| Objective:To observe the effect of Resveratrol(RES)on the proliferation and differentiation of MC3T3-E1 Subclone14 cells,and study its intervention on apoptosis induced by cadmium and the role of ERK1/2 signal in it.Methods:1.The effect of RES and cadmium on the viability of MC3T3-E1 cells by CCK-8 kit.2.The ALP kit was used to detect the effect of RES and cadmium on the synthesis and secretion of ALP in MC3T3-E1 cells.3.AnnexinV-FITC/PI double staining assay was used to detect the effect of RES and cadmium on the apoptosis rate of MC3T3-E1 cells.4.The effect of RES and cadmium on the gene expression of MC3T3-E1 ALP,COLI,BMP-2 and RUNX2 was detected by QPCR.5.The effect of RES and cadmium on the protein expression of COLI,BMP-2,RUNX2,ERK1/2 and p-ERK1/2 and after treatment with ERK1/2 inhibitor PD98059 was detected by Western-blot.Results:1.Compared with the control group,RES(2.5 μM,5 μM,10 μM)intervention for 72 h,cell viability significantly increased(P<0.01),the ALP synthesis and secretion of cell significantly increased(P<0.01).2.The mRNA expression of ALP,BMP-2 and RUNX2 was significantly increased after treatment MC3T3-E1 of RES(10 μM)for 2 h compared with the control group(P<0.05).Treated with RES(10 μM)at different time points(15,30,45 min)significantly increased the expression of BMP-2,RUNX2,and COLI protein(P<0.01).The phosphorylation level of ERK1/2 protein was significantly increased(P<0.01).After the ERK1/2 signal was blocked by ERK1/2 antagonist PD98059,the phosphorylation of ERK1/2 protein was significantly decreased(P<0.01),and the protein expression of COLI,BMP-2,and RUNX2 was significantly decreased(P<0.01).3.Compared with the control group,cadmium(5 μM,10 μM,20 μM)intervention for 48 h significantly reduced the viability of the cells(P<0.01).The ALP secretion of MC3T3-E1 cell was significantly decreased in cadmium(10 μM,20 μM)intervention for 2 h(P<0.01).4.Cd(10 μM)significantly inhibited the expression of ALP,COLI,RUNX2,and BMP-2 mRNA and protein in MC3T3-E1 cells(P<0.01).When cells were treated with cadmium (10 μM)at different time points(2,4,and 8 h),the phosphorylation level of ERK1/2 protein was significantly increased(P<0.01).5.After treated with cadmium(10 μM)for 48 h,cell morphology observations showed that the cells were reduced in size,disappeared in succession,detached from the surrounding cells,and floated in the culture medium,while RES(10 μM)and cadmium(10 μM)were used together to treat,part of the cells detachment,floating in the culture fluid,most cells still adhere to the wall,the state is normal.Apoptosis rate test showed that cadmium(10μM)induced MC3T3-E1 cell apoptosis(P<0.01),RES(10 μM)significantly improved the cell apoptosis induced by Cd.(P<0.01).6.The cell viability of the group treated with RES(10 μM)and cadmium(10 μM) significantly increased(P<0.05),and the ALP secretion of MC3T3-E1 cell significantly increased(P<0.01)compared with the cadmium group.7.The phosphorylation level of ERK1/2 protein was significantly decreased in the group treated with RES(10 μM)and cadmium(10 μM)(P<0.01)compared with the cadmium group.After blocking ERK1/2 signal with ERK1/2 antagonist PD98059(20 μM),the expression of p-ERK/ERK protein was significantly decreased in RES+cadmium group and cadmium+PD group compared with cadmium treatment group(P<0.01).The protein expression of RUNX2,BMP-2 was significantly increased(P<0.01).Compared with RES+cadmium group,the expression of p-ERK/ERK protein was significantly decreased in RES+cadmium+PD group(P<0.05),and the expression of COLI,RUNX2,BMP-2 protein was significantly increased(P<0.05).Conclusions:1.RES(2.5 μM,5 μM,10 μM)could promote the proliferation and differentiation of MC3T3-E1 cells.2.RES(10 μM)promoted differentiation of MC3T3-E1 cells by regulating ERK1/2 signaling.3.RES(10 μM)can protect Cd-induced apoptosis of MC3T3-E1 cells.4.Cadmium(10 μM)inhibited the differentiation of MC3T3-E1 cells,and RES(10 μM) could improve its inhibitory effect,and the improvement effect was related to ERK1/2 signal. |
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