Effect of sex combing protein 1 on proliferation and differentiation of osteoblasts in inflammatory microenvironment / 中国组织工程研究

BACKGROUND: Studies have shown that the loss of sex combing protein 1 (Asxl1) can lead to the occurrence of bone dysplasia and bone defects, but the relationship between this factor and bone destruction in the microenvironment of apical periodontitis has not been reported. OBJECTIVE: To study the effect of Asxl1 on proliferation and differentiation of osteoblasts in an inflammatory microenvironment. METHODS: MC3T3-E1 cells were excited by lipopolysaccharide to establish an in vitro inflammatory microenvironment. The best concentration and optimal action time of lipopolysaccharide were screened by cell counting kit-8 test. MC3T3-E1 cells were then stimulated with 20 mg/L lipopolysaccharide for 24 hours. The expression levels of Asxl1 protein and mRNA were detected by immunofluorescence and real-time PCR, respectively. After lipopolysaccharide stimulated the formation of inflammatory microenvironment, Asxl1-Si RNA was transfected for 24 hours, cell counting kit-8 was applied to detect the activity of cell proliferation, and real-time PCR was used to detect the expression levels of Asxl1 and osteogenic related genes ALP and RUNX2 mRNA. RESULTS AND CONCLUSION: After lipopolysaccharides stimulation of MC3T3-E1 cells, the expression levels of Asxl1 protein and mRNA were decreased. Under the inflammatory microenvironment, the proliferation activity of MC3T3-E1 cells transfected with Asxl1-Si RNA for 24 hours was significantly decreased, and the expression levels of Asxl1, ALP and RUNX2 mRNA were markedly decreased. These findings indicate that Asxl1 may influence the proliferation and differentiation of osteoblasts by involvement in the process of inflammatory reaction, thereby participating in bone destruction.

Effect of microrna-132-3p overexpression onproliferation and osteogenic differentiation of mc3t3-e1 cells / 中国组织工程研究

BACKGROUND: Mechanical stress can influence the proliferation and differentiation of MC3T3-E1 cells and trigger differential expression of miR-132-3p. However, further research is warranted concerning whether tensile stress can influence the proliferation and differentiation of osteoblasts by regulating miR-132-3p. OBJECTIVE: To determine the expression of osteogenic differentiation markers and miR-132-3p in MC3T3-E1 cells under 12% cyclic stretch and to explore the effect of miR-132-3p on cell proliferation and differentiation. METHODS: MC3T3-E1 cells were loaded with 0% and 12% tensile stress, and alkaline phosphatase activity, osteocalcin mRNA and miR-132-3p expression levels were detected. MC3T3-E1 cells were transiently transfected with miR-132-3p mimics and a negative control transfection group was set up. The expression of alkaline phosphatase, osteocalcin and Runx2 mRNA in transfected cells were detected by qRT-PCR, and the effect of miR-132-3p on cell proliferation were detected by cell counting kit-8 assay. RESULTS AND CONCLUSION: The alkaline phosphatase activity and osteocalcin mRNA expression were down-regulated in MC3T3-E1 cells under 12% stretch stress (P < 0.01), and the expression of miR-132-3p was significantly increased (P < 0.05). QRT-PCR results showed the expression levels of osteogenic differentiation markers alkaline phosphatase activity, osteocalcin, and Runx2 mRNA in miR-132-3p mimics group were significantly decreased after intracellular transfection of miR-132-3p (P < 0.05). Compared with the negative control transfection group, the cell proliferation in the miR-132-3p mimic group was decreased at 24, 48, and 72 hours after transfection (P < 0.001), and the most obvious reduction was observed after 48-hour transfection. These findings indicate that 12% cyclic tensile stress can negatively regulate the proliferation and differentiation ability of MC3T3-E1 cells by overexpressing miR-132-3p.

Promoting effect of osteopractic total flavone combined with nano-bone materials on proliferationand differentiation of MC3T3-E1 cells / 中国组织工程研究

BACKGROUND: Preliminary study has found that osteopractic total flavone can promote osteogenic differentiation of MC3T3-E1 cells on the surface of nano-bone material, but the underlying mechanism needs to be studied in depth. OBJECTIVE: To investigate the actin mechanism of osteopractic total flavone combined with nano-bone material on MC3T3-E1 cells. METHODS: MC3T3-E1 cells were co-cultured with nano-bone material, and 100 mg/L and 250 mg/L osteopractic total flavone were treated as drug intervention, including 10 μg/L transforming growth factor-β as positive control. The samples were divided into eight groups: (1) Normal group; (2) DKK1 group: Wnt pathway inhibitor DKK1 (0.1 mg/L) blocks Wnt/β-catenin signaling pathway; (3) DKK1+transforming growth factor-β group; (4) DKK1+100 mg/L osteopractic total flavone group; (5) DKK1+250 mg/L osteopractic total flavone group; (6) DKK1+ nano-hydroxyapatite/collagen+transforming growth factor-β group; (7) DKK1+nano-hydroxyapatite/collagen+100 mg/L osteopractic total flavone group; (8) DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group. Cells in each group were harvested after 24 and 48 hours of intervention. Immunofluorescence labeling was used to observe the binding of Wnt and LRP in osteoblasts in the Wnt/β-catenin pathway. The expression of β-catenin, LRP 5, GSK-3β, Cyclin D1, and RUNX2 was detected by real-time polymerase chain reaction and western blot assay. RESULTS AND CONCLUSION: (1) Confocal laser scanning microscope showed that obvious brown and yellow staining was shown in the DKK1+transforming growth factor-β group, DKK1+250 mg/L osteopractic total flavone group, DKK1+nano-hydroxyapatite/ collagen+transforming growth factor-β group, and DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group, indicating that Wnt and LRP combined better than other groups. (2) Real-time polymerase chain reaction and western blot assay results showed that osteopractic total flavone could promote the expression of β-catenin, LRP5 and RUNX2, and downregulated GSK3β expression. These findings confirm that osteopractic total flavone can promote the differentiation and proliferation of osteoblasts by activating the Wnt/β-catenin signaling pathway. Gene activation induced by osteopractic total flavone was dose-dependent.

Beta-ecdysterone promotes in vitro proliferation and osteogenic differentiation of MC3T3-E1 cells / 中国组织工程研究

BACKGROUND: β-ecdysterone as a “phytoestrogen” has the ability to stimulate protein synthesis, promote carbohydrate and lipid metabolism, relieve hyperglycemia and hyperlipidemia, protect endothelial cells from apoptosis and induce their proliferation. Some scholars have reported that it also plays an important role in the treatment of osteoporosis, fractures and other bone inflammatory diseases. OBJECTIVE: To observe the effect of β-ecdystrone on the proliferation of mouse pre-osteoblasts(MC3T3-E1 cells) in vitro, and to explore whether β-ecdysterone can induce osteogenic differentiation of MC3T3-E1 at a safe dose. METHODS: The fourth generation MC3T3-E1 cells were cultured in the osteogenic induction medium for 7, 10, 14, 21, and 28 days. The osteogenic differentiation proteins(alkaline phosphatase, type I collagen, osteopontin, and calcified nodules) were detected at different time points, to identify whether the cells have the ability of osteogenic differentiation. MC3T3-E1 cells were then seeded into the induction medium containing different final concentrations of β-ecdysterone(0.01, 0.1, 1, 10, 100 µmol/L). The proliferation activity of the cells was detected by cell counting kit-8 method at days 1, 2, 3, 4, 5, 6, and 7 after induction. The control group(general induction medium group) and the experimental group(general induction medium + β-ecdysterone) were cultured under the same conditions, and the expression levels of osteogenic marker proteins in each group of cells at different time periods were determined. RESULTS AND CONCLUSION: In the MC3T3-E1 cells stimulated by the osteogenic induction medium, alkaline phosphatase staining and type I collagen florescence staining showed higher expression at day 10 of induction, and this was also confirmed by detection of alkaline phosphatase activity(P 0.05). The expression of alkaline phosphatase and type I collagen was higher in the experimental group than in the control group at day 10 of induction. The expression of osteopontin and osteocalcin in the cells was higher at day 14 of induction, and there was no significant difference in the calcified nodule staining between the two groups at day 28 of induction. These findings indicate that β-ecdysterone can promote the proliferation of MC3T3-E1 cells in vitro and induce MC3T3-E1 cells to differentiate into osteoblasts at a safe dose.

Interaction between necroptosis and apoptosis in MC3T3-E1 cell death induced by dexamethasone / 南方医科大学学报

OBJECTIVE@#To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids.@*METHODS@#MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting.@*RESULTS@#At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells.@*CONCLUSIONS@#In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.

Effect of risperidone on proliferation and apoptosis of MC3T3-E1 cells

This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.

(2R,3S)-Pinobanksin-3-cinnamate promotes osteoblast differentiation through cAMP and cGMP pathways

Abstract Flavones have the potential of being used as a dietary supplement for bone health promotion beyond calcium and vitamin D. Recent studies have showed that flavones enhanced bone formation and inhibited bone resorption by affecting osteoblast and osteoclast differentiation through various cell signaling pathways. In this study, we investigated the effects of a new flavone (2R,3S)-pinobanksin-3-cinnamate, isolated from the metabolites of the endophytic fungus Penicillium sp. FJ-1 of Acanthus ilicifolius L., Acanthaceae, on osteoblast differentiation by using MC3T3-E1 cells. It was observed that (2R,3S)-pinobanksin-3-cinnamate promoted osteoblast differentiation, as evidenced by increased mineralization process and alkaline phosphatase activity, as well as expression of genes encoding the bone differentiation. Moreover (2R,3S)-pinobanksin-3-cinnamate treatment upregulated the gene expression of wingless-type MMTV integration site family, bone morphogenetic protein and runt-related transcription factor 2, and protein expression of phosphor-Smad1/5/8, β-catenin and runt-related transcription factor 2 in MC3T3-E1 cells. The osteoblast differentiation effects induced by (2R,3S)-pinobanksin-3-cinnamate were attenuated by the bone morphogenetic protein antagonist Noggin, and wingless-type MMTV integration site family signaling pathway inhibitors Dickkopf-1. Co-treatment with adenosine 30,50-cyclic monophosphate and guanosine 30,50-cyclic monophosphate pathway inhibitors, H89 and KT5823, respectively, reversed the (2R,3S)-pinobanksin-3-cinnamate-induced activations of p-Smad1/5/8, β-catenin, and runt-related transcription factor 2. Our data demonstrated that (2R,3S)-pinobanksin-3-cinnamate promoted the osteoblast differentiation of MC3T3-E1 cells, at least partially through the adenosine 30,50-cyclic monophosphate and guanosine 30,50-cyclic monophosphate signaling pathways, providing the scientific rational to develop (2R,3S)-pinobanksin-3-cinnamate against bone loss-associated diseases.

Hydroxyapatite Nanorod-Modified Sand Blasted Titanium Disk for Endosseous Dental Implant Applications

BACKGROUND: Sand blasted titanium (Ti) is commonly used in designing endosseous dental implants due to its biocompatibility and ability to form bonds with bone tissues. However, titanium implants do not induce strong interactions with teeth bones. To increase strong interactions between Ti disk implants and teeth bones, the L-glutamic acid grafted hydroxyapatite nanorods (nHA) were immobilized on albumin modified Ti disk implants (Ti-Alb). METHODS: For modification of Ti disk implants by nHA, the L-glutamic acid grafted nHA was synthesized and then immobilized on albumin modified Ti disk implants. Fourier transformed infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscope; energy dispersive spectroscopy and confocal laser scanning microscopy were used to confirm the modification of Ti disk implants. The bioactivity of nHA-modified Ti disk implants was evaluated by seeding MC3T3-E1 cells on Ti-nHA implants. RESULTS: Characterization techniques have confirmed the successful modification of Ti disk implants by L-glutamic acid grafted nHA. The nHA-modified Ti disk implants have shown enhanced adhesion, proliferation and cytotoxicity of MC3T3-E1 cells in comparison to pristine Ti implants. CONCLUSION: The modification of Ti implants by L-glutamic acid grafted nHA has produced highly osteogenic Ti disk plants in comparison to pristine Ti disk implants due to the formation of bioactive surfaces by hydroxyapatite nano rods on Ti disk implants. Ti-nHA disk implants showed enhanced adhesion, proliferation, and MC3T3-E1 cells viability in comparison to pristine Ti disk implants. Thus nHA might be to be useful to enhance the osseointegration of Ti implants with teeth bones.

Effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells / 中国修复重建外科杂志

Methods: The MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.

Effect of miR-21 down-regulated by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells / 中国修复重建外科杂志

Objective : To explore the effect and mechanism of miR-21 down-regulated which was induced by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells.

Estrogen-induced and Expression Repressed via Estrogen Receptor Beta in MC3T3-E1 Cells / 中华医学杂志(英文版)

<p><b>Background</b>Estrogen, as an important hormone in human physiological process, is closely related to bone metabolism. The aim of this study was to investigate the mechanism of estrogen on osteoblasts metabolism in MC3T3-E1 cells.</p><p><b>Methods</b>We treated the MC3T3-E1 cells with different concentrations of β-estradiol (0.01, 0.1, 1, and 10 nmol/L), observed the morphological changes of the cells, and detected the cell's proliferation and apoptosis of MC3T3-E1 cells. Two transcriptome libraries were constructed and sequenced. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to confirm the differentially expressed genes (DEGs), and then treated the MC3T3-E1 cells with estrogen receptor (ER) inhibitors α and β, respectively, and then examined the expression of Tgfbr1 and Bmpr1a genes. The promoter of Tgfbr1 and Bmpr1a gene was analyzed, and the ER response elements were identified. Finally, ChIP was used to verify the binding of ER to Tgfbr1 and Bmpr1a promoter.</p><p><b>Results</b>In the high-concentration β-estradiol treatment group (1 nmol/L and 10 nmol/L), there was no significant difference in the morphology of the cells under the microscope, 1 nmol/L and 10 nmol/L treated group appeared statistically significant difference in cell apoptosis and proliferation (P < 0.05 and P < 0.01, respectively). We found 460 DEGs compared with the control group. Among the DEGs, there were 66 upregulated genes and 394 downregulated genes. Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that many bone metabolism-related biological processes and cell signaling pathways were disordered. The qRT-PCR verification showed that the expressions of Tgfbr1- and Bmpr1a-related genes in bone metabolism pathway in the 10 nmol/L treatment group were significantly decreased (P < 0.05). ER β was involved in the inhibitory effect of Tgfbr1 and Bmpr1a genes. The bioinformatics of the promoter found that there were three ER response elements in the promoter of Tgfbr1, and there were two ER response elements in Bmpr1a promoter regions. ChIP experiments showed that estrogen could enhance the binding of ERs to Tgfbr1 and Bmpr1a genes.</p><p><b>Conclusions</b>Estrogen can promote the apoptosis and proliferation of osteoblasts simultaneously, and the mechanism may be the joint action of transforming growth factor-beta, Wnt, mitogen-activated protein kinase, and nuclear factor-kappaB bone metabolism-related signaling pathway. Estrogen inhibits the expression of Tgfbr1 and Bmpr1a genes through ER β and affects the metabolism of MC3T3-E1 osteoblasts.</p>

Glycyrrhiza uralensis (licorice) extracts increase cell proliferation and bone marker enzyme alkaline phosphatase activity in osteoblastic MC3T3-E1 cells / 한국영양학회지

PURPOSE: The Glycyrrhiza uralensis species (Leguminosae) as a medicinal biocompound, and one of its root components, isoliquritigenin (ISL), which is a flavonoid, has been reported to have anti-tumor activity in vitro and in vivo. However, its function in bone formation has not been studied yet. In this study, we tested the effect of Glycyrrhiza uralensis (ErLR) and baked Glycyrrhiza uralensis (EdLR) extracts on osteoblast proliferation, alkaline phosphatase (ALP) activity, and bone-related gene expression in osteoblastic MC3T3-E1 cells. METHODS: MC3T3-E1 cells were cultured in various levels of ErLR (0, 5, 10, 15, 20 µg/mL), EdLR (0, 5, 10, 15, 20 µg/mL), or ISL (0, 5, 10, 15, 20 µM) in time sequences (1, 5, and 20 days). Also, isoliquritigenin (ISL) was tested for comparison to those two biocompound extracts. RESULTS: MTT assay results showed that all three compounds (ErLR, EdLR, and ISL) increased osteoblastic-cell proliferation in a concentration-dependent manner for one day. In addition, both ErLR and EdLR compounds elevated the osteoblast proliferation for 5 or 20 days. Extracellular ALP activity was also increased as ErLR, EdLR, and ISL concentration increased at 20 days, which implies the positive effect of Glycyrrhiza species on osteoblast mineralization. The bone-related marker mRNAs were upregulated in the ErLR-treated osteoblastic MC3T3-E1 cells for 20 days. Bone-specific transcription factor Runx2 gene expression was also elevated in the ErLR- and EdLR-treated osteoblastic MC3T3-E1 cells for 20 days. CONCLUSION: These results demonstrated that Glycyrrhiza uralensis extracts may be useful for preventing osteoporosis by increasing cell proliferation, ALP activity, and bone-marker gene expression in osteoblastic cells.

Zinc upregulates bone-specific transcription factor Runx2 expression via BMP-2 signaling and Smad-1 phosphorylation in osteoblasts / 한국영양학회지

PURPOSE: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. METHODS: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn− (1 µM Zn) or Zn+ (15 µM Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. RESULTS: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn−. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn−. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn−, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn−, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn−. CONCLUSION: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

Neuropeptide Y stimulates osteoblastic differentiation of murine MC3T3-E1 cells related to activated Wnt signaling in vitro / 中华创伤骨科杂志

Objective To investigate the effect of neuropeptide Y (NPY) on the osteoblastic differentiation of murine MC3T3-E1 cells and its mechanism related to the Wnt signaling pathway.Methods The murine MC3T3-E1 cells were divided into 4 groups according to the stimulators added:phosphate buffered saline (PBS) (control) and different concentrations of NPY (10-8 mol/L,10-10 mol/L and 10-12 mol/L).The cellular proliferation was detected with MTT assay after 1,3,5,7 and 9 days.The cells were identified with cell immunochemistry and Western Blot to find out the most effective concentration of NPY at different time points under osteoblastic condition.The cells were then divided into 4 groups:PBS,NPY,NPY + NPY receptor antagonist,and NPY + DKK1.Western blot was used to determine the expression of β3-catenin and p-GSK-3β in each group.Nuclear signaling activity of β3-catenin was observed using immunofluorescence staining.Results NPY significantly improved the proliferation of MC3T3-E1 cells at 7 and 9 days (P ＜0.05).NPY (10s mol/L and 10-10 mol/L) groups and NPY (10-10 mol/L and 10-12 mol/L) groups significantly improved the ALP activity at 4 and 14 days respectively (P ＜ 0.05).At 4 days,the expression of ALP protein was significantly decreased in the NPY + DKK1 group and the NPY + NPY receptor antagonist group compared with that in the NPY group (P ＜ 0.05).Although the expression levels of [β-catenin and p-GSK-3β protein were uninfluenced in either case,NPY significantly stimulated the nuclear signaling activity of β3-catenin.Conclusions NPY may significantly increase the expression of ALP protein in MC3T3-E1 ceils during osteoblastic differentiation.This effect might be mediated through the canonical Wnt signaling pathway.

Icaritin, a Flavonoid Derived from the Herb Epimedium, Promotes Osteogenic Differentiation of MC3T3-E1 Cells

Osteoporosis is a metabolic bone disease that is characterized by low bone mass resulting from an increase in bone resorption relative to bone formation. The most current therapies for osteoporosis have focused on inhibiting bone resorption by osteoclasts. The purpose of this study is to develop new anabolic agents for treatment of osteoporosis that have fewer risks compared to conventional therapies. We searched the natural products that were derived from the traditional Asian medicines which have been used for treatment of bone related diseases. Icaritin is a flavonoid glycoside derived from the herb Epimedium which has beneficial effects on bone formation. To determine the effect of icaritin on bone formation, we examined the effect of icaritin on MC3T3-E1 cell proliferation and differentiation. For determining the effects of icaritin on proliferation, we performed the MTT assay using MC3T3-E1 cells. To evaluate whether icaritin could promote the osteogenic differentiation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity and mRNA expressions of Runx2, osteocalcin (OCN), RANKL, and osteoprotegerin (OPG) were determined. Icaritin increased MC3T3-E1 cell proliferation. Icaritin increased the ALP activity of MC3T3-E1 cells on 72 hour culture in osteogenic media. mRNA expression of Runx2 was increased after 24 hour culture with icaritin. mRNA expression of osteocalcin was increased after 72 hour culture with icaritin. In addition, icaritin increased the mRNA expressions of OPG and RANKL. However, icaritin increased the mRNA expression of OPG much more than that of RANKL, and then, it increased the OPG/RANKL ratio. These results suggest that icaritin promotes osteogenic differentiation of osteoblasts and decreases osteoclast formation regulated by osteoblasts.

Effect of puerarin on proliferation, differentiation andmineralization of MC3T3-E1 cells and expression of TRPM3 mRNA / 中国药理学通报

Aim To investigate the effect of puerarin on proliferation, differentiation, and mineralization of MC3T3-E1 cells and the expression of TRPM3 mRNA.Methods Proliferation, differentiation, and mineralization of puerarin in MC3T3-E1 cells were determined using CCK-8 assay, alkaline phosphatase(ALP) activity assay, and Alizarin Red Staining, respectively.Effect of puerarin on cell cycle and intracellular calcium concentration of MC3T3-E1 cells was detected by flow cytometry.RT-PCR was used to detect the effect of puerarin on TRPM3 mRNA expression.Resuls 0.1, 1, 10 μmol·L-1 puerarin significantly promoted the proliferation of MC3T3-E1 cells, reduced the proportion of cells in G1 phase, increased the proportion of G2 and S phase, of which 0.1 μmol·L-1 concentration effect was the most significant.Compared with control group, the ALP activity and mineralized nodule area of 0.1 μmol·L-1 puerarin group were significantly increased.The expression of TRPM3 mRNA and the intracellular calcium concentration were significantly decreased in 0.1 μmol·L-1 puerarin group.Conclusion Puerarin can promote the proliferation, differentiation, and mineralization of MC3T3-E1 cells, and reduce the expression level of TRPM3 mRNA and intracellular calcium concentration.

EFFECT OF ACTIVED RAW264.7 INDUCED BY H2O2 ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1 / 中国修复重建外科杂志

OBJECTIVE: To explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice. METHODS: MC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I). RESULTS: The results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P<0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P<0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P<0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). CONCLUSIONS: The appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.

Osteogenesis differentiation of MC3T3-E1 cells induced by miRNA-2861 mimic transfection mediated by polyethylenimine / 吉林大学学报(医学版)

Objective:To transfect the non-viral vector polyethylenimine (PEI)mediated miR-2861 mimic into the MC3T3-E1 cell line,and to explore the transfection efficiency of PEI/miR-2861 complex and its effects on the proliferation and osteogenesis differentiation in pre-osteoblasts. Methods:The proper amount of PEI was blended with miR-2861 mimic and negative control (NC)separately in a ratio of N∶P=10∶1,and they were divided into experiment group and NC group. The NC/PEI complex acted as the NC group was used to eliminate the interference of osteogenesis from the addition of double-stranded RNA mimic.MTT assay was used to determine the optimal concentration of PEI/miR-2861 mimic complex.The fluorescence imaging technique and bulge-loop RT-PCR were used to detect the transfection efficiency and mRNA expression of miRNA-2861 in the cells with different concentrations (10,30, 50,and 100 nmol · L-1 ), separately.The osteogenesis ability of MC3T3 cells was identified with RT-PCR and Alizarin red staining with the selected concentration of PEI/miR-2861 by transient transfection.Results:Compared with blank control group,the proliferation rates of MC3T3 cells in 100 nmol·L-1 PEI/miR-2861 group was decreased significantly at 72 h (P < 0. 05 ). With the increasing of transfected concentration the transfection efficiency of miRNA/PEI complex was increased gradually.The results of Alizarin red staining and quantitative analysis showed that calcium deposits were more and bigger in experiment group after induced for 21 d,while both in blank control group and NC group they were less.Conclusion:The miRNA-2861 mimic can be effectively transfected into the MC3T3-E1 cell line and expresses with a high level,which is mediated by PEI as the gene vector.miR-2861 mimic has a certain ability of promoting osteogenesis differentiation of MC3T3-E1 cells.

Effect of 1, 25-dihydroxy vitamin D3 combined with mechanical strain treatment on proliferation, differentiation, and expression of OPG and RANKL in osteoblastic MC3T3-E1 cells / 医用生物力学

Objective To investigate the effect of 1,25-(OH)2-vitamin D3 (VD3) or mechanical strain alone and their combined treatment on proliferation and differentiation of pre-osteoblast MC3T3-E1 cells in vitro, as well as gene and protein expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-кB ligand (RANKL) in those cells. Methods MC3T3-E1 cells were treated with 10 nmol/L VD3, intermitted mechanical strain or with a combination of these two factors. Cell proliferation was assessed with flow cytometry, and alkaline phosphatase (ALP) activity was measured using a fluorometric detection kit. The mRNA expression of ALP, runt-related transcriptional factor 2 (Runx2), OPG, and RANKL genes was determined by real-time PCR. The proteins expression of Runx2, OPG, and RANKL was determined by Western blotting. ResultsVD3 inhibited the proliferation of MC3T3-E1 cells, but the mechanical strain had no effect on cell proliferation. Mechanical strain, VD3, and the combined treatment enhanced the ALP activity of MC3T3-E1 cells as well as the protein expression of Runx2. The effect of combined treatment was less pronounced than the effect of VD3 or mechanical strain alone. Mechanical strain promoted the gene and protein expression of osteoprotegerin (OPG) and increased the ratio of OPG/RANKL. However, the combination of VD3 and mechanical strain led to a decrease in ratio of OPG/RANKL. Conclusions Mechanical strain might be effective in inducing osteogenic differentiation and increasing bone formation. A joint stimulation with VD3 and strain can decrease proliferation and osteogenic differentiation and increase RANKL expression, which might affect bone remodeling. This study supplies some new data, which might be important in theoretical and clinical research of osteoporosis (OP) and other related bone diseases.

Effect of gliclazide on the proliferation and differentiation in mouse osteoblastic-like cells MC3T3-E1 / 中华内分泌代谢杂志

[Summary] To investigate the effect of gliclazide on the proliferation and differentiation in MC3T3-E1 cells exposed to normal glucose concentration by applying CCK-8 and RT-PCR. Gliclazide improved the proliferation and stimulated COL-I, OPN and Runx2 mRNA expression in MC3T3-E1 cells, the best concentration of gliclazide was 20μmol/ L, the expression of COL-1 and OPN mRNA had a significant positive correlation with Runx2 binding activity.