高糖环境下药桑提取物脱氧野尻霉素对MC3T3-E1细胞的影响

Effect of 1-Deoxynojirimycin(DNJ)from mulberry extract on MC3T3-E1 cells under high glucose environment

目的研究药桑提取物脱氧野尻霉素(1-Deoxynojirimycin,DNJ)在高糖环境下对MC3T3-E1细胞的增殖和成骨分化的作用。方法取P4代MC3T3-E1细胞,在50 mmol/L的高糖环境下通过细胞毒性检测试剂盒(Cell dountingkit-8,CCK-8)检测不同浓度的DNJ(10 mmol/L、1 mmol/L、100μmol/L、10μmol/L、1μmol/L、100 nmol/L、10 nmol/L、1 nmol/L)干预对MC3T3-E1细胞增殖的影响,筛选出后续实验研究的DNJ浓度。取培养至对数生长期的MC3T3-E1细胞,按不同干预方式分为:空白组(完全培养基)、高糖组(50 mmol/L糖浓度的完全培养基)、实验组[50 mmol/L糖浓度的完全培养基+不同浓度DNJ(100、10、1μmol/L)溶液]。使用流式细胞技术检测细胞的凋亡和活性氧,采用ELISA试剂盒检测细胞上清液中AGEs和IL-1β、IL-6、TNF-α的含量,通过碱性磷酸酶染色及活性检测MC3T3-E1细胞的早期成骨能力的影响,通过茜素红染色和定量检测MC3T3-E1细胞的晚期成骨能力,采用RT-PCR检测IL-1β、IL-6、TNF-α、Bax、Bcl-2、IGF-1、ALP、OCN、OSX、Col-1和Runx2的mRNA表达情况。结果高糖环境下,100、10、1μmol/L的DNJ可以促进MC3T3-E1细胞增殖。与高糖组相比,实验组凋亡率、活性氧含量、细胞上清液中AGEs、IL-1β、IL-6和TNF-α含量降低,差异有统计学意义(P<0.05)。RT-PCR结果表明,与高糖组相比,实验组Bax、IL-1β、IL-6和TNF-α的mRNA表达降低,Bcl-2、IGF-1、ALP、OCN、OSX、Col-1和Runx2的mRNA表达升高,差异有统计学意义(P<0.05)。结论在高糖环境下,一定浓度范围内DNJ能够抑制MC3T3-E1细胞的凋亡和炎症因子的产生,促进成骨细胞的分化。

Objective To investigate the effect of 1-Deoxynojirimycin(DNJ)on the proliferation and osteogenic differentiation of MC3T3-E1 cells in high glucose environment.Methods The P4 generation MC3T3-E1 cells were taken,and the effects of different concentrations of DNJ(10 mmol/L,1 mmol/L,100μmol/L,10μmol/L,1μmol/L,100 nmol/L,10 nmol/L and 1 nmol/L)on the proliferation of osteogenic precursor cells were detected by CellDounting Kit-8(CCK-8)in a high glucose environment of 50 mmol/L,and the concentration of DNJ in subsequent experimental studies was screened.MC3T3-E1 cells cultured to logarithmic growth phase were selected and divided into blank group(complete medium),high glucose group(complete medium with 50 mmol/L glucose concentration)and experimental group[complete medium with 50 mmol/L glucose concentration+different concentrations of DNJ(100μmol/L,10μmol/L,1μmol/L)solution].Flow cytometry was used to detect cell apoptosis and reactive oxygen species production.The contents of AGEs,IL-1β,IL-6 and TNF-αwere detected by ELISA kit.The early osteogenic ability of MC3T3-E1 cells were detected by alkaline phosphatase staining and activity.The late osteogenic ability of MC3T3-E1 cells were detected by alizarin red staining and quantitative detection.The mRNA expressions of IL-1β,IL-6,TNF-α,Bax,Bcl-2,IGF-1,ALP,OCN,OSX,Col-1 and Runx2 were detected by RT-PCR.Results In the high glucose environment,100μmol/L,10μmol/L and 1μmol/L DNJ can significantly promote the proliferation of MC3T3-E1 cells,so it is used for subsequent research.Compared with the high glucose group,the apoptosis rate,reactive oxygen species and the contents of AGEs,IL-1β,IL-6 and TNF-αin the cell supernatant of the experimental group weredecreased,and the difference was statistically significant(P<0.05).The results of RT-PCR showed that compared with the high glucose group,the mRNA expression of Bax,IL-1β,IL-6 and TNF-αin the experimental group was significantly decreased,and the mRNA expression of Bcl-2,IGF-1,ALP,OCN,OSX,Col-1 and Runx2 was significantly increased,the difference was statistically significant(P<0.05).Conclusion In the high glucose environment,DNJ can inhibit the apoptosis of mouse pre-osteoblast MC3T3-E1 and the production of inflammatory factors within a certain concentration range,and promote the differentiation of osteoblasts.