Co-Culture of Bone Marrow Mesenchymal Stem Cell (BMSC) and Osteoclasts Regulates Cell Differentiation and Alleviates Osteoporosis by Up-Regulating miR-211

Bone marrow mesenchymal stem cells (BMSCs) can release a large amount of exosomes (EXO) during bone remodeling by osteoclasts. EXO contains miRNA-211, which has a variety of biological effects. However, little is known about whether miR-211 from BMSC-EXO affects the surrounding cells. Therefore, we aim to study the role of miRNA-211 derived from BMSC-EXO in regulating osteoclasts differentiation. Macrophage colony stimulating factor (M-CSF) and nuclear factor kappa B receptor activator (RANKL) were used to stimulate bone marrow macrophages (BMM) to obtain osteoclasts, which were treated with BMSC-EXO or LPS followed by analysis of osteoclast-related genes expression by PCR, ROS release by flow cytometry, actin ring formation by immunofluorescence, and osteoclast differentiation by anti-tartrate acid phosphatase (TRAP) staining. Finally, an in vivo experiment was conducted to verify BMSC-EXO’s effect on osteoporosis. BMSC-EXO significantly inhibited RNAKL-induced osteoclast differentiation of BMMs. During osteoclasts formation, BMSC-EXO inhibited ROS production induced by RANKL and the subsequent activation of NF-κB signaling pathway induced by ROS. In addition, BMSC-EXO significantly down-regulated the osteoclast genes including nuclear factor, cytoplasmic 1 (NFATc1), C-fos, tartrate-resistant acid phosphatase (TRAP) and osteoclast-associated immunoglobulin-like receptor (OSCAR) in activated T cells. BMSC-EXO inhibited ROS release by promoting miR-211 expression, thereby inhibiting the NF-κB signaling and ultimately participating in osteoclasts differentiation. In LPS-induced mouse osteoporosis models, BMSC-EXO inhibited LPS-induced bone loss and exerted a protective effect. In conclusion, microRNA-211 derived from BMSC-EXO can regulate osteoclasts differentiation, suggesting that it might be used as a potential approach for treating osteoporosis.

Ecophysiological properties of a cluster root-forming plant, Helicia cochinchinensis (Proteaceae) grown on Miyajima Island, Japan

Aims The family Proteaceae is one of the dominant families in nutrient-impoverished habitats in the Southern hemisphere, and less common in the Northern hemisphere. Helicia cochinchinensis Lour. is the only Proteaceae species in Japan. This study aimed to unveil the ecophysiological properties of H. cochinchinensis grown on Miyajima Island, Hiroshima, Japan.Methods Phosphorus (P) status and dynamics of soils in H. cochinchinensis habitats were measured. Plant P and nitrogen (N) concentrations of leaves were measured after digestion. Roots and rhizosheath soil were collected to assess root morphology and root exudates.Results Available P (Olsen-P) in soils in habitats of H. cochinchinensis was 0.46–3.7 mg P kg-1 soil. Citrate was the major carboxylate in root exudates and its concentration increased during cluster-root formation. Acid phosphatase activity was greater at the surface of cluster roots that on the surface of other roots and bulk soil, especially for mature cluster roots. Sparingly soluble organic P concentrations decreased in the rhizosheath soil of mature cluster roots. The P concentrations of H. cochinchinensis leaves were relatively low; 0.34–0.69 mg P g-1 DW and 0.15–0.29 mg P g-1 DW in mature and senesced leaves, respectively. The P demand of H. cochinchinensis was less than that of nearby trees, showing greater P-remobilization efficiency.Conclusions Phosphorus mobilization from unavailable P by cluster roots supported P uptake by H. cochinchinensis, and P remobilization from senescing leaves contributed to sustain growth under P-deficient conditions.

Tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis by modulating β-catenin signaling

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited therapeutic options. Tartrate-resistant acid phosphatase 5 (ACP5) performs a variety of functions. However, its role in IPF remains unclear. Here, we demonstrate that the levels of ACP5 are increased in IPF patient samples and mice with bleomycin (BLM)-induced pulmonary fibrosis. In particular, higher levels of ACP5 are present in the sera of IPF patients with a diffusing capacity of the lungs for carbonmonoxide (DLCO) less than 40% of the predicted value. Additionally, Acp5 deficiency protects mice from BLM-induced lung injury and fibrosis coupled with a significant reduction of fibroblast differentiation and proliferation. Mechanistic studies reveal that Acp5 is upregulated by transforming growth factor-β1 (TGF-β1) in a TGF-β receptor 1 (TGFβR1)/Smad family member 3 (Smad3)-dependent manner, after which Acp5 dephosphorylates p-β-catenin at serine 33 and threonine 41, inhibiting the degradation of β-catenin and subsequently enhancing β-catenin signaling in the nucleus, which promotes the differentiation, proliferation and migration of fibroblast. More importantly, the treatment of mice with Acp5 siRNA-loaded liposomes or Acp5 inhibitor reverses established lung fibrosis. In conclusions, Acp5 is involved in the initiation and progression of pulmonary fibrosis and strategies aimed at silencing or suppressing Acp5 could be considered as potential therapeutic approaches against pulmonary fibrosis.

Soil enzymatic activity under coffee cultivation with different water regimes associated to liming and intercropped brachiaria

ABSTRACT: This research evaluated the effects of coffee cultivation with two different water regimes associated or not with liming and the presence/absence of brachiaria as intercrop on the activities of the soil enzymes β-glucosidase, arylsulfatase and acid phosphatase. The study was carried out at the experimental farm of Embrapa Cerrados, using the cultivar IAC 144 (Coffea arabica L.), under a clayey dystrophic Cerrado Oxisol. Two water regimes (WR) were considered, WR1 with irrigation shifts throughout the year and WR3 with controlled water stress, for about 70 days, in the dry season. In each water regime, effects of lime application (with/without) and the presence/absence of brachiaria cultivated between the lines of coffee plants were evaluated. The activities of the enzymes β-glucosidase, arylsulfatase and acid phosphatase were evaluated during the rainy and dry seasons. Liming and intercropped brachiaria positively affected the activities of the three enzymes assessed in this study at varying degrees, depending on season and/or the WR. Our findings evidenced that intercropped brachiaria in coffee rows was the factor that most positively impacted soil enzymes activities.

Tanshinone I Mitigates Steroid-Induced Osteonecrosis of the Femoral Head and Activates the Nrf2 Signaling Pathway in Rats

Steroid-induced osteonecrosis of the femoral head (SIONFH) is a frequent orthopedic disease caused by long-term or high-dose administration of corticosteroids. Tanshinone I (TsI), a flavonoid compound isolated from Salvia miltiorrhiza Bunge, has been reported to inhibit osteoclastic differentiation in vitro. This study aimed to investigate whether TsI can ameliorate SIONFH. Herein, SIONFH was induced by intraperitoneal injection of 20 μg/kg lipopolysaccharide every 24 h for 2 days, followed by an intramuscular injection of 40 mg/kg methylprednisolone every 24 h for 3 days. Four weeks after the final injection of methylprednisolone, the rats were intraperitoneally administrated with low-dose (5 mg/kg) and high-dose (10 mg/kg) TsI once daily for 4 weeks. Results showed that TsI significantly alleviated osteonecrotic lesions of the femoral heads as determined by micro-CT analysis. Furthermore, TsI increased alkaline phosphatase activity and expressions of osteoblastic markers including osteocalcin, type I collagen, osteopontin, and Runt-related transcription factor 2 and decreased tartrate-resistant acid phosphatase activity and expressions of osteoclastic markers including cathepsin K and acid phosphatase 5. TsI also reduced inflammatory response and oxidative stress and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in the femoral heads. Taken together, our findings show that TsI can relieve SIONFH, indicating that it may be a candidate for preventing SIONFH.

Monovalent Copper Ions Inhibit Enzymatic Systems

The effect of monovalent copper ions on enzymatic systems has hardly been studied to date; this is due to the low stability of monovalent copper ions in aqueous solutions, which led to the assumption that their concentration is negligible in biological systems. However, in an anaerobic atmosphere, and in the presence of a ligand that stabilizes the monovalent copper ions over the divalent copper ions, high and stable concentrations of monovalent copper ions can be reached. Moreover, the cell cytoplasm has a substantial concentration of potential stabilizers that can explain significant concentrations of monovalent copper ions in the cytoplasm. This study demonstrates the effect of monovalent and divalent copper ions on DNA polymerase, ligaseT4 DNA, the restriction enzymes EcoP15I and EcoR I, acid phosphatase, and α and βamylase enzymes. These systems were chosen because they can be monitored under conditions necessary for maintaining a stable concentration of monovalent copper ions, and since they exhibit a wide range of dependency on ATP. Previous studies indicated that ATP interacts with monovalent and divalent copper ions and stabilizing monovalent copper ions over divalent copper ions. The results showed that monovalent copper ions dramatically inhibit DNA polymerase and acid phosphatase, inhibit ligaseT4 DNA and the restriction enzyme EcoP15I, moderately inhibit α and β amylase, and have no effect on the restriction enzyme EcoR I. From the results presented in this work, it can be concluded that the mechanism is not one of oxidative stress, even though monovalent copper ions generate reactive oxygen species (ROS). Molecular oxygen in the medium, which is supposed to increase the oxidative stress, impairs the inhibitory effect of monovalent and divalent copper ions, and the kinetics of the inhibition is not suitable for the ROS mechanism.ATP forms a complex with copper ions (di and monovalent ions, where the latter is more stable) in which the metal ion is bound both to the nitrogen base and to the oxygen charged on the phosphate groups, forming an unusually distorted complex. The results of this study indicate that these complexes have the ability to inhibit enzymatic systems that are dependent on ATP.This finding can provide an explanation for the strong antimicrobial activity of monovalent copper ions, suggesting that rapid and lethal metabolic damage is the main mechanism of monovalent copper ions’ antimicrobial effect.

Development of Inflammation in the Gastrointestinal Tract of Rats after Prolonged Administration of Alcohol

The purpose of the work was to experimentally study the chronic alcohol intoxication on the indicators of inflammation and lipid peroxidation in the gastrointestinal system. Materials and methods. Ethyl alcohol was added to the water for 2-month-old male rats, ranging from 5% to 15% for 108 days. In homogenates of mucous membranes of the gastrointestinal tract and liver, the activity of elastase enzymes, acid phosphatase and the concentration of malonic dialdehyde were determined, in serum – elastase activity and malonic dialdehyde content. Results and discussion. Biochemical research of one of the markers of inflammation (elastase activity) in rats found a probable increase of elastase activity in different parts of the digestive tract after prolonged alcohol consumption, regardless of the sex of the animals. Thus, in the serum of rats after the introduction of ethanol, the activity of elastase increased by 71.7%, in the oral mucosa – by 29.2%, in the gastric mucosa – by 55.5%, in the liver – by 29.0%. In the small and large intestine, the level of this marker of inflammation has changed slightly. The level of elastase activity shows the degree of accumulation of leukocytes in the tissues as a result of the development of the inflammatory process. Acid phosphatase activity in the oral mucosa of rats treated with ethanol increased by 47.4%, in the gastric mucosa – by 30.3%, in the mucous membrane of the small intestine – by 37.4%, in the mucous membrane of the colon – by 40.4%, in the liver – by 112.6%. Activation of acid phosphatase, along with other lysosomal enzymes, is the primary inflammatory response that triggers the production of mediators, which in turn cause secondary tissue alteration in subsequent stages of the inflammatory process. Therefore, the results obtained on the activation of acid phosphatase along with elastase indicate the presence of inflammation in the mucous membranes of the digestive tract, and especially in the liver of rats chronically treated with ethanol. The introduction of alcohol also led to an increase in the concentration of malonic dialdehyde in the mucous membranes: the oral cavity – by 20.3%, the stomach – by 32.3%, the small intestine – by 96.6%, the colon – by 50.2%, in the liver – by 39.4%, in serum – by 33.3%. A significant increase in the level of malonic dialdehyde in the tissues of the digestive tract of rats after long-term intake of ethanol is a sign of activation of lipid peroxidation and intensification of oxidative stress reactions. Conclusion. The results of the study of elastase activity indicate the development of inflammation in the mucous membranes of the gastrointestinal tract, liver and serum of rats under the influence of chronic administration of ethanol. Increased acid phosphatase activity in the tissues of the gastrointestinal tract after prolonged use of ethanol indicates damage to cell membranes, which is a consequence of inflammation. A significant increase in the level of malonic dialdehyde in the mucous membranes of the gastrointestinal tract, liver and serum of rats after chronic ethanol intake is a sign of intensification of oxidative stress reactions