PNS Can Promote The Proliferation And Differentiation Of Osteoblasts By Regulating The PI3K/Akt/Runx2 Pathway By MiR-204 In Osteoblasts

Objective:To investigate the effects and effecacy of PNS on the MC3T3 E1 cells and explore the PI3K/Akt/Runx2 signaling mechanism via the miR-204.Methods:Mouse MC3T3-E1 cells were cultured in vitro and divided into four groups:control group,treatment groups with different concentrations of PNS,and the concentrations of PNS in for each group were 0,10,20,50mg/ml.After 14 days of culture,the inverted phase microscope has been applied to examine the effect of PNS on the morphological changes of MC3T3-E1 cells;moreover,the expressions of osteocalcin and alkaline phosphatase in the supernatant were evaluated using enzyme-linked immunosorbent assay(ELISA)methods;next,the proliferation of PNS on MC3T3-E1 cells was determined by MTT assay,and flow cytometry was used to determine the effect of PNS on the apoptosis of MC3T3-E1 cells.RT-PCR and Western Blot methods were used to detect the expression of type I collagen,osteopontin(Ostepontin,OPN),Gla(Bone,protein,BGP)on mRNA and protein levels in each group.Then,the miR-204,PI3K,Akt,BCL-2,Runx2 expressions in each group were detected by RT-PCR method,and western blot method was applied to examine the expression of BCL-2,Bax,Runx2,and phosphorylated PI3K and Akt.Result:1.PNS does culture in vitro and promote MC3T3-E1 osteoblasts proliferation and differentiation.1.1 Cellular Morphology 10,20,50mg/L PNS has no obvious effect on MC3T3-E1 cellular morphology.1.2 Cell Proliferation Trough 24,48,72h,compared with comparison group,cell proliferation of PNS group 20 and 50mg/L rise a lot(P<0.05),its promotion on MC3T3-Elcell proliferation strengthens with the increase of intensity,and the difference has statistics meaning.However,the cell proliferation of 10mg/L treatment group has no obvious difference with the compassion group.1.3 Cell Apoptosis After 48h PNS stimulation and cultivation,cell apoptosis in 20 and 50mg/L group decline obviously(P<0.05),and the difference has statistic meaning compared to comparison group.Cell apoptosis in 50mg/L group is much lower than 20mg/L group.However,cell apoptosis of 10mg/L has no obvious difference with compassion group.1.4 Osteocalcin and Alkaline Level Phosphatase After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,osteocalcin and alkaline phosphatase of cell culture supernatant fluid in 20 and 50mg/L group rise obviously(P<0.05),and the difference has statistic meaning.Osteocalcin and alkaline phosphatase in 50mg/L group are much higher than 20mg/L group.However,cell apoptosis of 10mg/L has no obvious difference with compassion group.1.5 Marking Gene of Bone Formation COL1A1,OPN,BGP mRNA expression Level After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,cell COL1A1,OPN and BGP mRNA in 20 and 50mg/L group rise obviously(P<0.05),and the difference has statistic meaning.In addition,COL1A1,OPN and BGP expression level in 50mg/L group are much higher than 20mg/L group.However,cell COL1A1,OPN and BGP of 10mg/L group are all slightly higher than compassion group and the difference has no statistic meaning(P>0.05),1.6 Marking Gene of Bone Formation COL1A1,OPN,BGP Protein Expression Level After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,cell COL1A1,OPN and BGP level in 20 and 50mg/L group rise obviously(P<0.05),and the difference has statistic meaning.In addition,COL1A1,OPN and BGP expression level in 50mg/L group are much higher than 20mg/L group.However,cell COL1A1,OPN and BGP of 10mg/L group are all slightly higher than compassion group and the difference has no statistic meaning(P>0.05).2.PNS inhibits mir-204 expression of MC3T3-Elosteoblasts in order to improve MC3T3osteoblasts proliferation and differentiation.2.1 BCL-2 and Bax mRNA Level Expression After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,mRNA expression level of BCL-2 in20 and 50mg/L group rise obviously while mRNA of Bax decline obviously(P<0.05),and the differences both have statistic meaning.In addition,BCL-2 and Bax mRNA expression level in 50mg/L group are much higher than 20mg/L group.However,BCL-2 mRNA expression level of 10mg/L group is slightly higher than compassion group while mRNA of Bax declines slightly and the difference has no statistic meaning(P>0.05).2.2 BCL-2 and Bax Protein Level Expression After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,BCL-2 protein expression level in20 and 50mg/L group rise obviously while protein expression level of Bax declines obviously(P<0.05),and their differences both have statistic meaning.In addition,BCL-2 and Bax protein expression level in 50mg/L group are much higher than 20mg/L group.However,BCL-2 protein expression level of 10mg/L group is slightly higher than compassion group while protein expression level of Bax declines slightly and the difference has no statistic meaning(P>0.05).2.3 miR-204 Expression After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,miR-204 expression level in 20 and 50mg/L group decline obviously(P<0.05),and the difference has statistic meaning.In addition,miR-204 expression level in 50mg/L group are much lower than 20mg/L group.However,miR-204 expression level of 10mg/L group declines slightly than compassion group,but the difference has no statistic meaning(P>0.05).2.4 After the enhancement of mir-204 expression,PNS were reduced to the bone capacity of Mc3t3-E1 cells.After 48 hours 50 mg/L PNS cultivation compared with not transfection miR-204 in control group,the levels of osteocalcin and alkaline phosphatase were significantly reduced in the cells cultured with high expression of miR-204(P<0.05),PCR results showed that the levels of BGP,CoL1A1 and OPN were significantly reduced in the cells cultured with high expression of miR-204(P<0.05),the difference has statistic meaning.2.5 Signal Path Molecule PI3K,Akt and Runx2 mRNA Level Expression After 48h,10,20,50mg/L PNS cultivation,compared with comparison group,PI3Kand Akt expression level on mRNA layer in 10,20 and 50mg/L group have no obvious difference(P>0.05),while cell Runx2 mRNA level rise obviously in 20mg/L and 50mg/L group,and the difference has statistic meaning(P<0.05).However,Runx2 expression level of 50mg/L group is much higher than 20mg/L.Cell Runx2 mRNA level expression of 10mg/L rises slightly than the comparison group,but the difference has no statistic meaning(P>0.05).2.6 Effect of PI3K,Akt protein Phosphorylation and its Expression on Runx2 Protein Expression level After half an hour,10,20,50mg/L PNS cultivation,compared with comparison group,p-PI3K,p-Akt on Mma layer in 20 and 50mg/L group are much higher,and the difference has statistic meaning(P<0.05).In addition,p-PI3K,p-Akt in 50mg/L group are much higher than 20mg/L group.p-PI3K,p-Akt in 10mg/L group is slightly higher than comparison group but the difference has no statistic meaning(P>0.05).After 48 hours,10,20,50mg/L PNS cultivation,compared with comparison group,cell Runx2 protein level in 20 and 50mg/L group are much higher,and the difference has statistic meaning(P<0.05).In addition,Runx2 expression level in 50mg/L group is much higher than 20mg/L group.Runx2 in 10mg/L group is slightly higher than comparison group but the difference has no statistic meaning(P>0.05).Conclusion:PNS can promote the proliferation and differentiation of osteoblasts by regulating the PI3K/Akt/Runx2 pathway by miR-204 in osteoblasts.