RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border.However,the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown.Here,we show with GeneChip analysis that RUN and FYVE domain-containing protein 4(RUFY4)is strongly upregulated during osteoclastogenesis.Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo.Furthermore,deleting Rufy4 did not affect osteoclast differentiation,but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes,their trafficking to the membrane,and their secretion of cathepsin K into the extracellular space.Mechanistically,RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes.Consequently,Rufy4-deficient mice were highly protected from lipopolysaccharide-and ovariectomy-induced bone loss.Thus,RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Catalpa bignonioides extract improves exercise performance through regulation of growth and metabolism in skeletal muscles

Objective:To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo.Methods:Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell proliferation was assessed using a 5-bromo-2’-deoxyuridine(BrdU)assay kit.Western blot analysis was performed to determine the protein expressions of related factors.The effects of Catalpa bignonioides extract were investigated in mice using the treadmill exhaustion test and whole-limb grip strength assay.Chemical composition analysis was performed using high-performance liquid chromatography(HPLC).Results:Catalpa bignonioides extract increased the proliferation of C2C12 mouse myoblasts by activating the Akt/mTOR signaling pathway.It also induced metabolic changes,increasing the number of mitochondria and glucose metabolism by phosphorylating adenosine monophosphate-activated protein kinase.In an in vivo study,the extract-treated mice showed improved motor abilities,such as muscular endurance and grip strength.Additionally,HPLC analysis showed that vanillic acid may be the main component of the Catalpa bignonioides extract that enhanced muscle strength.Conclusions:Catalpa bignonioides improves exercise performance through regulation of growth and metabolism in skeletal muscles,suggesting its potential as an effective natural agent for improving muscular strength.

Anti-senescence and anti-wrinkle activities of 3-bromo-4,5-dihydroxybenzaldehyde from Polysiphonia morrowii Harvey in human dermal fibroblasts

Objective:To investigate the anti-senescence effect of 3-bromo-4,5-dihydroxybenzaldehyde(BDB)from Polysiphonia morrowii Harvey in human dermal fibroblasts(HDF).Methods:HDF were subjected to treatment of BDB and then treated with hydrogen peroxide(H2O2)to induce premature senescence.Senescence-associatedβ-galactosidase(SA-β-gal)activity in HDF was determined using the SA-β-gal staining method.Intracellular reactive oxygen species(ROS)production was measured using the 2’,7’-dichlorodihydrofluorescein diacetate assay.Western blotting assay was performed to assess the level of antioxidant enzyme glutathione peroxidase 1(GPX1).In addition,intracellular collagen and collagenase contents were analyzed using the respective ELISA kits.Elastase activity in HDF supernatants was measured from p-nitroaniline release and normalized using total protein content.Results:Treatment of HDF with H2O2 increased the activity of SA-β-gal,but BDB pre-treatment resulted in the reduction of SA-β-gal activity.Moreover,BDB significantly reduced H2O2-induced intracellular ROS production.BDB also markedly increased the level of GPX1,which was inhibited by 400μM of H2O2.Furthermore,in in vitro study,BDB significantly increased intracellular collagen content and decreased matrix metalloproteinase-1 and elastase activities in HDF.Conclusions:Our results demonstrate that BDB shows antisenescence and anti-wrinkle activities in vitro.

Dieckol isolated from Eisenia bicyclis extract suppresses RANKL-induced osteoclastogenesis in murine RAW 264.7 cells

Objective:To demonstrate the effect of dieckol from Eisenia bicyclis on osteoclastogenesis using RAW 264.7 cells.Methods:Murine macrophage RAW 264.7 cells were subjected to dieckol treatment,followed by treatment with receptor activator of nuclear factor kappa-B ligand(RANKL)to induce osteoclastogenesis.Tartrate-resistant acid phosphatase(TRAP)activity was examined using a TRAP activity kit.Western blotting analysis was conducted to examine the level of osteoclast-related factors,including TRAP and calcitonin receptor(CTR),transcriptional factors,including c-Fos,c-Jun,and nuclear factor of activated T cells cytoplasmic 1(NFATc1),nuclear factor kappa-B(NF-κB),extracellular signal-regulated kinase(ERK),and c-Jun N-terminal kinase(JNK).Immunofluorescence staining was conducted to examine the expression of c-Fos,c-Jun,and NFATc1.Results:Among the four phlorotannin compounds present in Eisenia bicyclis,dieckol significantly hindered osteoclast differentiation and expression of RANKL-induced TRAP and CTR.In addition,dieckol downregulated the expression levels of c-Fos,c-Jun,NFATc1,ERK,and JNK,and suppressed NF-κB signaling.Conclusions:Dieckol can suppress RANKL-induced osteoclastogenesis.Therefore,it has therapeutic potential in treating osteoclastogenesis-associated diseases.

Neuroprotection via maintenance or increase of antioxidants and neurotrophic factors in ischemic gerbil hippocampus treated with tanshinone I

Background Danshen （Radix Salvia miltiorrhizae） has been used as a traditional medicine in Asia for treatment of various microcirculatory disturbance related diseases. Tanshinones are mainly hydrophobic active components, which have been isolated from Danshen and show various biological functions. In this study, we observed the neuroprotective effect of tanshinone I （Tsl） against ischemic damage in the gerbil hippocampal CA1 region （CA1） after transient cerebral ischemia and examined its neuroprotective mechanism. Methods The gerbils were divided into vehicle-treated-sham-group, vehicle-treated-ischemia-group, Tsl-treated-sham- group, and Tsl-treated-ischemia-group. Tsl was administrated intraperitoneally three times （once a day for three days） before ischemia-reperfusion. The neuroprotective affect of Tsl was examined using H＆E staining, neuronal nuclei （NeuN） immunohistochemistry and Fluoro-Jade B staining. To investigate the neuroprotective mechanism of Tsl after ischemia- reperfusion, immunohistochemical （IHC） and Western blotting analyses for Cu, Zn-superoxide dismutase （SOD1）, Mn- superoxide dismutase （SOD2）, brain-derived neurotrophic factor （BDNF） and insulin-like growth factor-I （IGF-I） were performed.Results Treatment with Tsl protected pyramidal neurons from ischemia-induced neuronal death in the CA1 after ischemia-reperfusion. In addition, treatment with Tsl maintained the levels of SOD1 and SOD2 as determined by IHC and Western blotting in the CA1 after ischemia- reperfusion compared with the vehicle-ischemia-group. In addition, treatment with Tsl increased the levels of BDNF and IGF-I determined by IHC and Westem blotting in the Tsl-treated-sham-group compared with the vehicle-treated- sham-group, and their levels were maintained in the stratum pyramidale of the ischemic CA1 in the Tsl-treated- ischemia-group. Conclusion Treatment with Tsl protects pyramidal neurons of the CA1 from ischemic damage induced by transient cerebral ischemia via the maintenance of antioxidants and the increase of neurotrophic factors.

Protective Effect of Brassica napus L.Hydrosols against Inflammation Response in RAW 264.7 Cells

Objective:To demonstrate the anti-inflammatory activity of Brassica napus L.hydrosols(BNH)in lipopolysaccharide(LPS)-stimulated RAW 264.7 cells.Methods:Composition analysis of BNH was conducted via gas chromatography-mass spectrometry after BNH were extracted.The nitric oxide(NO)production was measured using the Griess assay.Prostaglandin E2(PGE2)production was evaluated with enzyme-linked immunosorbent assay.The effects of BNH on LPS-induced pro-inflammatory enzymes including inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)were evaluated using Western blot analysis.Furthermore,phosphorylation of nuclear factor-kappa B(N F-k B)and nuclear translocation of N F-k B p65 were evaluated with Western blot analysis and immunofluorescence staining,respectively.Results:Compared with LPS-stimulated cells,BNH markedly decreased the generation of NO and PGE_(2)in LPS-stimulated RAW 264.7 cells(P<0.01 or P<0.05).Moreover,BNH inhibited protein levels of iNOS and COX-2(P<0.01).Phosphorylation of NF-k B and nuclear translocation of NF-k B p65 was significantly inhibited by BNH(P<0.01 or P<0.05).Conclusion:The anti-inflammatory activities of BNH were mediated via blockage of the N F-k B signaling pathways in LPS-stimulated RAW 264.7 cells.