| Objective:To study the toxicity of nicotine on mouse embryonic osteoblast precursor cells（MC3T3-E1 cell line）and its effect on osteoblast differentiation,and to explore the effect of nicotine on osteoblast differentiation and BMP-2/Smad1 signaling pathway,in order to understand the mechanism of nicotine affecting osteoblast differentiation.Methods:1.Differentiation of MC3T3-E1 cells:MC3T3-E1 cells were induced to differentiate into osteoblasts,which were identified by alkaline phosphatase（ALP）and alizarin red（ARS）.2.CCK-8 was used to determine the proliferation activity of the cells.3.RNA was extracted from MC3T3-E1 cells cultured for 7 days under the action of different concentrations of nicotine by an osteogenic induction solution,RT-PCR was used to detect the gene expression of Runt-related transcription factor 2（Runx2）,osteocalcin（OCN）,alkaline phosphatase（ALP）,osteoblast-specific transcription factor（Osx）and bone morphogenetic protein-2（BMP-2）;Alizarin red staining was used to detect the effect of nicotine on the formation of calcium nodules.4.Effects of nicotine on osteoblast differentiation via BMP-2/Smad1 signaling pathway.Four groups of control experiments were designed:（1）MC3T3-E1 cells（without nicotine medium）（2）MC3T3-E1 cells+10-5 mol/L nicotine medium（3）MC3T3-E1 cells+10-4 mol/L nicotine medium（4）MC3T3-El cells+10-3 mol/L nicotine medium Results:1.CCK-8 experiment showed that nicotine could inhibit the proliferation of MC3T3-E1 cells in a time-and concentration-dependent manner.2.The results of ALP staining showed that MC3T3-E1 cells had osteogenic effect after 1 day of induction,and the staining effect was more significant with the increase of time.3.Alizarin red staining results showed that within a certain concentration range,the higher the concentration of nicotine,the stronger the inhibitory effect on the formation of calcium nodules in osteoblasts.4.MC3T3-E1 cells were co-cultured with osteogenic medium containing nicotine(10-5 mol/L,10-4 mol/L,10-3 mol/L)and MC3T3-E1 cells for 7 days,respectively,and the plate without nicotine containing osteogenic medium was used as control.The expression of osteoblast related genes Runx2,Osterix,OCN,BMP-2and ALP were determined at day 7.The results showed that the expressions of Runx2,Osterix,BMP-2 and ALP were inhibited in nicotine group in a dose-dependent manner;Compared with the control group,the expression of OCN gene in nicotine group was inhibited,but there was no significant difference between the nicotine groups of 10-4 mol/L and 10-3 mol/L.The results of this experiment indicated that nicotine has an inhibitory effect on the differentiation of osteoblasts.5.MC3T3-E1 cells were co-cultured for 14 days with the osteogenic induction solution containing nicotine at the concentrations of 10-5 mol/L,10-4 mol/L and 10-3mol/L,respectively,and the orifice plate containing the osteogenic induction solution of MC3T3-E1 cells without nicotine was used as a control to extract the total protein of the cells.The BMP-2,Runx2 and Smad1 proteins related to BMP-2/Smad1signaling pathway were detected by Western blotting.The results showed that the inhibition of BMP-2 and Runx2 protein expression increased with the increase of nicotine concentration,and the phosphorylation expression of Smad1 protein was inversely proportional to nicotine concentration,that is,the higher the nicotine concentration was,the lower the protein expression was.The results confirmed that different concentrations of nicotine may regulate the differentiation of osteoblasts through BMP-2/Smad1 signaling pathway.Conclusion:Nicotine can inhibit the differentiation of osteoblasts,and nicotine may regulate the differentiation of osteoblasts through BMP-2/Smad1 signaling pathway. |
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