Pharmacological evaluation of imidazole‐derived bisphosphonates on receptor activator of nuclear factor‐κB ligand‐induced osteoclast differentiation and function

Bisphosphonates (BPs) have been commonly used in the treatment of osteolytic bone lesions, such as osteoporosis and osteogenesis imperfecta. However, serious side‐effects can occur during the therapy. To search for novel potent BPs with lower side‐effects, a series of imidazole‐containing BPs (zoledronic acid [ZOL]; ZOL derivatives by substitution of the hydrogen at the 2‐position on the imidazole ring with a methyl [MIDP], ethyl [EIDP], n‐propyl [PIDP], or n‐butyl group [BIDP]) were developed and the effects on receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclast differentiation were investigated using the murine macrophage RAW 264.7 cells at the protein, gene, and morphological and functional levels. Influences of these BPs on the cell growth and proliferation of RAW 264.7 were also studied in order to determine cytotoxicity. The results showed that PIDP significantly inhibited the RANKL‐induced osteoclast formation in a dose‐dependent fashion without inducing cytotoxicity under the concentration of 12.5 μM. It exerted remarkable suppressive effects on the development of actin rings, the bone resorption, and the expressions of osteoclastogenesis‐related gene and protein markers. The down‐regulation of c‐Jun N‐terminal kinase (JNK), protein kinase B (Akt), and inhibitor of nuclear factor kappa‐B (IκB) phosphorylation in the early signaling event and subsequent inhibition of the expression of c‐Fos and nuclear factor of activated T cells (NFATc1) might be involved in these effects. All these results indicated that PIDP might be a promising drug to treat bone‐related disorders.     

Deoxyactein stimulates osteoblast function and inhibits bone‐resorbing mediators in MC3T3‐E1 cells

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic agents with minimal side effects is highly desirable. Cimicifuga racemosa has a long and diverse history of medicinal use and deoxyactein isolated from this species is one of the major constituents. In the present study, the effect of deoxyactein on the function of osteoblastic MC3T3‐E1 cells was studied. Deoxyactein caused a significant elevation of cell growth, alkaline phosphatase activity, collagen content, and mineralization in the cells (P < 0.05). Moreover, deoxyactein significantly (P < 0.05) decreased the production of reactive oxygen species (ROS) and osteoclast differentiation‐inducing factors such as TNF‐α, IL‐6 and receptor activator of nuclear factor‐κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. These results demonstrate that deoxyactein may have positive effects on skeletal structure. Copyright © 2011 John Wiley & Sons, Ltd.     

Actein alleviates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐mediated cellular dysfunction in osteoblastic MC3T3‐E1 cells

The environmental pollutant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is known to affect bone metabolism. We evaluated the protective effects of the triterpene glycoside actein from the herb black cohosh against TCDD‐induced toxicity in MC3T3‐E1 osteoblastic cells. We found that TCDD significantly reduced cell viability and increased apoptosis and autophagy in MC3T3‐E1 osteoblastic cells (P < .05). In addition, TCDD treatment resulted in a significant increase in intracellular calcium concentration, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cardiolipin peroxidation, whereas pretreatment with actein significantly mitigated these effects (P < .05). The effects of TCDD on extracellular signal‐related kinase (ERK), aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and cytochrome P450 1A1 levels in MC3T3‐E1 cells were significantly inhibited by actein. The levels of superoxide dismutase, ERK1, and nuclear factor kappa B mRNA were also effectively restored by pretreatment with actein. Furthermore, actein treatment resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblastic differentiation (ALP, type I collagen, osteoprotegerin, bone sialoprotein, and osterix). This study demonstrates the underlying molecular mechanisms of cytoprotection exerted by actein against TCDD‐induced oxidative stress and osteoblast damage.     

Curcumin ameliorates asthmatic airway inflammation by activating nuclear factor‐E2‐related factor 2/haem oxygenase (HO)‐1 signalling pathway

Previous studies have shown that curcumin alleviates asthma in vivo. However, the relationship between curcumin and the nuclear factor‐E2‐related factor 2 (Nrf2)/haem oxygenase (HO)‐1 pathway in asthma treatment remains unknown. The aim of the present study was to investigate the mechanisms of curcumin involved in the amelioration of airway inflammation in a mouse asthma model. Curcumin was administrated to asthmatic mice, and bronchoalveolar lavage fluid was collected. Inflammatory cell infiltration was measured by Giemsa staining. Immunoglobulin E production in bronchoalveolar lavage fluid was measured by enzyme–linked immunosorbent assay. Histological analyses were evaluated with haematoxylin‐eosin and periodic acid‐Schiff staining. Airway hyperresponsiveness was examined by whole‐body plethysmography. Nuclear factor‐E2‐related factor 2, HO‐1, nuclear factor‐κB and inhibitory κB/p‐inhibitory κB levels in lung tissues were detected by western blot, and Nrf2 activity was measured by electrophoretic mobility shift assay. Tumour necrosis factor‐α, interleukin (IL)‐1β, and IL‐6 levels in the small interfering RNA‐transfected cells were detected by enzyme–linked immunosorbent assay. Curcumin treatment significantly reduced immunoglobulin E production, attenuated inflammatory cell accumulation and goblet cell hyperplasia, and ameliorated mucus secretion and airway hyperresponsiveness. Nuclear factor‐E2‐related factor 2 and HO‐1 levels in lung tissues were significantly increased. Meanwhile, Nrf2 activity was enhanced. Nuclear factor‐κB and p‐inhibitory κB levels were elevated in the lung tissue of ovalbumin‐challenged mice. Both were restored to normal levels after curcumin treatment. Haem oxygenase‐1 and nuclear Nrf2 levels were enhanced in dose‐ and time‐dependent manners in curcumin‐treated RAW264.7 cells. Curcumin blocked lipopolysaccharide‐upregulated expression of tumour necrosis factor‐α, IL‐1β, and IL‐6. After the cells were transfected with HO‐1 or Nrf2 small interfering RNA, lipopolysaccharide‐induced pro‐inflammation cytokine expression was significantly restored. In summary, curcumin might alleviate airway inflammation in asthma through the Nrf2/HO‐1 pathway, potentially making it an effective drug in asthma treatment.     

Effect of Stem Cell Factor,Interleukin–6, Nitric Oxide and Transforming Growth Factor–β on the Osteoclast Differentiation Induced by 1α,25–(OH)2D3 in Primary Murine Bone Marrow Cultures

Abstract: Osteotropic hormones and cytokines are involved in the differentiation of osteoclast progenitors from haematopoietic stem cells to multinucleated osteoclasts which mediate bone resorption. Stem cell factor, interleukin–6, nitric oxide, and transforming growth factor–β are implicated in the regulation of bone resorption by osteoclast. We test whether stem cell factor, interleukin–6, nitric oxide, and transforming growth factor–β affect the generation of osteoclast–like multinucleated cells induced by 1α,25–(OH)₂D₃. 1α,25–(OH)₂D₃ increases the generation of osteoclast–like cells retaining osteoclast characteristics including multinuclearity and positive staining for tartrate–resistant acid phosphatase. Combined treatment of stem cell factor with interleukin–6 synergistically potentiates the ability of 1α,25–(OH)₂D₃ to generate tartrate–resistant acid phosphatase–positive multinucleated cells. However, either stem cell factor or interleukin–6 alone does not induce the generation of tartrate–resistant acid phosphatase–positive multinucleated cells. Transforming growth factor–β produces a biphasic effect on osteoclast generation induced by 1α,25–(OH)₂D₃. Transforming growth factor–β stimulates osteoclast generation at low concentration (0.1 ng/ml) whereas it suppresses the formation of osteoclast–like cell at higher concentration (1 ng/ml). Sodium nitroprusside, a donor of nitric oxide, almost completely inhibits the generation of 1α,25–(OH)₂D₃–induced osteoclast at high concentration (100 μM), but it significantly enhances the osteoclast generation at low concentrations (3 μM). These results suggest that stem cell factor, interleukin–6, transforming growth factor–β, and nitric oxide interact with 1α,25–(OH)₂D₃ to modulate the differentiation of hematopoietic precursors toward committed osteoclast precursors.     

The effect of naringenin on the role of nuclear factor (erythroid‐derived 2)‐like2 (Nrf2) and haem oxygenase 1 (HO‐1) in reducing the risk of oxidative stress‐related radiotoxicity in the spleen of rats

The present study was to evaluate the radiomitigative effect of naringenin (NRG) on the modulation of ionizing radiation (IR)‐induced spleen injury. Rats were exposed to 12 Gy (3Gy/two times/week). NRG (50mg/Kg), was orally given one hour after the first radiation dose, and daily continued during the irradiation period. Rats were sacrificed 1 day after the last dose of radiation. NRG showed a significant decrease of malondialdehyde, hydrogen peroxide with a significant elevation of superoxide dismutase, catalase and glutathione peroxidase activities and glutathione content. Moreover, NRG confirmed the intracellular defense mechanisms through activation of nuclear factor (erythroid‐derived 2)‐like2 (Nrf2) and haem oxygenase‐1 (HO‐1) levels and their protein expression. In addition, NRG deactivated the nuclear factor‐κB (NF‐κB) and reduced the pro‐inflammatory cytokines. Further, NRG showed positive modulation in the haematological values (WBCs, RBCs, Hb, Hct% and PLt). In conclusion, these results suggested that NRG reversed the IR‐induced redox‐imbalance in the rat spleen.     

Systemic RANK‐Fc protein therapy is efficacious against primary osteosarcoma growth in a murine model via activity against osteoclasts

Objectives Osteosarcoma (OS) is the most common primary malignant bone tumour, and mainly affects adolescents and young adults. Although there has been substantial improvement in management of OS with surgery and chemotherapy, further survival increase has not been achieved over the past two decades. Methods We focused on the receptor activator of nuclear factor κB ligand (RANKL)–osteoclast (OCL) system as a biological target for OS. RANKL is a critical factor for OCL formation and bone resorption activity. The primary lesion in bone and ensuing metastasis in OS both require the induction of OCLs. RANK‐Fc is a potent RANKL antagonist and inhibitor of OCL formation and activity. Key findings In an orthotopic model in Balb/c nu/nu mice, a twice weekly dosing regimen of 350 μg of RANK‐Fc per mouse subcutaneously (n= 5) reduced lung metastasis (P > 0.05), preserved bone structure and reduced tartrate‐resistant acid phosphatase (TRAP)⁺ OCLs (P < 0.005) in OS‐bearing bone. In vitro, RANK‐Fc suppressed OCL formation (P < 0.005), bone resorption activity (P < 0.005) and RANKL‐induced anti‐apoptosis (P < 0.5) of OCLs.     

The natural compound 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐d glucoside protects against adriamycin‐induced nephropathy through activating the Nrf2‐Keap1 antioxidant pathway

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐d ‐glucoside (THSG) is an active compound extracted from Polygonum multiflorum Thunb. This herb and radix Polygoni Multiflori preparata have been used to treat arteriosclerosis, hyperlipidemia, hypercholesterolemia, and diabetes for thousands of years. This study aimed to investigate the protective effects of THSG in an Adriamycin (AD)‐induced focal segmental glomerulosclerosis (FSGS) mouse model and the underlying mechanisms in an in vitro system. Mice were treated with THSG (2.5 and 10 mg/kg, oral gavage) for 24 consecutive days. On the third day, mice were intravenously given a single dose of AD (10 mg/kg). At the end of the experiment, plasma and kidney samples were harvested to evaluate the therapeutic effects of THSG. The potential mechanisms of THSG in protecting against AD‐induced cytotoxicity were examined using a real‐time polymerase chain reaction, immunoblots, lactate dehydrogenase assay, and a cellular oxidized‐thiol detection system in a mouse mesangial cell line. In this study, THSG showed concentration‐dependent protective effects in ameliorating the progression of AD‐induced FSGS. THSG suppressed albuminuria and hypercholesterolemia and reduced the status of lipid peroxidation in urine, plasma, and kidney tissue samples. Furthermore, THSG protected against podocyte damage, reduced renal fibrotic gene expressions, and alleviated the severity of glomerulosclerosis. Treatment of mouse mesangial cells with THSG induced nuclear factor erythroid‐derived 2‐like 2 (Nrf2) nuclear translocation, increased heme oxygenase‐1 and NAD(P)H:quinone oxidoreductase (NQO)‐1 gene expressions, and reduced cellular thiol oxidation and resistance to AD‐induced cytotoxicity. Silencing Nrf2 and its repressor protein, Kelch‐like ECH‐associated protein 1 (Keap1), abolished these protective effects of THSG. In conclusion, THSG can play a protective role in ameliorating the progression of FSGS in a mouse model through activation of the Nrf2‐Keap1 antioxidant pathway. Although a well‐designed therapeutic study is needed, THSG may be applied to manage chronic kidney disease.     

珊瑚共附生黄柄曲霉次级代谢产物研究

目的 研究东沙短足软珊瑚（Cladiella sp.）来源的黄柄曲霉菌（Aspergillus flavipes）中的活性次级代谢产物。方法 采用硅胶柱层析、凝胶柱层析、制备HPLC等分离手段对真菌发酵液乙酸乙酯提取物进行分离,运用现代波谱技术结合文献报道数据,对化合物的结构进行鉴定;采用核因子κB受体活化因子配基（RANKL）诱导小鼠骨髓单核巨噬细胞（bone marrow macrophage cells,BMMs）分化为成熟的破骨细胞,经抗酒石酸酸性磷酸酶（TRAP）特异性染色,对化合物抑制破骨细胞分化活性进行研究。结果 从该株真菌中分离得到4个细胞松弛素类化合物,其结构鉴定为trichalasins H,aspergilluchalasin,aspochalasin I和aspochalasin D。体外活性测试结果显示,化合物3和4可不同程度抑制BMMs向破骨细胞分化。结论 对化合物3和4抑制破骨细胞分化活性的报告是本文首次报道,对新型抗骨质疏松活性物质研究具有科学价值。     

6‐Ethyl‐N,N′‐bis(3‐hydroxyphenyl)[1,3,5]triazine‐2,4‐diamine (RS‐0466) Enhances the Protective Effect of Brain‐Derived Neurotrophic Factor on Amyloid β‐Induced Cytotoxicity in Cortical Neurones

Abstract: Amyloid β peptide in the senile plaques of patients with Alzheimer's disease is considered to be responsible for the pathology of Alzheimer's disease. We have previously reported that 6‐ethyl‐N,N′‐bis(3‐hydroxyphenyl)[1,3,5]triazine‐2,4‐diamine, RS‐0466, is capable of significantly inhibiting amyloid β‐induced cytotoxicity in HeLa cells. To determine various profiles of RS‐0466, we investigated whether RS‐0466 would enhance the neuroprotective effect of brain‐derived neurotrophic factor on amyloid β_1–42‐induced cytotoxicity in rat cortical neurones. Consistent with previous observations, brain‐derived neurotrophic factor ameliorated amyloid β_1–42‐induced cytotoxicity. Furthermore, co‐application of RS‐0466 enhanced the neuroprotective effect of brain‐derived neurotrophic factor. RS‐0466 also reversed amyloid β_1–42‐induced decrease of brain‐derived neurotrophic factor‐triggered phosphorylated Akt. These results raise the possibility that RS‐0466 or one of its derivatives has potential to enhance the neuroprotective effect of brain‐derived neurotrophic factor, and could serve as a therapeutic agent for patients with Alzheimer's disease.     

Xanthohumol ameliorates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin–induced cellular toxicity in cultured MC3T3‐E1 osteoblastic cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is an environmental contaminant. Xanthohumol is a prenylated flavonoid found in hops (Humulus lupulus) and beer. The aim of the current study was to explore the role of xanthohumol in modulating the toxicity of TCDD in MC3T3‐E1 osteoblastic cells. In cells treated with TCDD alone, intracellular Ca²⁺ concentrations, mitochondrial membrane potential disruption, reactive oxygen species production, cardiolipin peroxidation, nitric oxide release and cytochrome P450 1A1 expression were significantly increased. TCDD treatment increased the mRNA levels of extracellular signal‐regulated kinase 1 and nuclear factor kappa B, and significantly decreased the level of protein kinase B (AKT) in MC3T3‐E1 osteoblastic cells. However, the presence of xanthohumol alleviated the pathological effects of TCDD. In addition, xanthohumol treatment significantly increased the expression of genes associated with osteoblast differentiation (alkaline phosphatase, osteocalcin, osteoprotegerin and osterix). We conclude that xanthohumol has a beneficial influence and may antagonize TCDD toxicity in osteoblastic cells.     

Rhodium‐mediated [11C]carbonylation: a library of N‐phenyl‐N′‐{4‐(4‐quinolyloxy)‐phenyl}‐[11C]‐urea derivatives as potential PET angiogenic probes

As part of our ongoing investigation into the imaging of angiogenic processes, a small library of eight vascular endothelial growth factor receptor‐2 (VEGFR‐2)/platelet‐derived growth factor receptor β dual inhibitors based on the N‐phenyl‐N′‐4‐(4‐quinolyloxy)‐phenyl‐urea was labelled with ¹¹C (β⁺, t_1/2=20.4 min) in the urea carbonyl position via rhodium‐mediated carbonylative cross‐coupling of an aryl azide and different anilines. The decay‐corrected radiochemical yields of the isolated products were in the range of 38–81% calculated from [¹¹C]carbon monoxide. Starting with 10.7±0.5 GBq of [¹¹C]carbon monoxide, 1‐[4‐(6,7‐dimethoxy‐quinolin‐4‐yloxy)‐3‐fluoro‐phenyl]‐3‐(4‐fluoro‐phenyl)‐[¹¹C]‐urea (2.1 GBq) was isolated after total reaction time of 45 min with a specific activity of 92±4 GBq µmol^?1. Copyright © 2009 John Wiley & Sons, Ltd.     

Dexmedetomidine preconditioning for myocardial protection in ischaemia‐reperfusion injury in rats by downregulation of the high mobility group box 1‐toll‐like receptor 4‐nuclear factor κB signalling pathway

Pharmacological preconditioning reduces myocardial infarct size in ischaemia‐reperfusion (I‐R) injury. Dexmedetomidine, a selective α₂‐adrenoceptor agonist, has a proven cardioprotective effect when administered prior to I‐R, although the underlying mechanisms for this effect are not fully understood. We evaluated whether dexmedetomidine preconditioning could induce a myocardio‐protective effect against I‐R injury by inhibiting associated inflammatory processes through downregulation of the high mobility group box 1 (HMGB1)‐toll‐like receptor 4 (TLR4)‐nuclear factor κB (NF‐κB) signalling pathway. Seventy rats were randomly assigned to seven groups: a control and six test groups, involving I‐R for 30 and 120 minutes, respectively, in isolated rat hearts and different pretreatment protocols with dexmedetomidine (10 nmol/L) as well as yohimbine (1 μmol/L) and recombinant HMGB1 peptide (rHMGB1; 20 μg/L), alone or in combination with dexmedetomidine. Cardiac function was recorded; myocardial HMGB1, TLR4, and NF‐κB activities and levels of tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) were measured as were lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary outflow. Dexmedetomidine preconditioning significantly improved cardiac function (P<.05), downregulated the expression of HMGB1‐TLR4‐NF‐κB, reduced levels of TNF‐α and IL‐6 in isolated ventricles during I‐R injury, and significantly reduced CK and LDH levels in coronary outflow (P<.05). All of these effects were partially reversed by yohimbine (P<.05) or rHMGB1 (P<.05). Dexmedetomidine preconditioning alleviated myocardial I‐R injury in rats through inhibition of inflammatory processes associated with downregulation of the HMGB1‐TLR4‐NF‐κB signalling pathway via activation at α₂‐adrenergic receptors.