SPI1 exacerbates iron accumulation and promotes osteoclast formation through inhibiting the expression of Hepcidin.

BACKGROUND: Osteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts' effects on osteoporosis. METHODS: RAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl's iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: We found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. CONCLUSION: Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.

Giant cell tumors of the mobile spine with invasion of adjacent vertebrae: an unusual imaging finding.

BACKGROUND: Giant cell tumors of the mobile spine invasion of the adjacent vertebrae are an ignored imaging finding. METHODS: Nine patients with giant cell tumors of the mobile spine with invasion of the adjacent vertebrae confirmed by pathology were enrolled. Eight patients had pure giant cell tumors (GCTs), while one patient also had an aneurysmal bone cyst. All patients underwent conventional computed tomography, three-dimensional reconstruction, and conventional magnetic resonance imaging, while seven patients also underwent post-contrast magnetic resonance imaging. RESULTS: All patients showed GCTs of the mobile spine that arose from the vertebral body and extended to the vertebral arch. The tumors showed soft-tissue attenuation with no evidence of a mineralized matrix. Pathological fracture was seen in five patients. The margin of the original tumor showed partial sclerosis in four patients and involved an adjacent vertebral body with a sclerotic rim in two patients. The tumors showed a homogeneous and similar signal intensity to the normal spinal cord on T1WI (T1-weighted image) in five patients. The cystic area of the tumors was hyperintense on T2WI in the remaining four patients, while one patient showed hemorrhage that was hyperintense on T1WI. The solid components of the GCTs show marked enhancement in all cases, while the cystic area of the tumors was observed without enhancement on contrast-enhanced images in four patients. Bone destruction of the adjacent vertebral body showed a homogeneous signal on T1WI and T2WI and marked enhancement on contrast-enhanced images. CONCLUSIONS: Giant cell tumors of the mobile spine with invasion into adjacent vertebrae are an unusual imaging finding. Radiologists should be familiar with this imaging characteristic.

A universal laboratory method for determining physical parameters of radon migration in dry granulated porous media.

The particle size and heaped methods of exhalation media have important effects on physical parameters, such as the free radon production rate, porosity, permeability, and radon diffusion coefficient. However, existing methods for determining those parameters are too complex, and time-consuming. In this study, a novel, systematic determining method was proposed based on nuclide decay, radon diffusion migration theory, and the mass conservation law, and an associated experimental device was designed and manufactured. The parameters of uranium ore heap and sandy soil of radon diffusion coefficient (D), free radon production rate (α), media permeability (k), and porosity (ε) were obtained. At the same time, the practicality of the novel determining method was improved over other methods, with the results showing that accuracy was within the acceptable range of experimental error. This novel method will be of significance for the study of radon migration and exhalation in granulated porous media.

Modeling and experimental examination of water level effects on radon exhalation from fragmented uranium ore.

In this study, a one-dimensional steady-state mathematical model of radon transport in fragmented uranium ore was established according to Fick's law and radon transfer theory in an air-water interface. The model was utilized to obtain an analytical solution for radon concentration in the air-water, two-phase system under steady state conditions, as well as a corresponding radon exhalation rate calculation formula. We also designed a one-dimensional experimental apparatus for simulating radon diffusion migration in the uranium ore with various water levels to verify the mathematical model. The predicted results were in close agreement with the measured results, suggesting that the proposed model can be readily used to determine radon concentrations and exhalation rates in fragmented uranium ore with varying water levels.

Biomedical molecular of woody extractives of Cunninghamia Lanceolata biomass.

Extractives, important compounds from wood, provide abundant resources for woody medicine. In this study, the three extractives from Cunninghamia lanceolata wood were removed by method of three-stage extraction with alcohol, petroleum ether, and alcohol/petroleum ether and their chemical components were analyzed by gas chromatography-mass spectrometry (GC-MS). Thirteen chemical components were discovered in the first-stage extractives, including: 4-((1e)-3-hydroxy-1-propenyl)-2-methoxyphenol (36.80%), α-(2-phenylethenyl)-1-piperidineacetonitrile (15.39%). One-hundred chemical components were discovered in the second-stage extractives, including: [1s-(1α,4aα,10aß)]-1, 2,3,4,4a,9,10,10a-octahydro-1,4a- dimethyl-7-(1-methylethyl)-1- phenanthrenecar-boxylic acid (15.16%), 1,3-dimethoxy-5-[(1e)-2- phenylethenyl]-benzene (6.99%). Seven chemical components were discovered in the third-stage extractives, including: 1,3-dimethoxy -5-[(1E)-2-phenylethenyl]-benzene (32.88%), stigmasta-4,6,22-trien-3α-ol (17.83%). And both the main retention time of the first-stage and which of third-stage extractives are 20-30 minutes, and the main retention time of the second-stage extractives is <10 minutes. Besides, the three extractives contained many biomedical molecular, such as [1s-(1α,4aα,10aß)]-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenecar-boxylic acid, squalene, stigmast-4-en-3-one and γ-sitosterol and so on, which means that the three extractives from Cunninghamia lanceolata wood have huge potential in biomedicine.

[Manipulative treatment of vertebral artery type of cervical spondylosis].

OBJECTIVE: To compare the effects of the three manipulative methods in treating vertebral artery type of cervical spondylosis. METHODS: From December 2006 to December 2008, 300 patients (male 138 and female 162, the age from 18 to 76 years with an average of 38.6 years) with vertebral artery type of cervical spondylosis were randomly divided into group A, B, C (100 cases each group). Patients in group A, B were respectively treated with rotation-traction and rotation-turn manual reduction (one time per week, 30 min per time, 4 times a course of treatment; patients in group C were treated with traction (one time per day, 30 min per time, 10 times a course of treatment). Therapic time was a course of treatment in all groups. At 6 months after treatment, the clinical effects, cervical curvature, change of blood flow were respectively observed according sympotoms, X-ray, transcranial doppler sonography (TCD). RESULTS: All patients were followed up more than 6 months. The improving average of cervical curvature (C2-C7 Cobb angle): group A, B, C was respectively 1.82 +/- 0.88, 0.12 +/- 0.06, 0.56 +/- 0.22; group A was better than group B (P < 0.01) and group C (P < 0.05). There was no significantly difference between group B and C (P > 0.05);and there was significantly difference in three groups (P < 0.05). TCD detection: LVA, RVA, BA improving significantly in group A than group B (P < 0.01) and group C (P < 0.05); there was no significantly difference between group B and C (P > 0.05) and there was significantly difference among three groups (P < 0.05). Clinical effects: in group A, 36 cases obtained curing results, 36 excellent, 20 utility, 8 ineffective, the rate of excellent and good was 92%; in group B: 6 cases obtained curing results, 20 excellent, 10 utility, 64 ineffective, the rate of excellent and good was 36%;in group C,10 cases obtained curing results, 26 excellent, 8 utility, 56 ineffective, the rate of excellent and good was 44%; there was significantly difference in three groups (P < 0.05). CONCLUSION: All three methods can significantly relieve headache and vertigo through improving cervical curvature and VA, BA blood flow, but its long-term effect should be observed.

[C/EBP homologous protein-mediated endoplasmic reticulum stress-related apoptosis pathway is involved in abdominal aortic constriction-induced myocardium hypertrophy in rats.].

Endoplasmic reticulum stress (ERS) is an adaptive process in response to circumstantial changes, but excessive and/or prolonged ERS can induce cell apoptosis. C/EBP homologous protein (CHOP) is a very important marker participating in ERS-associated cell apoptosis, while the role of the myocyte apoptosis induced by CHOP remains unclear in the development of hypertrophy. The present study aimed to investigate the effect of CHOP-mediated ERS-associated apoptosis on myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and control group (n=40). The rats in model group received abdominal aortic constriction. Hemodynamic changes, whole heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/BW) were measured on 1 d, 3 d, 7 d, 14 d and 28 d after surgery, respectively. The mRNA expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT) and CHOP, which are important markers of ERS, were detected by RT-PCR, and Western blot was used to assess the protein level of GRP78, CRT, CHOP, and apoptosis-associated proteins, Bax and Bcl-2. The results obtained were as follows. Compared with control group, the blood pressure, LVW/BW, and HW/BW of rats in model group increased significantly and cardiac function enhanced compensatively on 7 d after surgery, and increased progressively during the experiment. As early as 1 d after surgery, the mRNA level of CRT in model group increased by 136% (P< 0.01) compared with control, while the protein expression increased by 69.2% on 7 d after surgery (P<0.01). Both mRNA and protein expression of GRP78 increased by 20% and 186% (P<0.01) respectively on 7 d after surgery, and the expression sustained high level during the experiment afterwards. Correlation analysis indicated a positive correlation between +dp/dt(max) and CRT protein expression (r=0.780, P<0.01) as well as GRP78 protein expression (r=0.694, P<0.01). Prolonged ERS triggered myocyte apoptosis, as both the mRNA and protein level of CHOP in model group increased by 22.2% (P<0.01) and 76.0% (P<0.01) respectively compared with control on 7 d after hypertrophy (14 d after surgery), and meanwhile, the protein expression of pro-apoptotic Bax increased by 41.1% (P<0.01) and anti-apoptotic Bcl-2 protein expression decreased by 25.5% (P<0.01). Correlation analysis indicated a positive correlation between CHOP and Bax expression (r=0.654, P<0.01), and a negative correlation between CHOP and Bcl-2 expression (r=-0.671, P<0.01). These results suggest that abdominal aortic constriction induces a significant up-regulation in ER molecular chaperones at early stage of post-surgery, indicating that ERS response is activated in the rat heart; while prolonged ERS could lead to myocyte apoptosis, and CHOP-mediated ERS-associated apoptosis may contribute to myocardial hypertrophy. We speculate that cell apoptosis may take part in the regulation of myocardial hypertrophy and heart failure, and determine the progression of decompensated hypertrophy.

[Regulation of rat airway smooth muscle cell proliferation by mitochondrial ATP-sensitive K(+) channel in asthmic rats.].

The objective of this paper was to investigate the effect and mechanism of mitochondrial ATP-sensitive K(+) (MitoK(ATP)) channel on the proliferation of airway smooth muscle cells (ASMCs) in asthmic rats. Thirty-six Sprague-Dawley (SD) rats were randomly assigned into 2 groups (18 in each): (1) Asthma group: the asthmic rat model was established by ovalbumin (OVA) sensitization and excitation; (2) Normal group: rats were subjected to inhalation of equal amount of normal saline. The rat ASMCs were isolated from fresh lung tissues and cultured respectively as follows: (1) CONTROL GROUP: normal ASMCs were cultured under normoxia for 24 h; (2) Diazoxide group: normal ASMCs were cultured under normoxia for 24 h with diazoxide (an opener of MitoK(ATP) channel); (3) 5-HD group: normal ASMCs were cultured under normoxia for 24 h with 5-hydroxydecanoate (5-HD) (an antagonist of MitoK(ATP) channel); (4) Asthma group: Asthmic ASMCs were cultured under normoxia for 24 h; (5) Asthma + diazoxide group: Asthmic ASMCs were cultured under normoxia with diazoxide for 24 h; (6) Asthma + 5-HD group: Asthmic ASMCs were cultured under normoxia with 5-HD for 24 h. The mitochondrial membrane potential (ΔΨm) was detected using Rhodamine 123 (R-123). The level of reactive oxygen species (ROS) was detected by DCF fluorescence. The expression of nuclear factor-kappa B (NF-κB) mRNA was examined by RT-PCR. The proliferation and apoptosis of rat ASMCs were examined respectively by MTT colorimetric assay and cell cycle analysis. The results were as follows. (1) After exposure to diazoxide for 24 h, the R-123 fluorescence intensity, the ROS level, NF-κB mRNA expression and the MTT absorbance value (A value) in normal ASMCs were significantly increased, and the apoptosis of rat ASMCs was significantly decreased compared to the control group (P<0.05). However, there was no significant changes in those indices after the normal ASMCs had been exposed to 5-HD for 24 h. (2) In Asthma and Asthma + diazoxide groups, the R-123 fluorescence intensity, ROS level and the MTT A value were markedly increased, and the apoptosis was markedly decreased compared to control group (P<0.05). These changes were more obvious in Asthma + diazoxide group than those in Asthma group (P<0.05). 5-HD partly weakened the effect of asthma on the R-123 fluorescence intensity, ROS level and the MTT A value and the apoptosis of rat ASMCs (P<0.05). R-123 fluorescence intensity and NF-κB mRNA expression were positively correlated with ROS level. NF-κB mRNA expression was positively correlated with the MTT A value and negatively correlated with the apoptosis of rat ASMCs. All the results suggest that the opening of MitoK(ATP) channel followed by a depolarization of ΔΨm contributes to the increase in ROS level and NF-κB mRNA expression in rat ASMCs and to the unbalance between cell proliferation and apoptosis of ASMCs induced by asthma. This might be a mechanism of the development of airway remodeling in asthma.

The phosphorylation of NS protein of wheat rosette stunt virus.

The genome of wheat rosette stunt virus (WRSV), a plant rhabdovirus, is a single negative strand RNA. It encodes five viral structural proteins: the glycoprotein (G), the matrix protein (M), the nucleocapsid protein (N), the large protein (L) and the non?structural protein (NS), which was later proved to be a viral structural protein too and existed in a variety of phosphorylation forms in case of vascular stomatitis virus (VSV). In this paper we demonstrated that NS protein of WRSV, either bound with the viral nucleocapsid or expressed in bacteria could be in vitro phosphorylated in presence of viral nucleocapsid. We concluded that the NS protein of WRSV was a phosphorylated protein and it might exist in both phosphorylated and dephosphorylated forms in virions. Our results excluded the possibility that the NS protein could be autophosphorylated. The L protein, the major component of viral RNA dependent RNA polymerase is associated with the protein kinase for phosphorylation of NS protein.

Sequence Analysis of the Trailer Region of Wheat Rosette Stunt Virus Genomic RNA.

According to a highly conserved 8 amino acid sequence of L proteins in Rhabdoviridae, a 24-mer degenerate oligonucleotide primer was designed to amplify the 5' terminus of the genomic RNA of wheat rosette stunt virus (WRSV), a plant rhabdovirus, by anchored PCR. The complete nucleotide sequence of the 5' trailer in WRSV genomic RNA was determined by deoxynucleotide sequencing of cloned cDNA derived from the anchored PCR products.

Cloning and Sequencing of the Gene Encoding NS Protein of Wheat Rosette Stunt Virus.

The cDNA fragment located downstream of the WRSV N protein gene was obtained from the cDNA library of WRSV by hybridization with a 24 nucleotides sequence fragment of the 3' end of mRNA of the N protein. The cDNA fragment was sequenced and on ORF encoding a 40 kD protein was found. The gene was expressed in E. coli DE3 and was identified by Western blot experiment as the NS gene of WRSV.

Cloning, Expression and Identification of Matrix Protein Gene of Wheat Rosette Stunt Virus (WRSV).

cDNA fragment located downstream of the WRSV NS protein gene was sequenced. Following an 80 nt untranslated region at the 5 'terminus, there is a 453 nt open reading frame(ORF) encoding a 17 kD protein, which was allowed to be expressed in E. coli using bacterial expression vector pGEX-3X and produced a 43 kD fusion protein. The result of Western blot showed that the fusion protein was able to react strongly with antibody raised against the purified WRSV particles. According to the similarities between the gene organizations of VSV and WRSV and between the molecular weights of deduced and expressed proteins, as well as the viral structural M protein, and to the result of Western blot, the 453 nt ORF was identified as WRSV M protein gene.