M2型巨噬细胞来源的小细胞外囊泡抑制内质网应激减轻慢性间歇性缺氧诱导的H9C2心肌细胞损伤

doi:10.16138/j.1673-6087.2023.06.008

摘要/Abstract

摘要：

目的：探讨M2型巨噬细胞来源的小细胞外囊泡（M2 macrophages-derived small extracellular vesicle, M2-sEV）对慢性间歇性缺氧（chronic intermittent hypoxia，CIH）诱导的H9C2心肌细胞损伤的影响及机制。方法：白介素-4（interleukin 4，IL-4）诱导巨噬细胞向M2型极化，实时荧光定量PCR（real time fluorogenic quantitative polymerase chain reaction, qRT-PCR）检测M2型标志物CD206、精氨酸酶-1（arginase-1,Arg-1）表达水平。提取并鉴定M2-sEV。将H9C2细胞分为对照组（CON组）、CIH组、CIH+M2-sEV组。CCK8法检测细胞活力，qRT-PCR和蛋白质印迹法检测缺氧诱导因子1α（hypoxia inducible factor 1 α, HIF-1α）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和转化生长因子-β（transforming growth factor-β，TGF-β）等炎症因子，活化的胱天蛋白酶3（cleaved caspase-3）、活化的胱天蛋白酶9、B细胞淋巴瘤2（B cell lymphoma-2, Bcl-2）和凋亡蛋白Bcl-2相关X蛋白（Bcl-2 associated X protein, Bax）等凋亡因子，内质网跨膜蛋白肌醇酶1α（inositol-requiring enzyme 1α，IRE1α）、转录因子剪接型X-盒结合蛋白1（spliced X-box binding protein 1，XBP1）、转录激活因子6（activating transcription factor 6, ATF6）和葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）等内质网应激因子。结果：成功极化M2型巨噬细胞，并获取M2-sEV。CCK8检测显示，M2-sEV可提高CIH下H9C2心肌细胞增殖活性。qRT-PCR和蛋白质印迹法显示，与CON组比，CIH组HIF-1α、IL-6、TNF-α、TGF-β、活化的胱天蛋白酶3、活化的胱天蛋白酶9、Bax、IRE1α、XBP1、ATF6、GRP78表达水平明显增高（均P<0.05），Bcl-2表达水平降低、Bax/Bcl-2比值增高（均P<0.05）；加入M2-sEV共孵育后，上述缺氧诱导因子、炎症因子、促凋亡蛋白、内质网应激因子等表达均显著降低（均P<0.05），抗凋亡蛋白Bcl-2表达增高、Bax/Bcl-2比值降低（均P<0.05）。结论：M2-sEV减轻CIH诱导的H9C2细胞损伤，其机制可能与下调内质网应激水平，抑制炎症反应和心肌细胞凋亡有关。

关键词: 阻塞性睡眠呼吸暂停低通气综合征, 间歇性缺氧, M2型巨噬细胞, 小细胞外囊泡, 内质网应激

Abstract:

Objective To investigate the effect and mechanism of M2 macrophages-derived small extracellular vesicle (M2-sEV) on injury of H9C2 cardiomyocytes induced by chronic intermittent hypoxia (CIH). Methods Interleukin-4(IL-4) was used to induce the polarization of RMa-bm macrophages to M2-type. The mRNA expression levels of M2-type markers CD206 and arginase-1（Arg-1） were detected by quantitative reverse transcription PCR (qRT-PCR). M2-sEV were extracted and identified. The signature proteins CD9, CD63 and CD81 of M2-sEV were detected by Western blotting(WB). H9C2 cells were randomly divided into control（CON） group, CIH group and CIH+M2-sEV group. CCK8 was used to detect cell viability, and qRT-PCR and WB were respectively used to detect mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α）, IL-6、tumor necrosis factor-α（TNF-α）、transforming growth factor-β（TGF-β） and apoptotic factors(cleaved caspase-3、cleaved caspase-9、Bcl-2、Bax) as well as endoplasmic reticulum stress factors (IRE1α、XBP1、ATF6、GRP78). Results M2-type macrophages were polarized successfully and M2-sEV were extracted successfully. CCK8 showed that M2-sEV increased the proliferation activity of H9C2 cardiomyocytes under CIH. Compared with the CON group, the expression level of HIF-1α, inflammatory factors (IL-6,TNF-α,TGF-β), pro-apoptotic proteins(cleaved caspase-3, cleaved caspase-9, Bax) and endoplasmic reticulum stress factors (IRE1α, XBP1, ATF6, GRP78) were significantly increased in the CIH group(P<0.05) in both mRNA and protein level. The protein level of Bcl-2 in the CIH group was decreased, and the ratio of Bax/Bcl-2 was increased in the CIH group (P<0.05). However, after co-incubating with M2-sEV, the expressions of HIF-1α, inflammatory factors, pro-apoptotic proteins and endoplasmic reticulum stress factors in CIH group were significantly decreased (P<0.05), and the expression of Bcl-2 and the ratio of Bax/Bcl-2 were also decreased (P<0.05). Conclusions M2-sEV can alleviate the injury of H9C2 cell induced by CIH, which is related to the downregulation of endoplasmic reticulum stress, inhibition of inflammation and reduction of apoptosis.

Key words: Obstructive sleep apnea hypopnea syndrome, Intermittent hypoxia, M2 macrophages, Small extracellular vesicles, Endoplasmic reticulum stress

中图分类号:

R563.5

何美娟, 何嫣婕, 王韵, 朱春雪, 黄汉鹏. M2型巨噬细胞来源的小细胞外囊泡抑制内质网应激减轻慢性间歇性缺氧诱导的H9C2心肌细胞损伤[J]. 内科理论与实践, 2023, 18(06): 416-423.

HE Meijuan, HE Yanjie, WANG Yun, ZHU Chunxue, HUANG Hanpeng. M2-sEV inhibit endoplasmic reticulum stress to alleviate chronic intermittent hypoxic-induced H9C2 cardiomyocyte injury[J]. Journal of Internal Medicine Concepts & Practice, 2023, 18(06): 416-423.

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项目	正向引物	反向引物
CD206	5’-GGTTCCGGTTTGTGGAGCAG-3’	5’-TCCGTTTGCATTGCCCAGTA-3’
Arg1	5’-TCCTTAGAGATTATCGGAGCG-3’	5’-GTCTTTGGCAGATATGCAGG-3’
TNF-α	5’-ATGTCTCAGCCTCTTCTCATTC-3’	5’-GCTTGTCACTCGAATTTTGAGA-3’
IL-6	5’-CTCCCAACAGACCTGTCTATAC-3’	5’-CCATTGCACAACTCTTTTCTCA-3’
TGF-β	5’-CCAGATCCTGTCCAAACTAAGG-3’	5’-CTCTTTAGCATAGTAGTCCGCT-3’
IRE1α	5’-GCATCACCAAGTGGAAGTATC-3’	5’-ACCATTGAGGGAGAGGCATAG-3’
XBP1	5’-GGTCTGCTGAGTCCGCAGCACTC-3’	5’-AGGCTTGGTGTATACATGG-3’
活化的胱天蛋白酶3	5’-GCACTGGAATGTCAGCTCGCAA-3’	5’-GCCACCTTCCGGTTAACACGAG-3’
Bax	5’-GGTTTCATCCAGGATCGAGCAGG-3’	5’-ACAAAGATGGTCACGGTCTGCC-3’
GAPDH	5’-GCCAAGGCTGTGGGCAAGGT-3’	5’-TCTCCAGGCGGCACGTCAGA-3’

项目	CON组	CIH组	CIH+M2-sEV组	P
HIF-1α	1	3.250±0.039¹⁾	2.250±0.047¹⁾²⁾	<0.000 1
活化的胱天蛋白酶3	1	1.170±0.033¹⁾	1.030±0.044¹⁾²⁾	<0.000 9
活化的胱天蛋白酶9	1	1.220±0.023¹⁾	1.090±0.035¹⁾²⁾	<0.000 5
Bax/Bcl-2	0.450±0.019	2.210±0.091¹⁾	1.040±0.106¹⁾²⁾	<0.000 1
IL-6	1	1.270±0.033¹⁾	1.080±0.039¹⁾²⁾	<0.000 3
TNF-α	1	1.470±0.063¹⁾	1.110±0.077¹⁾²⁾	<0.000 7
TGF-β	1	1.610±0.048¹⁾	0.097±0.053¹⁾²⁾	<0.000 1
GRP78	1	1.530±0.039¹⁾	1.260±0.050¹⁾²⁾	<0.000 1
ATF6	1	1.500±0.053¹⁾	1.170±0.056¹⁾²⁾	<0.000 1
IRE1α	1	2.370±0.060¹⁾	1.560±0.061¹⁾²⁾	<0.000 1
XBP1	1	1.630±0.063¹⁾	1.210±0.074¹⁾²⁾	<0.000 1

项目	CON组	CIH组	CIH+M2-sEV组	P
Bax	1	2.190±0.069¹⁾	1.650±0.080^{1) 2)}	<0.000 1
活化的胱天蛋白酶3	1	3.160±0.042¹⁾	1.740±0.032^{1) 2)}	<0.000 1
IL-6	1	21.590±0.196¹⁾	9.270±0.345^{1) 2)}	<0.000 1
TNF-α	1	4.470±0.105¹⁾	2.190±0.137^{1) 2)}	<0.000 1
TGF-β	1	12.860±0.203¹⁾	5.590±0.217^{1) 2)}	<0.000 1
IRE1α	1	3.960±0.052¹⁾	2.150±0.103^{1) 2)}	<0.000 1
XBP1	1	7.790±0.152¹⁾	3.040±0.196^{1) 2)}	<0.000 1