大黄素通过调控miR-29a-5p/VEGFB介导的细胞凋亡抑制多柔比星诱导的心脏毒性

Emodin Inhibits Doxorubicin-Induced Cardiotoxicity by Regulating miR-29a-5p/VEGFB Mediated Apoptosis

目的研究大黄素(Emodin)对抗多柔比星(Doxorubicin,DOX)诱导的心脏毒性的作用和可能的机制。方法使用低、中、高剂量(5、15、25μmol·L^(-1))的大黄素预孵育H9C2细胞8 h,然后使用2.5μmol·L^(-1) DOX处理心肌细胞16 h建立心脏毒性损伤模型。采用MTT法检测细胞存活率;试剂盒检测H9C2细胞中心肌肌钙蛋白(cTnT)、乳酸脱氢酶(LDH)、肌酸激酶(CK)、超氧化物歧化酶(SOD)、活性氧(ROS)、丙二醛(MDA)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)水平以及Caspase-3和Caspase-9活性;使用TUNEL法检测H9C2细胞凋亡水平;采用RT-qPCR法检测miR-29a-5p和血管内皮生长因子B(Vascular endothelial growth factor-B,VEGFB)表达量;Western blot检测VEGFB蛋白及其他凋亡相关因子表达水平;在转染miR-29a-5p模拟物和抑制剂后,RT-qPCR和Western blot法检测VEGFB对miR-29a-5p的靶向性;H9C2细胞转染miR-29a-5p模拟物,在大黄素15μmol·L^(-1)剂量下,MTT法检测H9C2细胞活力、RT-qPCR和Western Blot法检测VEGFB表达水平。结果与DOX组相比,大黄素低、中、高剂量组均显著提高了H9C2细胞活力(P<0.05,P<0.01),降低了cTnT、LDH、CK水平(P<0.05,P<0.01);RT-qPCR检测结果显示miR-29a-5p表达水平显著降低,VEGFB表达水平显著升高(P<0.05,P<0.01);细胞内ROS、MDA水平显著降低,GSH和GSH-Px水平显著升高(P<0.05,P<0.01);TUNEL阳性细胞显著减少,Caspase-3和Caspase-9活性显著降低,Western blot结果显示VEGFB表达水平显著升高,凋亡相关因子Bax表达水平显著降低、Bcl-2和Bcl-xl表达水平显著升高(P<0.05,P<0.01)。在转染miR-29a-5p模拟物和抑制剂后,VEGFB表达水平分别降低和升高(P<0.05,P<0.01)。转染miR-29a-5p模拟物同时给予H9C2细胞大黄素15μmol·L^(-1)治疗后,DOX诱导的H9C2细胞活力明显降低的现象被逆转,并且VEGFB表达水平明显降低的现象也被逆转(P<0.05,P<0.01)。结论大黄素通过调节miR-29a-5p/VEGFB介导的心肌细胞凋亡显著降低了DOX诱导的心脏毒性。

Objective To investigate the effect and possible mechanism of Emodin on Doxorubicin(DOX)-induced cardiotoxicity.Methods H9C2 cells were preincubated with low,medium and high doses(5,15 and 25μmol·L^(-1))of Emodin for 8 h,and then cardiomyocytes were treated with 2.5μmol·L^(-1) DOX for 16 h to establish a cardiotoxic injury model.MTT assay was used to detect the cell survival rate.The levels of cardiac troponin(cTnT),lactate dehydrogenase(LDH),creatine kinase(CK),superoxide dismutase(SOD),reactive oxygen species(ROS),malondialdehyde(MDA),reduced glutathione(GSH),glutathione peroxidase(GSH-Px)and the activities of Caspase-3 and Caspase-9 in H9C2 cells were detected by kit;RT-qPCR was used to detect miR-29a-5p and vascular endothelial growth factor-B(VEGFB)expression levels.The expression levels of VEGFB protein and other apoptosis-related factors were detected by Western blot.After transfection of miR-29a-5p mimics and inhibitors,the targeting of VEGFB to miR-29a-5p was detected by RT-qPCR and Western blot.H9C2 cells were transfected with miR-29a-5p mimics,Under the dose of Emodin 15μmol·L^(-1),the activity of H9C2 cells was detected by MTT method,and the expression levels of miR-29a-5p and VEGFB were detected by RT-qPCR and Western blot.Results Compared with the DOX group,Emodin low,medium and high dose groups significantly increased the activity of H9C2 cells(P<0.05,P<0.01),and decreased the levels of cTnT,LDH and CK(P<0.05,P<0.01);RT-qPCR indicated that the expression level of miR-29a-5p decreased significantly and the expression level of VEGFB increased significantly(P<0.05,P<0.01);The levels of ROS and MDA decreased significantly,and the levels of GSH and GSH-Px increased significantly(P<0.05,P<0.01);TUNEL positive cells decreased significantly,and the activities of Caspase-3 and Caspase-9 decreased significantly.Western blot showed that the expression level of VEGFB increased significantly,the expression level of apoptosis-related factor Bax decreased significantly,and the expression levels of Bcl-2 and Bcl-xl increased significantly(P<0.05,P<0.01).After transfection with miR-29a-5p mimics and inhibitors,the expression of VEGFB decreased and increased respectively(P<0.05,P<0.01).After transfection of miR-29a-5p mimics and treatment with Emodin 15μmol·L^(-1) of H9C2 cells,the significant decrease in the activity of H9C2 cells induced by DOX was reversed,and the significant decrease in the expression level of VEGFB was also reversed(P<0.05,P<0.01).Conclusion Emodin significantly reduces DOX-induced cardiotoxicity by regulating miR-29a-5p/VEGFB-mediated cardiomyocyte apoptosis.