Zishen-huoxue Decoction alleviates ischemic myocardial injury via Sirt5-β-tubulin mediated synergistic mechanism of "mitophagy-unfolded protein response" and mitophagy

Background and objective: Zishen Huoxue decoction (ZSHX) increases cardiomyocyte activity following hypoxic stress; However, its upstream therapeutic targets have not yet been clarified. Methods: Network pharmacological analysis and RNA sequencing were used to determine the pathological mechanism of myocardial ischemic injury and the primary pathway(s) via which ZSHX improves the viability of cardiomyocytes. Cardiomyocytes damaged by hypoxic stress were treated with ZSHX. A series of assays, including RT-PCR, laser confocal microscopy, immunofluorescence microscopy, and fluorescence co-localization, were conducted to confirm the regulatory effects of ZSHX on sirtuin 5 (SIRT5)-β-tubulin axis in post-hypoxia cardiomyocytes and the interplay mechanism of mitophagy and UPR^mt. Results: Network pharmacology and RNA sequencing analyses revealed that ZSHX target genes were closely associated with mitophagy and apoptosis in the mitochondrial pathway. In vitro, ZSHX inhibited pathological mitochondrial fission following hypoxic stress, regulated FUNDC1-related mitophagy, and increased the levels of mitophagy lysosomes and LC3II/TOM20 expression while inhibiting the over-activated mitochondrial unfolded protein response. Besides, ZSHX regulated the stability of beta-tubulin through SIRT5 and could regulate FUNDC1-related synergistic mechanisms of mitophagy and UPR^mt via SIRT5 and -β-tubulin axis. This targeting pathway may be necessary for cardiomyocytes to resist hypoxia. Conclusion: ZSHX can protect cardiomyocyte injury via the SIRT5-β-tubulin axis, which may be related to the synergistic protective mechanism of SIRT5-β-tubulin axis-related mitophagy and UPR^mt on cardiomyocytes.