Investigation of the reverse effect of Danhong injection on doxorubicin-induced cardiotoxicity in H9c2 cells: Insight by LC–MS based non-targeted metabolomic analysis

https://doi.org/10.1016/j.jpba.2018.02.012 Get rights and content

Highlights

  • The reverse effect of DHI on DOX-induced cardiotoxicity was analyzed by LC–MS based metabolomics.

  • The satisfactory effect of DHI exerted at the concentration of 10 μL/mL by MTT and real-time cell viability assays.

  • DHI restored DOX-induced disturbances of arginine, GSH, pantothenic acid, cytidine, inosine and 5′-methylthioadenosine.

  • DHI may exert the therapeutic effect by improving energy metabolism and attenuating oxidative stress.

Abstract

Although Danhong injection (DHI) has been clearly shown to attenuate ischemic myocardial injury and improve heart function, there is no research regarding its role in doxorubicin (DOX)-induced cardiomyopathy. In this study, we aimed to investigate the reverse effect of DHI on DOX-induced cardiotoxicity in H9c2 cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that DHI had no cytotoxicity towards the relevant cell line unless the concentration was as high as 50 μL/mL. The satisfactory cardioprotective effect of DHI exerted at the concentration of 10 μL/mL, which agreed well with the result of real-time cell viability assay. Then non-targeted metabolomics based on LC–MS was employed to characterize metabolic alterations in DOX-induced cells with DHI treatment. Multivariate analysis, including PCA and PLS-DA, revealed 31 altered metabolites after DOX treatment that were primarily related to the disturbance of amino acids and nucleotides metabolism. While DHI could intervene in some disturbed metabolic pathways, such as the metabolism of arginine, glutathione (GSH), pantothenic acid, cytidine, inosine and 5′-methylthioadenosine. These results suggested that DHI exerted the therapeutic effect by improving energy metabolism and attenuating oxidative stress. The present study can lay a foundation for further research on the promising therapeutic effect of DHI in managing DOX-induced cardiotoxicity.

Introduction

Doxorubicin (DOX), a typical anthracycline antineoplastic agent, has limited clinical applications due to its severe and irreversible myocardial damage [[1], [2]]. Previous studies proposed that DOX-induced cardiomyopathy was associated with a variety of pathways and mechanisms, including reactive oxygen species (ROS) formation, cardiomyocyte apoptosis, calcium dynamics interference and DNA damage [[1], [3]]. In recent years, enormous efforts have been devoted to the discovery of effective therapeutic drugs against the heart damage caused by DOX. Many researches have revealed that Traditional Chinese Medicines (TCM) or their bioactive components were effective agents in reducing DOX-induced cardiotoxicity, such as resveratrol, Clinopodium chinense and Sheng Mai Yin [[4], [5], [6]].

Danhong injection (DHI), composed of Radix Salviae miltiorrhizae (Danshen) and Flos Carthami tinctorii (Honghua), has been shown to exert cardioprotective effect and is widely used to prevent or treat cardiovascular diseases [7]. It has been proven that DHI could alleviate ischemic myocardial injury [8], but the effect of DHI against DOX-induced cardiomyopathy has been rarely explored. Researches reported that Danshen and one major component of DHI, salvianolic acid B, could reverse the cardiotoxicity caused by DOX [[9], [10]]. Consequently, it is essential to evaluate the potential cardioprotective effect of DHI against DOX-induced cardiomyopathy, which may provide a theoretical basis for the secondary development of DHI and give new insights for the study of integrated Chinese and Western Medicine.

Metabolomics, following the idea of genomics and proteomics, aims to monitor changes of endogenous metabolites with low molecular weight in different physiological or pathological states [11]. It has distinct advantages such as high throughput, high sensitivity and good reproducibility, which allows the successful applications in disease diagnosis, drug toxicity prediction, and nutritional intervention. As the multi-component and multi-target nature of TCM, it is difficult to thoroughly illuminate its pharmacological mechanisms. The metabolomic approach could provide global metabolic information in cells, biofluids and organisms, which matches well with the integrity and systemic conception of TCM [12].

In this study, we developed a classical cell model to investigate the therapeutic effect of DHI on DOX-induced cardiotoxicity for the first time. To reveal the possible pharmacological mechanisms underlying DHI’s actions, a non-targeted metabolomics strategy based on liquid chromatography-mass spectrometry (LC–MS) was conducted to profile metabolic alterations of DHI in DOX-induced cells.

Section snippets

Materials and reagents

DHI was obtained from Shandong Danhong Pharmaceutical Co., Ltd. (Shandong, China). DOX was produced by Haizheng Co., Ltd. (Zhejiang, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin and other cell culture reagents were purchased from Gibco BRL (Calsbad, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and formic acid (FA) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Cell viability assay

Fig. 1A and B exhibited the cytotoxicity of different concentrations of DHI and DOX in H9c2 cells. DOX induced significant cytotoxicity in a dose dependent manner (Fig. 1A) and the IC50 value was found to be approximately 0.5 μg/mL. The percentage of cell viability under DHI treatment was shown in Fig. 1B, and the results clearly demonstrated that DHI had no cytotoxicity towards the relevant cell line at experimental concentrations unless the concentration was as high as 50 μL/mL.

The effect of

Discussion

The present study demonstrated, for the first time, DHI could prevent DOX-induced cytotoxicity in H9c2 cells. Meanwhile, non-targeted metabolomics was utilized for identifying metabolic alterations and the PCA model provided a clear metabolic profile distinction among the four groups, suggesting an expected modulation of myocardial metabolism upon DOX-induced cardiomyopathy and the reverse effect of DHI. 31 metabolites were detected after DOX treatment which were related to the disturbance of

Conclusion

In this study, an LC–MS based non-targeted metabolomics strategy along with MTT and real-time cell viability assays were performed to systemically assess the reverse effect of DHI on DOX-induced cardiomyopathy. Our study demonstrated that DOX exposure could induce cardiotoxicity, which mainly involved in alternations of amino acids, purine and pyrimidine metabolism, indicating the energy disruption and oxidative stress induced by DOX. Meanwhile, DHI may be an effective agent in treating

Acknowledgement

This study was supported by National Natural Science Foundation of China (No. 81173021).

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