目的:探究小檗碱(berberine, BBR)对柔红霉素(daunorubicin, DNR)损伤心肌细胞的保护效应,以及CaN-NFAT通路在其中的作用。方法:将生长良好的H9C2心肌细胞按简单随机分组方法分为4组:CON组、DNR组、DNR+BBR组和DNR+VIVIT(通路抑制剂)组,显微镜观察H9C2心肌细胞形态;CCK-8法检测H9C2心肌细胞存活率;生化试剂盒检测H9C2心肌细胞损伤情况;Western Blot法检测NFATc4蛋白及凋亡蛋白Bax、Bcl-2表达。结果:(1)显微镜下细胞形态:与CON组相比,DNR组心肌细胞数量减少;与DNR组相比,DNR+BBR组、DNR+VIVIT组心肌细胞数量增加;(2)CCK-8法测细胞存活率:与CON组相比,DNR组心肌细胞存活率明显下降(P＜0.05),与DNR组相比,DNR+BBR组、DNR+VIVIT组心肌细胞存活率上升(P＜0.05);(3)肌酸激酶(creatine kinase,CK)含量及乳酸脱氢酶(lactate dehydrogenase,LDH)释放量:①与CON组相比,DNR组CK活性升高(P＜0.05);与DNR组相比,DNR+BBR、DNR+VIVIT组CK含量降低(P＜0.05);②与CON组相比,DNR组的LDH释放量升高(P＜0.05);与DNR组相比,DNR+BBR、DNR+VIVIT组LDH释放量降低(P＜0.05);(4)Western Blot:①与CON组相比,DNR组Bax表达、Bax/Bcl-2比值及NFATc4表达升高而Bcl-2表达下降(P＜0.05);②与DNR组相比,DNR+BBR组、DNR+VIVIT组Bax表达、Bax/Bcl-2及NFATc4表达下降而Bcl-2表达升高(P＜0.05)。结论:BBR可能通过调控CaN-NFAT信号通路抑制DNR诱导的心肌细胞损伤,减少细胞凋亡。
Objective: To investigate the protective effect of berberine (BBR) on daunorubicin (DNR)-induced cardiomyocyte injury and the role of CaN-NFAT pathway. Methods: H9C2 cardiomyocytes were randomly divided into 4 groups : CON group, DNR group, DNR + BBR group and DNR + VIVIT (pathway inhibitor) group. The morphology of H9C2 cardiomyocytes was observed by microscope. CCK-8 method was used to detect the survival rate of H9C2 cardiomyocytes. The injury of H9C2 cardiomyocytes was detected by biochemical kit. Western Blot was used to detect the expression of NFATc4 protein and apoptotic proteins Bax and Bcl-2. Results: (1)Cell morphology under the microscope: compared with the CON group, the number of cardiomyocytes in the DNR group decreased. Compared with the DNR group, the number of cardiomyocytes in the DNR+BBR group and the DNR+VIVIT group increased. (2)CCK-8 assay: compared with CON group, the survival rate of cardiomyocytes in DNR group was significantly decreased(P＜0.05). Compared with DNR group, the survival rate of cardiomyocytes in DNR+BBR group and DNR+VIVIT group was increased(P＜0.05). (3)Creatine kinase (CK) content and lactate dehydrogenase (LDH) release: ①Compared with the CON group, the CK activity of the DNR group increased(P＜0.05). Compared with the DNR group, the CK content in the DNR+BBR and DNR+VIVIT groups decreased(P＜0.05). ②Compared with the CON group, the LDH release in the DNR group was increased(P＜0.05). Compared with the DNR group, the LDH release in the DNR+BBR and DNR+VIVIT groups was decreased(P＜0.05). (4)Western Blot: ①Compared with the CON group, the expression of Bax, Bax/Bcl-2 ratio and NFATc4 in the DNR group increased, while the expression of Bcl-2 decreased(P＜0.05). ②Compared with DNR group, the expression of Bax, Bax/Bcl-2 and NFATc4 in DNR+BBR group and DNR+VIVIT group decreased, while the expression of Bcl-2 increased(P＜0.05). Conclusion: BBR may inhibit DNR induced cardiomyocyte injury and reduce apoptosis by regulating CaN-NFAT signaling pathway.
[1] Alizadehasl A, Ghadimi N, Kaveh S, et al. Prevention of anthracycline-induced cardiotoxicity: a systematic review and network meta-analysis[J]. Int J Clin Pharm, 2021, 43(1): 25-34.
[2] Stojak M, Mazur L, Opydo-Chanek M, et al. Effects of structural modifications of daunorubicin on in vitro antileukemic activity[J]. Anticancer Res, 2012, 32(12): 5271-5277.
[3] Matthews ER, Johnson OD, Horn KJ, et al. Anthracyclines induce cardiotoxicity through a shared gene expression response signature[J]. PLoS Genet, 2024, 20(2): e1011164.
[4] Fujiwara SI, Murahashi R, Nakashima H, et al. Effect of cumulative daunorubicin dose on cardiotoxicity after allogeneic stem cell transplantation[J]. Leuk Res, 2022, 121: 106951.
[5] Naz I, Masoud MS, Chauhdary Z, et al. Anti-inflammatory potential of berberine-rich extract via modulation of inflammation biomarkers[J]. J Food Biochem, 2022, 46(12): e14389.
[6] 冯晓明, 钱前, 李松, 等. 黄连及其提取物防治肺部感染的研究进展[J]. 环球中医药, 2024, 17(2): 359-364.
[7] Zhang L, Wu X, Yang R, et al. Effects of berberine on the gastrointestinal microbiota[J]. Front Cell Infect Microbiol, 2020, 10:588517.
[8] 玄欣彤. 中药黄连有效成分-小檗碱对高脂喂养ApoE -/-小鼠主动脉粥样硬化的影响[D]. 南京: 南京中医药大学, 2018.
[9] 李旸. 盐酸小檗碱对血脂异常伴动脉粥样硬化病人的疗效及安全性观察[D]. 长春: 吉林大学, 2019.
[10] 王春艳, 王桂茹, 李晶, 等. 盐酸小檗碱抗实验性心律失常的研究[J]. 中国老年学杂志, 2009, 29(6): 651-653.
[11] 杨静, 王巧云. 小檗碱和利多卡因抗实验性心律失常作用[J]. 中国现代药物应用, 2010, 4(3): 14-15.
[12] 叶路亮, 金华, 何彦虎, 等. 黄连素治疗心血管疾病研究概况[J]. 中医药临床杂志, 2023, 35(1): 174-178.
[13] Crabtree GR, Schreiber SL. SnapShot: Ca2+-calcineurin-NFAT signaling[J]. Cell, 2009, 138(1): 210, 210, e1.
[14] Nguyen T, Di Giovanni S. NFAT signaling in neural development and axon growth[J]. Int J Dev Neurosci, 2008, 26(2): 141-145.
[15] Wang Y, Liao J, Luo Y, et al. Berberine alleviates doxorubicin-induced myocardial injury and fibrosis by eliminating oxidative stress and mitochondrial damage via promoting Nrf-2 pathway activation[J]. Int J Mol Sci, 2023, 24(4):3257.
[16] 武彦昭, 张兰, 武子笑, 等. SIRT1/p66Shc介导小檗碱对阿霉素诱导心肌毒性拮抗作用研究[J]. 广州中医药大学学报, 2022, 39(6): 1374-1382.
[17] Huang Z, Han Z, Ye B, et al. Berberine alleviates cardiac ischemia/reperfusion injury by inhibiting excessive autophagy in cardiomyocytes[J]. Eur J Pharmacol, 2015, 762: 1-10.
[18] Zhang P, Wang Q, Lin Z, et al. Berberine inhibits growth of liver cancer cells by suppressing glutamine uptake[J]. Onco Targets Ther, 2019, 12: 11751-11763.
[19] Jabbarzadeh Kaboli P, Leong MP, Ismail P, et al. Antitumor effects of berberine against EGFR, ERK1/2, P38 and AKT in MDA-MB231 and MCF-7 breast cancer cells using molecular modelling and in vitro study[J]. Pharmacol Rep, 2019, 71(1): 13-23.
[20] Yang J, Wang HD, Lu DX, et al. Effects of neutral sulfate berberine on LPS-induced cardiomyocyte TNF-alpha secretion, abnormal calcium cycling, and cardiac dysfunction in rats[J]. Acta Pharmacol Sin, 2006, 27(2): 173-8.
[21] Szenczi O, Kemecsei P, Holthuijsen MF, et al. Poly(ADP-ribose) polymerase regulates myocardial calcium handling in doxorubicin-induced heart failure[J]. Biochem Pharmacol, 2005, 69(5): 725-32.
