Isopsoralen accelerates tibia fracture healing by promoting bone formation in mice / 陆军军医大学学报

Objective To determine the effect of isopsoralen(ISO)on the healing of tibia fracture in mice and explore its underlying mechanism.Methods Fifty male C57BL/6 mice(2 month old,20±2 g)were randomly divided into model group and ISO treatment group,with 25 animals in each group.From the 3rd day after modeling,the mice from the ISO group were given an intragastric gavage of 40 mg/kg ISO,once per day for 28 consecutive days,while those of the model group was given same volume of normal saline in same way.On the 7th,14th,21st,and 28th day after gavage,the tibia on the surgical side was taken,and the fracture area was quantified by bone volume/total volume(BV/TV)after micro-CT scanning.The healing and shaping of the fracture end were observed through HE staining.ELISA was used to detect the serum contents of bone alkaline phosphatase(BALP)and procollagen type I N-terminal peptide(PINP)on the 14th day of gavage.Western blotting was employed to determine the expression levels of Collagen Ⅰ,Runx2,BMP2,OSX,and VEGF in the tibial callus tissue in 7 and 14 d after gavage.Vascular perfusion was applied to observe the callus microvessels in 28 d to quantitatively analyze the vascular volume fraction and vessel diameter.Immunohistochemical staining was conducted to observe the expression of VEGF in the callus in 14 d after gavage.Results HE staining displayed that the ISO group had faster healing process than the model group.Micro-CT quantification results showed that the ISO group had higher BV/TV ratio in 7 d after gavage though no statistical difference,significantly higher ratio in 14 d(P＜0.05),but obviously lower ratio in 21 and 28 d after gavage(both P＜0.05)when compared with the model group.The serum contents of BALP and PINP were also remarkably higher in the ISO group than the model group(P＜0.05).Western blotting results indicated that the expression levels of Collagen Ⅰ,Runx2,BMP2,OSX and VEGF in the ISO group were higher than those in the model group(P＜0.05).The results of angiography revealed that the vascular volume fraction and vessel diameter were notably increased in the ISO group than the model group(both P＜0.05).Immunohistochemical assay showed that the expression of VEGF was higher in the ISO group than the model group(P＜0.05).Conclusion ISO can improve the activity of osteoblasts,increase the expression of osteogenesis-related proteins,and accelerate the angiogenesis to promote fracture healing.

Effects of sinusoidal electromagnetic fields at different exposure times on osteoblastic activity and the underlying signal transduction mechanisms / 中华老年医学杂志

Objective To investigate the signal transduction mechanisms of time-dependent effects of 50 Hz 1.8 mT sinusoidal electromagnetic fields(SEMFs)on osteoblastic activity.Methods Newborn rat calvarial osteoblasts(ROBs) were treated with 50 Hz 1.8mT SEMFs for 30,60,90,120,and 150 min,respectively.Intracellular alkaline phosphatase (ALP) activity was assayed,and protein expression of Runt-related transcription factor 2 (Runx2),Smad1/5/8 and mitogen-activated protein kinases(MAPKs)were examined by Western blot.The Dual-Luciferase Reporter Assay System was used to measure the activity of the Wnt pathway.Immunofluorescence staining and laser confocal microscopy were applied to examine the nuclear translocation of Smad1/5/8 and β-catenin.Changes in ALP activity were determined after inhibiting p38 MAPK using a specific inhibitor.Results ALP activity of ROBs increased after 30,60,90,120 and 150 min of SEMFs treatment,compared with the control group(51.41±5.21,59.47±4.02,67.56[4.68,63.69±3.92,and 58.16±3.61 vs.45.53± 3.24),and reached the highest value at 90 min and then started to decline.Protein expression of Runx-2,p-Smad1/5/8,p-p38 and β-catenin increased after SEMFs treatment,and reached the highest value at 90 min and then gradually returned to baseline levels.The values for Wnt pathway activity for the control group and with SEMFs treatment at 30,60,90,120 and 150 min were 0.49± 0.06,0.52 ± 0.09,0.75±0.05,0.77 ± 0.42,0.58 ± 0.08 and 0.42 ± 0.09,respectively.Wnt pathway activity,the nuclear translocation of β-catenin and Smad1/5/8 reached the highest level at 90 min.Values of ALP activity in the control group and the SEMFs group were 44.60±3.84 and 71.54±7.34,respectively,before specifically inhibiting p38 MAPK,and were 52.08±0.83 and 52.15±10.77,respectively,after p38 MAPK inhibition,indicating that ALP activity could not be increased with inhibition.Conclusions 50 Hz 1.8 mT SEMFs increase osteoblastic activity by activating the BMP-2/Smad1/5/8,Wnt/β-catenin and p38 MAPK signal pathways.The optimal duration of treatment is 90 min per day.

Effects of sinusoidal electromagnetic fields at different exposure times on osteoblastic activity and the underlying signal transduction mechanisms / 中华老年医学杂志

Objective@#To investigate the signal transduction mechanisms of time-dependent effects of 50 Hz 1.8 mT sinusoidal electromagnetic fields(SEMFs)on osteoblastic activity.@*Methods@#Newborn rat calvarial osteoblasts(ROBs)were treated with 50 Hz 1.8mT SEMFs for 30, 60, 90, 120, and 150 min, respectively.Intracellular alkaline phosphatase(ALP)activity was assayed, and protein expression of Runt-related transcription factor 2(Runx2), Smad1/5/8 and mitogen-activated protein kinases(MAPKs)were examined by Western blot.The Dual-Luciferase Reporter Assay System was used to measure the activity of the Wnt pathway.Immunofluorescence staining and laser confocal microscopy were applied to examine the nuclear translocation of Smad1/5/8 and β-catenin.Changes in ALP activity were determined after inhibiting p38 MAPK using a specific inhibitor.@*Results@#ALP activity of ROBs increased after 30, 60, 90, 120 and 150 min of SEMFs treatment, compared with the control group(51.41±5.21, 59.47±4.02, 67.56±4.68, 63.69±3.92, and 58.16±3.61 vs.45.53±3.24), and reached the highest value at 90 min and then started to decline.Protein expression of Runx-2, p-Smad1/5/8, p-p38 and β-catenin increased after SEMFs treatment, and reached the highest value at 90 min and then gradually returned to baseline levels.The values for Wnt pathway activity for the control group and with SEMFs treatment at 30, 60, 90, 120 and 150 min were 0.49±0.06, 0.52±0.09, 0.75±0.05, 0.77±0.42, 0.58±0.08 and 0.42±0.09, respectively.Wnt pathway activity, the nuclear translocation of β-catenin and Smad1/5/8 reached the highest level at 90 min.Values of ALP activity in the control group and the SEMFs group were 44.60±3.84 and 71.54±7.34, respectively, before specifically inhibiting p38 MAPK, and were 52.08±0.83 and 52.15±10.77, respectively, after p38 MAPK inhibition, indicating that ALP activity could not be increased with inhibition.@*Conclusions@#50 Hz 1.8 mT SEMFs increase osteoblastic activity by activating the BMP-2/Smad1/5/8, Wnt/β-catenin and p38 MAPK signal pathways.The optimal duration of treatment is 90 min per day.

Effect of low frequency low intensity electromagnetic fields on maturation and mineralization of rat skull osteoblasts in vitro / 浙江大学学报·医学版

Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P P PP0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all PP0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all PPPPConclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.

Icaritin promotes maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signal pathway / 浙江大学学报·医学版

Objective: To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. Methods: The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. Results: Real time RT-PCR showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related genes in icaritin group were significantly increased (PPCXCR4 gene was decreased (PPPPPConclusion: Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.