Neocryptotanshinone protects against myocardial ischemia-reperfusion injury by promoting autolysosome degradation of protein aggregates via the ERK1/2-Nrf2-LAMP2 pathway

Background: Aggrephagy is a critical compensatory mechanism for the elimination of misfolded proteins resulting from stress and depends on the autolysosome degradation of protein aggregates. However, there have been few mechanism research related to aggrephagy in myocardial ischemia/reperfusion (I/R) injury. Neocryptotanshinone (NCTS) is a fat-soluble active compound extracted from Salvia miltiorrhiza, and may be cardioprotective against I/R. However, the efficacy and specific mechanism of NCTS on I/R have not been studied.

Purpose: The current study aimed to investigate the molecular mechanism of NCTS involved in the therapeutic effect on I/R, with a special emphasis on the up-regulation of the ERK1/2-Nrf2-LAMP2 pathway to increase autolysosomal degradation during aggrephagy.

Methods: A rat model of myocardial I/R injury was constructed by left anterior descending (LAD) ligation-reperfusion. To verify cardiac protection, autolysosome clearance of protein aggregates, and their intracellular biological mechanism, an oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocyte model was created.

Results: NCTS was found to have a significant cardioprotective effect in I/R rats as evidenced by remarkably improved pathological anatomy, decreased myocardial damage indicators, and substantially enhanced cardiac performance. Mechanistically, NCTS might boost the levels of LAMP2 mRNA and protein, total and Ser40 phosphorylated Nrf2, and ^{Thr202/Tyr204}p-ERK1/2 protein. Simultaneously, the cytoplasmic Nrf2 level was reduced after NCTS administration, which was contrary to the total Nrf2 content. However, these beneficial changes were reversed by the co-administration with ERK1/2 inhibitor, PD98059. NCTS therapy up-regulated Rab7 protein content, Cathepsin B activity, and lysosomal acidity, while down-regulating autophagosome numbers, Ubiquitin (Ub), and autophagosome marker protein accumulations through the above signaling pathway. This might indicate that NCTS enhanced lysosomal fusion and hydrolytic capacity. It was also found that NCTS intervention limited oxidative stress and cellular Apoptosis both in vivo and in vitro.

Conclusions: We reported for the first time that NCTS promoted the autolysosome removal of protein aggregation both in vivo and in vitro, to exert the therapeutic advantages of myocardial I/R injury. This was reliant on the up-regulation of the ERK1/2-Nrf2-LAMP2 signaling pathway.

Aggrephagy; Autolysosme; ERK1/2-Nrf2; LAMP2; Myocardial I/R injury; Neocryptotanshinone.