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Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

ABSTRACT: Pinostrobin (PI), a natural flavonoid found in a variety of plants, is well known for its rich pharmacological activities. However, its osteogenic function remains unclear. The aim of this study is to evaluate the effect of PI on the proliferation, differentiation, and mineralization of murine pre-osteoblastic MC3T3-E1 cells in vitro using MTT, alkaline phosphatase (ALP) activity, the synthesis of collagen I (Col I) assay, and Von-Kossa staining, respectively. The expression of osteocalcin (OCN) mRNA in cells was detected by real-time PCR. The effect of PI on the differentiation of dexamethasone (DEX)-suppressed cells was also investigated. The results showed that PI greatly promoted the proliferation of MC3T3-E1 cells at 5-80 ?g/mL (p < 0.05 or p < 0.01), and caused a significant elevation of ALP activity, Col I content, and mineralization of osteoblasts at 10-40 ?g/mL (p < 0.05 or p < 0.01), and the expression levels of OCN gene were greatly upregulated after PI treatment (p < 0.01). Furthermore, PI could rescue the inhibition effect of cell differentiation induced by DEX. Taken together, these results indicated that PI could directly promote proliferation, differentiation, and mineralization of MC3T3-E1 cells and has potential for use as a natural treatment for osteoporosis.

SUBMITTER: Gu C

PROVIDER: S-EPMC6151515 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

Gu Chengbo C Fu Linan L Yuan Xiaohan X Liu Zhiguo Z

Molecules (Basel, Switzerland) 20171016 10

Pinostrobin (PI), a natural flavonoid found in a variety of plants, is well known for its rich pharmacological activities. However, its osteogenic function remains unclear. The aim of this study is to evaluate the effect of PI on the proliferation, differentiation, and mineralization of murine pre-osteoblastic MC3T3-E1 cells in vitro using MTT, alkaline phosphatase (ALP) activity, the synthesis of collagen I (Col I) assay, and Von-Kossa staining, respectively. The expression of osteocalcin (OCN) ...[more]

PMID: 29035339

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Project description:BackgroundIn mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact.Methods(1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention.Results(1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group's alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05).ConclusionThe present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.

Project description:Eucommia leaves are dry leaves of Eucommia ulmoides which have long been considered as a functional health food for the treatment of hypertension, hypercholesterolemia, fatty liver, and osteoporosis. With the recent development of Chinese medicine, Eucommia leaves are widely used for tonifying the kidneys and strengthening bone. However, the specific molecular mechanism of Eucommia leaves for strengthening bone remains largely unknown. Osteoblasts are the main functional cells of bone formation; thus, it is essential to study the effect of Eucommia leaves on osteoblasts to better understand their mechanism of action. In the present study, we prepared an aqueous extract of Eucommia leaves (ELAE) and determined its content by high-performance liquid chromatography (HPLC). The effects of ELAE on MC3T3-E1 cells were investigated by CCK-8 assay, alkaline phosphatase (ALP), and Alizarin red S staining assays, combined with RNA sequencing (RNA-seq) and qRT-PCR validation. We demonstrated that ELAE had a significant promoting effect on the proliferation of MC3T3-E1 cells and significantly enhanced extracellular matrix synthesis and mineralization, which were achieved by regulating various functional genes and related signaling pathways. ELAE significantly increased the expression level of genes promoting cell proliferation, such as Rpl10a, Adnp, Pex1, Inpp4a, Frat2, and Pcdhga1, and reduced the expression level of genes inhibiting cell proliferation, such as Npm1, Eif3e, Cbx3, Psmc6, Fgf7, Fxr1, Ddx3x, Mbnl1, and Cdc27. In addition, ELAE increased the expression level of gene markers in osteoblasts, such as Col5a2, Ubap2l, Dkk3, Foxm1, Col16a1, Col12a1, Usp7, Col4a6, Runx2, Sox4, and Bmp4. Taken together, our results suggest that ELAE could promote osteoblast proliferation, differentiation, and mineralization and prevent osteoblast apoptosis. These findings not only increase our understanding of ELAE on the regulation of bone development but also provide a possible strategy to further study the prevention and treatment of osteogenic related diseases by ELAE.

Dataset Information

Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

Publications

Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

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