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中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (06) : 321 -327. doi: 10.3877/cma.j.issn.2095-1221.2020.06.001

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论著

柚皮苷对高糖作用下MC3T3-E1细胞活力和Akt通路相关因子表达的影响
林春淑1, 舒晓春1, 肖菲娜1, 彭虹1, 容婵2, 江健3, 胡洁华1, 孟晓军1,()   
  1. 1. 519000 珠海,广东中山大学附属第五医院健康管理中心
    2. 528300 佛山,广东南方医科大学附属顺德医院内科
    3. 518107 深圳,广东中山大学附属第七医院科研中心
  • 收稿日期:2020-03-21 出版日期:2020-12-01
  • 通信作者: 孟晓军

Effect of naringin on MC3T3-E1 cell viability and expression of Akt pathway-related factors in high glucose environment

Chunshu Lin1, Xiaochun Shu1, Feina Xiao1, Hong Peng1, Chan Rong2, Jian Jiang3, Jiehua Hu1, Xiaojun Meng1,()   

  1. 1. Health Management Center, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China
    2. Internal medicine, Shunde Hospital Affiliated to Southern Medical University, Foshan 528330, China
    3. Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, China
  • Received:2020-03-21 Published:2020-12-01
  • Corresponding author: Xiaojun Meng
  • About author:
    Corresponding author:Meng Xiaojun, Email:
引用本文:

林春淑, 舒晓春, 肖菲娜, 彭虹, 容婵, 江健, 胡洁华, 孟晓军. 柚皮苷对高糖作用下MC3T3-E1细胞活力和Akt通路相关因子表达的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2020, 10(06): 321-327.

Chunshu Lin, Xiaochun Shu, Feina Xiao, Hong Peng, Chan Rong, Jian Jiang, Jiehua Hu, Xiaojun Meng. Effect of naringin on MC3T3-E1 cell viability and expression of Akt pathway-related factors in high glucose environment[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(06): 321-327.

目的

探讨柚皮苷(NG)对高糖环境下MC3T3-E1细胞活力的影响及可能的分子机制。

方法

体外培养小鼠MC3T3-E1细胞,实验分5组:对照组(正常无血清培养基)、高糖组(含25 mmol/L葡萄糖)、0.1 μmol/L +高糖组(0.1 μmol/L NG + 25 mmol/L葡萄糖)、1 μmol/L +高糖组(1 μmol/L NG+25 mmol/L葡萄糖)、10 μmol/L +高糖组(10 μmol/L NG+25 mmol/L葡萄糖)。药物干预后,采用CCK-8法检测细胞活力;实时荧光定量PCR(qPCR)法检测细胞成骨特异性转录因子(Runx2)、胰岛素样生长因子-1(IGF-1)、蛋白激酶(Akt1)mRNA的表达;蛋白质印迹法(Western blot)检测细胞碱性磷酸酶(ALP)、Akt1、IGF-1蛋白的表达。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。

结果

CCK8检测结果显示,与对照组比较,高糖组细胞OD值(12 h:0.90±0.01比0.80±0.01,24 h:1.00±0.05比0.84±0.01,48 h:1.09±0.03比0.90±0.01)均降低,差异有统计学意义(P < 0.01);与高糖组比较,0.1 μmol/L+高糖组细胞OD值(24 h:0.84±0.01比0.93±0.05,48 h:0.90±0.01比0.99±0.01)、1 μmol/L +高糖组和10 μmol/L+高糖组OD值(12 h:0.80±0.01比0.92±0.01、1.01±0.32,24 h:0.84±0.01比1.01±0.04、1.16±0.03,48 h:0.90±0.01比1.12±0.02、1.20±0.02)均升高,差异有统计学意义(P < 0.05)。与对照组比较,高糖组细胞Runx2、IGF-1、Akt1的mRNA的表达水平(24 h:1.00比0.34±0.02、1.00比0.34±0.01、1.00比0.15±0.02)、(48 h:1.00比0.72±0.03、1.00比1.09±0.07、1.00比0.38±0.04)降低,差异有统计学意义(P < 0.01)。与高糖组比较,1 μmol/L +高糖组和10 μmol/L+高糖组细胞Runx2、IGF-1、Akt1的mRNA表达水平(24 h:0.34±0.02比0.62±0.09、0.64±0.05,0.34±0.01比0.77±0.03、1.02±0.07,0.15±0.02比0.24±0.08、0.4±0.09)、(48 h:0.72±0.03比1.27±0.02、1.37±0.02,1.09±0.07比2.44±0.19、2.73±0.04,0.38±0.04比0.86±0.06、1.43±0.03)均升高,差异有统计学意义(P < 0.05)。与对照组比较,高糖组细胞ALP、Akt1、IGF-1蛋白表达水平(48 h:1.00比0.72±0.02、1.00比0.89±0.03、1.00比0.09±0.01)均降低,差异有统计学意义(P < 0.05);与高糖组比较,0.1 μmol/L+高糖组、1 μmol/L+高糖组和10 μmol/L+高糖组ALP、Akt1、IGF-1蛋白表达水平(48 h:0.72±0.02比1.92±0.02、2.30±0.30、3.09±0.10,0.89±0.03比1.50 ± 0.03、1.43±0.04、1.40±0.13,0.09±0.01比1.75±0.01、2.30±0.31、2.07±0.07)均升高,差异有统计学意义(P < 0.05)。

结论

NG逆转高糖诱导的MC3T3-E1细胞活力减退;同时改善高糖的抑制作用,促进MC3T3-E1细胞IGF-1、AKt-1、Runx2 mRNA和IGF-1、AKt-1、ALP蛋白的表达。

Objective

To explore the effect of naringin (NG) on MC3T3-E1 cell viability under high glucose environment and its possible molecular mechanism.

Methods

Cultured in vitro, mouse MC3T3-E1 cells were were divided into 5 groups, including normal control group, high glucose group (25 mmol/L glucose) , 0.1 μmol/L NG+25 mmol/L glucose group, 1 μmol/L NG +25 mmol/L glucose group, 10 μmol/L NG +25 mmol/L glucose group. After drug intervention, the cell viability was measured by CCK-8 assay. The mRNA expression of IGF-1, Akt1 and Runx2 were detected by qPCR. The protein expression of IGF-1, Akt1 and ALP were determined by Western blot. Univariate analysis of variance was used for comparison among groups, and LSD-t test was used for pairwise comparison between groups.

Results

The results of CCK8 assay showed that compared with the control group, the OD values of the high glucose group (12 h: 0.90±0.01 vs 0.80±0.01, 24 h: 1.00±0.05 vs 0.84±0.01, 48 h: 1.09±0.03 vs 0.90±0.01) were all reduced, and the difference was statistically significant (P < 0.01) . Compared with the high glucose group, the cell OD values were increased in 0.1 μmol/ L+glucose group (24 h: 0.93±0.05, 48 h: 0.99±0.01) , 1 μmol/L+ glucose group (12 h: 0.92±0.01, 24 h: 1.01±0.04, 48 h: 1.12±0.02) and 10 μmol/L+ glucose group (12 h: 1.01±0.32, 24 h: 1.16±0.03, 48 h: 1.20±0.02) , the difference was statistically significant (P < 0.05) . Compared with the control group, the high-glucose group decreased in Runx2, IGF-1, and Akt1 expression levels (24 h: 1.00 vs 0.34±0.02, 1.00 vs 0.34±0.01, 1.00 vs 0.15±0.02; 48 h: 1.00 vs 0.72±0.03, 1.00 vs 1.09±0.07, 1.00 vs 0.38±0.04) , and the differences were statistically significant. Compared with the high glucose group, 0.1 μmol/L+ glucose group had no significant difference in the gene expression. While the cell mRNA expression of Runx2, IGF-1 and Akt1 were increased in both 1 μmol/L+glucose group (24 h: 0.62±0.09, 0.77±0.03, 0.24±0.08; 48 h: 1.27±0.02, 2.44±0.19, 0.86±0.06) and 10 μmol/L+glucose group (24 h: 0.64±0.05, 1.02±0.07, 0.40±0.09; 48 h: 1.37±0.02, 2.73±0.04, 1.43±0.03) the differences were statistically significant. Compared with the control group, the cell expression levels of ALP, Akt1 and IGF-1 protein in the high glucose group were reduced (48 h: 1.00 vs 0.72±0.02, 1.00 vs 0.89±0.03, 1.00 vs 0.09±0.01) , and the differences were statistically significant (P < 0.05) . Compared with the high glucose group, after 48 hours of drug intervention, the expression of ALP, Akt1 and IGF-1 protein increased in 0.1 μmol/L+glucose group (1.92±0.02, 1.50±0.03, 1.75±0.01) , 1 μmol/L+glucose group (2.30±0.30, 1.43±0.04, 2.30±0.31) , 10 μmol/ L+GLUgroup (3.09±0.10, 1.40±0.13, 2.07±0.07) , the difference was statistically significant (P < 0.05) .

Conclusion

NG reversed the viability reduction of MC3T3-E1 cells induced by high glucose, while improved the inhibition of high glucose and promoted the expression of IGF-1, Akt-1, Runx2 mRNA and IGF-1, Akt-1, ALP protein in MC3T3-E1 cells.

表1 引物序列信息
表2 NG对高糖环境下MC3T3-E1的CCK-8检测结果( ± s
表3 干预24 h后不同浓度NG对高糖环境下MC3T3-E1细胞Runx2、IGF-1、Akt1 mRNA表达的影响( ± s
表4 干预48 h后不同浓度NG对高糖环境下MC3T3-E1细胞Runx2、IGF-1、Akt1 mRNA表达的影响( ± s
图1 免疫印迹检测不同时间点各组MC3T3-E1细胞IGF-1、Akt1、ALP蛋白的表达
表5 干预24 h后不同浓度NG对高糖环境下MC3T3-E1细胞ALP、Akt1、IGF-1蛋白表达的影响( ± s
表6 干预48 h后不同浓度NG对高糖环境下MC3T3-E1细胞ALP、Akt1、IGF-1蛋白表达的影响( ± s
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