甘草次酸通过HIF-1α/BNIP3介导的线粒体自噬改善索拉非尼诱导的心肌细胞损伤及凋亡*

doi:10.3870/j.issn.1672-0741.24.12.008

摘要/Abstract

摘要： 目的基于缺氧诱导因子-1α(HIF-1α)/腺病毒E1B相互作用蛋白3(BNIP3)信号通路,探究甘草次酸对索拉非尼诱导的心肌细胞损伤及凋亡的影响。方法 MTT检测筛选甘草次酸作用于大鼠心肌细胞H9c2的浓度,随后将实验分为Control组、索拉非尼组、索拉非尼+甘草次酸组、索拉非尼+甘草次酸+HIF-1α抑制剂YC-1组、索拉非尼+甘草次酸组+YC-1+自噬激动剂雷帕霉素(RAPA)组。试剂盒测定乳酸脱氢酶(LDH)释放量、丙二醛(MDA)水平,荧光探针法测定细胞内总活性氧(ROS)水平,流式细胞术检测细胞凋亡,免疫荧光法检测LC3蛋白的表达及BNIP3与LC3的细胞内共定位,Western blot检测线粒体自噬相关蛋白LC3Ⅱ/Ⅰ、p62、Beclin-1的表达,qRT-PCR及Western blot分别检测HIF-1α、BNIP3 mRNA及蛋白的表达水平。结果相对于索拉非尼组,添加100、200 μmol/L甘草次酸处理的H9c2细胞活力显著提升(均P<0.05)。相对于Control组,索拉非尼组H9c2细胞的LDH释放量、MDA及ROS水平显著上调,细胞的凋亡率显著升高,细胞中LC3Ⅱ/Ⅰ、Beclin-1蛋白的表达水平明显降低,p62水平显著上调,细胞中LC3荧光强度减弱,LC3和BNIP3共定位荧光强度减弱,HIF-1α、BNIP3 mRNA及蛋白的表达显著下调(均P<0.05);相对于索拉非尼组,索拉非尼+甘草次酸组H9c2细胞的LDH释放量、MDA及ROS水平显著下调,细胞的凋亡率显著降低,细胞中LC3Ⅱ/Ⅰ、Beclin-1蛋白的表达水平显著上调,p62水平显著下调,细胞中LC3荧光强度增强,LC3和BNIP3共定位荧光强度增强,HIF-1α、BNIP3 mRNA及蛋白的表达显著上调(均P<0.05);YC-1能显著逆转以上指标(均P<0.05),RAPA能显著抑制YC-1的作用(均P<0.05)。结论甘草次酸能够通过激活HIF-1α/BNIP3信号通路促进线粒体自噬抑制索拉非尼诱导的心肌细胞损伤及凋亡。

关键词: 甘草次酸, HIF-1α/BNIP3, 线粒体自噬, 索拉非尼, 心肌细胞

Abstract: Objective The effects of glycyrrhetinic acid on sorafenib-induced cardiomyocyte injury and apoptosis were investigated via the hypoxia-inducible factor-1α(HIF-1α)/adenovirus E1B 19 kDa-interacting protein 3(BNIP3)signaling pathway. Methods MTT was used to determine the effects of glycyrrhetinic acid on H9c2 cells in rat cardiomyocytes.The experiment was divided into control group,sorafenib group,sorafenib+glycyrrhetinic acid group,sorafenib+glycyrrhetinic acid+HIF-1α inhibitor YC-1 group,and sorafenib+glycyrrhetinic acid+YC-1+autophagy agonist rapamycin(RAPA)group.A kit was used to determine LDH release and MDA levels.The fluorescent probe method was used to determine the level of total ROS in the cells.Apoptosis was detected via flow cytometry.Immunofluorescence was used to detect the protein expression of LC3 and the colocalization of BNIP3 and LC3.The expression levels of the mitochondrial autophagy-related proteins LC3 II/I,p62 and Beclin-1 were detected via Western blotting.RT-qPCR and Western blotting were used to detect the mRNA and protein expression levels of HIF-1α and BNIP3,respectively. Results Compared with that in the sorafenib group,the viability of H9c2 cells in the 100 and 200 μmol/L glycyrrhetinic acid groups was significantly increased(P<0.05).Compared with those in the control group,LDH release and the MDA and ROS levels in H9c2 cells in the sorafenib group were significantly up-regulated(P<0.05);the apoptosis rate of cells was significantly increased(P<0.05);the expression levels of LC3 II/I and Beclin-1 protein were significantly down-regulated(P<0.05);and the levels of p62 were significantly up-regulated(P<0.05);the fluorescence intensity of LC3 in H9c2 cells was weakened,and the co-localization of LC3 and BNIP3 in H9c2 cells was reduced,the mRNA and protein expression levels of HIF-1α and BNIP3 were significantly down-regulated(P<0.05).Compared with those in the sorafenib group,LDH release and the MDA and ROS levels in H9c2 cells in the sorafenib+glycyrrhetinic acid group were significantly down-regulated(P<0.05);the apoptosis rate of H9c2 cells was significantly reduced(P<0.05);the expression levels of LC3 II/I and Beclin-1 proteins were significantly up-regulated(P<0.05);the p62 levels were significantly down-regulated(P<0.05);the fluorescence intensity of LC3 in H9c2 cells was enhanced,the colocalization of LC3 and BNIP3 in H9c2 cells was enhanced,the mRNA and protein expression levels of HIF-1α and BNIP3 were significantly up-regulated(P<0.05).YC-1 significantly reversed these effects(P<0.05),and RAPA significantly inhibited the effect of YC-1(P<0.05). Conclusion Glycyrrhetinic acid can promote mitophagy by activating the HIF-1α/BNIP3 signaling pathway and can inhibit sorafenib-induced cardiomyocyte injury and apoptosis.

Key words: glycyrrhetinic acid, HIF-1α/BNIP3, mitophagy, sorafenib, cardiomyocyte

中图分类号:

R542.2

张显林, 王丽珍, 张洁, 张利芳. 甘草次酸通过HIF-1α/BNIP3介导的线粒体自噬改善索拉非尼诱导的心肌细胞损伤及凋亡^*[J]. 华中科技大学学报（医学版）, 2026, 55(1): 68-75.

Zhang Xianlin, Wang Lizhen, Zhang Jie et al. Glycyrrhetinic Acid Ameliorates Sorafenib-induced Cardiomyocyte Injury and Apoptosis through HIF-1α/BNIP3-mediated Mitophagy[J]. Acta Medicinae Universitatis Scientiae et Technologiae Huazhong, 2026, 55(1): 68-75.

参考文献

[1] Fan Q Q,Tian H,Cheng J X,et al.Research progress of sorafenib drug delivery system in the treatment of hepatocellular carcinoma:An update[J].Biomed Pharmacother,2024,177:117118.
[2] Touyz R M,Herrmann J.Cardiotoxicity with vascular endothelial growth factor inhibitor therapy[J].NPJ Precis Oncol,2018,2:13.
[3] 曹蓉,刘灵,郭林,等.甘草次酸调控铁死亡缓解舒尼替尼肝损伤的机制研究[J].中南药学,2024,22(8):1993-1998.
Cao R,Liu L,Guo L,et al.Glycyrrhetinic acid alleviates sunitinib-induced liver injury via regulating ferroptosis and related mechanism[J].Central South Pharmacy,2024,22(8):1993-1998.
[4] 侯佳华,韦秋,黄露,等.甘草次酸拮抗阿霉素心脏毒性的作用研究[J].南开大学学报:自然科学版,2022,55(6):7-14.
Hou J H,Wei Q,Huang L,et al.Study on the antagonistic affect of glycyrrhetinic acid on the cardiotoxicity of doxorubicin[J].Acta Scientiarum Naturalium Universitatis Nankaiensis,2022,55(6):7-14.
[5] Brogyanyi T,Kejík Z,Veselá K,et al.Iron chelators as mitophagy agents:Potential and limitations[J].Biomed Pharmacother,2024,179:117407.
[6] 梁凡凡,高晨盈,马文卓,等.索拉非尼致心肌线粒体动力学、自噬失衡与凋亡的作用及机制研究[C]//首届东方药理论坛暨2018年全国心脑血管药理学术会议、上海市药理学会第十九届学术年会论文集.上海:中国药理学会,2018:109-110.
[7] Wang X,Wu Z,Zhang Y,et al.Autophagy induced by hypoxia in pulpitis is mediated by HIF-1α/BNIP3[J].Arch Oral Biol,2024,159:105881.
[8] Zhu N,Li J,Li Y,et al.Berberine protects against simulated ischemia/reperfusion injury-induced H9c2 cardiomyocytes apoptosis in vitro and myocardial ischemia/reperfusion-induced apoptosis in vivo by regulating the mitophagy-mediated HIF-1α/BNIP3 pathway[J].Front Pharmacol,2020,11:367.
[9] 邢燕,历飞,林大勇,等.甘草次酸通过PI3K-AKT途径抑制H₂O₂所致大鼠心肌细胞氧化损伤[J].现代生物医学进展,2018,18(6):1044-1049.
Xing Y,Li F,Lin D Y,et al.Glycyrrhetinic acid decreases H₂O₂-induced oxidative injury of H9c2 cells through PI3K-AKT pathway[J].Progress in Modern Biomedicine,2018,18(6):1044-1049.
[10] 姜卉,魏甜,李建平,等.葛根素对索拉非尼心肌毒性的保护及作用机制[J].山东大学学报:医学版,2022,60(8):14-22.
Jiang H,Wei T,Li J P,et al.Protective effects and molecular mechanism of puerarin on sorafenib-induced cardiotoxicity[J].Journal of Shandong University:Health Sciences,2022,60(8):14-22.
[11] Shen Y,Chen G,Zhuang L,et al.ARHGAP4 mediates the Warburg effect in pancreatic cancer through the mTOR and HIF-1α signaling pathways[J].Onco Targets Ther,2019,12:5003-5012.
[12] Shangguan J,Liu G,Xiao L,et al.Meteorin-like/meteorin-β p-rotects against cardiac dysfunction after myocardial infarction in mice by inhibiting autophagy[J].Exp Ther Med,2024,28(1):293.
[13] Li Y,Wang W,Gao R,et al.Genome-wide prioritization reveals novel gene signatures associated with cardiotoxic effects of tyrosine kinase inhibitors[J].Oncol Lett,2021,21(2):94.
[14] Qin S,Bi F,Gu S,et al.Donafenib versus sorafenib in first-line treatment of unresectable or metastatic hepatocellular carcinoma:A randomized,open-label,parallel-controlled phase II-III trial[J].J Clin Oncol,2021,39(27):3002-3011.
[15] Zamorano J L,Lancellotti P,Rodriguez Muñoz D,et al.2016 ESC Position Paper on cancer treatments and cardiovascular toxicity developed under the auspices of the ESC Committee for Practice Guidelines:The task force for cancer treatments and cardiovascular toxicity of the European Society of Cardiology(ESC)[J].Eur Heart J,2016,37(36):2768-2801.
[16] Ma W,Liu M,Liang F,et al.Cardiotoxicity of sorafenib is mediated through elevation of ROS level and CaMKII activity and dysregulation of calcium homoeostasis[J].Basic Clin Pharmacol Toxicol,2020,126(2):166-180.
[17] Chen L,Gong J,Yong X,et al.A review of typical biological activities of glycyrrhetinic acid and its derivatives[J].RSC Adv,2024,14(10):6557-6597.
[18] Han D,Wang F,Jiang Q,et al.Enhancing cardioprotection th-rough neutrophil-mediated delivery of 18β-glycyrrhetinic acid in myocardial ischemia/reperfusion injury[J].Adv Sci,2024,11(42):2406124.
[19] Shi M,Zhang S,Rong J,et al.Identification of 18β-glycyrrhetinic acid as an AGT inhibitor against LPS-induced myocardial dysfunction via high throughput screening[J].Biochem Pharmacol,2024,223:116127.
[20] Cheng Y,Wu X,Nie X,et al.Natural compound glycyrrhetinic acid protects against doxorubicin-induced cardiotoxicity by activating the Nrf2/HO-1 signaling pathway[J].Phytomedicine,2022,106:154407.
[21] 马治.甘草次酸保护顺铂诱导H9c2心肌细胞损伤的机制研究[D].天津:天津中医药大学,2022.
Ma Z.Mechanism of glycyrrhetinic acid protecting cisplatin-induced H9c2 cardiomyocytes injury[D].Tianjin:Tianjin University of Traditional Chinese Medicine,2022.
[22] Zeng J,Chen H,Liu X,et al.Cuproptosis in microsatellite stable colon cancer cells affects the cytotoxicity of CD8⁺T through the WNT signaling pathway[J].Chem Biol Interact,2024,403:111239.
[23] He Q,Lu S,Wang J,et al.Lactobacillus salivarius and berberine alleviated yak calves’diarrhea via accommodating oxidation resistance,inflammatory factors,and intestinal microbiota[J].Animals,2024,14(16):2419.
[24] Hushmandi K,Einollahi B,Aow R,et al.Investigating the interplay between mitophagy and diabetic neuropathy:uncovering the hidden secrets of the disease pathology[J].Pharmacol Res,2024,208:107394.
[25] Song Z,Song H,Liu D,et al.Overexpression of MFN2 alleviates sorafenib-induced cardiomyocyte necroptosis via the MAM-CaMKIIδ pathway in vitro and in vivo[J].Theranostics,2022,12(3):1267-1285.
[26] 刘爱华,冷锦红,刘子英,等.加味桂枝茯苓汤通过PINK1/Parkin信号通路调控线粒体自噬途径减轻PDPN大鼠坐骨神经损伤的作用机制[J].中国实验方剂学杂志,2024,30(21):42-51.
Liu A H,Leng J H,Liu Z Y,et al.Mechanism of Jiawei G-uizhi Fuling decoction in alleviating sciatic nerve injury in PDPN rats by regulating mitophagy through PINK1/parkin signaling pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2024,30(21):42-51.
[27] Sato T,Takeda N.The roles of HIF-1α signaling in cardiovascular diseases[J].J Cardiol,2023,81(2):202-208.
[28] Neikirk K,Marshall A G,Santisteban M M,et al.BNIP3 as a new tool to promote healthy brain aging[J].Aging Cell,2024,23(2):e14042.
[29] Xia J,Chen C,Sun Y,et al.Panax quinquefolius saponins and panax notoginseng saponins attenuate myocardial hypoxia-reoxygenation injury by reducing excessive mitophagy[J].Cell Biochem Biophys,2024,82(2):1179-1191.
[30] Zhang Y N,Pang Y X,Liu D W,et al.JMJD5 attenuates oxygen-glucose deprivation and reperfusion-induced injury in cardiomyocytes through regulation of HIF-1α-BNIP3[J].Kaohsiung J Med Sci,2022,38(1):38-48.

甘草次酸通过HIF-1α/BNIP3介导的线粒体自噬改善索拉非尼诱导的心肌细胞损伤及凋亡^*

Glycyrrhetinic Acid Ameliorates Sorafenib-induced Cardiomyocyte Injury and Apoptosis through HIF-1α/BNIP3-mediated Mitophagy

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 10

编辑推荐

Metrics

本文评价

[1]	程月月, 郭苒, 张鹤骞, 吴晓爽, 康永安, 祁春晖, 杨静怡, 刘其伟, 赵迪, 高社干, 齐义军. 牙龈卟啉单胞菌通过HIF-1α增加线粒体自噬活性抑制食管鳞癌细胞铁死亡[J]. 华中科技大学学报（医学版）, 2025, 54(4): 470-476.
[2]	戴婷, 徐亚宁, 谌赟, 蔡毅, 刘敏. 亚精胺通过miR-451介导自噬缓解阿霉素诱导的心肌细胞衰老[J]. 华中科技大学学报（医学版）, 2024, 53(6): 791-795.
[3]	叶子丰, 邝高艳, 院一蔚, 邱礼国, 许晓彤, 文志, 卢敏. 线粒体自噬对骨性关节炎的影响[J]. 华中科技大学学报（医学版）, 2024, 53(6): 858-864.
[4]	顾浩南, 王淇, 孙惠美, 周易, 梁华敏. IL-6对小鼠胚胎心肌细胞增殖能力的影响及发育依赖性变化[J]. 华中科技大学学报（医学版）, 2023, 52(6): 743-748.
[5]	文莉雅 , 雅各布·马西卡 , 王子赫, 王松, 梁华敏. 单细胞/单细胞核 RNA 测序分析心脏细胞转录谱推进心脏细胞类型和心脏发育研究[J]. 华中科技大学学报（医学版）, 2023, 52(4): 554-557.
[6]	张瑶, 杨少娟, 薛佳佳, 江燕丽, 刘侃玲. SGLT2i通过HIF1α/NRF2/HO-1通路对缺氧-复氧心肌细胞老化的保护作用[J]. 华中科技大学学报（医学版）, 2023, 52(3): 301-306.
[7]	邓方方, 李继勇, 张力, 邹高锐, 陈治军, 乐薇. 七氟醚后处理通过调节FTO/KCNJ2对缺氧/复氧所致心肌细胞损伤的保护作用[J]. 华中科技大学学报（医学版）, 2023, 52(2): 174-180.
[8]	周云洁, 宋歌, 路永平, 韩丽华△. miR-219a-5p通过下调 KLF4减轻 H2O2 诱导的人心肌细胞氧化损伤[J]. 华中科技大学学报（医学版）, 2021, 50(6): 723-.
[9]	张荣城, 吴玉塘. 下调lncRNA KCNQ1OT1抑制 H2O2诱导的心肌细胞凋亡和氧化损伤[J]. 华中科技大学学报（医学版）, 2021, 50(6): 734-.
[10]	王文华, 刘洋, 吕洋, 赵亚如, 王海萍△. 骨髓间充质干细胞向心肌细胞分化的研究进展[J]. 华中科技大学学报（医学版）, 2021, 50(6): 811-816.