Elsevier

Toxicology in Vitro

Volume 65, June 2020, 104817
Toxicology in Vitro

Tetramethylpyrazine alleviates iron overload damage in vascular endothelium via upregulating DDAHII expression

https://doi.org/10.1016/j.tiv.2020.104817Get rights and content

Highlights

  • Tetramethylpyrazine (TMP) can protect HUVECs against iron-overloaded injury.

  • Protective mechanism of TMP may be associated with ROS/ADMA/DDAH II/eNOS/NO pathway.

  • Mitochondria are the target organelles of TMP's protection.

Abstract

Iron overload causes vascular endothelium damage. It has been thought to relate excessive reactive oxygen species (ROS) generation. Tetramethylpyrazine (TMP), an active ingredient of Ligusticum chuanxiong Hort, protects various cells by inhibiting oxidative stress and cascade reaction of apoptosis. However, whether TMP can increase DDAHII activity and expression against endothelial cell damage induced by iron overload, and the protective mechanism has not been elucidated. In this study, 50 μM iron dextran and 25 μM TMP were used to co-treat HUVECs for 48 h. TMP could increase cell viability and decrease LDH activity, enhance DDAHII expression and activity, p-eNOS/eNOS ratio, NO content, and reduce ADMA level. TMP also showed a strong antioxidant activity with inhibited ROS generation and oxidative stress. Moreover, TMP attenuated mitochondrial membrane potential loss, inhibited mitochondrial permeability transition pore openness, and decreased apoptosis induced by iron overload. While mentioned above, the protective effects of TMP were abolished with the addition of pAD/DDAHII-shRNA. The effects of TMP against iron overload were similar to the positive control groups, L-arginine, a competitive substrate of ADMA, or edaravone, free radical scavenger. These results signify that TMP alleviated iron overload damage in vascular endothelium via ROS/ADMA/ DDAHII/eNOS/NO pathway.

Introduction

Iron is an essential nutritional element, which is widely contained in blood, muscle tissue, and some critical enzymes (Comporti, 2002). In cases of excessive iron intake, iron can be highly toxic to biological molecules as an active oxidizing agent (Yao et al., 2005). The toxic effects of iron overload have been shown to involve oxidative reactions and redox cycling (Comporti, 2002). Iron is known to induce over-production of superoxide and hydroxyl radicals via Fenton and Haber-Weis reactions, hydroxyl being the most active and toxic form of reactive oxygen species (ROS) (Valko et al., 2005). Excessive ROS generation induces by iron overload can lead to endothelial dysfunction and reduced nitric oxide bioavailability (Marques et al., 2019; Bertoli et al., 2018; Ribeiro Júnior et al., 2017; Marques et al., 2015; Kraml, 2017; Vinchi et al., 2014). Some studies have reported that the patients of hereditary/ hemolytic/hemorrhagic diseases or hemodialysis might suffer from vascular endothelium damage caused by either iron overload or iron accumulation (Kelaidi et al., 2018; Levin et al., 2018; Cash et al., 2014; Kukongviriyapan et al., 2008; Day et al., 2003; Lekakis et al., 1999).

As endogenous nitric oxide synthase (eNOS) is phosphorylated, or its uncoupling function is inhibited, nitric oxide (NO) signaling and its vasodilation function will be significantly improved (Balat et al., 2009). Asymmetric dimethylarginine (ADMA), an inhibitor of NO synthesis, performs a series of inflammatory effects by mediating NOS uncouple and the subsequent ROS generation (Mangoni, 2009). L-arginine (L-Arg), the substrate for NO synthesis, dramatically reduces ADMA induced target cellulare injury (Molinari et al., 2018). Dimethylarginine dimethylaminohydrolase (DDAH) is an essential ADMA scavenger (Chen et al., 2008). It is reported that DDAH can effectively reverse the occurrence of cardio-cerebrovascular events (Balat et al., 2009; Lin et al., 2002). DDAHII, one of the two isoforms of DDAH, is rich in vascular endothelial cells while it has the characteristic of susceptibility to ROS (Chen et al., 2009; Stuhlinger et al., 2001). Thus, this article attempted to provide new evidence to elucidate the relation between DDAH, ROS, and iron overload in umbilical vein endothelial cells (HUVECs).

Tetramethylpyrazine (TMP), the primary active substance of Ligusticum wallichii Franchat (chuanxiong), it has multiple targets and many biological functions (Donkor et al., 2016). Recently, many studies have shown that TMP has an excellent application prospect in the prevention and treatment of cardio-cerebro- vascular diseases (Yang et al., 2019; Zhang et al., 2018b; Huang et al., 2018; Zhao et al., 2016; Zhang et al., 2016). Interestingly, TMP was reported that reduced iron overload injury by inhibiting hepcidin expression and attenuating the iron deposition in tissues (Sun et al., 2018). Hence, we interrogated whether TMP protects HUVECs from iron overload damage and the potential mechanisms devoted to finding a phytochemical for iron overload related diseases.

Section snippets

Materials

TMP (purity ≥98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Iron dextran (ID), L-Arg, ciclosporin A (CsA), and edaravone (Eda) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Adenovirus pAD/DDAHII-shRNA and pAD/scrRNAi, a negative control adenovirus, were obtained from Gene Chem Co., Ltd. (Shanghai, China). Antibodies directed against DDAHII, p-eNOS (Ser 1177), eNOS, and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly,

TMP treatment against iron overload damage of HUVECs

Cell viability and LDH activity were regarded broadly as biomarkers of cell toxicity (Zhang et al., 2018a). Compared with the control group, cell viability decreased, and LDH activity increased with iron treatment (p < .01, Fig. 1). After treated with various concentrations of TMP, HUVECs viability was higher, and LDH activity was lower than that of the iron group in a concentration-dependent manner (p < .01). Taking the above results into consideration, 25 μM TMP was used in the following

Discussion

Iron, a trace element, is dispensable for oxygen delivery, cellular respiration and energy generation, mitochondria electron transport chain and so on (Andrews and Schmidt, 2007). Notably, excessive iron, which to produce free radicals, can also be toxic (Kraml, 2017). Therefore, the homeostasis of systemic iron levels could be broken by the occurrence of hematological disease and metabolic disorders, that a particular interest in the field of cardiovascular disease caused by iron overload is

Conclusion

Taken together, our data argue that TMP increases DDAHII expression and activity, declines ADMA level, increase p-eNOS/eNOS ratio and NO content, restores mitochondrial function. Of course, as an effective antioxidant, TMP can also alleviate ROS generation, enhance the activity of endogenous antioxidant enzymes, ameliorate oxidative stress caused by iron overload, and thereby protect vascular endothelium resist iron overload damage.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by grants from the Natural Science Foundation of China (№: 21467017, 81660583, 81803534) and Jiangxi applied research and cultivation program (20181BBG78059).

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    These authors contributed equally to this work.

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