PMID- 38301300 OWN - NLM STAT- MEDLINE DCOM- 20240214 LR - 20240214 IS - 1618-095X (Electronic) IS - 0944-7113 (Linking) VI - 125 DP - 2024 Mar TI - Suxiao Jiuxin Pill alleviates myocardial ischemia/reperfusion-induced autophagy via miR-193a-3p/ALKBH5 pathway. PG - 155359 LID - S0944-7113(24)00024-2 [pii] LID - 10.1016/j.phymed.2024.155359 [doi] AB - BACKGROUND: Myocardial ischemia/reperfusion injury (MIRI) poses a formidable challenge to cardiac reperfusion therapy due to the absence of effective clinical interventions. Methylation of N6-methyladenosine (m(6)A), which is the most common post-transcriptional modifications occurring within mammalian mRNA, is believed to be involved in MIRI by modulating autophagy. MicroRNAs (miRNAs) play a crucial role in regulating gene expression at the post-transcriptional level and have been implicated in the regulation of m(6)A methylation. Suxiao Jiuxin Pill (SJP) is extensively used in China for the clinical treatment of angina pectoris and confers benefits to patients with acute coronary syndrome who have received percutaneous coronary intervention. However, the precise mechanisms underlying SJP intervention in MIRI remain unclear. PURPOSE: This study aimed to demonstrate, both in vivo and in vitro, that SJP could alleviate autophagy in MIRI by regulating miR-193a-3p to target and upregulate the demethylase ALKBH5. METHODS: An in vitro hypoxia/reoxygenation model was established using H9c2 cells, while an in vivo MIRI model was established using Wistar rats. A lentivirus harboring the precursor sequence of miR-193a-3p was employed for its overexpression. Adeno-associated viruses were used to silence both miR-193a-3p and ALKBH5 expressions. Cardiac function, infarct size, and tissue structure in rats were assessed using echocardiography, triphenyl tetrazolium chloride (TTC) staining, and HE staining, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was employed to detect the levels of apoptosis in rat cardiac tissue. m(6)A methylation levels were assessed using colorimetry. GFP-RFP-LC3B was used to monitor autophagic flux and transmission electron microscopy was used to evaluate the development of autophagosomes. Western Blot and qRT-PCR were respectively employed to assess the levels of autophagy-related proteins and miR-193a-3p. RESULTS: SJP alleviated autophagy, preserved cardiac function, and minimized myocardial damage in the hearts of MIRI rats. SJP attenuated autophagy in H/R H9C2 cells. Elevated levels of miR-193a-3p were observed in the cardiac tissues of MIRI rats and H/R H9C2 cells, whereas SJP downregulated miR-193a-3p levels in these models. ALKBH5, a target gene of miR-193, is negatively regulated by miR-193a-3p. Upon overexpression of miR-193a-3p or silencing of ALKBH5, m(6)A methylation decreased, and the autophagy-attenuating effects of SJP and its components, senkyunolide A and l-borneol, were lost in H/R H9C2 cells, whereas in MIRI rats, these effects were not abolished but merely weakened. Further investigation indicated that the METTL3 inhibitor STM2475, combined with the silencing of miR-193a-3p, similarly attenuated autophagy in the hearts of MIRI rats. This suggests that a reduction in m(6)A methylation is involved in autophagy alleviation. CONCLUSION: We demonstrated that SJP mitigates autophagy in MIRI by downregulating miR-193a-3p, enhancing ALKBH5 expression, and reducing m(6)A methylation, a mechanism potentially attributed to its constituents, senkyunolide A and l-borneol. CI - Copyright (c) 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved. FAU - Wang, Dongyuan AU - Wang D AD - Branch of National Clinical Research Center for Chinese Medicine Cardiology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; Cardiovascular Research Institute of Traditional Chinese Medicine, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China. FAU - Wang, Dan AU - Wang D AD - Branch of National Clinical Research Center for Chinese Medicine Cardiology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; Cardiovascular Research Institute of Traditional Chinese Medicine, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China. FAU - Jin, Qipeng AU - Jin Q AD - Branch of National Clinical Research Center for Chinese Medicine Cardiology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; Cardiovascular Research Institute of Traditional Chinese Medicine, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China. FAU - Wang, Xiaolong AU - Wang X AD - Branch of National Clinical Research Center for Chinese Medicine Cardiology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; Cardiovascular Research Institute of Traditional Chinese Medicine, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China. Electronic address: wxlqy0214@163.com. LA - eng PT - Journal Article DEP - 20240124 PL - Germany TA - Phytomedicine JT - Phytomedicine : international journal of phytotherapy and phytopharmacology JID - 9438794 RN - L88RA8N5EG (isoborneol) RN - 0 (MicroRNAs) RN - EC 2.1.1.62 (METTL3 protein, human) RN - EC 2.1.1.- (Methyltransferases) RN - EC 1.14.11.- (ALKBH5 protein, human) RN - EC 1.14.11.- (AlkB Homolog 5, RNA Demethylase) RN - 0 (Camphanes) SB - IM MH - Humans MH - Rats MH - Animals MH - *Myocardial Reperfusion Injury/drug therapy/metabolism MH - Rats, Wistar MH - *Myocardial Ischemia/drug therapy/metabolism MH - *MicroRNAs/genetics/metabolism MH - Autophagy MH - Reperfusion MH - Apoptosis MH - Myocytes, Cardiac/metabolism MH - Mammals/genetics/metabolism MH - Methyltransferases/metabolism/pharmacology MH - AlkB Homolog 5, RNA Demethylase/metabolism MH - *Camphanes OTO - NOTNLM OT - ALKBH5 OT - Autophagy OT - Myocardial ischemia/reperfusion injury OT - Suxiao Jiuxin Pill OT - m(6)A methylation OT - miR-193a-3p COIS- Declaration of competing interest The authors declare that the research was conducted without any commercial or financial relationships that could be perceived as potential conflicts of interest. EDAT- 2024/02/01 18:41 MHDA- 2024/02/12 05:43 CRDT- 2024/02/01 18:00 PHST- 2023/10/17 00:00 [received] PHST- 2024/01/02 00:00 [revised] PHST- 2024/01/11 00:00 [accepted] PHST- 2024/02/12 05:43 [medline] PHST- 2024/02/01 18:41 [pubmed] PHST- 2024/02/01 18:00 [entrez] AID - S0944-7113(24)00024-2 [pii] AID - 10.1016/j.phymed.2024.155359 [doi] PST - ppublish SO - Phytomedicine. 2024 Mar;125:155359. doi: 10.1016/j.phymed.2024.155359. Epub 2024 Jan 24.