fm, qN, qP, Y(II), Y(NPQ) and Y(NO)) and by performing an RNA-sequencing analysis. M. fortunei could accumulate up to 2249.10 µg/g DW Cd in roots under a 15-day 1000 µmol/L Cd treatment, with little Cd translocated into the leaves (maximum 138.26 µg/g DW). The M. fortunei leaves could maintain their normal physiological functions with no phytosynthesis damage and few changes in metal contents or differentially expressed genes. M. fortunei roots showed a decrease in Zn concentration, with potential Cd-tolerance mechanisms such as heavy metal transporters, vesicle trafficking and fusion proteins, antioxidant systems, and primary metabolites like plant hormones, revealed by differentially expressed functional genes. In conclusion, M. fortunei may serve as a potential cadmium-hypertolerant fern that sequesters and detoxifies most cadmium in the roots, with a minimum root-to-shoot Cd translocation to guarantee the physiological functions in the more vulnerable leaves.

Full-Length Transcriptome Sequencing and Modular Organization Analysis of the Naringin/Neoeriocitrin-Related Gene Expression Pattern in Drynaria roosii.

Drynaria roosii (Nakaike) is a traditional Chinese medicinal fern, known as 'GuSuiBu'. The effective components, naringin and neoeriocitrin, share a highly similar chemical structure and medicinal function. Our HPLC-tandem mass spectrometry (MS/MS) results showed that the accumulation of naringin/neoeriocitrin depended on specific tissues or ages. However, little was known about the expression patterns of naringin/neoeriocitrin-related genes involved in their regulatory pathways. Due to a lack of basic genetic information, we applied a combination of single molecule real-time (SMRT) sequencing and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of D. roosii. According to the SGS data, the differentially expressed gene (DEG)-based heat map analysis revealed that naringin/neoeriocitrin-related gene expression exhibited obvious tissue- and time-specific transcriptomic differences. Using the systems biology method of modular organization analysis, we clustered 16,472 DEGs into 17 gene modules and studied the relationships between modules and tissue/time point samples, as well as modules and naringin/neoeriocitrin contents. We found that naringin/neoeriocitrin-related DEGs distributed in nine distinct modules, and DEGs in these modules showed significantly different patterns of transcript abundance to be linked to specific tissues or ages. Moreover, weighted gene co-expression network analysis (WGCNA) results further identified that PAL, 4CL and C4H, and C3H and HCT acted as the major hub genes involved in naringin and neoeriocitrin synthesis, respectively, and exhibited high co-expression with MYB- and basic helix-leucine-helix (bHLH)-regulated genes. In this work, modular organization and co-expression networks elucidated the tissue and time specificity of the gene expression pattern, as well as hub genes associated with naringin/neoeriocitrin synthesis in D. roosii. Simultaneously, the comprehensive transcriptome data set provided important genetic information for further research on D. roosii.

Comparisons of cadmium bioaccumulation potentials and resistance physiology of Microsorum pteropus and Echinodorus grisebachii.

To better monitor and remediate environments contaminated by cadmium (Cd), plants are used as hyperaccumulators or biomonitors; however, few have been identified for aquatic Cd pollution. In our study, two aquatic ornamental plants, Microsorum pteropus (Blume) Copel. and Echinodorus grisebachii Small, were studied for their Cd accumulation capacity, morphological characteristics, and leaf physiological indexes. Microsorum pteropus (Blume) Copel. leaf has the potential to hyperaccumulate Cd (166 mg/kg dry weight for 1 mg/L exposure), with no significant physiological difference under exposure. Echinodorus grisebachii Small had sensitive diagnostic responses to Cd toxicity, such as significant decreases in Chl (a + b) and Chl-a/b, increased peroxidase (POD) activity, greater malondialdehyde (MDA) content, and increased soluble sugar content. These results suggest that Microsorum pteropus (Blume) Copel. could have the potential to be a Cd hyperaccumulator, while Echinodorus grisebachii Small could serve as a biomonitor for Cd-contaminated water bodies.

Lipid production in aquatic plant Azolla at vegetative and reproductive stages and in response to abiotic stress.

The aquatic plant Azolla became increasingly popular as bioenergy feedstock because of its high growth rate, production of biomass with high levels of biofuel-producing molecules and ability to grow on marginal lands. In this study, we analysed the contribution of all organs of Azolla to the total yield of lipids at vegetative and reproductive stages and in response to stress. Triacylglycerol-containing lipid droplets were detected in all (vegetative and reproductive) organs with the highest level in the male microsporocarps and microspores. As a result, significantly higher total yields of lipids were detected in Azolla filiculoides and Azolla pinnata at the reproductive stage. Starving changed the yield and composition of the fatty acid as a result of re-direction of carbon flow from fatty acid to anthocyanin pathways. The composition of lipids, in regard the length and degree of unsaturation of fatty acids, in Azolla meets most of the important requirements for biodiesel standards. The ability of Azolla to grow on wastewaters, along with their high productivity rate, makes it an attractive feedstock for the production of biofuels.

Detection of chlorophylls in spores of seven ferns.

Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, "cryptochlorophyllous spores", is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.

Copper phytoextraction by Salvinia cucullata: biochemical and morphological study.

The present study investigated the effect of copper on photosynthesis, antioxidant potential, and anatomical response of aquatic fern, Salvinia cucullata, with a view to ascertain its phytoremediation potential. Plants were exposed in hydroponics for 21 days to different Cu concentrations (10, 15, 20, and 30 mg/L). Significant declines in chlorophyll, carotenoids, and soluble proteins, as a function of Cu proportion were observed. Lipid peroxidation was also evident, which implied reactive oxygen species (ROS) generation. However, both root and leaf tissues responded remarkably to the ROS produced, by inducing superoxide dismutase (1.6-6.5 times), catalase (1.5-5.4 times), guaicol peroxidase (1.5-7.2 times), and ascorbyl peroxidase (1.3-4.7 times) over the control. The plant showed best phytoremedial activity within Cu range of 10-15 mg/L, with maximum accumulation of 2956 ± 82.6 µg/g dw., at 15 mg Cu/L and showed efficient root to shoot translocation (translocation factor, TF > 1) at this range, which is the stipulated minimum requirement to be a hyperaccumulator. The capacity of metal extraction from environment to leaf (extraction coefficient, EC) was also high (EC = 73-197). However, at higher doses (20-30 mg/L), the plant resorted to an exclusion strategy, whereby, more metal accumulation was observed in root than in leaf. The plant conferred suitable remediation attributes by showing minimal root and leaf anatomical damages along with high Ca peaks in both the tissues, and rapid leaf stomatal closure, all of which probably helped in the Cu induced stress mitigation. Due to its widespread availability, fast growth, ability to grow in myriads of polluted environment, and having hardy physiology, this plant can be suggested for use as a suitable Cu phytoremediator.