Oleuropein alleviates myocardial ischemia-reperfusion injury by suppressing oxidative stress and excessive autophagy via TLR4/MAPK signaling pathway

Background: Myocardial ischemia/reperfusion injury (MIRI) is an important complication of reperfusion therapy, and has a lack of effective prevention and treatment methods. Oleuropein (OP) is a natural strong antioxidant with many protective effects on cardiovascular diseases, but its protective effect on MIRI has not yet been studied in depth.

Methods: Tert-Butyl hydroperoxide (tBHP) was used to establish an in vitro oxidative stress model. Cell viability was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and lactate dehydrogenase (LDH). Flow cytometry and fluorescence assays were performed for evaluating the ROS levels and mitochondrial membrane potential (MMP). Immunofluorescence analysis detected the NRF2 nuclear translocation and autophagy indicators. Further, Western blotting and quantitative real-time PCR were performed to evaluate the expression levels of proteins and mRNAs. Molecular docking, CETSA, and molecular interaction analysis explored the binding between OP and TLR4. The protective effects of OP in vivo were determined using a preclinical MIRI rat model.

Results: OP protected against tBHP-treated injury, reduced ROS levels and reversed the damaged MMP. Mechanistically, OP activated NRF2-related antioxidant pathways, inhibited autophagy and attenuated the TLR4/MAPK signaling pathway in tBHP-treated H9C2 cells with a high binding affinity to TLR4 (K_D = 37.5 µM). The TLR4 inhibitor TAK242 showed a similar effect as OP. In vivo, OP could alleviate cardiac ischemia/reperfusion injury and it ameliorated adverse cardiac remodeling. Consistent with in vitro studies, OP inhibited TLR4/MAPK and autophagy pathway and activated NRF2-dependent antioxidant pathways in vivo.

Conclusion: This study shows that OP binds to TLR4 to regulate oxidative stress and autophagy for protecting damaged cardiomyocytes, supporting that OP can be a potential therapeutic agent for MIRI.