Study On The Effect Of Gubenzenggufang Drug Containing Serum In Promoting Osteogenic Differentiation Of BMSCs By Regulating Notch Pathway

Objective: To observe the effect of Gubenzenggu drug-containing serum in promoting the proliferation and osteogenic differentiation of rat BMSCs cultured in vitro,and then to explore the relationship between the promoting effect of Gubenzenggu drug-containing serum in BMSCs' s osteogenic differentiation and the function of Notch signaling pathway.Methods: The theory of serum pharmacology was used to prepare Gubenzenggu drug-containing serum.In the first experiment,the rat BMSCs were divided into 5%?10%,?20%,?30% drug-containing serum group and blank control group,then we applied the MTT method to observe the effect on the proliferation in rat BMSCs of every group;In the Second experiment,the rat BMSCs were divided into 5%? 10%?20%?30% drug-containing serum groups and the traditional induction group.First,we induced the adipogenic differentiation of BMSCs,and the Oil red O staining was used in each group to analyze the inhibitory effect of Gubenzenggu drug-containing serum on adipogenic differentiation of rat BMSCs after corresponding intervention.Second,we induced the osteogenic differentiation of BMSCs.After the intervention of each group,we used alizarin red for calcified nodule staining,and the ALP activity was detected in each group of cells and cell culture.In the third experiment,we know that the optimal drug concentration is 20% drug-containing serum after analyzing the above experimental results.So the BMSCs were divided into six groups:traditional induction group,20% drug-free serum group,20% drug-containing serum group,traditional induced + DAPT group,20% drug-free serum + DAPT group and 20%drug-containing serum + DAPT group.Real-TimePCR method was used to detect the expression of Notch1 and CBF1 m RNA in each group to confirm that the Notch pathway was blocked after adding DAPT,and then the expressions of Col-1,BGP and RUNX2 in each group were detected by Western-Blot.,At last we combined with all the above results to analyze the relationship between the promoting effect of Gubenzenggu drug-containing serum in BMSCs' s osteogenic differentiation and the function of Notch signaling pathway.Result:Different concentrations of Gubenzenggu drug-containing serum affect the proliferation of BMSCs at different time points have different characteristics.Compared with the control group,the 30% drug-containing serum group had significant difference(P <0.05)at 24 h,indicating that the 30% drug-containing serum group had the effect of promoting the proliferation of BMSCs at this time point.At 48 h,72h and 96 h,the serum concentrations of10%,20% and 30% were significantly higher than those of blank group(P <0.05),indicating that 10%,20%,30% concentration of drug-containing serum can further promote the proliferation of BMSCs,and 20% concentration of drug-containing serum has the strongest promotion.At120 h,compared with the control group,20%,?30% drug-containing serum grous were significantly different(P <0.05).But 30% drug-containing serum group showed a decreasing trend at 120 h compared with the trend at 96 h.After adipogenic induction of BMSCs,the fat conversion rates in the 5%?10%?20% and 30% drug-containing serum groups were significantly lower than those in the traditional induction groups(P <0.05),of which 20% drug-containing serum group was significantly lower than the other concentration groups(P <0.05);After the osteogenic induction of BMSCs,compared with the traditional induction group,the activity of ALP in the serum of various concentrations of drug-containing serum showed an upward trend,20%,?30% drug-containing serum groups were significantly different(P <0.05).Moreover,ALP activity in 20% drug-containing serum group was the highest among all other groups(P <0.05).Compared with the traditional induction group,20% drug-free serum group,20% drug-containing serum group respectively,their corresponding groups including traditional induced + DAPT group,20%drug-free serum + DAPT group and 20% drug-containing serum + DAPT group all has a significantly decreased expression of Notch1 and CBF1 mRNA(P <0.05);Compared with the traditional induction group,the expression of Notch1 and CBF1 mRNA in 20% drug-free serum showed no significant difference(P> 0.05),Compared with the traditional induction group and 20% drug-free serum group,the Notch1 mRNA in 20%drug-containing serum group was significantly increased(P <0.05)But the CBF1 mRNA was significantly decreased(P <0.05),Compared with the traditional induced + DAPT group and the 20% drug-free serum+ DAPT group,the expression of Notch1 mRNA in 20% drug-containing serum + DAPT group showed no significant difference(P> 0.05),while the expression of CBF1 mRNA decreased significantly(P < 0.05);Compared with the traditional induction group,20%drug-free serum group and 20% drug-containing serum groupr espectively,the expressions ofRUNX2 ?Col?and BGP in the traditional induced + DAPT group,20%drug-free serum +DAPT group and 20%drug-containing serum + DAPT group was significantly increased(P<0.05).Compared with the traditional induction group and 20% drug-free serum group,the expression of RUNX2?Col-?and BGP in 20% drug-containing serum group increased significantly(P <0.05).Compared with the traditional induced + DAPT group and the 20%drug-free serum + DAPT group,the expression of Col?,BGP and RUNX2 in 20%drug-containing serum + DAPT group were significantly decreased(P <0.05).Conclusion:1.Gubenzenggu drug-containing serum can promote the proliferation and osteogenic differentiation of BMSCs and inhibit the adipogenic differentiation of BMSCs.The 20%concentration drug-containing serum has the best effect compared with the other groups.2.The Notch pathway mainly plays the role of inhibiting the osteogenic differentiation of BMSCs in this experiment.3.In the process of osteogenic differentiation of BMSCs,Gubenzenggu drug-containing serum can up-regulate the expression of Notch1 mRNA and down-regulate the expression of CBF1 mRNA in Notch pathway.When used in combination with DAPT,Gubenzenggu drug-containing serum's ability to up-regulate Notch1 diminished,and the synergism may down-regulate the expression of CBF1 mRNA.4.In the process of osteogenic differentiation of BMSCs,Gubenzenggu drug-containing serum can promote the expression of RUNX2?Col?and BGP which may be related to its founction of up-regulating Notch1 mRNA and down-regulating CBF1 mRNA in Notch pathway.