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Objective To investigate the effects and its mechanism of isoflavones extracted from chickpea sprouts (ICS) on the proliferation and the differentiation of the osteogenic precursor MC3T3-E1 cells. Methods CCK-8 was used to detect the proliferation of MC3T3-E1 cells at different concentrations of ICS (0, 0. 1, 0. 5, 1, 5, 10, 50, 100 mg/L) at 24, 48, 72 h. Different concentrations of ICS (0, 0.5, 1195 mg/L) were added into the osteogenic induction medium to observe its effects on the osteogenic differentiation: Alkaline phosphatase staining (ALP) assay was performed to observe the enzyme activities after 7-day induction; Alizarin red staining was performed on the 21st day to observe the calcium deposition; RT-qPCR was used to detect the mRNA expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and type 1 collagen (COL1) in each group on the 7th day of induction; Western Blot was used to detect the expression levels of RUNX2 and COL1 on the 7th day of induction, and estrogen receptor (ER) inhibitor ICI 182, 780 (1 μmol/L) was also added to detect the effect on the protein expression of the osteogenic differentiation-related proteins RUNX2 and COL1. Results ICS promoted cell proliferation in a certain concentration range (1-10 mg/L), and had some toxicity to cells when the concentration was greater than 10 mg/L (P< 0. 05). Compared with the control group (0 mg/L), ICS at concentrations of 0. 5,1,5 mg/L could promote alkaline phosphatase activity, with significant differences in a dose-dependent manner; ICS could promote calcium deposition, with a significant increase in the number of calcium nodules with increasing dose, with statistical differences; ICS could up-regulate the mRNA transcription levels of osteogenic differentiation-related genes RUNX2, OCN and COLT (P<0. 05), and promote the expression of osteogenic differentiation-related proteins COL1 and RUNX2 (P<0. 05). The estrogen receptor inhibitor ICI 182 780 inhibited the promoting effect of ICS on osteogenic differentiation-related proteins COL1 and RUNX2 (P < 0. 05). Conclusion ICS can promote the proliferation of MC3T3-E1 osteogenic precursor cells in mice, and improve the osteogenic differentiation of MC3T3-E1 cells by up-regulating the expression levels of osteogenic differentiation-related genes and proteins. The use of estrogen receptor inhibitor ICI 182,780 can inhibit the osteogenic differentiation of ICS. It is speculated that ICS promotes osteogenic differentiation through ER-related pathway. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of microRNA (miR) - 196a on osteogenic differentiation of MC3T3-El cells by target regulation of his tone deacetylase 9 (HDAC9). Methods MC3T3-El cells were divided into control group (Cont) group, induction group, miR-196a-mimics-NC group, miR-196a-mimics group, miR-196a-inhibitor-NC group, miR-196a-inhibitor group, miR-196a-mimics+pCMV-HDAC9-NC group, and miR-196a-mimics+pCMV-HDAC9 group. Osteogenic induction was performed after transfection according to the group. Quantitative fluorescent PCR was performed to measure the expression of miR-196a and HDAC9 in MC3T3-El cells. Alkaline phosphatase (ALP) activity was measured with a commercial kit. Alizarin red staining was performed to observe the degree of mineralization. Western blotting was performed to measure the expressions of HDAC9, ALP, Runt-related transcription factor 2 (RUNX2), collagen I (COLl), osteopontin (OPN), Histone H3, and Histone H3 (acetyl K9, K 14 and K23). Results Compared to those in the Cont group, the expression of miR-196a, the expressions of ALP, Runx2, COLl, and OPN proteins, ALP activity, mineralization degree, and the acetylation levels of Histone H3 K9, K14, and K23 sites in MC3T3-E I cells in the induction group increased (P < 0. 05), but the expression of HDAC9 mRNA and protein decreased (P < 0. 05). Transfection of miR-196a-mimics was able to significantly increase miR-196a expression, to decrease HDAC9 expression, and to increase ALP, Runx2, COL 1, and OPN protein expression, ALP activity, mineralization degree, and Histone H3 acetylation. Transfection of miR-196a-inhibitor had the opposite effect. MiR-196a was able to target down-regulation of HDAC9 expression. Overexpression of HDAC9 was able to partially reverse the promoting effect of miR-196a mimics on osteogenic differentiation of MC3T3-E I cells. Conclusion MiR-196a target down-regulates HDAC9 expression, increases hi stone acetylation level, and promotes osteogenic differentiation of MC3T3-El cells. [ABSTRACT FROM AUTHOR]

Objective To explore the effect and mechanism of miR-381-3p on the osteogenic differentiation of MC3T3-E1 cells. Methods The lentivirus-transfected cells were divided into NC mimic miR-381-3p group, mimic miR-381-3p group, and NC inhibitor miR-381-3p group and inhibitor miR-381-3p group. Real-time fluorescent quantitative PCR (qRT-PCR) method was used to detect the expression levels of miR-381-3p in different groups to verify the transfection efficiency. After inducing osteogenic differentiation of MC3T3-E1 cells, the levels of alkaline phosphatase (ALP) activity in the cells of each group were detected. Alizarin red staining was used to detect the osteogenic mineralization ability of each group of cells. qRT-PCR was used to determine the expression levels of ALP, Runx2, PPARγ, and ETS1 mRNA in each group of cells. Western blotting was used to determine the protein levels of collagen Ⅰ, ALP, Runx2, PPARγ, ETS1, and P-ERK1/2 in each group of cells. MAPK/ERK pathway inhibitor (PD98059) was used to intervene in each group of cells to detect the target protein. Results Compared to those in NC mimic miR-381-3p group, mimic miR-381-3p group, and NC inhibitor miR-381-3p group, the levels of ALP, Runx2, and ETS1 mRNA in the cells of inhibitor miR-381-3p group increased significantly. The expression levels of collagen Ⅰ, ALP, Runx2, ETS1, and P-ERK1/2 protein also significantly increased, but the expression levels of PPARγ mRNA and protein were down-regulated. Compared to those in the other three groups, ALP activity and calcification ability of the cells in the inhibitor miR-381-3p group showed significantly high expression. The protein experiment results after the inhibition of the MAPK pathway also confirmed that miR-381-3p participated in the regulation of the osteogenic differentiation process of MC3T3-E1 cells. Conclusion miR-381-3p inhibits the osteogenic differentiation ability of MC3T3-E1 cells by down-regulating the expression level of ERK1/2/ETS1 signaling pathway. [ABSTRACT FROM AUTHOR]

Objective The aim of this study was to explore the protective effect and related mechanism of tetrahydroxystilbene glucoside (TSG) on apoptosis of osteoblast precursor cells (MC3 T3-E1) induced by H2O2. Methods MC3 T3-E1 cells pretreated with different concentrations of TSG were treated with 300 μmol/L H2O2 for 24 h. The experiment consisted of 6 groups, including the control group, H2O2, H2O2+TSG (0.1 μmol/L), H2O2+TSG (1 μmol/L), H2O2+TSG (10 μmol/L) and H2O2+NAC (1 mmol/L)(anti-oxidant positive control group). We evaluated the protective effect of TSG on the apoptosis of MC3 T3-E1 cells by MTT assay and Hoechst33258 staining. We tested the production of ROS and MDA by fluorescence microplate reader and MDA assay kit to investigate the oxidative stress status. We tested the production of Bcl-2 and Bax by Western-blotting and the gene expression of Bcl-2 and Bax by RT-PCR. Results Cell death induced by H2O2 alone was observed under microscope, and Hoechst33258 staining showed more cells with nuclear pyknosis and nuclear enrichment, which was improved after being pretreated with different concentrations of TSG. H2O2 alone could lead to a large degree of apoptosis in MC3 T3-E1 cells, and the apoptosis rate decreased after pretreatment with different concentrations of TSG. At the same time, TSG(1-10 μmol/L) could significantly reduce the level of ROS and MDA (P<0.05) and improve the oxidative stress status in MC3 T3-E1 cells. The result of Western-blotting and real-time quantitative RT-PCR demonstrated that pretreatment with TSG could decrease the expression of pro-apoptosis protein Bax and increase the expression of anti-apoptosis protein Bcl-2. Conclusion The apoptosis of MC3 T3-E1 cells increased after induced by H2O2 and decreased by pretreatment with TSG. The mechanism of the protective effect could be through decreasing the expression of pro-apoptosis protein Bax and increasing the expression of anti-apoptosis protein Bcl-2. [ABSTRACT FROM AUTHOR]

Objective To observe the correlation between endogenous lipid accumulation and apoptosis of MC3T3-E1 cells affected by Betulin and dexamethasone in vitro. Methods MC3T3-E1 cells were used as the research object. The cells were treated by Betulin and dexamethasone alone or by the combination of both. The lipid synthesis in the cells was determined by triglyceride content examination and Nilered staining. The osteoblast′s apoptosis rate was detected by flow cytometry; and Caspase-3 enzyme activity by ELISA. Spearman rank correlation was used to analyze the correlation between lipid content and cell apoptosis. Results The cell apoptosis rate, the activity of Caspase-3 and the lipid content in the cells were significantly increased when affected by 0.5 uM/L and 1 uM/L dexamethasone (p<0.05); but Betulin could significantly decreased the apoptosis rate and the activity of Caspase-3, as well as the lipid accumulation in the cells. Spearman rank correlation analysis indicated that the lipid content was positively correlated with the rate of apoptosis of osteoblasts, p<0.05, r=0.412. Conclusion Dexamethasone can obviously promotes the apoptosis of osteoblast cells and lipid accumulation, which were positively correlated; Betulin can significantly reduce the bad effect of dexamethasone suggested that hormone induced apoptosis at least in part correlated with the intracellular lipid accumulation. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of trifolium pretense-derived total isoflavones containing serum on the proliferation,differentiation,and mineralization of MC3T3-E1 osteoblastic cells in vitro. Methods Rats were intragastrically administered with the t. pretense-derived total isoflavones to prepare drug containing serum. The proliferation of MC3T3-E1 osteoblastic cells was determined using MTT method. The activity of alkaline phosphatase and osteocalcin were determined using ELISA. The mineralized nodules were observed using Alizarin red staining method. Results Different concentrations of total isoflavones containing serum significantly promoted the proliferation and increased the activity of alkaline phosphatase of MC3T3-E1 osteoblastic cells. The best concentration of the serum was 5%. 5% of total isoflavones containing serum significantly promoted the secretion of osteocalcin in MC3T3-E1 osteoblastic cells at 6th day and 9th day, and promoted the formation of mineralized nodules in MC3T3-E1 osteoblastic cells at 6th day,12th, and 18th day. Conclusion The t. pratense-derived total isoflavones containing serum significantly promotes the proliferation, the activity of alkaline phosphatase, the secretion of osteocalcin, and the mineralization of MC3T3-E1 osteoblastic cells, indicating that it can significantly promote the maturation of MC3T3-E1 osteoblastic cells. The total isoflavones has the potential to develop a new medicine for osteoporosis. [ABSTRACT FROM AUTHOR]