Study on the mechanism of cardiomyocyte energy metabolism regulated by Echinocystic acid derivative in improving myocardial ischemia-reperfusion injury

Background: Myocardial ischemia-reperfusion injury (MI/RI) is a major complication that occurs after reperfusion therapy for acute myocardial infarction. Mitochondrial dysfunction and excessive inflammatory response are the major reasons underlying MI/RI. Echinocystic acid (Ech) is a natural triterpenoid compound with multiple pharmacological activities, such as anti-inflammation and antioxidation. However, whether it can protect against MI/RI and the specific molecular mechanism underlying its action remain unclear.

Methods: We established MI/RI rat models and administered Ech to determine the effects of Ech on cardiac function and tissue damage via echocardiography, detection of serum biomarkers, and cardiac tissue staining. We also assessed the morphological characteristics of mitochondria by transmission electron microscopy and evaluated the expression of proteins related to mitochondrial dynamics (DRP1, p-DRP1, MFN2, and OPA1) by conducting Western blotting assays to determine the role of Ech in regulating myocardial cell energy metabolism. By conducting Bulk RNA-Seq, we screened key pathways and predicted the binding of Ech to its targets by molecular docking. In vitro, an H9C2 cell oxygen-glucose deprivation/reoxygenation (OGD/R) model was constructed and treated with Ech and the Toll-like Receptor 8 (TLR8) agonist resiquimod. EdU, JC-1, Western blotting, and metabolite detection assays were performed to elucidate the mechanism underlying the TLR8/NF-κB/HIF-1α pathway in the anti-MI/RI effect of Ech.

Results: We found that Ech significantly improved the cardiac function of MI/RI rats, reduced the area of myocardial infarction, alleviated myocardial tissue pathological damage and destruction of mitochondrial structure, and decreased the levels of cTn-I and CK-MB in serum. In both in vivo and in vitro experiments, Ech reversed the imbalance in mitochondrial dynamics induced by MI/RI or OGD/R, which manifested as a decrease in the expression of p-DRP1, an increase in the expression of MFN2 and OPA1, and improvement in energy metabolism via an increase in ATP production and reduction of L-lactic acid accumulation. RNA-seq analysis showed that Ech inhibited the abnormally activated Toll-like Receptor, NF-κB, and Other inflammatory and metabolic pathways in myocardial tissue. Further investigation revealed that Ech directly interacted with TLR8 and attenuated the excessive activation of the TLR8/NF-κB/HIF-1α signaling pathway; resiquimod (TLR8 Agonist) partially reversed the protective effects of Ech on cell proliferation, mitochondrial function, and energy metabolism of H9C2 cells in vitro.

Conclusion: Ech alleviates MI/RI by targeting TLR8 to inhibit the NF-κB/HIF-1α pathway, improving the balance of mitochondrial dynamics and cellular energy metabolism.