Abstract：
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 (m6A), 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 m6A 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.
OBJECTIVE: 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. m6A 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, m6A 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 m6A methylation is involved in autophagy alleviation.
CONCLUSIONS: We demonstrated that SJP mitigates autophagy in MIRI by downregulating miR-193a-3p, enhancing ALKBH5 expression, and reducing m6A methylation, a mechanism potentially attributed to its constituents, senkyunolide A and l-borneol.
OBJECTIVE: 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. m6A 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, m6A 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 m6A methylation is involved in autophagy alleviation.
CONCLUSIONS: We demonstrated that SJP mitigates autophagy in MIRI by downregulating miR-193a-3p, enhancing ALKBH5 expression, and reducing m6A methylation, a mechanism potentially attributed to its constituents, senkyunolide A and l-borneol.
摘要：
背景：由于缺乏有效的临床干预措施，心肌缺血/再灌注损伤（MIRI）对心脏再灌注治疗提出了巨大的挑战。N6-甲基腺苷（m6A）的甲基化，这是哺乳动物mRNA中最常见的转录后修饰，被认为通过调节自噬参与MIRI。MicroRNAs(miRNAs)在转录后水平调控基因表达中起着至关重要的作用,并参与m6A甲基化的调控。速效救心丸（SJP）在中国广泛用于心绞痛的临床治疗，并为接受经皮冠状动脉介入治疗的急性冠状动脉综合征患者带来益处。然而,SJP干预MIRI的确切机制尚不清楚.
目的：本研究旨在证明，在体内和体外，SJP可以通过调节miR-193a-3p靶向和上调去甲基酶ALKBH5来减轻MIRI中的自噬。
方法：使用H9c2细胞建立体外缺氧/复氧模型，而使用Wistar大鼠建立了体内MIRI模型。将携带miR-193a-3p的前体序列的慢病毒用于其过表达。使用腺相关病毒来沉默miR-193a-3p和ALKBH5表达。心功能,梗死面积,使用超声心动图评估大鼠的组织结构，氯化三苯基四唑（TTC）染色，HE染色,分别。末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）用于检测大鼠心脏组织中的凋亡水平。使用比色法评估m6A甲基化水平。GFP-RFP-LC3B用于监测自噬通量，透射电子显微镜用于评估自噬体的发育。分别采用WesternBlot和qRT-PCR评估自噬相关蛋白和miR-193a-3p的水平。
结果：SJP减轻了自噬，保留的心脏功能，减少MIRI大鼠心脏的心肌损伤。SJP在H/RH9C2细胞中减弱自噬。在MIRI大鼠心脏组织和H/RH9C2细胞中观察到miR-193a-3p水平升高,而SJP在这些模型中下调miR-193a-3p水平。miR-193的靶基因ALKBH5受miR-193a-3p负调控。miR-193a-3p过表达或ALKBH5沉默后，m6A甲基化降低，以及SJP及其组分的自噬减弱作用，senkyunolideA和l-冰片，在H/RH9C2细胞中丢失，而在MIRI大鼠中，这些影响没有被废除，只是被削弱了。进一步的研究表明，METTL3抑制剂STM2475与miR-193a-3p的沉默结合，MIRI大鼠心脏的自噬也同样减弱。这表明m6A甲基化的减少与自噬缓解有关。
结论：我们证明SJP通过下调miR-193a-3p减轻MIRI中的自噬，增强ALKBH5表达，减少m6A甲基化，一种可能归因于其成分的机制，senkyunolideA和l-冰片。
目的：本研究旨在证明，在体内和体外，SJP可以通过调节miR-193a-3p靶向和上调去甲基酶ALKBH5来减轻MIRI中的自噬。
方法：使用H9c2细胞建立体外缺氧/复氧模型，而使用Wistar大鼠建立了体内MIRI模型。将携带miR-193a-3p的前体序列的慢病毒用于其过表达。使用腺相关病毒来沉默miR-193a-3p和ALKBH5表达。心功能,梗死面积,使用超声心动图评估大鼠的组织结构，氯化三苯基四唑（TTC）染色，HE染色,分别。末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）用于检测大鼠心脏组织中的凋亡水平。使用比色法评估m6A甲基化水平。GFP-RFP-LC3B用于监测自噬通量，透射电子显微镜用于评估自噬体的发育。分别采用WesternBlot和qRT-PCR评估自噬相关蛋白和miR-193a-3p的水平。
结果：SJP减轻了自噬，保留的心脏功能，减少MIRI大鼠心脏的心肌损伤。SJP在H/RH9C2细胞中减弱自噬。在MIRI大鼠心脏组织和H/RH9C2细胞中观察到miR-193a-3p水平升高,而SJP在这些模型中下调miR-193a-3p水平。miR-193的靶基因ALKBH5受miR-193a-3p负调控。miR-193a-3p过表达或ALKBH5沉默后，m6A甲基化降低，以及SJP及其组分的自噬减弱作用，senkyunolideA和l-冰片，在H/RH9C2细胞中丢失，而在MIRI大鼠中，这些影响没有被废除，只是被削弱了。进一步的研究表明，METTL3抑制剂STM2475与miR-193a-3p的沉默结合，MIRI大鼠心脏的自噬也同样减弱。这表明m6A甲基化的减少与自噬缓解有关。
结论：我们证明SJP通过下调miR-193a-3p减轻MIRI中的自噬，增强ALKBH5表达，减少m6A甲基化，一种可能归因于其成分的机制，senkyunolideA和l-冰片。
