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Background/Aims: Epigallocatechin gallate (EGCG) has established protective actions against myocardial ischemia/reperfusion (I/R) injury by regulating autophagy. However, little is known about the mechanisms of EGCG in posttranscriptional regulation in the process of cardioprotection. Here we studied whether microRNAs play a role in EGCG-induced cardioprotection. Methods: The myocardial I/R injury in vitro and in vivo model were made, with or without EGCG pretreatment. The upregulation and silencing of microRNA-384-5p (miR-384) and Beclin-1 in H9c2 cell lines were established. Rats were transfected with miR-384 specific shRNA. Dual-luciferase reporter gene assay was conducted to verify the relationship between miR-384 and Beclin-1. TTC staining was performed to analyze the area of myocardial infarct size. Cell viability was monitored by cell counting kit-8 (CCK-8). The release of cardiac troponin-I (cTnI) was examined by ELISA. The levels of autophagy-related genes or proteins expression were evaluated by qRT-PCR or Western blotting. Autophagosomes of myocardial cells were detected by transmission electron microscopy and laser scanning confocal microscope. Results: I/R increased both autophagosomes and autolysosomes, thereby increasing autophagic flux both in vitro and in vivo. Pretreatment with EGCG attenuated I/R-induced autophagic flux expression, accompanied by an increase in cell viability and a decrease in the size of myocardial infarction. MiR-384 expression was down-regulated in H9c2 cell lines when subjected to I/R, while this suppression could be reversed by EGCG pretreatment. The dual-luciferase assay verified that Beclin-1 was a target of miR-384. Both overexpression of miR-384 and knocking down of Beclin-1 significantly inhibited I/R-induced autophagy, accompanied by the activation of PI3K/Akt pathway, thus enhanced the protective effect of EGCG. However, these functions were abrogated by the PI3K inhibitor, LY294002. Conclusion: We confirmed that EGCG has a protective role in microRNA-384-mediated autophagy by targeting Beclin-1 via activating the PI3K/Akt signaling pathway. Our results unveiled a novel role of EGCG in myocardial protection, involving posttranscriptional regulation with miRNA-384.

Background: Ischemia-derived exosomes can restrict excessive autophagy by transferring microRNA-30a (miR30a) to cells. Reports have confirmed that epigallocatechin gallate (EGCG) alleviates acute myocardial infarction (AMI) by regulating autophagy; however, research evaluating the communication with cardiomyocytes and exosomes is lacking. This study aimed to explore whether exosomes derived from EGCGtreated cardiomyocytes mitigated AMI by adjusting miR30a to inactivate apoptosis and autophagy. Methods: Exosomes were extracted from cardiomyocytes, cultured either in control or AMI condition, with or without EGCG pretreatment. The exosome characteristics were analyzed by nanoparticle tracking analyses and transmission electron microscopy. The change in miR30a in cells and exosomes was demonstrated by qRT-PCR. H9c2 or stable miR30a knockdown (miR30a KD) cell lines were incubated with exosomes derived from EGCG-treated cardiomyocytes in vitro or in vivo. The effect of EGCG and exosomes on I/ R-induced cardiomyocyte apoptosis and autophagy was assessed. Results: EGCG improved the activity of cardiomyocytes, and increased average diameter, concentration, miR30a mRNA level, and specific protein expression in AMIderived exosomes produced by cardiomyocytes. Moreover, the coincubation of AMI cells with EGCG or exosomes derived from EGCG-treated cardiomyocytes attenuated cardiomyocyte apoptosis and autophagy. Conclusions: The findings showed that EGCG upregulates miR30a, which was efficiently transferred via exosomes between cardiomyocytes, thereby contributing to the suppression of apoptosis and autophagy. By focusing on the cardiomyocyte microenvironment, we identified a new target of EGCG alleviating AMI by regulating apoptosis and autophagy.

The aim of this study was to characterize hepato carcinogenesis in diethylnitrosamine (DEN)-treated hamsters. Syrian golden hamsters (n=36) were administered DEN by hypodermic injection and addition to drinking water. Morphological analyses, including light microscopy and immunohistochemistry of α-fetal protein (AFP), were performed on liver and lung tissues. Primary cell culture and tumor transplantation were carried out to evaluate the potential application in hepatocellular carcinoma (HCC) research. From 25 to 50 weeks of treatment, liver tumors, including macronodular HCC and ascites, were found in one-third (4/12) of the animals treated with DEN. HCC was characterized by poor differentiation, frequent mitosis, AFP reaction, vessel invasion and potential application in primary cell culture and xenotransplantation. Pre-neoplastic lesions were hyperplastic nodules comprised of clear cells, bile duct proliferation, fatty metamorphosis and multilocular cysts. The DEN-treated hamsters also showed lung tumors consisting of AFP-negative, well-differentiated neoplastic cells. Characterization of DEN-induced HCC in hamsters provides insights into human hepatocarcinogenesis. This animal model has potential applications in HCC research.

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