Induction of subject-ventilator asynchrony by variation of respiratory parameters in a lung injury model in pigs.

BACKGROUND: Subject-ventilator asynchrony (SVA) was shown to be associated with negative clinical outcomes. To elucidate pathophysiology pathways and effects of SVA on lung tissue histology a reproducible animal model of artificially induced asynchrony was developed and evaluated. METHODS: Alterations in ventilator parameters were used to induce the three main types of asynchrony: ineffective efforts (IE), auto-triggering (AT), and double-triggering (DT). Airway flow and pressure, as well as oesophageal pressure waveforms, were recorded, asynchrony cycles were manually classified and the asynchrony index (AIX) was calculated. Bench tests were conducted on an active lung simulator with ventilator settings altered cycle by cycle. The developed algorithm was evaluated in three pilot experiments and a study in pigs ventilated for twelve hours with AIX = 25%. RESULTS: IE and AT were induced reliably and fail-safe by end-expiratory hold and adjustment of respiratory rate, respectively. DT was provoked using airway pressure ramp prolongation, however not controlled specifically in the pilots. In the subsequent study, an AIX = 28.8% [24.0%-34.4%] was induced and maintained over twelve hours. CONCLUSIONS: The method allows to reproducibly induce and maintain three clinically relevant types of SVA observed in ventilated patients and may thus serve as a useful tool for future investigations on cellular and inflammatory effects of asynchrony.

Interleukin-12 sustained release system promotes hematopoietic recovery after radiation injury.

The continuous production of mature blood cell lineages is maintained by hematopoietic stem cells but they are highly susceptible to damage by ionizing radiation (IR) that induces death. Thus, devising therapeutic strategies that can mitigate hematopoietic toxicity caused by IR would benefit acute radiation syndrome (ARS) victims and patients receiving radiotherapy. Herein, we describe the preparation of an injectable hydrogel formulation based on Arg-Gly-Asp-alginate (RGD-Alg) and Laponite using a simple mixing method that ensured a slow and sustained release of interleukin-12 (IL-12) (RGD-Alg/Laponite@IL-12). The local administration of RGD-Alg/Laponite@IL-12 increased survival rates and promoted the hematopoietic recovery of mice who had received sublethal-dose irradiation. Local intra-bone marrow (intra-BM) injection of RGD-Alg/Laponite@IL-12 hydrogel effectively stimulated IL12 receptor-phosphoinositide 3-kinase/protein kinase B (IL-12R-PI3K/AKT) signaling axis, which promoted proliferation and hematopoietic growth factors secretion of BM mesenchymal stem/stromal cells. This signaling axis facilitates the repair of the hematopoietic microenvironment and plays a pivotal role in hematopoietic reconstitution. In conclusion, we describe a biomaterial-sustained release of IL-12 for the treatment of irradiated hematopoietic injury and provide a new therapeutic strategy for hematopoietic ARS.

Mitochondrial Targeted Cerium Oxide Nanoclusters for Radiation Protection and Promoting Hematopoiesis.

Purpose: Mitochondrial oxidative stress is an important factor in cell apoptosis. Cerium oxide nanomaterials show great potential for scavenging free radicals and simulating superoxide dismutase (SOD) and catalase (CAT) activities. To solve the problem of poor targeting of cerium oxide nanomaterials, we designed albumin-cerium oxide nanoclusters (TPP-PCNLs) that target the modification of mitochondria with triphenyl phosphate (TPP). TPP-PCNLs are expected to simulate the activity of superoxide dismutase, continuously remove reactive oxygen species, and play a lasting role in radiation protection. Methods: First, cerium dioxide nanoclusters (CNLs), polyethylene glycol cerium dioxide nanoclusters (PCNLs), and TPP-PCNLs were characterized in terms of their morphology and size, ultraviolet spectrum, dispersion stability and cellular uptake, and colocalization Subsequently, the anti-radiation effects of TPP-PCNLs were investigated using in vitro and in vivo experiments including cell viability, apoptosis, comet assays, histopathology, and dose reduction factor (DRF). Results: TPP-PCNLs exhibited good stability and biocompatibility. In vitro experiments indicated that TPP-PCNLs could not only target mitochondria excellently but also regulate reactive oxygen species (ROS)levels in whole cells. More importantly, TPP-PCNLs improved the integrity and functionality of mitochondria in irradiated L-02 cells, thereby indirectly eliminating the continuous damage to nuclear DNA caused by mitochondrial oxidative stress. TPP-PCNLs are mainly targeted to the liver, spleen, and other extramedullary hematopoietic organs with a radiation dose reduction factor of 1.30. In vivo experiments showed that TPP-PCNLs effectively improved the survival rate, weight change, hematopoietic function of irradiated animals. Western blot experiments have confirmed that TPP-PCNLs play a role in radiation protection by regulating the mitochondrial apoptotic pathway. Conclusion: TPP-PCNLs play a radiologically protective role by targeting extramedullary hematopoietic organ-liver cells and mitochondria to continuously clear ROS.

Effects of Positive End-expiratory Pressure on Pulmonary Perfusion Distribution and Intrapulmonary Shunt during One-lung Ventilation in Pigs: A Randomized Crossover Study.

BACKGROUND: During one-lung ventilation (OLV), positive end-expiratory pressure (PEEP) can improve lung aeration but might overdistend lung units and increase intrapulmonary shunt. The authors hypothesized that higher PEEP shifts pulmonary perfusion from the ventilated to the nonventilated lung, resulting in a U-shaped relationship with intrapulmonary shunt during OLV. METHODS: In nine anesthetized female pigs, a thoracotomy was performed and intravenous lipopolysaccharide infused to mimic the inflammatory response of thoracic surgery. Animals underwent OLV in supine position with PEEP of 0 cm H2O, 5 cm H2O, titrated to best respiratory system compliance, and 15 cm H2O (PEEP0, PEEP5, PEEPtitr, and PEEP15, respectively, 45 min each, Latin square sequence). Respiratory, hemodynamic, and gas exchange variables were measured. The distributions of perfusion and ventilation were determined by IV fluorescent microspheres and computed tomography, respectively. RESULTS: Compared to two-lung ventilation, the driving pressure increased with OLV, irrespective of the PEEP level. During OLV, cardiac output was lower at PEEP15 (5.5 ± 1.5 l/min) than PEEP0 (7.6 ± 3 l/min) and PEEP5 (7.4 ± 2.9 l/min; P = 0.004), while the intrapulmonary shunt was highest at PEEP0 (PEEP0: 48.1% ± 14.4%; PEEP5: 42.4% ± 14.8%; PEEPtitr: 37.8% ± 11.0%; PEEP15: 39.0% ± 10.7%; P = 0.027). The relative perfusion of the ventilated lung did not differ among PEEP levels (PEEP0: 65.0% ± 10.6%; PEEP5: 68.7% ± 8.7%; PEEPtitr: 68.2% ± 10.5%; PEEP15: 58.4% ± 12.8%; P = 0.096), but the centers of relative perfusion and ventilation in the ventilated lung shifted from ventral to dorsal and from cranial to caudal zones with increasing PEEP. CONCLUSIONS: In this experimental model of thoracic surgery, higher PEEP during OLV did not shift the perfusion from the ventilated to the nonventilated lung, thus not increasing intrapulmonary shunt.

Enteric α-Defensin Contributes to Recovery of Radiation-Induced Intestinal Injury by Modulating Gut Microbiota and Fecal Metabolites.

The effect of ionizing radiation on the gastrointestinal tract is a common complication of abdominal and pelvic radiotherapy. However, the pathological features of radiation enteropathy and its effective medical intervention regimen is still a global challenge. Here, we explored the role and mechanism of enteric alpha-defensins (EαDs) in protecting against radiation enteropathy. To address this, we utilized EαDs-deficiency mice, in which the matrix metallopeptidase 7 to activate Paneth cell α-defensins was knockout (KO) mice, and the complementary wild-type (WT) control mice for this study. Remarkably, the KO mice were more susceptible to 5.0 Gy total-body irradiation, resulting in worse clinic scores and lower survival rate, compared with the wild-type mice. Histological examination indicated that the KO mice were subjected to slow recovery of intestinal villus and mucosa function, characterized by the reduced expression of TFF3, Glut1 and Muc2. In addition, compared with the wild-type controls, the KO mice experienced serious inflammation response in intestinal tissue, indicated by the remarkably increased expression level of IL-1ß, IL-6 and IL-12. Using high-throughput sequencing analysis, we found that the intestinal bacterial community of the KO mice was more prone to dysbiosis than that of the WT mice, with significantly increased abundance of opportunistic pathogenic bacteria, such as Streptococcus sp. and Escherichia-Shigella sp., whereas remarkably decreased probiotics harboring Lactobacillus sp., Desulfovibrio sp. etc. Fecal metabolomics analysis indicated that the relative abundance of 31 metabolites arose significantly different between WT and KO mice on day 10 after radiation exposure. A subset of differential metabolites to regulate host metabolism and immunity, such as acetic acid, acetate, butanoic acid, was negatively correlated with the alteration of gut microbiota in the irradiated KO mice. This study provides new insight into EαDs contribution to the recovery of radiation-induced intestinal damage, and suggests a potential novel target to prevent the adverse effects of radiotherapy.

Modulation of the hippo-YAP pathway by cyclic stretch in rat type 2 alveolar epithelial cells-a proof-of-concept study.

Background: Mechanical ventilation (MV) is a life supporting therapy but may also cause lung damage. This phenomenon is known as ventilator-induced lung injury (VILI). A potential pathomechanisms of ventilator-induced lung injury may be the stretch-induced production and release of cytokines and pro-inflammatory molecules from the alveolar epithelium. Yes-associated protein (YAP) might be regulated by mechanical forces and involved in the inflammation cascade. However, its role in stretch-induced damage of alveolar cells remains poorly understood. In this study, we explored the role of YAP in the response of alveolar epithelial type II cells (AEC II) to elevated cyclic stretch in vitro. We hypothesize that Yes-associated protein activates its downstream targets and regulates the interleukin-6 (IL-6) expression in response to 30% cyclic stretch in AEC II. Methods: The rat lung L2 cell line was exposed to 30% cyclic equibiaxial stretch for 1 or 4 h. Non-stretched conditions served as controls. The cytoskeleton remodeling and cell junction integrity were evaluated by F-actin and Pan-cadherin immunofluorescence, respectively. The gene expression and protein levels of IL-6, Yes-associated protein, Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1), and connective tissue growth factor (CTGF/CCN2) were studied by real-time polymerase chain reaction (RT-qPCR) and Western blot, respectively. Verteporfin (VP) was used to inhibit Yes-associated protein activation. The effects of 30% cyclic stretch were assessed by two-way ANOVA. Statistical significance as accepted at p < 0.05. Results: Cyclic stretch of 30% induced YAP nuclear accumulation, activated the transcription of Yes-associated protein downstream targets Cyr61/CCN1 and CTGF/CCN2 and elevated IL-6 expression in AEC II after 1 hour, compared to static control. VP (2 µM) inhibited Yes-associated protein activation in response to 30% cyclic stretch and reduced IL-6 protein levels. Conclusion: In rat lung L2 AEC II, 30% cyclic stretch activated YAP, and its downstream targets Cyr61/CCN1 and CTGF/CCN2 and proinflammatory IL-6 expression. Target activation was blocked by a Yes-associated protein inhibitor. This novel YAP-dependent pathway could be involved in stretch-induced damage of alveolar cells.

Effects of Body Position and Hypovolemia on the Regional Distribution of Pulmonary Perfusion During One-Lung Ventilation in Endotoxemic Pigs.

Background: The incidence of hypoxemia during one-lung ventilation (OLV) is as high as 10%. It is also partially determined by the distribution of perfusion. During thoracic surgery, different body positions are used, such as the supine, semilateral, lateral, and prone positions, with such positions potentially influencing the distribution of perfusion. Furthermore, hypovolemia can impair hypoxic vasoconstriction. However, the effects of body position and hypovolemia on the distribution of perfusion remain poorly defined. We hypothesized that, during OLV, the relative perfusion of the ventilated lung is higher in the lateral decubitus position and that hypovolemia impairs the redistribution of pulmonary blood flow. Methods: Sixteen juvenile pigs were anesthetized, mechanically ventilated, submitted to a right-sided thoracotomy, and randomly assigned to one of two groups: (1) intravascular normovolemia or (2) intravascular hypovolemia, as achieved by drawing ~25% of the estimated blood volume (n = 8/group). Furthermore, to mimic thoracic surgery inflammatory conditions, Escherichia coli lipopolysaccharide was continuously infused at 0.5 µg kg-1 h-1. Under left-sided OLV conditions, the animals were further randomized to one of the four sequences of supine, left semilateral, left lateral, and prone positioning. Measurements of pulmonary perfusion distribution with fluorescence-marked microspheres, ventilation distribution by electrical impedance tomography, and gas exchange were then performed during two-lung ventilation in a supine position and after 30 min in each position and intravascular volume status during OLV. Results: During one-lung ventilation, the relative perfusion of the ventilated lung was higher in the lateral than the supine position. The relative perfusion of the non-ventilated lung was lower in the lateral than the supine and prone positions and in semilateral compared with the prone position. During OLV, the highest arterial partial pressure of oxygen/inspiratory fraction of oxygen (PaO2/F I O 2) was achieved in the lateral position as compared with all the other positions. The distribution of perfusion, ventilation, and oxygenation did not differ significantly between normovolemia and hypovolemia. Conclusions: During one-lung ventilation in endotoxemic pigs, the relative perfusion of the ventilated lung and oxygenation were higher in the lateral than in the supine position and not impaired by hypovolemia.

Comparative effects of flow vs. volume-controlled one-lung ventilation on gas exchange and respiratory system mechanics in pigs.

BACKGROUND: Flow-controlled ventilation (FCV) allows expiratory flow control, reducing the collapse of the airways during expiration. The performance of FCV during one-lung ventilation (OLV) under intravascular normo- and hypovolaemia is currently unknown. In this explorative study, we hypothesised that OLV with FCV improves PaO2 and reduces mechanical power compared to volume-controlled ventilation (VCV). Sixteen juvenile pigs were randomly assigned to one of two groups: (1) intravascular normovolaemia (n = 8) and (2) intravascular hypovolaemia (n = 8). To mimic inflammation due to major thoracic surgery, a thoracotomy was performed, and 0.5 µg/kg/h lipopolysaccharides from Escherichia coli continuously administered intravenously. Animals were randomly assigned to OLV with one of two sequences (60 min per mode): (1) VCV-FCV or (2) FCV-VCV. Variables of gas exchange, haemodynamics and respiratory signals were collected 20, 40 and 60 min after initiation of OLV with each mechanical ventilation mode. The distribution of ventilation was determined using electrical impedance tomography (EIT). RESULTS: Oxygenation did not differ significantly between modes (P = 0.881). In the normovolaemia group, the corrected expired minute volume (P = 0.022) and positive end-expiratory pressure (PEEP) were lower during FCV than VCV. The minute volume (P ≤ 0.001), respiratory rate (P ≤ 0.001), total PEEP (P ≤ 0.001), resistance of the respiratory system (P ≤ 0.001), mechanical power (P ≤ 0.001) and resistive mechanical power (P ≤ 0.001) were lower during FCV than VCV irrespective of the volaemia status. The distribution of ventilation did not differ between both ventilation modes (P = 0.103). CONCLUSIONS: In a model of OLV in normo- and hypovolemic pigs, mechanical power was lower during FCV compared to VCV, without significant differences in oxygenation. Furthermore, the efficacy of ventilation was higher during FCV compared to VCV during normovolaemia.

Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression.

BACKGROUND/AIMS: Bone morphogenetic proteins (BMPs) and BMP receptors widely participate in osteolytic metastasis of breast cancer, while their role in tumor-stromal interaction is largely unknown. In this study, we investigated whether BMP receptor type 1a (BMPR1a) can alter the interaction between metastatic cancer cells and osteoclast precursors. METHODS: Adenovirus-mediated RNA interference was used to interrupt target genes of human breast cancer cell lines and nude mice were injected intratibially with the cancer cells. Tumor-bearing mice were examined by bioluminescence imaging and microCT. Sections of metastatic legs were measured by a series of staining methods. Murine bone marrow mononuclear cells or RAW264.7 cells were cultured with conditioned media of breast cancer cells. RT-PCR, Western blotting and ELISA were used to test mRNA and protein expressions of target molecules. RESULTS: Expression of BMPR1a of MDA-MB-231-luc cells at tumor-bone interface was apparently stronger than that of cancer cells distant from the interface. Mice injected with BMPR1a-knockdown MDA-MB-231-luc cells showed reduced tumor growth and bone destruction compared with control groups. Knockdown (KD) of BMPR1a of MDA-MB-231-luc cells or MCF-7 cells decreased the level of receptor activator for NF-κB ligand (RANKL). Level of RANKL in MDA-MB-231-luc cells or MCF-7 cells was reduced by p38 inhibitor. Compared with control group, knockdown of p38 of breast cancer cells decreased cancer-induced osteoclastogenesis. CONCLUSION: Knockdown of BMPR1a of breast cancer cells suppresses their production of RANKL via p38 pathway and inhibits cancer-induced osteoclastogenesis, which indicates that BMPR1a might be a possible target in breast cancer-induced osteolytic metastasis.

Tetrandrine inhibits the proliferation of human osteosarcoma cells by upregulating the PTEN pathway.

Tetrandrine (TET) is a natural product isolated from the Chinese herb Stephania tetrandra S. Moore and has been reported to have antiproliferation and apoptosis-inducing activity in various malignant tumor cells. However, the exact molecular mechanisms underlying these effects remain unclear. In the present study, we tested the antiproliferation effect of TET on osteosarcoma (OS) 143B cells and explored the possible potential molecular mechanism in this process. Using CCK-8 assay and flow cytometry, we found that TET inhibited proliferation, induced apoptosis and arrested the cell cycle of the 143B cells. Using a xenograft tumor model of human OS, tetrandrine was found to inhibit tumor growth in vivo. TET increased the protein level of phosphatase and tensin homolog (PTEN) and decreased its phosphorylation as detected by western blot analysis and immunohistochemistry.Overexpression of PTEN strengthened the anticancer effect of TET, while knockdown of PTEN attenuated it. Meanwhile, TET activated p38 MAPK and increased its phosphorylation. Our findings suggest that TET may be a potential anticancer drug for OS. In addition, its effects may be mediated by the upregulation of PTEN. Moreover the expression alteration of PTEN and p-PTEN was mediated by the TET-induced activation of p38 MAPK in a direct or indirect manner.

Ursolic acid inhibits proliferation and induces apoptosis by inactivating Wnt/ß-catenin signaling in human osteosarcoma cells.

Although multiple chemotherapeutic agents have been used for osteosarcoma (OS) treatment, their mechanisms need further study. Ursolic acid (UA), a pentacyclic triterpenoid, can reduce cell proliferation and induce apoptosis in various cancer cells, such as OS. However, the exact mechanism underlying this function remains unclear. In this study, we investigated the anti­proliferative effect of UA in human OS 143B cells and dissected the possible molecular mechanism underlying this effect. We demonstrated that UA can reduce cell proliferation, induce apoptosis and arrest cell cycle in 143B cells, as well as inhibit OS tumor growth in a mouse xenograft model. Using a luciferase reporter assay, we found that the Wnt/ß­catenin signaling is inhibited by UA in 143B cells. Correspondingly, the expression level and nuclear translocation of ß­catenin are both decreased by UA. Exogenous expression of ß­catenin attenuates the anticancer effect of UA in 143B cells, while knockdown of ß­catenin enhances this effect. UA increases the expression level of p53 in a concentration­dependent manner, and inhibition of p53 reduces the anticancer effect of UA in 143B cells. Moreover, inhibition of p53 partly reverses the UA­induced downregulation of ß­catenin, as do the targets of Wnt/ß­catenin signaling, such as c­Myc and cyclin D1. Our findings indicated that UA can inhibit the proliferation of 143B OS cells through inactivation of Wnt/ß-catenin signaling, which may be mediated partly by upregulating the expression of p53.

Adenovirus-mediated siRNA targeting CXCR2 attenuates titanium particle-induced osteolysis by suppressing osteoclast formation.

BACKGROUND Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. MATERIAL AND METHODS Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. RESULTS Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. CONCLUSIONS CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening.

IGF1 potentiates BMP9-induced osteogenic differentiation in mesenchymal stem cells through the enhancement of BMP/Smad signaling.

Engineered bone tissue is thought to be the ideal alternative for bone grafts in the treatment of related bone diseases. BMP9 has been demonstrated as one of the most osteogenic factors, and enhancement of BMP9-induced osteogenesis will greatly accelerate the development of bone tissue engineering. Here, we investigated the effect of insulin-like growth factor 1 (IGF1) on BMP9-induced osteogenic differentiation, and unveiled a possible molecular mechanism underling this process. We found that IGF1 and BMP9 are both detectable in mesenchymal stem cells (MSCs). Exogenous expression of IGF1 potentiates BMP9-induced alkaline phosphatase (ALP), matrix mineralization, and ectopic bone formation. Similarly, IGF1 enhances BMP9-induced endochondral ossification. Mechanistically, we found that IGF1 increases BMP9-induced activation of BMP/Smad signaling in MSCs. Our findings demonstrate that IGF1 can enhance BMP9-induced osteogenic differentiation in MSCs, and that this effect may be mediated by the enhancement of the BMP/Smad signaling transduction triggered by BMP9. [BMB Reports 2016; 49(2): 122-127].

Evodiamine inhibits the proliferation of human osteosarcoma cells by blocking PI3K/Akt signaling.

Osteosarcoma (OS) is the most common non-hematologic primary malignancy of bone, and multiple chemotherapeutic agents have been applied in the treatment of OS for over 40 years. Nevertheless, due to the poor prognosis of OS, it is essential to develop a novel treatment strategy. Evodiamine (EVO), a quinolone alkaloid extracted from the fruit of Evodia rutaecarpa, has been demonstrated to inhibit tumor cell proliferation. Thus, the main aim of the present study was to investigate the anti-proliferative and apoptosis-inducing effects of evodiamine (EVO) on human OS 143B cells, but also the possible mechanisms underlying these effects. The results of crystal violet staining, flow cytometry, western blot analysis and an in vivo experiment demonstrated that EVO exhibits significant inhibitory effects on cell proliferation, exhibits apoptosis-inducing effects and arrests the cell cycle in 143B cells. According to our findings of polymerase chain reaction (PCR), western blot analysis and recombinant adenoviral transfection, we confirmed that EVO upregulates both the protein and gene levels of phosphatase and tensin homolog (PTEN) in a concentration-dependent manner in 143B cells. Overexpression of PTEN reinforced the anti-proliferative effect of EVO in the 143B cells, while knockdown of PTEN upregulated PI3K/Akt signaling transduction and reversed the inhibitory effect of EVO on 143B cell proliferation. Further analysis indicated that EVO upregulated the expression of PTEN by inactivating PI3K/Akt signaling by decreasing phosphorylated Akt1/2. Based on the above results, we conclude that PTEN/PI3K/Akt signaling is involved in the inhibitory effect on human OS 143B cell proliferation by EVO.

Oridonin inhibits the proliferation of human osteosarcoma cells by suppressing Wnt/ß-catenin signaling.

It has been reported that oridonin (ORI) can inhibit proliferation and induce apoptosis in various types of cancer cell lines. However, the exact mechanism for this function remains unclear. In this study, we investigated the proliferation inhibitory effect of ORI on human osteosarcoma (OS) 143B cells and dissected the possible molecular mechanism(s) underlying this effect. We demonstrated that ORI can inhibit proliferation, induce apoptosis and arrest the cell cycle in 143B cells. Using luciferase reporter assay, we found that the Wnt/ß-catenin signaling was inhibited in 143B cells by ORI. Accordingly, the total protein levels and nuclear translocation of ß-catenin were reduced by ORI treatment. ORI increased glycogen synthase kinase 3ß (GSK3ß) activity and upregulated Dickkopf-1 (Dkk-1) expression. We found that Dkk-1 overexpression or ß-catenin knockdown can potentiate the proliferation inhibitory effect of ORI in 143B cells, while ß-catenin overexpression attenuated this effect. Using the xenograft tumor model of human OS, we demonstrated that ORI effectively inhibited the growth of tumors. Histological examination showed that ORI inhibited cancer cell proliferation, decreased the expression of PNCA and ß-catenin. Our findings suggest that ORI can inhibit 143B OS cell proliferation by downregulating Wnt/ß-catenin signal transduction, which may be mediated by upregulating the Dkk-1 expression and/or enhancing the function of GSK3ß. Therefore, ORI can be potentially used as an effective adjuvant agent for the clinical management of OS.

BMP9 and COX-2 form an important regulatory loop in BMP9-induced osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can self-renew and differentiate into osteogenic, chondrogenic, adipogenic and myogenic lineages. It's reported that bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic BMPs to initiate the commitment of MSCs to osteoblast lineage. Cyclooxygenase-2 (COX-2) is critical for bone fracture healing and osteogenic differentiation in MSCs. However, the relationship between COX-2 and BMP9 in osteogenesis remains unknown. Herein, we investigate the role of COX-2 in BMP9-induced osteogenesis in MSCs. We demonstrate that COX-2 is up-regulated as a target of BMP9 in MSCs. Both COX-2 inhibitor (NS-398) and COX-2 knockdown siRNAs can effectively decrease alkaline phosphatase (ALP) activities induced by BMP9 in MSCs. NS-398 also down-regulates BMP9-induced expression of osteopontin and osteocalcin, so does the matrix mineralization. The in vivo studies indicate that knockdown of COX-2 attenuates BMP9-induced ectopic bone formation. In perinatal limb culture assay, NS-398 is shown to reduce the hypertropic chondrocyte zone and ossification induced by BMP9. Mechanistically, knockdown of COX-2 significantly inhibits the BMP9 up-regulated expression of Runx2 and Dlx-5 in MSCs, which can be rescued by exogenous expression of COX-2. Furthermore, knockdown of COX-2 apparently reduces BMP9 induced BMPR-Smad reporter activity, the phosphorylation of Smad1/5/8, and the expression of Smad6 and Smad7 in MSCs. NS-398 blocks the expression of BMP9 mediated by BMP9 recombinant adenovirus. Taken together, our findings suggest that COX-2 plays an important role in BMP9 induced osteogenic differentiation in MSCs; BMP9 and COX-2 may form an important regulatory loop to orchestrate the osteogenic differentiation in MSCs.

The effect of adenovirus-mediated siRNA targeting BMPR-II on UHMWPE-induced osteoclast formation.

Aseptic loosening (AL) is the single most common complication of total joint arthroplasty. The critical factor may contribute to loosening is the adverse tissue response to wear debris. A growing body of literature suggests that BMPs influence the formation and activity of osteoclasts, and BMP signaling plays an important role in the osteoclast formation. In this study, we have employed an RNA interference approach by transfecting a small interfering RNA (siRNA) specific for BMPR-II, to determine the possible importance of this receptor as a target for UHMWPE (Ultra high molecular weight polyethylene) induced osteoclastogenesis in the air pouch model in vivo. Meanwhile, in order to further elucidation of the mechanism of BMPR-II signaling pathway in osteoclast formation, we investigated the effects of siBMPR-II toward RANKL induced osteoclast differentiation in vitro. The present study showed that locally injection of adenovirus-mediated siRNA targeting BMPR-II appears to be a feasible and effective candidate to treat or prevent wear debris-associated osteolysis. Furthermore, we revealed that the effects of BMPR-II signaling on osteoclast formation are mediated directly by osteoclast itself, as well as indirectly by altered expression of RANKL and OPG in osteoblast.

Protective effect of atorvastatin on radiation-induced vascular endothelial cell injury in vitro.

Vascular endothelial cells are very sensitive to ionizing radiation, and it is important to develop effective prevent agents and measures in radiation exposure protection. In the present study, the protective effects of atorvastatin on irradiated human umbilical vein endothelial cells (HUVEC) and the possible mechanisms were explored. Cultured HUVEC were treated by atorvastatin at a final concentration of 10 µ mol/ml for 10 minutes, and then irradiated at a dose of 2 Gy or 25 Gy. Twenty-four hours after irradiation, apoptosis of HUVEC was monitored by flow cytometry, and the expression of thrombomodulin (TM) and protein C activation in HUVEC was respectively assessed by flow cytometry and spectrophotometry. After treatment with atorvastatin for 24 h, the rate of cell apoptosis decreased by 6% and 16% in cells irradiated with 2 Gy and 25 Gy, respectively. TM expression increased by 77%, 59%, and 61% in untreated cells, 2 Gy irradiation-treated cells, and 25 Gy irradiation-treated cells, respectively. The protein C levels in 2 Gy and 25 Gy irradiation-treated cells were reduced by 23% and 34% when compared with untreated cells, but up-regulated by 79% and 76% when compared with cells which were irradiated and treated with atorvastatin. In conclusion, these data indicate that atorvastatin exerts protective effects on irradiated HUVEC by reducing apoptosis by up-regulating TM expression and enhancing protein C activation in irradiated HUVEC.