丁香酚对大鼠心肌缺血再灌注损伤的抑制作用及其机制

Inhibitory effect of eugenol on myocardial ischemia-reperfusion injury in rats and its mechanism

目的观察丁香酚对大鼠心肌缺血再灌注损伤(MIRI)的抑制作用并分析其机制。方法Wistar大鼠随机分为假手术组、MIRI组、丁香酚低剂量组、丁香酚中剂量组、丁香酚高剂量组、阳性对照组,每组10只。丁香酚低、中、高剂量组及阳性对照组分别给予50、100、200 mg/(kg·d)丁香酚及30 mg/(kg·d)盐酸地尔硫[艹卓]灌胃14 d,假手术组、MIRI组给予羧甲基纤维素钠溶液灌胃。除假手术组外,各组均于末次给药1 h后采用结扎冠状动脉左前降支法建立大鼠MIRI模型,假手术组开胸后在冠状动脉左前降支处仅穿线不结扎。采用全自动生化分析仪检测各组血清心肌损伤标志物肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH);摘取大鼠心脏,TTC染色后计算心肌梗死面积比。采用转录组测序筛选丁香酚作用于MIRI的差异表达基因,对转录组测序筛选出的差异表达基因进行GO功能及KEGG信号通路富集分析。结果血清心肌损伤标志物CK、CK-MB、LDH水平MIRI组>丁香酚低剂量组、丁香酚中剂量组、丁香酚高剂量组、阳性对照组>假手术组,心肌梗死面积比假手术组>丁香酚低剂量组、丁香酚中剂量组、丁香酚高剂量组、阳性对照组>MIRI组(P均<0.05)。与MIRI组比较,丁香酚低剂量组共有1035个差异表达基因,其中594个基因上调、441个基因下调;丁香酚中剂量组共有513个差异表达基因,其中197个基因上调、316个基因上调;丁香酚高剂量组共有1962个差异表达基因,其中1151个基因上调、811个基因下调。丁香酚各剂量组与MIRI组差异表达基因的GO分析结果显示,丁香酚作用于MIRI的生物过程主要涉及免疫反应、氧气输送、急性期反应等;细胞组分主要涉及细胞外空间、染色体、细胞外基质等;分子功能主要涉及趋化因子的活动、载氧活性、钙离子结合等。丁香酚各剂量组与MIRI组差异表达基因的KEGG分析结果显示,丁香酚作用下差异表达基因主要涉及FOXO信号通路、HIF-1信号通路、PI3K-AKT信号通路、JAK-STAT信号通路等,其中以HIF-1信号通路富集得分最高且差异最明显。结论丁香酚可抑制大鼠MIRI,其机制可能与调控HIF-1信号通路有关。

Objective To investigate the inhibitory effect of eugenol on myocardial ischemia-reperfusion injury(MIRI)in rats and to analyze its mechanism.Methods Wistar rats were randomly divided into sham operation group,MIRI model group,low-dose eugenol group,medium-dose eugenol group,high-dose eugenol group,and positive control group,with 10 rats in each group.Rats in the low-dose eugenol group,medium-dose eugenol group,and high-dose eugenol group were given 50,100,and 200 mg/(kg•d)eugenol and 30 mg/(kg•d)diltiazepine hydrochloride by gavage for 14 d,respectively,while rats in the sham operation group and MIRI model group were given sodium carboxymethyl cellulose(CMC-Na)solution by gavage.MIRI models were established 1 h after the last administration by ligation of the left anterior descending branch of coronary artery in all groups,except the sham operation group.The left anterior descending branch of coronary artery was only lapped without ligation in sham operation group after chest opening.Serum markers of myocardial injury,such as creatine kinase(CK),creatine kinase isoenzyme(CK-MB),and lactate dehydrogenase(LDH),were detected by automatic biochemical analyzer.The myocardial infarction area ratio was calculated by 2,3,5-triphenyltetrazolium chloride(TTC)staining after heart extraction.The differentially expressed genes of MIRI induced by eugenol were screened by transcriptome sequencing.The screened differentially expressed genes underwent Gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis.Results Serum myocardial injury markers CK,CK-MB,and LDH levels were as follows:MIRI group>low-dose eugenol group,mediumdose eugenol group,high-dose eugenol group,and positive control group>sham operation group,and the myocardial infarction area ratio was in the following order:sham-operation group>low-dose eugenol group,medium-dose eugenol group,high-dose eugenol group,and positive control group>MIRI group(all P<0.05).Compared with the MIRI group,there were a total of 1035 differentially expressed genes in the low-dose eugenol group,of which 594 genes were up-regulated and 441 genes were down-regulated;there were a total of 513 differentially expressed genes in the medium-dose eugenol group,of which 197 genes were up-regulated and 316 genes were up-regulated;and there were a total of 1962 differentially expressed genes in the high-dose eugenol group,of which 1151 genes were up-regulated and 811 genes were downregulation.GO analysis of the differentially expressed genes in the eugenol dose groups and the MIRI group showed that the biological processes of eugenol in MIRI were mainly related to immune response,oxygen transport,acute phase response pairs,etc.;the cellular components were mainly related to the extracellular space,chromosomes,and extracellular matrix,etc.;and the molecular functions were mainly related to the activities of chemokines,oxygen-carrying activity,and calcium-ion binding,etc.KEGG analysis of the differentially expressed genes in the eugenol dose groups and the MIRI group showed that the differentially expressed genes under the action of eugenol were mainly involved in signaling pathways such as the forkhead box O(FOXO)signaling pathway,hypoxia inducible factor-1(HIF-1)signaling pathway,PI3K-AKT signaling pathway,and JAK-STAT signaling pathway and other signaling pathways;among them,the HIF-1 signaling pathway had the highest enrichment score and the most significant difference.Conclusion Eugenol can inhibit the MIRI of rats,and its molecular mechanism may be related to regulating the HIF-1 signaling pathway.