Abstract
Doxorubicin (Dox) with cardiotoxicity and endotheliotoxicity limits its clinical application for cancer. The toxicitic mechanism involves excess ROS generation. 14-3-3s have the protective effects on various injured tissues and cells. Tetramethylpyrazine (TMP) is an alkaloid extracted from the rhizome of Ligusticum wallichii and has multiple bioactivities. We hypothesize that TMP has the protective effects on vascular endothelium by upregulating 14-3-3γ. To test the hypothesis, Dox-induced endotheliotoxicity was used to establish vascular endothelium injury models in mice and human umbilical vein endothelial cells. The effects of TMP were assessed by determining thoracic aortic strips' endothelium-dependent dilation (EDD), as well as LDH, CK, caspase-3, SOD, CAT, GSH-Px activities and MDA level in serum, apoptotic rate, and histopathological changes of vascular tissue (in vivo). Also, cell viability, LDH and caspase-3 activities, ROS generation, levels of NAD+/NADH and GSH/GSSG, MMP, mPTP opening, and apoptotic rate were evaluated (in vitro). The expression of 14-3-3γ and Bcl-2, as well as phosphorylation of Bad (S112), were determined by Western blot. Our results showed that Dox-induced injury to vascular endothelium was decreased by TMP via upregulating 14-3-3γ expression in total protein and Bcl-2 expression in mitochondria, activating Bad (S112) phosphorylation, maintaining EDD, reducing LDH, CK, and caspase-3 activities, thereby causing a reduction in apoptotic rate, and histopathological changes of vascular endothelium (in vivo). Furthermore, TMP increased cell viability and MMP levels, maintained NAD+/NADH, GSH/GSSG balance, decreased LDH and caspase-3 activities, ROS generation, mPTP opening, and apoptotic rate (in vitro). However, the protective effects to vascular endothelium of TMP were significantly canceled by pAD/14-3-3γ-shRNA, an adenovirus that caused knockdown 14-3-3γ expression, or ABT-737, a specific Bcl-2 inhibitor. In conclusion, this study is the first to demonstrate that TMP protects the vascular endothelium against Dox-induced injury via upregulating 14-3-3γ expression, promoting translocation of Bcl-2 to the mitochondria, closing mPTP, maintaining MMP, inhibiting RIRR mechanism, suppressing oxidative stress, improving mitochondrial function, and alleviating Dox-induced endotheliotoxicity.
Highlights
Doxorubicin (Dox) is a broad-spectrum, high efficiency, low cost and convenient use of anticancer antibiotic [1]
We have found that TMP could up-regulate 14-3-3γ expression, improve mitochondrial function, and reduce apoptosis induced by LPS to cardiomyocytes [20]
The aims of the current study were to investigate by in vivo and in vitro 1) Whether TMP protected vascular endothelium against endotheliotoxicity induced by Dox; 2) Whether up-regulation of 14-3-3γ expression, phosphorylation of Bad (S112) and subsequent translocation of Bcl-2 to the mitochondria were involved in the protection of TMP against endotheliotoxicity induced by Dox; 3) Whether the change of 14-3-3γ/Bcl-2 caused by TMP could affect mitochondrial oxidative stress that vascular endothelium induced by Dox endotheliotoxicity; 4) Whether improvement of mitochondrial function mediated by 14-3-3γ/Bcl-2 was involved in TMP protecting vascular endothelium against endotheliotoxicity induced by Dox
Summary
Doxorubicin (Dox) is a broad-spectrum, high efficiency, low cost and convenient use of anticancer antibiotic [1]. The damage of Dox to vascular endothelium, and so-called endotheliotoxicity has attracted considerable attention [3]. Many studies have found that there are various reasons for Dox’s cardiotoxicity or endotheliotoxicity [3, 4]. One of the most important reason is that Dox itself may induce oxidative stress, resulting in excessive reactive. Oxidative Medicine and Cellular Longevity oxygen species (ROS) generation [3,4,5,6]. We have shown that Dox toxicity can cause excessive ROS generation, resulting in severe myocardial damage [7, 8]. Inhibiting oxidative stress and reducing ROS generation may alleviate cardiotoxicity or endotheliotoxicity induced by Dox [9,10,11,12,13]. Phytochemicals are candidate subjects [7, 8, 10,11,12,13]
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