南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (8): 1205-1211.doi: 10.12122/j.issn.1673-4254.2022.08.13

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和厚朴酚可体外减轻阿霉素诱导的心肌毒性：基于激活AMPK/Nrf2信号通路抑制细胞焦亡

熊凤梅，刘瑞萍，李 洋，孙 娜   

  1. 西安市儿童医院药剂科，营养科，陕西 西安 710003；西安医学院基础部基础医学研究所，陕西 西安 710021
  • 出版日期:2022-08-20 发布日期:2022-09-05

Honokiol reduces doxorubicin-induced cardiotoxicity in vitro by inhibiting pyroptosis via activating AMPK/Nrf2 signaling

XIONG Fengmei, LIU Ruiping, LI Yang, SUN Na   

  1. Department of Pharmacy, Department of Nutrition, Xi'an Children's Hospital, Xi'an 710003, China; Institute of Basic Medical Science, Xi'an Medical University, Xi'an 710021, China
  • Online:2022-08-20 Published:2022-09-05

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摘要： 目的 探讨和厚朴酚（HKL）改善阿霉素（DOX）诱导的H9c2心肌细胞毒性的作用及机制。方法 采用DOX处理H9c2细胞心肌毒性模型，随机分为4组：正常对照组、DOX处理组（DOX）、HKL和DOX共处理组（DOX+HKL）以及HKL、腺苷酸活化蛋白激酶（AMPK）抑制剂和DOX共处理组（DOX+HKL+AMPK抑制剂）。24 h后观察细胞形态变化，用CCK-8法检测细胞活力，RT-PCR检测炎性因子肿瘤坏死因子α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）的mRNA水平，Western blot检测裂解的半胱氨酸天冬氨酸蛋白酶3（caspase-3）、细胞色素c和细胞焦亡分子NOD样受体热蛋白结构域相关蛋白3（NLRP3）、半胱氨酸天冬氨酸蛋白酶1（Caspase-1）、凋亡相关斑点样蛋白（ASC）以及p-AMPK、红系衍生核因子相关因子2（Nrf2）的表达，免疫荧光染色观察NLRP3和p-AMPK的表达。结果 和正常对照组相比，DOX组H9c2细胞变得肿胀且细胞活力明显降低（P< 0.05），TNF-α、IL-6和IL-1β的mRNA水平显著增加（P<0.05），同时裂解的caspase-3、细胞色素c、NLRP3、Caspase-1和ASC表达增加（P<0.05），p-AMPK和Nrf2表达降低（P<0.05），NLRP3荧光染色强度增加，p-AMPK荧光强度降低；和DOX组相比，DOX+HKL组细胞肿胀减轻，细胞活力明显增加（P<0.05），TNF-α、IL-6和IL-1β的mRNA水平显著降低（P<0.05），裂解的caspase-3、细胞色素c、NLRP3、Caspase-1和ASC表达降低（P<0.05），而p-AMPK和Nrf2表达增加（P<0.05），另外NLRP3荧光染色强度降低，p-AMPK荧光强度增加；和DOX+HKL组相比，AMPK抑制剂可以逆转HKL对DOX心肌的保护作用。结论 HKL可通过抑制细胞焦亡减轻DOX引起的H9c2心肌细胞毒性损伤，这一作用与激活AMPK调控Nrf2信号有关。

关键词: 和厚朴酚；阿霉素；心肌毒性；细胞焦亡

Abstract: Objective To investigate the effect of honokiol (HKL) for reducing doxorubicin (DOX)-induced cardiotoxicity in H9c2 cells and the underlying mechanisms. Methods H9c2 cells were divided into control group, DOX group, HKL + DOX group, and HKL+compound C+DOX group. After 24 h of corresponding treatment, the cells were examined for morphological changes and cell viability using CCK-8 assay. The mRNA expressions of the inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were detected by RT-PCR, and the protein levels of cleaved caspase-3, cytochrome c, NOD-like receptor pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), p-AMPK and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) were detected with Western blotting; the expressions of NLRP3 and p-AMPK also detected with immunofluorescence staining. Results DOX treatment caused swelling and significantly lowered the viability of H9c2 cells (P<0.05), resulting also in increased mRNA expressions of TNF-α, IL-6 and IL-1β (P<0.05) and protein expressions of cleaved caspase-3, cytochrome c, NLRP3, caspase-1 and ASC (P<0.05) but reduced protein levels of p-AMPK and Nrf2 (P<0.05); fluorescence staining showed significantly increased NLRP3 expression and decreased expression of p-AMPK in DOX-treated cells (P<0.05). All these changes in COX-treated cells were significantly alleviated by HKL treatment (P<0.05). The application of compound C obviously mitigated the protective effects of HKL against DOX-induced cardiotoxicity in H9c2 cells. Conclusions HKL can alleviate DOX-induced cardiotoxicity by inhibiting pyroptosis in H9c2 cells, and this effect is mediated by activation of AMPK to regulate Nrf2 signaling.

Key words: honokiol; doxorubicin; cardiotoxicity; pyroptosis