| ObjectiveTo observe the effect of Gubenzengu prescription medicated serum on the proliferation and differentiation of MC3T3-E1,explore the mechanism of action of Gubenzengu prescription medicated serum in treating OP through TGF-β/Smad signaling pathway,and analyze the molecular mechanism and effect targets of regulating the proliferation and differentiation of MC3T3-E1.Method1.Different concentrations of Gubenzenggu prescription medicated serum(5%,10%,15%,20%,25%)were prepared,and the control group was set.Cell proliferation was measured by CCK-8 method,and cell osteoblast differentiation was measured by ALP activity detection and alizarin red staining,so as to select the optimal concentration to promote MC3T3-E1 proliferation and differentiation.2.After selecting the optimal concentration,MC3T3-E1 was randomly divided into 4groups: Blank serum group,optimal concentration drug-containing serum group,blank serum+SB43154 group,optimal concentration drug-containing serum +SB43154 group,Western blot detection of bone formation related proteins RUXN2,BMP-2 and TGF-β/Smad signaling pathway related proteins TGF-β1,Smad2,Smad3.Real-time PCR was used to detect mrna expressions of osteogenic genes RUXN2,BMP-2 and TGF-β1,Smad2 and Smad3 related genes in TGF-β/Smad signaling pathway,so as to explore the mechanism of action of Gubenzenggu prescription in treating OP based on TGF-β/Smad signaling pathway.Results1.Cell proliferation measured by CCK-8 assay: According to different intervention measures,compared with the control group,the OD value of 10%,20% and 25%drug-containing serum groups increased,and the differences were statistically significant(P<0.05),the OD value of 5% drug-containing serum group increased,but the difference was not statistically significant(P>0.05),and the OD value showed an upward trend,reaching the highest level in 20% drug-containing serum group.OD value decreased in 25%drug-containing serum group.According to the different intervention time,there was no significant difference in OD value of each group at 0h of intervention(P>0.05).The OD value of 48 h after intervention was higher than that of 0h,24 h and 72 h,and the difference was statistically significant(P<0.05).After 72 hours of intervention,the OD value of 20%drug-containing serum group was higher than that of 15% drug-containing serum group and25% drug-containing serum group,but the difference was not statistically significant(P>0.05).The results showed that 20% serum had the best effect on MC3T3-E1 proliferation after 48 days of intervention.2.ALP activity detection of cell differentiation: Compared with the blank serum group,the ALP activity of 10% drug-containing serum group,15% drug-containing serum group,20% drug-containing serum group,and 25% drug-containing serum group increased,and the differences were statistically significant(P<0.05),and the ALP activity of 5%drug-containing serum group increased slightly,but the differences were not statistically significant(P>0.05).ALP activity increased with the increase of drug-containing serum concentration,reached the highest in the 20% drug-containing serum group,and decreased when the concentration reached 25%.At the same time,there was significant difference between the 20% drug-containing serum group and other groups(P<0.05).3.Alizarin red staining was used to determine cell mineralization: the image showed that the number of red calcium nodules in 5% drug-containing serum group,10% drug-containing serum group,15% drug-containing serum group,20% drug-containing serum group,and 25%drug-containing serum group was significantly higher than that in blank serum group,and the number and density of calcium nodules in 20% drug-containing serum group was significantly higher than that in other groups.The optimal concentration for promoting osteogenic differentiation of MC3T3-E1 was 20%.4.Expression of related proteins detected by Western-blot: Compared with the 20%blank serum group,the protein expressions of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in the 20% drug-containing serum group and the 20% drug-containing serum +SB43154group were increased.The protein expression levels of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in 20% drug-containing serum group were significantly increased,and the difference was statistically significant(P<0.05).However,the protein expression of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in 20% blank serum +SB43154 group was slightly decreased,but the difference was not statistically significant(P>0.05).Compared with 20% drug-containing serum group,the protein expressions of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in 20%drug-containing serum +SB43154 group were significantly decreased,and the difference was statistically significant(P<0.05).5.Expression of related genes detected by Real-time PCR: Compared with 20% blank serum group,the expression of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 m RNA in 20%drug-containing serum group and 20% drug-containing serum +SB43154 group increased.The m RNA expression levels of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in 20%drug-containing serum group were significantly increased,and the differences were statistically significant(P<0.05).However,the expression of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 m RNA in 20% blank serum +SB43154 group was slightly decreased,but the difference was not statistically significant(P>0.05).Compared with 20% drug-containing serum group,the m RNA expression of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in 20%drug-containing serum +SB43154 group was significantly decreased,and the difference was statistically significant(P<0.05).The m RNA expression of RUXN2,BMP-2,TGF-β1,Smad2 and Smad3 in 20% drug-containing serum +SB43154 group was higher than that in 20%blank serum group and 20% blank serum +SB43154 group,and the differences were statistically significant(P<0.05).Conclusion1.Gubenzengu prescription medicated serum can promote the proliferation and differentiation of MC3T3-E1,and the best effect is achieved when the concentration is 20%.2.Gubenzengu prescription medicated serum can up-regulate the expression of RUNX2,BMP-2,Tgf-β1,Smad2,Smad3 protein and m RNA in MC3T3-E1 through TGF-β/Smad signaling pathway,and regulate bone metabolism,and its mechanism of action is achieved by activating TGF-β/Smad signaling pathway. |
