根皮苷通过下调miR-125a-5p减轻缺氧/复氧诱导的H9C2细胞氧化应激和凋亡

Phlorizin allevistes oxidative stress and apoptosis of rat cardiac myocytes H9C2 induced by hypoxia/reoxygenation by down-regulating miR-125a-5p

目的探讨根皮苷对缺氧/复氧(H/R)诱导的大鼠心肌细胞H9C2凋亡和氧化应激的影响及可能机制。方法体外培养H9C2细胞,用16、32、64μmol/L根皮苷预处理或转染anti-miR-125a-5p、anti-miR-NC、miR-125a-5p模拟物、模拟物阴性对照后建立H/R模型。CCK-8法测定细胞增殖,流式细胞术评估细胞凋亡,Western blot测定B淋巴细胞瘤-2(Bcl-2)蛋白和Bcl-2相关X(Bax)蛋白表达,比色法检测乳酸脱氢酶(LDH)释放量和超氧化物歧化酶(SOD)活性,实时荧光定量PCR(q RT-PCR)检测细胞中微小RNA-125a-5p(miR-125a-5p)表达。结果与H/R组比较,低、中及高剂量根皮苷处理后细胞抑制率、凋亡率、Bax蛋白表达、LDH含量、miR-125a-5p表达依次降低,SOD活性、Bcl-2蛋白表达依次增加(P<0.05)。抑制miR-125a-5p表达后,H/R处理细胞的抑制率、凋亡率、Bax蛋白表达及LDH含量降低,SOD活性、Bcl-2蛋白表达增加(P<0.05)。miR-125a-5p过表达逆转了根皮苷对H/R刺激H9C2细胞凋亡、增殖及氧化应激的作用。结论根皮苷可能通过降低miR-125a-5p表达来减轻H/R诱导的H9C2细胞的氧化应激和凋亡。

Objective To investigate the effect of phlorizin on the apoptosis and oxidative stress of rat cardiomyocytes H9C2 induced by hypoxia/reoxygenation(H/R)and its possible mechanism.Methods H9C2 cells were cultured in vitro.H/R model was established after pretreatment with different doses(16,32,64μmol/L)of phlorizin or transfection with anti-miR-125a-5p,anti-miR-NC,miR-125a-5p mimics and negative controls.Proliferation was detected by CCK-8,and apoptosis was detected by flow cytometry.The protein expression levels of B lymphoblastoma-2 associated X protein(Bax)and B lymphocytoma-2(Bcl-2)were detected by Western blot assay.The release of lactate dehydrogenase(LDH)and the activity of superoxide dismutase(SOD)were detected by colorimetric method.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of miR-125a-5p.Results Compared with the H/R group,inhibition rate,apoptosis rate,Bax protein expression,LDH content and miR-125a-5p expression were decreased after low,medium and high doses of phlorizin treatment(P<0.05),and SOD activity,Bcl-2 protein expression were increased(P<0.05).After inhibiting the expression of miR-125a-5p,the inhibition rate,apoptosis rate,Bax protein expression and LDH content of H9C2 cells induced by H/R were decreased(P<0.05),and SOD activity,Bcl-2 protein expression were increased(P<0.05).Overexpression of miR-125a-5p reversed the effect of phloridin on H/R-induced proliferation,apoptosis and oxidative stress of H9C2 cells.Conclusion Phlorizin may reduce H/R-induced apoptosis and oxidative stress in H9C2 cells by decreasing the expression of miR-125a-5p.