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Osteoprotegerin (OPG), secreted by osteoblasts, is a marker of bone turnover. OPG can inhibit osteoclastic differentiation by binding receptor activator of nuclear factor-κB ligand (RANKL). In this study, we found that rutaecarpine (RUT) had the up-regulating OPG activity, and it could significantly increase OPG protein levels in both mouse embryonic osteogenic precursor MC3T3-E1 and human osteosarcoma U-2OS cells. Osteoblastogenic differentiation calcified nodules staining results showed that RUT significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Osteoclastic differentiation tartrate resistant acid phosphatase (TRAP) staining results showed that RUT obviously inhibited the osteoclast differentiation of mouse macrophages RAW264.7 induced by RANKL. In vivo studies showed that low-dose RUT group (5 mg·kg-1·day-1) and high-dose RUT group (45 mg·kg-1·day-1) treatments for 3 months significantly increased bone density in ovariectomized (OVX) rats; calcein double labeling experiment and toluidine blue staining results indicated that low-dose RUT group promoted bone formation and decreased bone loss in vivo; immunohistochemistry results showed that low-dose RUT group increased the expression of OPG in rat femur. All animal procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. In summary, this study demonstrated that RUT could up-regulate OPG expression and had promoting osteoblastic differentiation and inhibiting osteoclastic differentiation effects in vitro and in vivo.
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The purpose of this research is to study the anti-atherosclerotic effects and mechanisms of berberine (BBR) in high fat diet (HFD) fed ApoE-/- mice, and then to lay a solid foundation of the clinical studies of BBR treatment. The hyperlipidemic ApoE-/- mice model was established by feeding HFD for 12 weeks. Mice were randomly divided into control group (chow diet), model group, BBR group (BBR-L: 50 mg·kg-1, BBR-H: 150 mg·kg-1) and atorvastatin (5 mg·kg-1) group. Mice were intragastric administration with BBR in 0.5% sodium salt of caboxy methyl cellulose. After 12 weeks, enface aortas were stained with oil red O, and the lesions area were analyzed by Image J software. The inflammatory factor levels were detected by suspension microarray kits. Liver total cholesterol (TC), triglyceride (TG) and free fatty acids (FFA) were determined by commercial kits. Western blot was performed to examine the inflammatory pathway related and cholesterol and lipid transport related proteins' expression. All animal experiments were performed in accordance with the Regulation on the Administration of Laboratory Animals of Institute of Medicinal Biotechnology. After 12 weeks treatment, compared with model group, BBR treatment significantly reduced the lesions area of en face aortas and obviously inhibited serum proinflammatory factors such as IL-1β and IL-6 compared with model group. In addition, BBR treatment obviously reduced liver TC, TG and FFA levels compared with model group. Furthermore, mechanic study showed that BBR significantly inhibited MAPKs and NF-κB pathways, and increased cholesterol and lipid regulated proteins expression such as p-AMPK, LDLR, ABCA1 and SR-BI. In conclusion, BBR can obviously reduce enface aortas lesions in ApoE-/- mice, which is related to inhibit inflammation and liver cholesterol and lipid accumulation.
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The aim of this paper was to study the curative effect of Huotan Jiedu Tongluo (HTJDTL) decoction on a rabbit model with early atherosclerosis (AS),and furtherly to explore whether it could inhibit the BH4/eNOS uncoupling ROS or not. Twenty-four Japanese white rabbits were randomly divided into sham operation group, model group, HTJDTL decoction group and atorvastatin group. Rabbit models with early atherosclerosis were established by high fat diet, nitrogen drying and carotid artery balloon injury. The rabbits were sacrificed at 7th days after balloon injury and several parameters were measured. The pathological morphology of the common carotid artery was observed by HE staining. The blood lipids were detected by peroxidase method. The ratio of vascular eNOS dimer and monomer was measured by Western blot. The ELISA and biochemical technology were respectively used for testing BH4 and ROS levels in serum. The results showed that compared with the sham operation group, the model group had mild stenosis of the common carotid artery lumen, uneven intimal hyperplasia, lipid deposition in the intima and media, and obvious hyperplasia of the adventitia with inflammatory cell infiltration. The HTJDTL decoction could significantly inhibit the intimal hyperplasia compared with the model group, meanwhile, reduce the lipid deposition of the media and the infiltration of the adventitial cells. Compared with the sham operation group, the blood lipids and ROS of the model animals significantly increased, but BH4 and the ratio of eNOS dimer/monomer decreased. Compared with the model group, HTJDTL decoction significantly reduced the TC, ox-LDL and ROS levels, and also up-regulated eNOS dimer/monomer ratio, but it increased BH4 trend without statistical difference. According to the results, it was found that HTJDTL decoction couldsignificantly prevent and improve the vascular remodeling of rabbits model with early atherosclerosis. The mechanism of decoction may largely be related to the inhibition of BH4/eNOS uncoupling and the reduction of oxidative stress.
Тема - темы
Animals , Rabbits , Atherosclerosis , Drug Therapy , Carotid Arteries , Pathology , Drugs, Chinese Herbal , Pharmacology , Nitric Oxide Synthase Type III , Metabolism , Oxidative Stress , Random Allocation , Signal TransductionРеферат
Bone morphogenetic protein 2 (BMP2) plays a key role in bone development and reestablishment. In the study, we screened up-regulators of BMP2 among 20 000 compounds through a cell-based high throughput screening model and a positive compound E40071[2-(4-(5-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-7-yl) piperazin-1-yl)ethan-1-ol] was found as the positive hit. The EC50 value of E40071 was 2.73 μmol·L-1. In vitro, E40071 upregulated the mRNA levels of BMP2 and the downstream transcription factors, Runx2 and Osx in MC3T3-E1 (subclone 14). Protein expression of Runx2 was up-regulated by E40071 through induction of Smad1/5/8 phosphorylation. The alkaline phosphatase (ALP) activity was increased by E40071. Moreover, E40071 promoted the mineralization of MC3T3-E1 (subclone 14) by Alizarin red S staining. In addition, E40071 markedly inhibited osteoclast differentiation of mice macrophage Raw264.7 induced by RANKL and reduced the expression of osteoclast differentiation markers, including MMP9 and NFATc1. The results suggest that E40071 is able to promote bone formation activity of osteoblasts and inhibit differentiation of osteoclasts.
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This study was designed to identify inducers of ATP-binding cassette transporter A1(ABCA1) and CD36 and lysosomal integral membrane protein-II analogous-1(CLA-1) and to evaluate the in vitro effect of the active compound on lipid metabolism. Among 20000 compounds screened, E23869 was found as a positive hit using cell-based high throughput screening models. The up-regulating activities of E23869 in ABCA1p-LUC and CLA-1p-LUC HepG2 cells were 196% and 198%, respectively. The EC50 values of E23869 in ABCA1p-LUC and CLA-1p-LUC HepG2 cells were 0.25 μmol·L-1 and 0.66 μmol·L-1, respectively. E23869 significantly upregulated the protein levels of ABCA1, scavenger receptor class B type I (SR-BI)/CLA-1 and ATP-binding cassette transporter G1(ABCG1) in both macrophages RAW264.7 and L02 cells by Western blotting analysis. Foam cell assay showed that E23869 inhibited lipids accumulations in macrophages RAW264.7. Cholesterol efflux assay showed that E23869 induced HDL-mediated cholesterol efflux in macrophages RAW264.7. Moreover, E23869 up-regulated ABCA1, SR-BI/CLA-1 and ABCG1 expressions through activation of PPARα and PPARγ. In addition, E23869 weakly promoted in vitro differentiation of mouse preadipocytes 3T3-L1. In conclusion, E23869 up-regulated ABCA1, SR-BI/CLA-1 and ABCG1 expressions to promote cholesterol efflux, which is a good leading compound for regulation of lipid metabolism.