雏菊叶龙胆酮通过调节Nrf2/HO-1和caspase-3通路减轻阿霉素诱导的H9c2心肌细胞损伤

Bellidifolin protects against doxorubicin-induced injury in H9c2 cardiomyocytes by regulating Nrf2/HO-1 and caspase-3 pathways

目的:探究雏菊叶龙胆酮(bellidifolin)对阿霉素(adriamycin;又称多柔比星,doxorubicin, DOX)诱导的H9c2心肌细胞损伤的作用及机制。方法:培养大鼠H9c2心肌细胞,采用CCK-8法筛选出DOX和雏菊叶龙胆酮的作用浓度和时间。将H9c2细胞随机分为正常对照组、DOX组和DOX+雏菊叶龙胆酮组进行相应处理,TUNEL染色检测细胞凋亡,Western blot检测Kelch样环氧氯丙烷相关蛋白1(Kelch-like ECH-associated protein 1, Keap1)、核因子E2相关因子2(nuclear factor E2-related factor 2, Nrf2)、血红素氧合酶1(heme oxygenase-1, HO-1)、谷氨酰半胱氨酸连接酶修饰亚基(glutamate cysteine ligase modifier subunit, GCLM)、NAD(P)H醌氧化还原酶1[NAD(P)Hquinone oxidoreductase 1, NQO1]、Bax和caspase-3蛋白的表达。结果:0.5~40μmol/L DOX处理H9c2细胞24 h,细胞活力呈剂量依赖性下降(P<0.05)。其中5μmol/L DOX处理24 h使细胞活力下降到59%。50μmol/L雏菊叶龙胆酮预处理H9c2细胞10 h可显著提高DOX诱导的细胞活力(P<0.05)。与对照组相比,DOX组H9c2细胞中Keap1、Nrf2、HO-1、GCLM和NQO1蛋白表达及Bcl-2/Bax比值显著下降(P<0.05),而caspase-3蛋白表达显著升高(P<0.05),细胞凋亡比例增多(P<0.05);雏菊叶龙胆酮预处理可以显著提高DOX处理的H9c2细胞中Keap1、Nrf2、HO-1、GCLM和NQO1蛋白表达及Bcl-2/Bax比值(P<0.05),降低caspase-3蛋白表达并减少细胞凋亡(P<0.05)。结论:雏菊叶龙胆酮可能通过激活Nrf2/HO-1通路、抑制caspase-3通路来减轻DOX诱导的H9c2心肌细胞损伤。

AIM:To investigate the effect of bellidifolin(Bel) on doxorubicin(DOX)-induced cytotoxicity in H9c2 cardiomyocytes and its mechanism. METHODS:Rat H9c2 cardiomyocytes were cultured, and then the action concentrations and time of DOX and Bel were determined by CCK-8 assay. Subsequently, H9c2 cardiomyocytes were randomly divided into control group, DOX group and DOX+Bel group. The protein levels of Kelch-like ECH-associated protein 1(Keap1), nuclear factor E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), glutamate cysteine ligase modifier subunit(GCLM), NAD(P)H-quinone oxidoreductase 1(NQO1), Bcl-2, Bax and caspase-3 were detected by Western blot analysis. Cell apoptosis was detected by TUNEL staining. RESULTS:After treatment of H9c2 cells with DOX(0. 5~40μmol/L) for 24 h, the cell viability decreased in a dose-dependent manner(P<0. 05). Cell viability fell to 59% with the exposure to 5 μmol/L DOX for 24 h. Pre-treatment of H9c2 cells with 50 μmol/L Bel for 10 h improved DOX-simulated cell viability significantly(P<0. 05). Compared with control group, the protein expression levels of Keap1, Nrf2, HO-1, GCLM and NQO1 and the ratio of Bcl-2/Bax were significantly decreased(P<0. 05), the protein level of caspase-3 was markedly increased(P<0. 05), and the cell apoptosis was increased by DOX(P<0. 05). Compared with DOX group, pretreatment with Bel significantly increased the protein expression levels of Keap1, Nrf2, HO-1, GCLM and NQO1 and the ratio of Bcl-2/Bax(P<0. 05), and reduced caspase-3 protein level and cell apoptosis of DOX-simulated H9c2 cardiomyocytes(P<0. 05). CONCLUSION:Bellidifolin attenuates DOX-induced injury of H9c2 cardiomyocytes by activating Nrf2/HO-1 pathway and inhibiting caspase-3 signaling pathway.