Purpose

Doxorubicin (Dox) is clinically limited due to its dose-dependent cardiotoxicity. Andrographolide (Andro) has been confirmed to exert cardiovascular protective activities. This study aimed to investigate protective effects of Andro in Dox-induced cardiotoxicity (DIC).

Methods

The cardiotoxicity models were induced by Dox in vitro and in vivo. The viability and apoptosis of H9c2 cells and the myocardial function of c57BL/6 mice were accessed with and without Andro pretreatment. Network pharmacology and RNA-seq were employed to explore the mechanism of Andro in DIC. The protein levels of Bax, Bcl2, NLRP3, Caspase-1 p20, and IL-1β were qualified as well.

Results

In vitro, Dox facilitated the downregulation of cell viability and upregulation of cell apoptosis, after Andro pretreatment, the above symptoms were remarkably reversed. In vivo, Andro could alleviate Dox-induced cardiac dysfunction and apoptosis, manifesting elevation of LVPWs, LVPWd, EF% and FS%, suppression of CK, CK-MB, c-Tnl and LDH, and inhibition of TUNEL-positive cells. Using network pharmacology, we collected and visualized 108 co-targets of Andro and DIC, which were associated with apoptosis, PI3K-AKT signaling pathway, and others. RNA-seq identified 276 differentially expressed genes, which were enriched in response to oxidative stress, protein phosphorylation, and others. Both network pharmacology and RNA-seq analysis identified Tap1 and Timp1 as key targets of Andro in DIC. RT-QPCR validation confirmed that the mRNA levels of Tap1 and Timp1 were consistent with the sequenced results. Moreover, the high expression of NLRP3, Caspase-1 p20, and IL-1β in the Dox group was reduced by Andro.

Conclusions

Andro could attenuate DIC through suppression of Tap1 and Timp1 and inhibition of NLRP3 inflammasome activation, serving as a promising cardioprotective drug.

Graphical Abstract

Framework of network pharmacology and RNA-seq approach in Andro on DIC.

中文翻译：

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探索穿心莲内酯对多柔比星引起的心脏毒性的保护作用的综合网络药理学和 RNA-seq 方法

目的

阿霉素（Dox）由于其剂量依赖性心脏毒性而在临床上受到限制。穿心莲内酯（Andro）已被证实具有心血管保护活性。本研究旨在探讨 Andro 对 Dox 诱导的心脏毒性 (DIC) 的保护作用。

方法

Dox在体外和体内诱导心脏毒性模型。在有和没有Andro预处理的情况下评估H9c2细胞的活力和凋亡以及c57BL/6小鼠的心肌功能。采用网络药理学和RNA-seq探讨Andro在DIC中的作用机制。Bax、Bcl2、NLRP3、Caspase-1 p20 和 IL-1β 的蛋白水平也合格。

结果

在体外，Dox促进细胞活力下调和细胞凋亡上调，Andro预处理后，上述症状明显逆转。在体内，Andro可以减轻Dox引起的心脏功能障碍和细胞凋亡，表现为LVPWs、LVPWd、EF%和FS%的升高，CK、CK-MB、c-Tnl和LDH的抑制，以及TUNEL阳性细胞的抑制。利用网络药理学，我们收集并可视化了 Andro 和 DIC 的 108 个共同靶点，这些靶点与细胞凋亡、PI3K-AKT 信号通路等相关。RNA-seq 鉴定了 276 个差异表达基因，这些基因因氧化应激、蛋白质磷酸化等而富集。网络药理学和 RNA-seq 分析均确定 Tap1 和 Timp1 是 Andro 在 DIC 中的关键靶点。RT-QPCR验证证实Tap1和Timp1的mRNA水平与测序结果一致。此外，Andro 降低了 Dox 组中 NLRP3、Caspase-1 p20 和 IL-1β 的高表达。

结论

Andro 可以通过抑制 Tap1 和 Timp1 以及抑制 NLRP3 炎性体激活来减轻 DIC，是一种有前途的心脏保护药物。

图形概要

DIC 上 Andro 的网络药理学和 RNA-seq 方法框架。