Currently, there is no effective treatment for MIRI [21], and its therapeutic methods and related mechanisms still need to be further elucidated. Here, we identified the therapeutic effect of ZXGD in mice with MIRI and explored its underlying mechanism. Luckily, we firstly revealed that ZXGD could protect myocardial mitochondrial function through the autophagy-ACBP-TSPO axis. This may provide insights into the clinical management of MIRI.
The development of MIRI involves several different mechanisms, including oxidative stress, apoptosis, and mitochondrial damage [22]. In past studies, ZXGD and the herbs contained therein have been proven to be involved in the treating mechanism of MIRI. ZXGD had an exceptional ability to attenuate cardiomyocyte injury induced by hypoxia/reoxygenation, concerning oxidative stress, cell apoptosis, mitochondrial dysfunction, and energy acquisition [23]. Among them, the herb combination of Gualou and Xiebai has a positive effect on chronic myocardial ischemia in rats via the regulation of energy homeostasis and apoptosis [24]. Moreover, naringenin, the active ingredient of Zhishi, reduces apoptosis, inflammation, and oxidative stress while improving mitochondrial energy metabolism, ultimately mitigating myocardial ischemia injury and promoting angiogenesis of ischemic myocardium [25]. Stabilizing the mitochondrial structure in MIRI is crucial to reducing ROS production [26, 27]. Here, we found that ZXGD could ameliorate the impairment of cardiac function caused by ischemia-reperfusion, and this effect was closely related to the protection of the morphology and function of mitochondria.
Autophagy assumes a pivotal function in the elimination of misfolded proteins and the preservation of intracellular homeostasis, thereby conferring advantageous implications for numerous pathological conditions, and is a biological process that involves multiple proteins working together including mTOR, ULK1, ATG3, ATG7, p62, LC3A and LC3B [28, 29]. However, autophagy is not always beneficial. On the one hand, excessive autophagy itself not only could lead to cell death (autophagic cell death), but also be closely linked to other types of cell death [30, 31]. Increased autophagy leads to the degradation of ferritin, lipid droplets, circadian proteins, and GPX4, which induces the onset of ferroptosis, and this phenomenon is reversed in autophagy-deficient cells [32, 33]. During the reperfusion phase of MIRI, the increase in autophagy levels is mainly mediated by beclin1 [34]. Phosphorylation of beclin1 leads to its direct binding to SLC7A11 and inhibits System Xc activity, thereby inducing ferroptosis [35]. On the other hand, excessive autophagy leads to excessive mitochondrial clearance, which induces cell death. Healthy mitochondria are essential for cardiac function during MIRI. Excessive mitophagy could lead to excessive mitochondrial clearance and exacerbate mitochondrial dysfunction, which is detrimental to the cell, especially cardiomyocytes [36, 37]. It has been proposed that autophagy may enable excessive degradation of certain cell survival factors and organelles to induce cell death [38]. Therefore, we determined by network analysis and molecular docking that the major components of ZXGD may regulate myocardial tissue autophagy levels by modulating the AKT1-mTOR signaling pathway and autophagy-related proteins. Among these compounds, irigenin and naringenin have an excellent affinity to AKT1 and autophagy-related proteins and are, therefore, considered important compounds for the treatment of MIRI by ZXGD. AKT1 has been shown to play a critical role in MIRI in previous studies [39]. AKT1 activation has a positive regulatory role in both mitochondrial damage and apoptosis during MIRI [40, 41]. However, few studies have focused on the role of AKT1-mTOR pathway-mediated autophagy in MIRI. To avoid the effects of AKT1 on other pathways, we used 3-MA and rapamycin to modulate autophagy levels in MIRI mice and verified that inhibition of autophagy could protect the myocardial mitochondrial function of the mice with MIRI.
Autophagy is not only capable of influencing the degradation of intracellular substances, but it is also closely related to cellular secretory functions (autophagy-dependent secretion) [42]. Past studies have shown that autophagy is necessary for the secretion of ACBP [43, 44]. Interestingly, we found elevated ACBP content in the myocardium of ZXGD-intervened mice, validating this idea. In addition, ACBP is essential for the regulation of normal mitochondrial function [45].
Certain proteins within the mitochondria have a crucial role in regulating the mitochondrial damage that results from MIRI. Among them, TSPO is important in maintaining normal mitochondrial function. The knockdown of TSPO leads to increased mitochondrial fragmentation, increased glucose uptake and lactate conversion, decreased oxidative phosphorylation, and increased glycolysis [46]. Additionally, TSPO has a significant association with the generation of ROS and the reinstatement of mitochondrial membrane potential [47]. 40-Chlorodiazepine (a ligand of TSPO) reduces mitochondrial sterol and oxysterol concentrations and improves mitochondrial function during MIRI in a rat model of high cholesterol [48]. Coordinated control of fatty acid uptake, beta-oxidation, and mitochondrial oxidative phosphorylation is essential for normal cardiac activity [49], and ACBP, a traditional receptor for TSPO, plays an essential role in mitochondrial energy metabolism. LCACoAs could be directly donated by ACBP to carnitine palmitoyl transferase I (CPTI), thereby enhancing mitochondrial β-oxidation [50]. However, the effect of ACBP action on TSPO on mitochondrial function still needs further elucidation [51]. Notably, when the autophagy-dependent paracrine pathway of ACBP is inhibited, it increases its intracellular content, closely related to mTOR [8, 44]. Similarly, we found that ZXGD could exert mitochondrial protection by inhibition of autophagy, which promotes ACBP aggregation in tissues and activates the ACBP-TSPO signaling. The enhancement of the ACBP-TSPO signaling may play an essential role in the maintenance of mitochondrial function in the myocardium during MIRI. This provides new evidence for the protective effect of the ACBP-TSPO signaling axis on mitochondrial function.
In the present study, we found that ZXGD could activate the autophagy-ACBP-TSPO axis to improve myocardial mitochondrial function in mice with MIRI, partly through activating the AKT1-mTOR pathway. This finding not only reveals the relationship between autophagy and mitochondrial function during MIRI but also provides new evidence of ZXGD in the therapy of cardiovascular diseases.