miR-495-3p联合黄氏总皂苷对高糖诱导的成骨细胞增殖、凋亡的影响及其机制研究

Effect of miR-495-3p combined with total saponins of Astragalus membranaceus on proliferation and apoptosis of osteoblasts induced by high glucose and its mechanism

目的探讨微小RNA-495-3p(miR-495-3p)联合黄芪总皂苷(TSA)在高糖诱导的成骨细胞增殖、凋亡中的作用及其机制。方法 2018年2月至2019年4月,将商购的小鼠成骨细胞MC3T3-E1用随机数字表法分为对照(NC)组,高糖组,高糖+不同浓度TSA组,高糖+miRNA阴性对照(miR-con)组,高糖+miR-495-3p组,高糖+TSA+抗miRNA阴性对照(anti-miR-con)组,高糖+TSA+抗miR-495-3p(anti-miR-495-3p)。噻唑蓝(MTT)法检测细胞增殖,流式细胞仪检测细胞凋亡,实时定量PCR(qRTPCR)检测miR-495-3p表达,Western blotting检测细胞周期蛋白D1(CyclinD1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleave-caspase-3)蛋白水平。结果与NC组比较,高糖组MC3T3-E1细胞存活率[(52.26±5.26)%比(100.26±10.03)%]和miR-495-3p[(0.25±0.03)比(1.00±0.10)]、CyclinD1表达量明显降低,细胞凋亡率[(35.33±3.53)%比(10.06±1.51)%]和Cleave-caspase-3蛋白水平显著升高(P<0.05)。与高糖组比较,0.5μg/mL、1.0μg/mL、2.0μg/mL、5.0μg/mL、10μg/mL浓度TSA显著提高MC3T3-E1细胞存活率[(63.26±6.02)%、(72.56±7.25)%、(90.46±8.88)%、(100.04±10.01)%、(102.56±10.23)%比(52.26±5.26)%],2.0μg/mL TSA显著降低细胞凋亡率[(18.56±1.66)%比(35.33±3.53)%]和Cleave-caspase-3水平,明显提高miR-495-3p[(0.95±0.09)比(0.25±0.03)]、CyclinD1表达量(P<0.05),这与高糖+miR-495-3p组促进MC3T3-E1增殖,抑制凋亡的作用相同。低表达miR-495-3p可以逆转TSA促进高糖诱导的MC3T3-E1细胞增殖作用和抑制细胞凋亡作用。结论黄芪总皂苷通过miR-495-3p促进高糖诱导的成骨细胞增殖,并抑制其凋亡。

Objective To investigate the effects of microRNA-495-3p(miR-495-3p)combined with total saponins of Astragalus membranaceus(TSA)on the proliferation and apoptosis of osteoblasts induced by high glucose and its mechanism.Methods From February 2018 to April 2019,purchased mouse osteoblasts MC3T3-E1 were randomly assigned into control(NC)group,high glucose group,high-glucose+different concentration TSA group,high glucose+miRNA negative control(miR-con)group,high glucose+miR-495-3p group,high glucose+TSA+anti-miRNA negative control(anti-miR-con)group,high glucose+TSA+anti-miR-495-3p(anti-miR-495-3p)group.Methyl thiazolyl tetrazolium(MTT)assay was used to detect cell proliferation,flow cytometry was applied to evaluate cell apoptosis,Quantitative real-time PCR(qRT-PCR)was used to determine the expression of miR-495-3p,and Western blotting was employed to analyze the levels of CyclinD1(CyclinD1)and active cysteine-containing Cleave-caspase-3 protein.Results Compared with NC group,the survival rate[(52.26±5.26)%vs(100.26±10.03)%]of MC3T3-E1 cells and the expressions of miR-495-3p[(0.25±0.03)vs(1.00±0.10)]and CyclinD1 in high glucose group were evidently decreased,while the apoptotic rate[(35.33±3.53)%vs(10.06±1.51)%]and the level of Cleave-caspase-3 protein were greatly increased(P<0.05).Compared with the high glucose group,0.5μg/mL,1.0μg/mL,2.0μg/mL,5.0μg/mL and 10μg/mL TSA significantly improved the survival rate of MC3T3-E1 cells[(63.26±6.02)%,(72.56±7.25)%,(90.46±8.88)%,(100.04±10.01)%,(102.56±10.23)%vs(52.26±5.26)%].2.0μg/mL TSA obviously reduced the apoptosis rate[(18.56±1.66)%vs(35.33±3.53)%]and the level of Cleave-caspase-3 significantly increased the expression of miR-495-3p[(0.95±0.09)vs(0.25±0.03)]and CyclinD1(P<0.05),which was the same as the high glucose+miR-495-3p group in facilitating the proliferation of MC3T3-E1 and inhibiting apoptosis.Low expression of miR-495-3p reversed the effect of TSA on promoting the proliferation of MC3T3-E1 cells induced by high glucose and suppressing apoptosis.Conclusion Total saponins of Astragalus membranaceus stimulates the proliferation and inhibits apoptosis of osteoblasts induced by high glucose through miR-495-3p.