Study on the effect of mulberry leaf water extract on osteogenic differentiation of pre-osteoblasts in high glucose environment
Objective To investigate the effect of mulberry leaf water extract on osteogenic differentiation of MC3T3-E1 cells in high glucose environment,and to provide a theoretical basis for the treatment of diabetic periodontitis(DP)in clinical practice.Methods The fourth generation MC3TE-E1 cells were treated with different concentrations of glucose(5 mM,10 mM,20 mM,30 mM,40 mM,50 mM,60 mM,70 mM,100 mM)for 1 d,3 d,5 d,7 d.The appropriate high glucose concentration was screened by CCK-8 method to construct a high glucose environment in vitro.The effects of different concentrations of mulberry leaf aqueous extract(10 mg/mL,1 mg/mL,100 μg/mL,10 μg/mL,1 μg/mL,100 ng/mL,10 ng/mL,1 ng/mL)on the proliferation of MC3T3-E1 cells in high glucose environment were detected and the appropriate concentration of mulberry leaf aqueous extract was screened.Cell Counting Kit-8(CCK-8),flow cytometry,Enzyme Linked Immunosorbent Assay(ELISA)and real-time quantitative PCR(RT-PCR)were used to detect the expression of inflammatory,apoptosis and DM related indicators to evaluate the mulberry leaf water extract in the high glucose ring.After osteogenic induction,Alkaline Phosphatase(ALP)staining,ALP activity,Alizarin Red S Staining(ARS)and quantitative and RT-PCR were used to evaluate the effect of mulberry leaf water on osteogenic differentiation of MC3T3-E1 in high glucose environment.Results ①The results of CCK8 showed that the proliferation of cells was significantly inhibited when the glucose concentration was more than 50 mM,so 50 mM glucose was selected to construct a high glucose cell model in vitro.In the high glucose environment,compared with other groups,1,10,100μg/mL mulberry leaf water extract was the optimal concentration to promote the proliferation of MC3T3-E1 cells.②The results of apoptosis showed that the apoptosis rate of the high glucose group was significantly higher than that of the control group,and the apoptosis caused by high glucose stimulation could be alleviated by adding mulberry leaf water extract(P＜0.05).The results of reactive oxygen species(ROS)showed that the aqueous extract of mulberry leaves could inhibit the production of ROS in cells under high glucose environment to a certain extent(P＜0.05).③The results of reactive oxygen species(ROS)showed that the aqueous extract of mulberry leaves could inhibit the production of ROS in cells under high glucose environment to a certain extent(P＜0.05).④ELISA results showed that compared with the control group,the expression of related inflammatory factors in the high glucose group was significantly higher than that in the control group.The addition of mulberry leaf water extract could inhibit the expression of related inflammatory factors.⑤The results of RT-PCR showed that the aqueous extract of mulberry leaves could regulate the ratio of Bax/Bcl-2 to inhibit apoptosis in high glucose environment,and the expression of related inflammatory genes was basically consistent with the results of ELISA(P＜0.05).⑥The results of alkaline phosphatase staining,ALP activity and alizarin red staining showed that compared with the control group,the ALP activity and the formation of mineralized nodules in the high glucose group were significantly reduced,and the addition of mulberry leaf water extract could be significantly alleviated(P＜0.05).⑦The results of RT-PCR showed that compared with the control group,the aqueous extract of mulberry leaves could alleviate the decrease of osteogenic related gene expression caused by high glucose stimulation(P＜0.001).Conclusion ①The water extract of mulberry leaves can alleviate the damage and apoptosis of MC3T3-E1 cells induced by high glucose stimulation.②The water extract of mulberry leaves can promote the osteogenic differentiation of MC3T3-E1 cells in high glucose environment.
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维普
