Aloe-emodin relieves zidovudine-induced injury in neonatal rat ventricular myocytes by regulating the p90rsk/p-bad/bcl-2 signaling pathway

https://doi.org/10.1016/j.etap.2020.103540Get rights and content

Highlights

  • Aloe-emodin protect against AZT-induced cardiomyocyte apoptosis.

  • Aloe-emodin regulates p90rsk/p-bad/bcl-2 signal pathway.

  • The application of AZT with AE produce superior effects for HIV-1 infection therapeutics.

Abstract

Background/Aims

Zidovudine (3′-azido-2′,3′-deoxythymidine; AZT) is a first-line drug for treatment of human immunodeficiency virus infection (HIV). However, its application is limited by cardiotoxicity due to cardiomyocyte injury. This study investigated whether Aloe-emodin (AE), an anthraquinone compound, protects against AZT-induced cardiomyocyte toxicity.

Methods

MTT, JC-1 assays and TUNEL were examined to verify the protective effect of AE against AZT-induced cardiomyocyte injury. Western blotting was performed to explore the anti-apoptotic effect of AE using anti-apoptotic proteins p90rsk, p-bad, and bcl-2 and pro-apoptotic proteins apaf-1, cleaved-caspase-3, and cytochrome c.

Results

We observed a protective effect of AE against cell viability decrease and TUNEL positive cells increase induced by AZT, which was counteracted by BI-D1870. Western blot analysis found that AE significantly inhibited cardiomyocyte apoptosis by activating p90rsk/p-bad/bcl-2 signaling pathway. Furthermore, BI-D1870 counteracted the anti-apoptotic effect of AE.

Conclusions

Taken together, these results indicate that AE attenuated AZT-induced cardiomyocyte apoptosis by activating p90rsk.

Introduction

Nucleoside reverse transcriptase inhibitors (NRTIs) are a category of drugs that are used worldwide in the clinical management of acquired immunodeficiency syndrome (AIDS)-associated symptoms caused by human immunodeficiency virus-1 (HIV-1) infection. Since the advent of the first AIDS drug, AZT or zidovudine, a number of agents acting on different drug targets, such as HIV enzymes (e.g. reverse transcriptase, protease, and integrase) and host cell factors critical for HIV infection have been added to our armamentarium to combat HIV/AIDS (Maeda et al., 2019). However, the zidovudine (3′-azido-2′,3′-deoxythymidine; AZT) is the first-line NRTI-type antiretroviral agent, long-term administration of AZT induces severe cardiotoxicity that leads to cardiomyopathy (Kohler et al., 2009).

Previous studies have shown that multiple interrelated mechanisms are involved in AZT-induced cardiotoxicity, including triggering the Fas-dependent cell-death pathway (Purevjav et al., 2007), inducing oxidative stress (Bialkowska et al., 2000; de la Asuncion et al., 1998; Deavall et al., 2012; Banerjee et al., 2013) and mitochondrial dysfunction (Liu et al., 2014), and increasing release of cytochrome c (CytC) from mitochondria (Matteucci et al., 2015). Mitochondria are major sites for generation of cellular ROS as well as toxicity targets of oxidative stress (Liu et al., 2014; Drose and Brandt, 2012; Sena and Chandel, 2012). Ruth Roberts found that oxidative stress causes further mitochondrial dysfunction, which was observed following long-term administration of AZT (Deavall et al., 2012). CytC released from mitochondria has a close relationship to mitochondrial functions and mitochondrial apoptosis-related pathways (Sun et al., 2017; Wang et al., 2017). Phosphorylated p90rsk (p-p90rsk) targets a Ser residue at position 112 of phosphorylated bad (p-bad), preventing bad from interacting with bcl-xL and bcl-2 and inhibiting translocation of the pro-apoptotic protein bax to the mitochondria, which in turn inhibits the release of CytC (Cheng et al., 2014, 2016; Ibata-Ombetta et al., 2003; Jia et al., 2014). CytC then promotes the formation of the apoptosome, which ultimately cleaves and activates caspase-3, leading to cardiomyocyte apoptosis mediated by the mitochondrial apoptosis pathway (Sena and Chandel, 2012). A recent report indicated that Nrf2/ARE signaling exerts chondroprotection by diminishing oxidative stress and inhibiting apoptosis via activation of the ERK1/2/ELK1-P70S6K-p90rsk signaling axis in osteoarthritic chondrocytes (Khan et al., 2018).Moreover, the work by Liu and colleagues indicated that doxorubicin-induced ROS activated signal-regulated kinase 1/2 in H9C2 cardiomyocytes (Liu et al., 2015). So, we supposed that p90rsk may play an important role in AZT-induced cardiomyocyte apoptosis.

Aloe-emodin (AE) is a hydroxyanthraquinone (Mijatovic et al., 2005; Tseng et al., 2017), which has numerous pharmacological effects including antioxidant action and cardiovascular protection (Bai et al., 2017). A recent study reported the anti-apoptotic effect of AE via activation of ERK in retinal ganglion cells (Lin et al., 2007). Evidence has been reported that chrysophanol, an anthraquinone compound structurally similar to AE, targets p90rsk, increases phosphorylation of p90rsk in choriocarcinoma cells, and induces cell apoptosis (Lim et al., 2017). This indicates that AE may participate in mediating the p90rsk/p-bad signaling pathway.

Taken together, we proposed a hypothesis that AE may protect against AZT-induced cardiomyocyte apoptosis by activating p90rsk, which further reduces CytC expression. Our work provides experimental evidence of the joint application of AZT and AE for HIV-1 infection therapeutics.

Section snippets

Neonatal rat ventricular myocytes (NRVMs) culture

NRVMs were isolated and cultured from 1 to 3-day-old Sprague-Dawley rats, in which use of animals complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Anon, 2011) and was pre-approved by the Experimental Animal Ethic Committee of the Harbin Medical University (Animal Experimental Ethical Inspection Protocol, No. 2009104). Briefly, hearts were quickly minced and digested with 0.25 % (v/v) trypsin (Beyotime, Jiangsu, China). The

AE relieves AZT-induced cardiomyocyte injury

Firstly, we aimed to establish the model of AZT-induced cardiomyocyte injury by treating NRVMs with 50 μM AZT for different durations (0, 6, 12, 24, 36 h). In our model, we saw that cell viability and MMP in the 6 h group had no changes compared with the 0 h group. However, cell viability and MMP were decreased in the 12 h, 24 h, and 36 h groups compared with the 0 h group in a time-dependent manner. Furthermore, the decreased degree of cell viability and MMP in the 24 h and 36 h groups was

Discussion

Long-term administration of NRTIs such as AZT causes mitochondrial compromise in cardiomyocytes (Koczor and Lewis, 2010). Damaged mitochondria lead to inadequate supply of cellular energy, further cardiomyocyte injury, and death. This implies the clinical necessity of mitochondrial protection for patients that need long-term treatment with AZT, as suggested by Liu and colleagues (Liu et al., 2012). Consistent with this, in the present study we reconfirmed AZT-induced mitochondrial injury in

CRediT author statement

Wei Zhao and Zhimin Du designed the experiments and wrote the manuscript. Wei Zhao, Yan Bai, Lihui Zhao, Tingting Chen, Yuyang Zheng, Burong Feng, Huiwei Jiang, Wei Zhao, Yue Sun and Yingfu Chen performed experiments. Ye Yuan and Pengzhou Hang reviewed the manuscript. ZhiminDu provided experimental materials and reviewed this manuscript.

Declaration of Competing Interest

The authors declare no conflict of interests

Acknowledgements

This work was supported by the National Natural Science Fund of China (81673424) and Special funds for Scientific Research Transformation of Heilongjiang Academy of Medical Sciences (CR201814).

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