OBJECTIVETo study the expression characteristics and biological significance of nuclear factor NF-kappaB p50 and NF-kappaB p65 in osteoporosis development. Adult mice were ovariectomized as models of experimental osteoporosis. In this way, we can explore the molecular mechanism of osteoporosis and understand the significance during the morbility of osteoporosis.

METHODSFour-month female BALB/c mice were randomly divided into ovariectomized group and sham-operated group. 12 weeks after surgery, the mice were sacrificed after the measurement of bone mineral density (BMD). The NF-kappaB level in bone tissue were determined by immunohistochemistry and in situ hybridization techniques.

RESULTSCompared with the sham-operated group, bone density, rarefaction of trabecula and the number of osteoblast decreased in the ovariectomized group, while the number of osteoclast increased in the ovariectomized group. The immunohistochemistry showed that NF-kappaB expressed in both groups. It mainly localized in the cytoplasm of osteoblast, osteocytes and marrow stroma cell. The expression level in the ovariectomized group was higher than that in the sham-operated group and was negatively correlated with the BMD (P < 0.01 or P < 0.05). In situ hybridization demonstrated that the expression levels of NF-kappaB 50mRNA and NF-kappaB 65mRNA in ovariectomized group were significantly higher than that of the sham-operated group (P < 0.05).

CONCLUSIONThe expression level of NF-kappaB significantly increased in the bone tissue of ovariectomized mice, and its abnormal expression was significantly correlated with BMD.

Animals ; Bone Density ; Bone and Bones ; metabolism ; Disease Models, Animal ; Female ; Immunohistochemistry ; In Situ Hybridization ; Mice ; Mice, Inbred BALB C ; NF-kappa B p50 Subunit ; biosynthesis ; genetics ; Osteoporosis ; etiology ; metabolism ; physiopathology ; Ovariectomy ; RNA, Messenger ; genetics ; Transcription Factor RelA ; biosynthesis ; genetics

OBJECTIVETo explore the effect of fluvastatin on vascular endothelial growth factor (VEGF) in rats with osteoporosis in the process of fracture healing.

METHODSFractures at the intermediate piece of the femur were made on 72 Sprague Dawley (SD) rats (weighing initially 290-340 g and aged 6 months) with osteoporosis after ovariectomy for three months, then these rats were divided randomly into the medication administration group (the experimental group) and the control group, 36 rats each. In the experimental group, the rats received fluvastatin lavage (10 mg/kg per day) since the next day of operation lasting for 6 weeks, and the rats in the control group received placebo. Then the expression of VEGF and VEGF mRNA in bony callus of the two groups was measured respectively with immunohistochemistry and in situ hybridization on days of 3rd, 7th, 14th, 21st, 28th, and 42nd, and image analysis was made with real-color image analysis machine.

RESULTSNo difference was found in the cellular localization of VEGF and VEGF mRNA gene expression between the experimental group and the control group in process of fracture healing and their expression modes were almost similar. On the 14th day postoperatively, the positive extent of positive cells in the experimental group was higher than that of the control group (P < 0.05).

CONCLUSIONFluvastatin can promote the VEGF level in rats with osteoporosis in process of fracture healing.

Animals ; Fatty Acids, Monounsaturated ; pharmacology ; Fracture Healing ; Immunohistochemistry ; In Situ Hybridization ; Indoles ; pharmacology ; Osteoporosis ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; analysis ; genetics

Isomers are widely distributed in Chinese herbal medicines,and can be discriminated by energy-resolved mass spectrometry( ER-MS). However,ER-MS was performed through direct injection of reference compounds with syringe pump,which encountered a significant technical barrier for high-throughput and automated measurements. Herein,online ER-MS was conducted using LC-MS platform,and a pair of isomers,kaempferol vs luteolin,were employed as a case study to illustrate and assess the utility of online ER-MS for isomeric discrimination. High-resolution tandem mass spectrometry data of both flavonoids were acquired on LC-QE-Orbitrap-MS,and the fragmentation pathways responsible for the primary fragment ions were proposed. The primary signal in MS1 occurred at m/z 285( [M-H]-),and the primary signals of either compound generated by retro-Diels-Alder fragmentation were observed at m/z 151 and 133. The spectral information was subsequently transferred onto LC-Qtrap-MS platform to carry out online ER-MS. Two precursor-to-product ion transition candidates were constructed as m/z 285>151 and 285>133,and either afterward derived a set of pseudo-ion transitions( PITs) and so forth,exactly corresponding to a series of progressive collision energies( eg-5,-8,-11 e V,and so on). All PITs were typed into the monitoring list of multiple reaction monitoring program to generate the peak area datasets. Either dataset was normalized using the highest values in the set and imported into Graph Pad Prism software to plot the Gaus-sian-shaped curve that was termed as the break-down graph. The apex of the regressive curve was termed as optimal collision energy( OCE). The OCE values corresponding to m/z 285>151 were calculated as-29. 06 e V and-35. 71 e V for kaempferol and luteolin,respectively. In the case of m/z 285>133,the OCEs were yielded as-44. 15 e V for kaempferol and-49. 01 e V for luteolin. With re-ference to their chemical structures,the location of hydroxyl group was regarded to be responsible for the differences of either m/z 285>151 or 285>133 between the isomers,attributing to their different bond properties. Above all,online ER-MS offers an eligible tool for isomeric discrimination,and provides meaningful information for the accurate chemical composition characterization based on LC-MS,which is not limited to Chinese herbal medicines.
Chromatography, Liquid ; Flavonoids ; Kaempferols ; Luteolin ; Tandem Mass Spectrometry

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Ten fractions(A-J) were prepared by separation of Longxue Tongluo Capsules(LTC) by using silica gel column chromatography and orthogonal experimental design,showing similar chemical profiles with different abundances of peaks.These ten samples were assessed with UHPLC-QE OrbitrapHRMS for 97 common peaks.For the pharmacological activity experiment,three kinds of in vitro cell models including lipopolysaccharide(LPS)-induced BV-2 microglial cells NO release model,oxygen-glucose deprivation/reoxygenation(OGD/R)-treated HUVEC vascular endothelial cells injury model,and OGD/R-treated PC-12 nerve cells injury model were employed to evaluated the bioactivity of each fraction.Based on the contribution of each identified component,grey relation analysis and partial least squares(PLS) analysis were performed to establish component-activity relationship of LTC,identify the potential active components.After that,validation of the potential active components in LTC was carried out by using the same models.The results indicated that 4 phenolic compounds including 7,4'-dihydroxyhomoisoflavanone,loureirin C,4,4'-dihydroxy-2,6-dimethoxydihydrochalcone,and homoisosocotrin-4'-ol,might be the active components for anti-neuroinflammation effect;five phenolic compounds such as 3,5,7,4'-tetrahydroxyhomoisoflavanone,loureirin D,7,4'-dihydroxyhomoisoflavane,and 5,7-dihydroxy-4'-methoxy-8-methyflavane,might have positive effects on the vascular endothelial injury;three phenolic compounds including 5,7,4'-trihydroxyflavanone,7,4'-dihydroxy-5-methoxyhomoisoflavane,and loureirin D,might be the active components in LTC against neuronal injury.
Brain Ischemia ; drug therapy ; Capsules ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Microglia ; drug effects ; Oxygen