红黄煎剂通过调节AKT/FoxO3a通路改善阿霉素造成的心肌细胞凋亡
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摘要
目的:阿霉素是一种临床常用的化疗药物,其心脏毒性制约了它的临床使用,为了了解红黄煎剂在体外对阿霉素造成的心脏损伤降的作用,并探讨机制,做如下体外实验。方法:体外培养H9c2心肌细胞。用不同浓度红黄煎剂(HHD),阿霉素(DOX),红黄煎剂+阿霉素分别处理H9c2心肌细胞24 h,MTT法检测细胞活力,TUNEL和DAPI双染法检测细胞凋亡率,Western Blot法检测细胞凋亡相关蛋白。然后Si-Fox O3a转染H9c2心肌细胞,免疫荧光及Western Blot法确定转染效果,western及MTT法检测红黄煎剂,阿霉素,红黄煎剂+阿霉素对Si-Fox O3a转染的H9c2心肌细胞的作用。结果:不同浓度(0~2 mg·m L-1)的红黄煎剂作用与H9c2细胞,发现对细胞有一定的促进增值的作用(P <0. 05),阿霉素作用与H9c2细胞,其IC 50为(3. 03±0. 11)μM,加用红黄煎剂以后发现其抑制曲线下移,其IC 50为(7. 07±0. 48)μM,其差异有统计学意义(P <0. 01)。TUNEL&DAPI双染法检测细胞凋亡,发现HHD+DOX将单用DOX组的凋亡率从(43. 23±5. 22)%下调到(25. 22±3. 21)%。其差异有统计学意义(P <0. 01)。红黄煎剂能显著下调阿霉素引起的Caspase-3的激活(P <0. 05),同时能上调生存蛋白Bcl-2(P <0. 05)。同时红黄煎剂能够提高H9c2细胞中的Akt/Fox O3a磷酸化的水平。转染siFox O3a后细胞增值率下降(P <0. 05),并且减弱了HHD促进细胞增值的作用(P <0. 05) Western Blot检测也证明了HHD在转染细胞系中的保护作用降低(P <0. 05)。结论:红黄煎剂能降低阿霉素诱导的心肌细胞凋亡,其可能是通过调节AKT/Fox O3a产生的。
Objectie:Doxorubicin( DOX),an anthracycline-based antitumor drug,has been used in clinic. However,its cardiac toxicity limits its clinical use. Honghuang Decoction( HHD) is a formula that has been used to treat doxorubicin-induced cardiotoxicity for many years. Therefore,in this study,we aimed to investigate the underlying mechanisms in vitro. Methods: H9 c2 cells were randomly assigned to three groups and treated with doxorubicin,Honghuang Decoction,respectively. Cell growth was determined by the MTT cell proliferation and cytotoxicity assay kit. Cell apoptosis was assessed by using TUNEL and DAPI staining. The expression of c-caspase 3/caspase 3,bcl-2,Fox O3 a,and AKT was examined by Western blotting. The Fox O3 a gene in H9 c2 cells was knocked down by small interfering RNA(siRNA) to prove that HHD reduced cell apoptosis induced by doxorubicin via AKT/Fox O3 a pathway. Results: Our study proved the HHD reduced cell apoptosis in H9 c2 cell lines. This protective effect was achieved by HHD via Akt/Fox O3 a pathway. Conclusion: It can be suggested that HHD had the protective effect of doxorubicin-induced myocardial apoptosis via Akt/Fox O3 a pathway.
Objectie:Doxorubicin( DOX),an anthracycline-based antitumor drug,has been used in clinic. However,its cardiac toxicity limits its clinical use. Honghuang Decoction( HHD) is a formula that has been used to treat doxorubicin-induced cardiotoxicity for many years. Therefore,in this study,we aimed to investigate the underlying mechanisms in vitro. Methods: H9 c2 cells were randomly assigned to three groups and treated with doxorubicin,Honghuang Decoction,respectively. Cell growth was determined by the MTT cell proliferation and cytotoxicity assay kit. Cell apoptosis was assessed by using TUNEL and DAPI staining. The expression of c-caspase 3/caspase 3,bcl-2,Fox O3 a,and AKT was examined by Western blotting. The Fox O3 a gene in H9 c2 cells was knocked down by small interfering RNA(siRNA) to prove that HHD reduced cell apoptosis induced by doxorubicin via AKT/Fox O3 a pathway. Results: Our study proved the HHD reduced cell apoptosis in H9 c2 cell lines. This protective effect was achieved by HHD via Akt/Fox O3 a pathway. Conclusion: It can be suggested that HHD had the protective effect of doxorubicin-induced myocardial apoptosis via Akt/Fox O3 a pathway.
引文
[1]曲敬琨,张佳,张靖,等.蒽环类药物心脏毒性防治药物研究进展[J].中国肿瘤临床,2014,41(22):1474-1477.
[2]冯佳,罗英豪,王思雄.阿霉素脂质体与传统制剂治疗晚期乳腺癌效果及毒性的meta分析[J].中国老年学杂志,2017,37(20):5074-5077.
[3]秦聪,吴星恒.蒽环类药物的心脏毒性机制[J].实用临床医学,2017,18(6):104-107.
[4]俸艳英,阳志军.蒽环类药物的心脏毒性及其防治进展[J].中国癌症防治杂志,2015,7(3):235-237.
[5]江雪沁.三黄煎剂改善蒽环类药物心脏毒性症状的临床研究[D].南京:南京中医药大学,2016.
[6]乐音子,卞卫和,姚昶,等.三黄煎剂干预化疗期乳腺癌患者血液和心脏毒性的疗效[J].中国老年学杂志,2016,36(7):1629-1632.
[7]周君,姚昶,卞卫和,等.三黄抗氧化方改善乳腺癌术后蒽环类药物化疗心脏毒性临床观察[J].辽宁中医药大学学报,2016,18(7):154-157.
[8]宋祎.振荡运动联合黄芪注射液对雌鼠乳腺癌化疗相关性疲乏的影响[D].锦州:锦州医科大学,2016.
[9]Xu Y,Chen X,Chen X,et al.San Huang Decoction downregulates Aurora kinase A to inhibit breast cancer cell growth and enhance chemosenstivity to anti-tumor drugs[J].Pathology-Research and Practice,2016,212(8):696-703.
[10]Liu M H,Zhang Y,He J,et al.Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/Fox O3a pathway[J].International Journal of Molecular Medicine,2016,37(6):1661-1668.
[11]陈东伟.中医药联合化疗治疗乳腺癌临床疗效的有效性及安全性的系统评价[D].沈阳:辽宁中医药大学,2016.
[12]郝炜,刘胜.中医药防治蒽环类抗肿瘤药物所致心脏毒性研究进展[J].上海中医药杂志,2016(6):99-102.
[13]罗霞,程黎明.抗癌药物引起心脏毒性的生物标志物研究进展[J].内科急危重症杂志,2017,23(1):65-69.
[14]Liu M H,Zhang Y,He J,et al.Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/Fox O3a pathway[J].International Journal of Molecular Medicine,2016,37(6):616-619.
[15]黄岸清.基于PI3K/Akt/Fox O3a通路白藜芦酵保护糖尿病心肌病大鼠心功能的机制研究[D].广州:南方医科大学,2017.
[16]吴茂林,翟昌林,张亚梅,等.西洋参茎叶总皂苷对大鼠心肌缺血再灌注损伤后FOXO3a/Bim信号通路的影响[J].中华中医药学刊,2014,32(11):2763-2766.
[17]周波,钱坤,胡明珠,等.PI3K/Akt/Fox O3a/Bim信号通路介导硫化氢后处理对缺氧H9c2心肌细胞的保护作用[J].中国药理学通报,2017,33(7):971-976.
[18]白璐,卢今奇,孙卓,等.三氧化二砷通过Fox O3a抑制乳腺癌血管生成的体外实验[J].南京医科大学学报(自然科学版),2017,37(7):842-846.
[19]李昂,邢雅琪,李晓霞,等.氧化应激中ROS对FOXO3a转录因子的调控作用研究进展[J].中国药理学通报,2016,32(9):1203-1207.
[20]陈欣欣,胡小戊,夏婷,等.snoRNA来源的pi-sno81对乳腺癌中FOXO3a表达的影响[J].中国当代医药,2017,24(18):8-12.
[21]刘米华.PI3K/Akt/Fox O3a信号通路在FGF-2拮抗心肌细胞凋亡中的作用研究[D].衡阳:南华大学,2014.
[2]冯佳,罗英豪,王思雄.阿霉素脂质体与传统制剂治疗晚期乳腺癌效果及毒性的meta分析[J].中国老年学杂志,2017,37(20):5074-5077.
[3]秦聪,吴星恒.蒽环类药物的心脏毒性机制[J].实用临床医学,2017,18(6):104-107.
[4]俸艳英,阳志军.蒽环类药物的心脏毒性及其防治进展[J].中国癌症防治杂志,2015,7(3):235-237.
[5]江雪沁.三黄煎剂改善蒽环类药物心脏毒性症状的临床研究[D].南京:南京中医药大学,2016.
[6]乐音子,卞卫和,姚昶,等.三黄煎剂干预化疗期乳腺癌患者血液和心脏毒性的疗效[J].中国老年学杂志,2016,36(7):1629-1632.
[7]周君,姚昶,卞卫和,等.三黄抗氧化方改善乳腺癌术后蒽环类药物化疗心脏毒性临床观察[J].辽宁中医药大学学报,2016,18(7):154-157.
[8]宋祎.振荡运动联合黄芪注射液对雌鼠乳腺癌化疗相关性疲乏的影响[D].锦州:锦州医科大学,2016.
[9]Xu Y,Chen X,Chen X,et al.San Huang Decoction downregulates Aurora kinase A to inhibit breast cancer cell growth and enhance chemosenstivity to anti-tumor drugs[J].Pathology-Research and Practice,2016,212(8):696-703.
[10]Liu M H,Zhang Y,He J,et al.Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/Fox O3a pathway[J].International Journal of Molecular Medicine,2016,37(6):1661-1668.
[11]陈东伟.中医药联合化疗治疗乳腺癌临床疗效的有效性及安全性的系统评价[D].沈阳:辽宁中医药大学,2016.
[12]郝炜,刘胜.中医药防治蒽环类抗肿瘤药物所致心脏毒性研究进展[J].上海中医药杂志,2016(6):99-102.
[13]罗霞,程黎明.抗癌药物引起心脏毒性的生物标志物研究进展[J].内科急危重症杂志,2017,23(1):65-69.
[14]Liu M H,Zhang Y,He J,et al.Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/Fox O3a pathway[J].International Journal of Molecular Medicine,2016,37(6):616-619.
[15]黄岸清.基于PI3K/Akt/Fox O3a通路白藜芦酵保护糖尿病心肌病大鼠心功能的机制研究[D].广州:南方医科大学,2017.
[16]吴茂林,翟昌林,张亚梅,等.西洋参茎叶总皂苷对大鼠心肌缺血再灌注损伤后FOXO3a/Bim信号通路的影响[J].中华中医药学刊,2014,32(11):2763-2766.
[17]周波,钱坤,胡明珠,等.PI3K/Akt/Fox O3a/Bim信号通路介导硫化氢后处理对缺氧H9c2心肌细胞的保护作用[J].中国药理学通报,2017,33(7):971-976.
[18]白璐,卢今奇,孙卓,等.三氧化二砷通过Fox O3a抑制乳腺癌血管生成的体外实验[J].南京医科大学学报(自然科学版),2017,37(7):842-846.
[19]李昂,邢雅琪,李晓霞,等.氧化应激中ROS对FOXO3a转录因子的调控作用研究进展[J].中国药理学通报,2016,32(9):1203-1207.
[20]陈欣欣,胡小戊,夏婷,等.snoRNA来源的pi-sno81对乳腺癌中FOXO3a表达的影响[J].中国当代医药,2017,24(18):8-12.
[21]刘米华.PI3K/Akt/Fox O3a信号通路在FGF-2拮抗心肌细胞凋亡中的作用研究[D].衡阳:南华大学,2014.