M2-sEV inhibit endoplasmic reticulum stress to alleviate chronic intermittent hypoxic-induced H9C2 cardiomyocyte injury

doi:10.16138/j.1673-6087.2023.06.008

Abstract

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

CLC Number:

R563.5

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.

Figures/Tables 9

References 31

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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