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

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)、0.1μmol/L+高糖组(0.1μmol/L NG+25 mmol/L葡萄糖)、1μmol/L+高糖组(1μmol/L NG+25 mmol/L葡萄糖)、10μmol/L+高糖组(10μmol/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,0.1μmol/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)、1μmol/L+高糖组和10μmol/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,1μmol/L+高糖组和10μmol/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,0.1μmol/L+高糖组、1μmol/L+高糖组和10μmol/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。

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

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