间充质干细胞来源的外泌体中miR-143-3p抑制自噬并逆转心肌缺血再灌注损伤的机制研究

Mechanism of miR-143-3p in exosomes derived from mesenchymal stem cells in inhibiting autophagy and reversing myocardial ischemia-reperfusion injury

目的 探讨间充质干细胞(MSCs)来源的外泌体(Exos)中miR-143-3p调控自噬并逆转心肌缺血再灌注(I/R)损伤的机制。方法 分别将miR-143-3p NC、miR-143-3p mimic转染到MSCs。从MSCs提取Exos,将Exos与H9c2细胞共培养,进行缺氧/复氧(H/R)处理,分为H/R组(不转染)、MSC组(只加入Exos)、miR-143-3p-NC组(加入Exos miR-143-3p-NC)、miR-143-3p mimic组(加入Exos miR-143-3p mimic)。将处理过的H9c2细胞注射到大鼠心肌组织,25只大鼠随机分为5组,即对照组、H/R组、MSC组、miR-143-3p-NC组、miR-143-3p mimic组。RT-PCR检测细胞miR-143-3p表达;Western blot检测兔抗人自噬相关蛋白3B-Ⅰ(LC3B-Ⅰ)、自噬相关蛋白3B-Ⅱ(LC3B-Ⅱ)及p62蛋白;透射电子显微镜、纳米颗粒示踪分析技术(NTA)及PKH-26标记Exo示心肌细胞摄取能力;自噬双标腺病毒(mRFP-GFP-LC3)实验检测自噬流;酶联免疫反应法测定大鼠血清心肌肌钙蛋白I(cTnI)、肌酸磷酸激酶(CK)含量;2,3,5-氯化三苯基四氮唑(TTC)染色检测心肌梗死面积;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色检测心肌细胞凋亡。结果 H/R组H9c2细胞自噬流高于对照组,MSC组细胞自噬流低于H/R组,差异均有统计学意义(P<0.01)。H/R组细胞LC3B-Ⅱ与LC3B-Ⅰ比值(LC3B-Ⅱ/LC3B-Ⅰ)高于对照组,p62表达量低于对照组,差异有统计学意义(P<0.01)。MSC组细胞LC3B-Ⅱ/LC3B-Ⅰ低于对照组、H/R组,p62表达量高于对照组、H/R组,差异有统计学意义(P<0.01)。MSCs-Exos中miR-143-3p表达水平为(1.6±0.2),高于MSCs的(0.9±0.1),差异有统计学意义(P<0.01)。H/R组H9c2细胞miR-143-3p水平为(0.32±0.03),低于对照组H9c2细胞的(1.02±0.09),差异有统计学意义(P<0.01);与H/R组比较,MSC组、miR-143-3p mimic组细胞miR-143-3p水平升高,差异有统计学意义(P<0.01)。miR-143-3p mimic组细胞LC3B-Ⅱ/LC3B-Ⅰ低于MSC组、miR-143-3p-NC组,p62表达量高于MSC组、miR-143-3p-NC组,差异有统计学意义(P<0.01)。H/R组大鼠心肌组织梗死面积、凋亡率、cTnI及CK水平高于对照组,差异有统计学意义(P<0.01);MSC组大鼠心肌组织梗死面积、凋亡率、cTnI及CK水平低于H/R组,差异有统计学意义(P<0.01);miR-143-3p mimic组大鼠心肌组织梗死面积、凋亡率、cTnI及CK水平低于MSC组、miR-143-3p-NC组,差异有统计学意义(P<0.01)。结论 MSCs-Exos来源的miR-143-3p可通过调控自噬减轻心肌I/R损伤。

Objective To investigate the mechanism of miR-143-3 p in exosome(Exos) derived from mesenchymal stem cells(MSCs) in regulating autophagy and reversing myocardial ischemia-reperfusion(I/R) injury.Methods MSCs were transfected with miR-143-3 p NC and miR-143-3 p mimic respectively.Exos was extracted from MSCs,and was co-cultured with H9 c2 cells for hypoxia/reoxygenation(H/R) treatment,and then they were divided into H/R group(no transfection),MSC group(processed with Exos only),miR-143-3 p-NC group(processed with Exos miR-143-3 p-NC) and miR-143-3 p mimic group(processed with Exos miR-143-3 p mimic).The processed H9 c2 cells were injected into myocardial tissues of rats,and 25 rats were randomly divided into 5 groups:control group,H/R group,MSC group,miR-143-3 p-NC group and miR-143-3 p mimic group.The expression of miR-143-3p was detected by RT-PCR;rabbit anti-human autophagy related protein 3B-Ⅰ( LC3B-Ⅰ),autophagy related protein 3B-Ⅱ( LC3B-Ⅱ) and p62 protein were detected by western blot;transmission electron microscopy,nanoparticle tracer analysis( NTA) and PKH-26 labeled Exo showed the uptake capacity of cardiomyocyte;autophagy flow was detected by autophagy double labeled adenovirus( mRFP-GFP-LC3);the contents of serum cardiac troponin I( c Tn I) and creatine kinase( CK) were measured by enzyme-linked immunosorbent assay;myocardial infarct area was detected by2,3,5-triphenyltetrazolium chloride( TTC) staining;deoxyribonucleotide terminal transferase mediated nick end labeling( TUNEL) staining was used to detect cardiomyocyte apoptosis.Results The autophagy flow of H9c2 cells in the H/R group was significantly higher than that in the control group( P < 0.01),while cellular autophagy flow of the MSC group was significantly lower than that in the H/R group( P < 0.01).The ratio of LC3B-Ⅱ to LC3B-Ⅰ( LC3B-Ⅱ/LC3B-Ⅰ) in the H/R group was significantly higher than that in the control group,while the expression of p62 was significantly lower than that in the control group( P < 0.01).LC3B-Ⅱ/LC3B-Ⅰ in the MSC group was significantly lower than that in the control group and the H/R group,while the expression of p62 was significantly higher than that in the control group and the H/R group( P < 0.01).The expression level of miR-143-3p in MSCs-Exos was( 1.6 ± 0.2),which was significantly higher than( 0.9 ±0.1) of MSCs( P < 0.01).Level of miR-143-3p in H9c2 cells in the H/R group was( 0.32 ±0.03),which was significantly lower than( 1.02 ± 0.09) in H9c2 cells in the control group( P <0.01);compared with the H/R group,the level of miR-143-3p in the MSC group and miR-143-3p mimic group was significantly higher( P < 0.01).LC3B-Ⅱ/LC3B-Ⅰ of cells in the miR-143-3p mimic group was significantly lower than that in the MSC group and miR-143-3p-NC group,while the expression of p62 was significantly higher than that in the MSC group and the miR-143-3p-NC group( P < 0.01).The infarct size,apoptosis rate,c Tn I and CK levels of myocardial tissue in the H/R group were significantly higher than those in the control group( P < 0.01);the infarct size,apoptosis rate,c Tn I and CK levels of myocardial tissue in the MSC group were significantly lower than those in the H/R group( P < 0.01);the infarct size,apoptosis rate,c Tn I and CK levels of myocardial tissue in the miR-143-3p mimic group were significantly lower than those in the MSC group and miR-143-3p-NC group( P < 0.01).Conclusion MSCs-Exos derived miR-143-3p can alleviate myocardial I/R injury by regulating autophagy.