桑叶水提物对高糖环境下前成骨细胞成骨分化的作用研究

Study on the effect of mulberry leaf water extract on osteogenic differentiation of pre-osteoblasts in high glucose environment

目的探讨桑叶水提物对高糖环境下MC3T3-E1细胞成骨分化的影响,为临床中糖尿病牙周炎(diabetic periodontitis,DP)的治疗提供一定的理论基础。方法取第4代MC3TE-E1细胞,将用不同浓度的葡萄糖(5 mM、10 mM、20 mM、30 mM、40 mM、50 mM、60 mM、70 mM、100 mM)干预MC3T3-E1细胞1 d、3 d、5d、7 d,用CCK-8法筛选出合适的高糖浓度构建体外高糖环境。继续检测高糖环境下不同浓度的桑叶水提物(10 mg/mL、1 mg/mL、100μg/mL、10μg/mL、1μg/mL、100 ng/mL、10 ng/mL、1 ng/mL)干预MC3T3-E1细胞后对细胞增殖的影响并筛选出合适的桑叶水提物浓度;通过细胞增殖毒性检测(cell dountingkit-8,CCK-8)、流式细胞技术、酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)和实时定量PCR(real-time PCR,RT-PCR)检测炎性、凋亡和糖尿病等相关指标的表达,用以评价桑叶水提物在高糖环境下对MC3T3-E1的影响。成骨诱导后,通过碱性磷酸酶(alkaline phosphatase,ALP)染色、ALP活性、茜素红染色(alizarin red staining,ARS)及定量以及RT-PCR用以评价桑叶水提物在高糖环境下对MC3T3-E1成骨分化的影响。结果①CCK8结果表明,在葡萄糖浓度为50 mM以上时对细胞的增殖有明显的抑制作用,故选用50 mM的葡萄糖用以构建体外高糖细胞模型。在高糖环境下,与其他组相比,1、10、100μg/mL的桑叶水提物为促进MC3T3-E1细胞增殖的适宜浓度;②凋亡结果表明,高糖组的凋亡率明显高于对照组,加入桑叶水提物后能缓解因高糖刺激引起的细胞凋亡(P<0.05)。③活性氧(reactive oxygen species,ROS)结果表明,桑叶水提物均可在一定程度上抑制高糖环境下细胞内的ROS的产生(P<0.05);④ELISA结果表明,与对照组相比,高糖组相关炎症因子的表达明显高于对照组,加入桑叶水提物后可以抑制相关炎症因子的表达。⑤通过RT-PCR结果表明,在高糖环境下桑叶水提物可以通过调控凋亡相关基因的表达来抑制凋亡,相关炎症基因的表达与ELISA的结果基本一致(P<0.05)。⑥通过碱性磷酸酶染色,ALP活性和茜素红染色结果表明,与对照组相比,高糖组的ALP活性和矿化结节的形成量均明显降低,加入桑叶水提物后可明显缓解(P<0.05);⑦通过RT-PCR结果表明,与对照组相比,桑叶水提物可以缓解因高糖刺激引起的成骨相关基因表达降低(P<0.001)。结论(1)桑叶水提取物可以缓解因高糖刺激引起的MC3T3-E1细胞损伤和凋亡。(2)桑叶水提取物可以促进高糖环境下MC3T3-E1细胞的成骨分化。

Objective To investigate the effect of mulberry leaf water extract on osteogenic differentiation o MC3T3-E1 cells in high glucose environment,and to provide a theoretical basis for the treatment of diabetic periodontiti(DP)in clinical practice.Methods The fourth generation MC3TE-E1 cells were treated with different concentration 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 glucos environment in vitro.The effects of different concentrations of mulberry leaf aqueous extract(10 mg/mL,1 mg/mL100μg/mL,10μg/mL,1μg/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 wa screened.Cell Counting Kit-8(CCK-8),flow cytometry,Enzyme Linked Immunosorbent Assay(ELISA)and realtime 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,Alkalin 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 glucos environment.Results①The 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 optima concentration to promote the proliferation of MC3T3-E1 cells.②The results of apoptosis showed that the apoptosis rate o the high glucose group was significantly higher than that of the control group,and the apoptosis caused by high glucos 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 o 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 inhibi the expression of related inflammatory factors.⑤The results of RT-PCR showed that the aqueous extract of mulberry leave 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 phosphatas staining,ALP activity and alizarin red staining showed that compared with the control group,the ALP activity and th formation of mineralized nodules in the high glucose group were significantly reduced,and the addition of mulberry lea water extract could be significantly alleviated(P<0.05).⑦The results of RT-PCR showed that compared with the contro group,the aqueous extract of mulberry leaves could alleviate the decrease of osteogenic related gene expression caused by high glucose stimulation(P<0.001).Conclusion①The 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 leave can promote the osteogenic differentiation of MC3T3-E1 cells in high glucose environment.