| ObjectiveTo clarify the effects of Guilu Erxian Glue containing serum on the proliferative activity,osteogenic differentiation and m RNA expression levels of key proteins and genes in the MAPK/ERK1/2 signaling pathway of SD rats;to reveal the intervention effect of Guilu Erxian Glue on the osteogenic differentiation of bone marrow mesenchymal stem cells and its regulatory mechanism from the perspective of in vitro cellular level and MAPK/ERK1/2signaling pathway.Methods1.Preparation of Guilu Erxian Glue containing serum PrescriptionSixty SD rats of SPF grade at 8 weeks of age were randomly divided into 30 rats in the drug-containing serum group and 30 rats in the blank serum group,and the drug-containing serum group was gavaged with 3.15g/kg/d of Guilu Erxian Glue,while the blank serum group was gavaged with equal doses of distilled water.After 7d of continuous gavage,blood was collected from the abdominal aorta to prepare the drug-containing serum.2.Screening of effective time of action of Guilu Erxian Glue containing serum on the growth of bone marrow mesenchymal stem cellsThe F3-generation cells were divided into 5 groups,and the absorbance values at 450 nm were measured by enzyme marker after adding 10%Guilu Erxian Glue containing serum for12 h,24h,48 h,72h and 96 h of intervention,respectively.The highest absorbance value of CCK-8 was used as the best intervention time point for this experiment.3.Intervention and index detection of osteogenic differentiation induced by bone marrow mesenchymal stem cellsThe F3 generation cells were divided into fetal bovine serum group,blank serum group,Guilu Erxian Glue containing serum group,and classical induction group.The conditioned medium of each group was used for osteogenic differentiation intervention,and the cell culture of each group was tested after intervention for 7d.ALP activity level in the liquid;Alizarin red staining and quantitative analysis were performed after 14 d of intervention;ALP,Runx2,ETS1,PPARγ m RNA expression levels were detected by RT-q PCR after 14 d of intervention;Fetal bovine serum was detected by Western-Blot after 14 d of intervention Group,blank serogroup,Guilu Erxian Glue containing serum group and classic induction group,and after pathway inhibitor intervention,Guilu Erxian Glue containing serum + MAPK inhibitor group,MAPK inhibitor group and Guilu Erxian Glue containing serum The expression levels of ALP,Runx2,ETS1,PPARγ,collagen I and P-ERK1/2 protein in the cells of the drug serum group.Results1.The effect of 10% Guilu Erxian Glue containing serum on the proliferation activity of bone marrow mesenchymal stem cells:According to the results of CCK-8 experiment,the pro-proliferative activity of BMMSCs was significant(P<0.01)after 72 h of intervention with 10% Guilu Erxian Glue containing serum,and the subsequent intervention experiments were conducted with 10% Guilu Erxian Glue containing serum,and the fluid was changed once at 72 h during the intervention period.2.The effect of Guilu Erxian Glue containing serum on alkaline phosphatase activity of bone marrow mesenchymal stem cells:Compared with the fetal bovine serum group and the blank serum group,the drug-containing serum intervention in the Guilu Erxian Glue group for 7d increased the activity of alkaline phosphatase in bone marrow mesenchymal stem cells(P<0.05),but the classical induction group increased the activity of alkaline phosphatase in bone marrow mesenchymal stem cells more significantly compared with the Guilu Erxian Glue containing serum group(P<0.05).3.Effect of Guilu Erxian Glue containing serum on osteogenic differentiation and mineralized nodule formation of bone marrow mesenchymal stem cells:Compared with the fetal bovine serum group and the blank serum group,calcified nodules were significantly increased after 14 d of osteogenic induction of bone marrow mesenchymal stem cells with Guilu Erxian Glue containing serum.The results of quantitative analysis of calcified nodules by alizarin red staining also showed that the osteogenic differentiation ability of Guilu Erxian Glue containing serum group was stronger than that of fetal bovine serum group and blank serum group(P<0.05).4.Comparison of m RNA expression of ALP,Runx2,ETS1 and PPARγ in each group:Compared with the fetal bovine serum group and the blank serum group,the expression of ALP,Runx2 and ETS1 m RNA was significantly higher in the cells of the Guilu Erxian Glue containing serum group and the classical induction group(P<0.05);compared with the blank serum group,the expression of PPARγ m RNA was down-regulated in the cells of the Guilu Erxian Glue containing serum group and the classical induction group(P<0.05).5.Protein expression of ALP,collagen Ⅰ,Runx2,ETS1,PPARγ and P-ERK1/2 in each group:Compared with the blank serum group,the expression levels of P-ERK1/2,ALP,collagen Ⅰ and Runx2 proteins were increased in the cells of the Guilu Erxian Glue containing serum group and the classical induction group(P<0.05).Compared with the blank serum group and fetal bovine serum group,the expression level of PPARγ protein in the cells of Guilu Erxian Glue containing serum group and classically induced group was significantly downregulated(P<0.05).After the pathway inhibitor intervention,the expression levels of P-ERK1/2,ALP,collagen Ⅰ,Runx2 and ETS1 were significantly higher in the Guilu Erxian Glue containing serum group compared with the Guilu Erxian Glue containing serum +MAPK pathway inhibitor group and MAPK pathway inhibitor group(P<0.05);meanwhile,the expression levels of PPARγ protein in the MAPK pathway inhibitor group were significantly higher than those in the Guilu Erxian Glue containing serum group(P<0.05).The level of PPARγ protein expression in the MAPK pathway inhibitor group was significantly higher than that in the Guilu Erxian Glue containing serum group(P<0.05).ConclusionsGuilu Erxian Glue containing serum activated the MAPK/ERK1/2/ETS1 signaling pathway related factors ALP,collagen Ⅰ,Runx2,ETS1 and P-ERK1/2 by up-regulating their expression and down-regulating PPARγ expression.MAPK/ERK1/2/ETS1 signaling pathway and promote the differentiation of BMMSCs to osteoblasts. |
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