外源性心肌球样细胞外囊泡Y RNA片段改善小鼠心肌缺血再灌注损伤的作用机制研究

doi:10.11958/20222017

天津医药 ›› 2023, Vol. 51 ›› Issue (6): 601-606.doi: 10.11958/20222017

外源性心肌球样细胞外囊泡Y RNA片段改善小鼠心肌缺血再灌注损伤的作用机制研究

刘德昭(), 罗小志, 黄锋

广西医科大学第一附属医院心血管内科（邮编530021）

收稿日期:2022-12-08 修回日期:2023-01-09 出版日期:2023-06-15 发布日期:2023-06-20
通讯作者: ^△E-mail：huangfeng3000@126.com
作者简介:刘德昭（1996），男，硕士在读，主要从事心肌缺血再灌注损伤方面研究。E-mail：liudezhao7673@126.com
基金资助:
国家自然科学基金资助项目(82070279);广西医学高层次骨干人才“139”计划项目(G201903007);广西高等学校千名中青年骨干教师培育计划项目(2018062);广西心脑血管疾病防治精准医学重点实验室(22-035-18);广西心脑血管疾病临床医学研究中心(AD17129014)

Mechanism of action of EV-YF1 based on artificially synthesized Y RNA fragments from extracellular vesicles of cardiosphere-derived cells on improving myocardial ischemia-reperfusion injury in mice

LIU Dezhao(), LUO Xiaozhi, HUANG Feng

Department of Cardiovascular Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China

Received:2022-12-08 Revised:2023-01-09 Published:2023-06-15 Online:2023-06-20
Contact: ^△E-mail：huangfeng3000@126.com

刘德昭, 罗小志, 黄锋. 外源性心肌球样细胞外囊泡Y RNA片段改善小鼠心肌缺血再灌注损伤的作用机制研究[J]. 天津医药, 2023, 51(6): 601-606.

LIU Dezhao, LUO Xiaozhi, HUANG Feng. Mechanism of action of EV-YF1 based on artificially synthesized Y RNA fragments from extracellular vesicles of cardiosphere-derived cells on improving myocardial ischemia-reperfusion injury in mice[J]. Tianjin Medical Journal, 2023, 51(6): 601-606.

摘要/Abstract

摘要：

目的探讨心肌球样细胞外囊泡Y RNA片段（EV-YF1）改善心肌缺血再灌注损伤的作用机制。方法将18只C57BL/6J小鼠按随机数字表法分为Sham组、MIRI组和EV-YF1组，每组6只。MIRI组与EV-YF1组建立心肌缺血再灌注模型，再灌注10 min后夹闭主动脉，EV-YF1组左心室心腔内注射EV-YF1 5 μg，MIRI组左心室注射等体积不加入EV-YF1的DharmaFECT4 。24 h后伊文思蓝/氯化三苯基四氮唑（TTC）双染色检测心肌梗死面积；HE染色观察心肌组织病理学形态；酶联免疫吸附试验检测外周血白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）；实时荧光定量PCR检测心肌组织IL-1β、IL-6、TNF-α mRNA表达水平；Western blot检测心肌组织B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关的X蛋白（Bax）、胱天蛋白酶（Caspase）-3蛋白表达水平；TUNEL染色检测心肌细胞凋亡情况。结果与Sham组相比，MIRI组可见明显梗死区域，心肌组织损伤明显，外周血中IL-1β、IL-6和TNF-α水平明显上调，心肌组织中IL-1β、IL-6和TNF-α mRNA表达升高，Bcl-2、Bax、Caspase-3蛋白表达升高（P<0.05），TUNEL核染阳性细胞数量明显增多；与MIRI组相比，EV-YF1组小鼠MIRI后梗死面积比值缩小，心肌损伤减轻，血清IL-1β、IL-6和TNF-α水平降低，心肌组织中IL-1β、IL-6、TNF-α mRNA表达降低，Bcl-2蛋白表达升高，而Bax和Caspase-3蛋白表达下降（P<0.05），TUNEL核染阳性细胞数量减少。结论 EV-YF1可通过减轻炎症反应及抑制心肌细胞凋亡，改善小鼠心肌缺血再灌注损伤。

关键词: 细胞外囊泡, 心肌再灌注损伤, 细胞凋亡, 白细胞介素6, 肿瘤坏死因子α, EV-YF1

Abstract:

Objective To investigate the mechanism of extracellular vesicular Y RNA fragment (EV-YF1) in ameliorating myocardial ischemia/reperfusion injury (MIRI). Methods Eighteen C57 BL/6J mice were divided into the Sham group, the MIRI group and the EV-YF1 group according to random number table method, with six mice in each group. Myocardial ischemia-reperfusion model was established in the MIRI group and the EV-YF1 group. Ten minutes after reperfusion, the aorta was clipped in the EV-YF1 group, and 5 μg EV-YF1 was injected into the left ventricle. DharmaFECT4 without EV-YF1 was injected into the left ventricle of the MIRI group. Evans Blue/triphenyltetrazole chloride (TTC) double staining was used to detect myocardial infarction size 24 h after modeling. Myocardial histopathology was observed by HE staining. Interleukin (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in peripheral blood were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of IL-1β, IL-6 and TNF-α in myocardial tissue were detected by real-time quantitative PCR. The expression levels of B-lymphoblastoma -2 (Bcl-2), Bcl-2 related X protein (Bax) and Caspase-3 protein in myocardial tissue were detected by Western blot assay. Myocardial cell apoptosis was detected by TUNEL staining. Results Compared with the Sham group, the MIRI group showed obvious infarct areas, significant myocardial tissue damage, significant upregulation of IL-1β, IL-6 and TNF-α levels in peripheral blood. IL-1β, IL-6 and TNF-α mRNA expression in myocardial tissue were elevated. Bcl-2, Bax and Caspase-3 protein expression were increased (P < 0.05), and the number of TUNEL nuclear staining positive cells was significantly increased. Compared with the MIRI group, the ratio of infarct area was reduced after MIRI in the EV-YF1 group, myocardial injury was alleviated, serum levels of IL-1β, IL-6 and TNF-α were significantly decreased, and mRNA expression levels of IL-1β, IL-6 and TNF-α in myocardial tissue were decreased. Bcl-2 protein expression level was increased, while Bax and Caspase-3 protein expression levels were decreased (P<0.05). The number of TUNEL nuclear staining positive cells was decreased. Conclusion EV-YF1 can ameliorate myocardial ischemia-reperfusion injury in mice by reducing inflammation and inhibiting myocardial apoptosis.

Key words: extracellular vesicles, myocardial reperfusion injury, apoptosis, interleukin-6, tumor necrosis factor-alpha, EV-YF1

中图分类号:

R542.22

图/表 9

表1 qPCR引物序列

Tab.1 Primer sequences for qPCR

基因名称	引物序列（5'→3'）	产物大小（bp）
IL-1β	上游：GAAGAAGAGCCCATCCTCTGT 下游：TGTTCACGGAGCCTGTAG	129
IL-6	上游：ACACTCTTCACACCCCTCTCCTTC 下游：GACCCTCACTCCTTCCCTTCTATCC	122
TNF-α	上游：CACCACGCTCTTCTGTCTACTGAAC 下游：CCATTAGCCCACTTCTTTCCCTCAC	147
GAPDH	上游：TGAGCAAGAGAGGCCCTATC 下游：AGGCCCCTCCTGTTATTATG	101

图1 各组小鼠MIRI后危险区域与梗死面积变化（伊文思蓝/TTC双染色）

Fig.1 Changes of risk area and infarct size in mice after MIRI in each group (Evans Blue/TTC double staining)

表2 各组小鼠MIRI后梗死面积比值与危险区域比值比较（n=6，%，$\bar{x}±s$）

Tab.2 Comparison of infarct area ratio and risk area ratio after MIRI between the three groups of mice

组别	梗死面积比值	危险区域比值
Sham组	2.89±0.38	56.92±1.35
MIRI组	48.48±1.21^a	57.42±1.44
EV-YF1组	33.86±0.86^ab	58.59±0.67
F	693.900^＊＊	0.508

图2 各组小鼠心脏HE染色（×200）

Fig.2 HE staining of mouse hearts in three groups (×200)

表3 各组小鼠外周血炎性因子水平比较（n=6，ng/L，$\bar{x}±s$）

Tab.3 Comparison of inflammatory factors in peripheral blood between the three groups of mice

组别	IL-1β	IL-6	TNF-α
Sham组	86.2±5.9	257.7±25.9	235.5±19.4
MIRI组	482.3±35.8^a	753.7±31.5^a	815.6±28.1^a
EV-YF1组	350.1±21.0^ab	494.8±25.8^ab	516.7±34.4^ab
F	69.480^＊＊	79.230^＊＊	107.600^＊＊

表4 各组小鼠心肌组织IL-1β、IL-6和TNF-α mRNA表达水平比较（n=6，$\bar{x}±s$）

Tab.4 Comparison of mRNA expression levels of IL-1β, IL-6 and TNF-α in myocardial tissue between the three groups of mice

组别	IL-1β	IL-6	TNF-α
Sham组	1.00±0.08	1.00±0.13	1.00±0.26
MIRI组	8.98±0.42^a	2.43±0.01^a	3.93±0.45^a
EV-YF1组	2.29±0.31^ab	1.39±0.04^ab	1.56±0.22^ab
F	197.900^＊＊	56.190^＊＊	22.630^＊＊

图3 小鼠心肌组织中Bax、Bcl-2、Caspase-3蛋白表达水平

Fig.3 Bax, Bcl-2, Caspase-3 protein expression levels in mouse myocardial tissue

表5 各组小鼠心肌组织凋亡相关蛋白相对表达量比较（n=6，$\bar{x}±s$）

Tab.5 Comparison of relative expression of apoptosis-related proteins in myocardial tissue between the three groups of mice

组别	Bcl-2	Bax	Caspase-3
Sham组	0.360±0.034	0.905±0.059	0.248±0.028
MIRI组	0.720±0.034^a	1.542±0.125^a	0.899±0.085^a
EV-YF1组	2.757±0.209^ab	1.054±0.144^ab	0.648±0.078^ab
F	109.600^＊＊	8.379^＊＊	23.060^＊＊

图4 各组心肌组织TUNEL染色结果（×400）

Fig.4 TUNEL staining results of myocardial tissue in each group (×400)

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外源性心肌球样细胞外囊泡Y RNA片段改善小鼠心肌缺血再灌注损伤的作用机制研究

Mechanism of action of EV-YF1 based on artificially synthesized Y RNA fragments from extracellular vesicles of cardiosphere-derived cells on improving myocardial ischemia-reperfusion injury in mice

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