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

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

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。

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 NLRP3expression 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.