| Background:The osteoblast precursor 3T3-E1 cells are derived from the cell lines of newborn mouse skulls and long dry bones,and visible mineralized nodules can be observed after culture,among which MC3T3 subclones 4 and 14 can form well mineralized extracellular matrix,which can prove that 3T3-E1 cells have the ability to induce and differentiate into bone cells in vitro and are a classic model for studying the differentiation of osteoblasts in vitro.At present,3T3-E1 cells are widely used as experimental objects to explore the mechanism of osteoblast differentiation.There have been studies on the effects of hypoglycemic drugs,anti-oxidative stress drugs and traditional Chinese medicine on 3T3-E1 cells,but few studies have been conducted on lipid-regulating drugs and estrogen receptor modulators.Objective:To observe the effects of atorvastatin calcium and raloxifene on the proliferation and differentiation of osteoblast precursor cells(3T3-E1),and to compare the effects of atorvastatin calcium and raloxifene on the proliferation and differentiation of 3T3-E1 cells.Methods:1.the morphology and growth state of 3T3-E1 were observed under microscope.2.3T3-E1 cells were cultured in logarithmic phase.The control group(without atorvastatin calcium)was set up.The experimental group was added with different concentrations of atorvastatin calcium(10-8mol/L,10-7mol/L,10-6mol/L,10-5mol/L)to continue to culture cells.24 hours later,the osteoblast precursor cells 3T3-E1 were observed by CCK-8 colorimetry.3T3-E1 osteoblast precursor cells were cultured to logarithmic growth stage and then set up control group.The experimental group was treated with different concentrations of raloxifene(10 9 mol/L,10-8 mol/L,10-7 mol/L,10-6 mol/L),and cultured 3T3-E1 osteoblast cells were detected by CCK8 kit 24 hours later.The proliferation of bone precursor cells 3T3-E1.3.3T3-E1 cells were cultured in logarithmic phase.The control group(without atorvastatin calcium)was set up.The experimental group was added with different concentrations of atorvastatin calcium(10-8mol/L,10-7mol/L,10-6mol/L,10-5mol/L)to continue to culture cells.Alkaline phosphatase(ALP)activity was measured by alkaline phosphatase assay kit 24 hours later.The 3T3-E1 osteoblast precursor cells cultured to logarithmic phase were divided into 2 groups,and ALP activity was measured by alkaline phosphatase assay kit 24 hours later to understand the differentiation of 3T3-E1 osteoblast precursor cells.4.Added 10-6mol/L atorvastatin calcium to 3T3-E1 cells,which had the most obvious effect on cell proliferation and differentiation.After 24 hours of intervention,total RNA was extracted from cell samples by Trizol method.Quantitative Real-time PCR was used to detect the expression of PERK,Bip and eIF2alpha,which are the markers of endoplasmic reticulum stress signaling pathway.At the same time,3T3-E1 osteoblast precursor cells cultured to logarithmic growth phase were added with 10-7mol/L raloxifene,which had the strongest effect on cell proliferation and differentiation.At the same time,the blank control group was set up.RNA was extracted 24 hours later.The expression of PERK,Bip and eIF2alpha in cells was detected by qRT-PCR.5.3T3-E1 cells were cultured in logarithmic phase,and the control group was set up.In the experimental group,10-6mol/L atorvastatin calcium and 10-7mol/L raloxifene,which had the strongest proliferation and differentiation ability to 3T3-E1 cells,were added to the culture medium,and the absorbance of 3T3-E1 cells was measured by CCK-8 colorimetry 24 hours later.At logarithmic phase,the control group was set up.The cells were cultured with 10-6mol/L atorvastatin calcium and 10-7mol/L raloxifene,which had the strongest proliferation and differentiation ability to 3T3-E1 cells,respectively.Alkaline phosphatase assay kit was used to determine the activity of alkaline phosphatase(ALP)24 hours later;3T3-E1 cells were cultured to logarithm.At the third stage,the control group was set up.The cells were cultured with 10-6mol/L atorvastatin calcium and 10-7mol/L raloxifene,which had the strongest proliferation and differentiation ability to 3T3-E1 cells.The expressions of PERK,Bip and eIF2alpha in cells were detected by qRT-PCR 24 hours later.6.Statistical processing:SPSS 21.0 software was used for statistical processing.The experimental data were expressed as mean(+standard deviation(+s).One-way ANOVA was used for comparison among groups.LSD-t test was used for comparison between two groups.The difference was statistically significant(P<0.05).Result:1.Microscopically,3T3-E1 cells cultured in vitro were observed to be adherent,showing spindle-shaped,polygonal and irregular shapes.Some of the cells protruded and had the growth characteristics of osteoblasts.2.Different concentrations of atorvastatin calcium(10-8 mol/L,10-7 mol/L,10-6 mol/L,10-5 mol/L)could promote the proliferation of 3T3-E1 cells(P<0.05).Within a certain concentration range,with the increase of atorvastatin calcium intervention concentration,the cell proliferation ability increased(P<0.05).Among them,atorvastatin calcium(10-6 mol/L)had the most significant effect.Compared with the blank control group,the addition of raloxifene could promote the proliferation of 3T3-E1 in osteoblast precursor cells(P<0.05).Within a certain concentration range,the concentration of raloxifene increased,and the degree of cell proliferation increased accordingly(P<0.05),and raloxifene(10-7 mol/1)promoted the proliferation of osteoblast precursor cells(P<0.05).The proliferation ability is the strongest.3.After the intervention of different concentrations of atorvastatin calcium(10-8 mol/L,10-7 mol/L,10-6 mol/L,10-5 mol/L),ALP activity increased in each group,and the most obvious increase was in atorvastatin calcium(10-6 mol/L)(P<0.05);compared with the blank control group,the addition of raloxifene did not increase ALP activity.ALP activity increased in all groups after the same concentration treatment(P<0.05),and changed with the different concentration of raloxifene,and raloxifene group(10-7 mol/1)increased ALP activity significantly.4.Atorvastatin calcium with final concentration of 10-6 mol/L intervened 3T3-E1 cells for 24 hours.RT-PCR results showed that the expression of Bip,PERK and eIF2alpha gene in 3T3-E1 cells was higher than that in control group(P<0.05).The expression of Bip,PERK and eIF2alpha in 3T3-E1 osteoblasts treated with 10-7 mol/L raloxifene and raloxifene increased(P<0.05).).5.10-6 mol/L atorvastatin calcium and 10-7 mol/1 raloxifene intervened 3T3-E1 cells for 24 hours.OD values of cells in both groups were higher than those in the control group,but there was no statistical difference between the two groups.6.The optimal concentration of atorvastatin calcium and raloxifene to interfere with 3T3-E1 cells,compared with the control group,the alkaline phosphatase increased significantly,and the effect of atorvastatin calcium on 3T3-E1 differentiation was more significant than that of raloxifene(P<0.05).7.The optimal concentration of atorvastatin calcium and raloxifene interfered with 3T3-E1 cells.Compared with the control group,the expression levels of Bip,PERK and eIF2alpha increased(P<0.05).Compared with the experimental group,the expression of Bip and PERK in atorvastatin calcium group was more obvious(P<0.05),while the expression of eIF2alpha had no statistical significance.Conclusion:1.Microscopically,Raloxifen acted on osteoblast precursor cell 3T3-E1,which conformed to the growth characteristics of osteoblast cell lines.2.Atorvastatin calcium and raloxifene can promote the proliferation of osteoblast precursor cells(3T3-E1),and in a certain range with the increase of concentration,the ability to promote cell proliferation is more obvious.3.Atorvastatin calcium and raloxifene can increase the osteogenic activity of osteoblast precursor cells(3T3-E1).Within a certain concentration range,the osteogenic activity of high concentration group is higher than that of relatively low concentration group.4.At the level of mRNA,atorvastatin calcium and raloxifene can up-regulate the expression of PERK,Bip and eIF2alpha,which are the markers of endoplasmic reticulum stress signaling pathway,suggesting that their mechanism of promoting proliferation and differentiation of 3T3-E1 cells may be related to endoplasmic reticulum stress signaling pathway.5.The optimal concentration of atorvastatin calcium and raloxifene had no significant difference in the proliferation of 3T3-E1 cells,but the effect of atorvastatin calcium on osteogenic activity and expression of endoplasmic reticulum stress marker genes was better than that of raloxifene. |
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