As₂O₃ is a traditional Chinese medicine that has been used in China for more than 2,400 years. In recent years, reports have emerged that As₂O₃ may be effective at treating a wide array of cancers, and it has been used as a more suitable drug for the treatment of acute promyelocytic leukemia (Wang X. et al., 2021; Zong et al., 2021). However, As₂O₃ can cause various diseases such as cardiovascular disorder, and As₂O₃ toxicity presented significant obstacles to its clinical applications (Emadi and Gore, 2010). It has been reported that As₂O₃-induced cardiotoxicity is regulated by reactive oxygen species (ROS) (Ficker et al., 2004; Ghosh et al., 2009), which in turn leads to pro-/anti-oxidant imbalances, such as increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). Elevation of MDA is a very useful indicator of oxidative stress (C. Zhang et al., 2021). SOD, GSH and CAT are all important free radical scavengers that exert antioxidant functions and protect cells from damage (Birari et al., 2020). However, there is no definitive clinical drug to treat As₂O₃-induced cardiotoxicity.

As a highly conserved serine/threonine-protein kinase, AMP-activated protein kinase (AMPK) has a triggered effect on the metabolism of bioenergy (Salt and Hardie, 2017). When AMPK is activated, it could monitor the function of mitochondria and the energy status of the cell (Bechard et al., 2010) and regulate the activity of the silent information regulator 1 (SIRT1). Then the peroxisome proliferator-activated receptor-gamma coactivator one alpha (PGC-1α) is activated after activation of AMPK and SIRT1. The AMPK/SIRT1/PGC-1α pathway plays an important role in oxidative stress, and mitochondrial biosynthesis (Canto and Auwerx, 2009; Birari et al., 2020).

Ginger is a widely used spice that exhibits several health benefits. It is used as a home remedy in the treatment of stomach disorders with significant value (Haniadka et al., 2013; Seif et al., 2021). The gingerol-like compounds are the main bioactive substances in the non-volatile, pungent components of ginger. 6-Gingerol (6G) is one of the major components of total gingerols extracted from ginger (Luo et al., 2021), and its chemical structure of 6G is shown in Figure 1. Specifically, 6G has been shown to be potentially efficacious against cancer (Kapoor et al., 2016; Luna-Dulcey et al., 2018) and to possess anti-inflammatory and anti-apoptotic properties (Krell and Stebbing, 2012; Zhang et al., 2017). Previous studies by our group have shown the protective effect of 6G in vitro and ex vivo models, and proposed that the cardioprotective and antioxidant properties of 6G may be a result of decreased intracellular Ca²⁺ via the suppression of Ca²⁺ influx and contractility in ventricular myocytes and inhibition of the TLR4/MAPKs/NF-κB pathway (Han et al., 2019; Han et al., 2020). These studies indicate that 6G possesses cardioprotective potential, while the exact effects and potential protective mechanisms of 6G in As₂O₃-induced cardiotoxicity remain to be fully elucidated. Hence, the present study aimed to explore the effects and underlying pharmacological mechanisms of 6G on As₂O₃-induced oxidative stress, inflammation, and apoptosis of cells.

Herein, a mouse model of As₂O₃ induced-cardiotoxicity was established to explore whether 6G regulates oxidative stress, inflammation, and apoptosis via the AMPK/SIRT1/PGC-1α signaling pathway. To date, the role of 6G as an anti-cardiotoxic agent has not been demonstrated in As₂O₃-induced cardiotoxicity. Thus, our study explores the antioxidant and cardioprotective properties of 6G against As₂O₃-induced cardiotoxicity.

As₂O₃, used in ancient Chinese medicine for more than 2,000 years, plays a vital role in the treatment of various diseases such as cancer (Hoonjan et al., 2018). Ginger is one of the oldest spices and is also used as a traditional Chinese medicine due to its many health benefits. The 6G extracted from ginger is the most pharmacologically active compound in gingerol (Luettig et al., 2016; de Lima et al., 2018). Our previous experimental findings have shown that 6G can effectively inhibit inflammation and apoptosis in cardiac fibrosis and myocardial injury (El-Bakly et al., 2012; Han et al., 2020). Furthermore, 6G was shown to be capable of reversing impaired insulin signaling in arsenic-intoxicated mice (Chakraborty et al., 2012). Moreover, consistent with previous research, our study found that 6G treatment can alleviate As₂O₃-induced heart injury in mice by reducing oxidative stress, the inflammatory response, and apoptosis. In addition, mice treated with 6G obtained these effects via activation of the AMPK/SIRT1/PGC-1α pathway.

The current study may help guide future research on 6G or suitable derivatives for the effective prevention of As₂O₃-induced cardiotoxicity. Evidence has amassed that heart rate and ST height are frequently used as important indicators for normal cardiac functioning and that elevated heart rate and ST segment changes indicate damage of the myocardium (Zenger et al., 2021). As₂O₃-induced myocardial injury is manifested by increased heart rate and ST-segment elevation (Sonawane et al., 2018). Our results showed that As₂O₃ treatment significantly increased heart rate and ST height, however, 6G reversed these pathological changes, suggesting that 6G protects myocardium from As₂O₃-induced injury (Figure 3). Meanwhile, the 6G-alone administration group was designed to observe changes in mice before and after 6G treatment. Our results showed no significant changes in mice treated with 6G alone compared to the Con group, indicating that 6G is a safe therapeutic agent.

The utilization of As₂O₃ in cancer patients causes various cardiotoxic effects (Yu et al., 2017). Serum levels of LDH and CK are indicators of myocardial damage and are frequently used to estimate cardiotoxicity (Saad et al., 2020). During cardiovascular disease or injury, the levels of CK-MB and CTnI in cardiac tissues are dramatically increased (Wang R. et al., 2021). We preliminarily analyzed the toxic effects of arsenic trioxide on the heart by the level of cardiac diagnostic markers. The data from our study demonstrate that As₂O₃ exposure increased serum levels of LDH, CK, CK-MB and CTnI. Elevated levels of these markers indicate cardiac damage, however, treatment with 6G can significantly reduce the levels of these indicators (Figure 4). We further observed the histopathological damage caused by As₂O₃. Results in the present study indicated that L-6G and H-6G treatment had curative effects on histopathological changes (Figure 5). Consistent with previous research, the protective effect of 6G, as determined by heart function markers, has been reported against ISO-induced cardiotoxicity (Han et al., 2020). 6G ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenol)-3-decanone) extracted from ginger is the most pharmacologically active compound in gingerol, and the active part of the molecule is an aliphatic chain molecule containing a hydroxyl group (Yang et al., 2010; Wang et al., 2014). Due to its cardioprotective effect, 6G can be considered a promising molecule for the treatment of drug and chemical-induced cardiotoxicity and/or other cardiovascular diseases.

Production of reactive oxygen species, depletion of antioxidants, impairment of mitochondrial function, and induction of apoptosis are involved in the pathogenesis of cardiotoxicity caused by As₂O₃. Mitochondria are the major site of ROS production (Bugger and Pfeil, 2020), and exposure of As₂O₃ is able to induce overproduction of intracellular ROS, which triggers oxidative stress-related cascades, inflammatory responses, apoptosis, and cellular membrane damage (Zhong et al., 2021). The high levels of intracellular ROS in As₂O₃-treated mice were associated with the impaired mitochondrial reduction of molecular oxygen (Ahangarpour et al., 2017). The levels of SOD, CAT and GSH in the tissues indicated the extent of antioxidant defense. The results of the present study also demonstrate that As₂O₃ can generate large amounts of ROS, which leads to a significant decrease in the levels of antioxidants (SOD, GSH and CAT), as well as an increase in the levels of lipid peroxides MDA. This suggests that As₂O₃ induces cardiac injury by inducing oxygen radical production and reducing endogenous protective antioxidant capacity (Figure 6). Mitochondrial dysfunction is the main cause of As₂O₃-induced damage. 6G treatment could reduce the swelling of mitochondria and the breakage of mitochondrial membrane (Figure 7). Our results suggest that 6G could maintain mitochondrial function.

Excessive production of ROS and induction of oxidative stress-related signaling cascades result in the overproduction of pro-inflammatory mediators. TNF-α and IL-6, both of which play key roles in the inflammatory process (Forrester et al., 2018). Inflammatory cytokines were found to be upregulated in As₂O₃-induced cardiotoxicity. Our results are in consistent with previous studies. Notably, TNF-α and IL-6 were significantly downregulated by 6G, suggesting that 6G exerts a significant anti-inflammatory effect by inhibiting the levels of TNF-α and IL-6 (Figure 8).

Oxidative stress caused by As₂O₃ results in dysfunction of mitochondria and apoptosis of cardiomyocytes, both of which contribute to the development of myocardial damage (Zhu and Zuo, 2013). It is well known that the apoptosis-related regulatory proteins Bax, Bcl-2, Caspase-3 and cleaved-Caspase-3 are closely associated with As₂O₃-induced cardiotoxicity. As a classical anti-apoptotic mediator, Bcl-2 can inhibit the activity of Caspase-3 which, in turn, inhibits cellular apoptosis. It is noteworthy that Bax upregulates the permeability of mitochondrial membranes, causing cytochrome ϲ release, which induces cellular apoptosis through activation of Caspase-3 and cleaved-Caspase-3 (Polat and Karaboga, 2019). We also found that As₂O₃ induced apoptotic damage to the heart, as evidenced by notable increases in Caspase-3, Bax, and Bcl-2 expression and the Bax/Bcl-2 ratio in the heart of As₂O₃-treated mice. However, in the groups treated with 6G, Bax, Caspase-3, and cleaved-Caspase-3 levels were downregulated and the Bax/Bcl-2 ratio decreased in heart tissue, while Bcl-2 expression in heart tissue was upregulated relative to As₂O₃-treated mice (Figure 9).

The signaling cascade mediated by AMPK, SIRT1 and PGC-1α can inhibit ROS production and inflammatory cytokines (Thirupathi and de Souza, 2017). Thus, activation of the cascade may be an effective therapy against As₂O₃-induced damage. Multiple studies have shown that AMPK could affect the activity of the downstream molecules SIRT1 and PGC-1α, as well as the activation of AMPK and PGC-1α, which could also be regulated by SIRT1 (Tian et al., 2019). PGC-1α plays an important role in cardioprotective therapies and is highly expressed in cardiac myocytes (Lehman et al., 2000). Furthermore, PGC-1α is a key factor for myocardial mitochondrial biogenesis and for regulating the expression of the downstream proteins involved in oxidative stress (Choi et al., 2017; Di et al., 2018). SIRT1, a histone deacetylase, plays an important role in regulating a wide range of critical biological functions, including oxidative stress, energy metabolism, apoptosis, and autophagy. Our present study found that As₂O₃ significantly decreased the protein expressions of AMPK, SIRT1, and PGC-1α, whereas 6G upregulated the expressions of AMPK, SIRT1, and PGC-1α, enhanced SOD activities, and suppressed the production of ROS and MDA to maintain mitochondrial function and control oxidative stress (Figures 10, 11).

In line with these findings, the data generated by our study suggests that 6G treatment exerts cardioprotective effects by attenuating oxidative stress, inflammation, and apoptosis. 6G may be a promising therapeutic agent for the prevention of As₂O₃-induced cardiotoxicity, and the AMPK/SIRT1/PGC-1α pathway may be an effective target. Our experiments provide guidance for future studies on the efficacy of 6G or suitable derivatives in preventing the cardiotoxicity of anticancer drugs.