飞燕草素葡萄糖苷通过下调微小RNA-106a保护缺氧复氧引起的心肌细胞损伤

Delphinidin glucoside protects cardiomyocytes from hypoxia-reoxygenation-induced injury by downregulating miR-106a

目的探讨飞燕草素葡萄糖苷(DPg)对缺氧复氧(H/R)引起的心肌细胞损伤的影响及其机制。方法2018年4月至2019年10月,用H9C2细胞建立心肌细胞H/R损伤模型,用常规培养的细胞作为对照组;用终浓度为50μmol/L、100μmol/L、1000μmol/L的DPg培养液处理H9C2细胞24 h,而后进行H/R处理,分别记为H/R+50μmol/LDPg组、H/R+100μmol/LDPg组、H/R+1000μmol/LDPg组;抗微小RNA-106a(anti-miR-106a)阴性对照(anti-miR-con)、anti-miR-106a质粒转染至H9C2细胞后进行H/R处理记为H/R+anti-miR-con组,H/R+anti-miR-106a组。miR-106a阴性对照(miR-con)、miR-106a分别转染至H9C2细胞中同时用100μmol/L的DPg处理24 h,而后进行H/R处理,记为H/R+DPg+miR-con组,H/R+DPg+miR-106a组。MTT法检测细胞活性;蛋白质印迹法(Westernblotting)检测活化胱天蛋白酶-3(cleaved-caspase-3)、细胞周期蛋白D1(cyclinD1)蛋白表达水平;流式细胞术检测细胞凋亡;实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测miR-106a表达水平。结果与对照组相比,H/R组心肌细胞存活率显著降低,细胞凋亡率显著升高[(18.35±1.83)%比(7.05±0.71)%],cleaved-caspase-3表达水平显著升高,miR-106a表达水平显著升高[(3.56±0.36)比(1.00±0.11)](P<0.05);DPg处理的心肌细胞存活率显著升高;且100μmol/LDPg组于H/R组,细胞凋亡率显著降低[(10.25±1.03)%比(18.35±1.83)%],cleaved-caspase-3表达水平显著降低,miR-106a表达水平显著降低[(1.53±0.15)比(3.56±0.36)](P<0.05)。miR-106a低表达抑制H/R引起的心肌细胞凋亡;高表达miR-106a逆转了DPg对H9C2细胞增殖促进和凋亡抑制的作用。结论DPg可促进细胞增殖、抑制细胞凋亡,保护H/R引起的心肌细胞损伤;其机制可能与miR-106a有关。

Objective To investigate the effect and mechanism of delphinidin glucoside(DPg)on cardiomyocyte injury induced by hypoxia-reoxygenation(H/R).Methods From April 2018 to October 2019,H9C2 cells were used to establish a cardiomyocyte H/R in⁃jury model,and conventionally cultured cells were used as the control(NC)group;H9C2 cells were treated with DPg at concentrations of 50μmol/L,100μmol/L,and 1,000μmol/L for 24 h and then treated with H/R,which were denoted as the H/R+50μmol/L DPg group,H/R+100μmol/L DPg group,H/R+1,000μmol/L DPg group;anti-microRNA-106a(anti-miR-106a)negative control(anti-miR-con),anti-miR-106a plasmid was transfected into H9C2 cells and then treated with H/R,which was recorded as the H/R+anti-miR-con group and H/R+anti-miR-106a group.MiR-106a negative control(miR-con)and miR-106a were transfected into H9C2 cells simultane⁃ously with 100μmol/L DPg for 24 h,followed by H/R treatment,which was recorded as the H/R+DPg+miR-con group and H/R+DPg+miR-106a group.Cell viability was detected by MTT assay;cleaved caspase-3 and cyclin D1 protein expressions was detected by West⁃ern blotting;cell apoptosis was detected by flow cytometry;and qRT-PCR was used to detect the expression of miR-106a.Results Compared with the NC group,the survival rate of cardiomyocytes in the H/R group was significantly decreased,the apoptosis rate was significantly increased[(18.35±1.83)%vs.(7.05±0.71)%],the expression of cleaved caspase-3 was significantly increased,and the ex⁃pression of miR-106a was significantly increased[(3.56±0.36)vs.(1.00±0.11)](P<0.05);the survival rate of cardiomyocytes treated with DPg was significantly increased,while the apoptosis rate was significantly reduced in the 100μmol/L DPg group compared with the H/R group[(10.25±1.03)%vs.(18.35±1.83)%];the expression level of cleaved-caspase-3 was significantly reduced,which was simi⁃lar to the change in miR-106a[(1.53±0.15)vs.(3.56±0.36)](P<0.05).Low expression of miR-106a inhibits H/R-induced cardiomyocyte apoptosis,while high expression of miR-106a reverses the effects of DPg on the proliferation promotion and apoptosis inhibition of H9C2 cells.Conclusion DPg can promote cell proliferation,inhibit cell apoptosis and protect cardiomyocytes from H/R-induced inju⁃ry,and the mechanism may be related to miR-106a.