Study on the role of charged multivesicular body proteins 5 in regulating pyroptosis of vascular endothelial cells

doi:10.16138/j.1673-6087.2024.03.02

Abstract

Abstract:

Objective To investigate the expression of charged multivesicular body proteins 5(CHMP5) in pyroptosis of human umbilical vein endothelial cell (HUVEC) and the effect of knocking down CHMP5 on HUVEC pyroptosis. Methods Using polyinosinic-polycytidylic acid (Poly I:C) to stimulate HUVEC to establish an in vitro model of double-stranded RNA (dsRNA) induced HUVEC injury in viral sepsis. HUVEC were randomly divided into control, Lipo, Poly I:C, siNC and siCHMP5+Poly I:C groups, and the expression of CHMP5 in each group was detected by real-time fluorescent quantitative PCR (qRT-PCR) to verify the transfection efficiency. Lactate dehydrogenase (LDH) cytotoxicity assay kit was used to detect the release rate of LDH, ELISA was used to detect the secretion of interleukin-1β(IL-1β) and transmission electron microscopy (TEM) was used to detect the ultrastructure of HUVEC to observe the integrity of the cell membrane, and Western blotting was used to detect the expression level of the cleaved caspase-3(Casp-3) and gasdermin E N-terminal(GSDME-N), and immunofluorescence staining was used to detect the expression and localization of the related proteins in each group of cells. Results In comparison with the control group, the HUVEC in the Poly I:C group were swollen and showed multiple cell membrane ruptures, and cleaved Casp-3, GSDME-N and CHMP5 protein expression was up-regulated. Compared with the control group and the siNC group, knocking down CHMP5 by RNAi technology resulted in increased release of LDH and IL-1β, up-regulation of the expression level of pyroptosis-related proteins, alteration of the distribution of GSDME protein recruitment, and significant aggravation of endothelial cell pyroptosis in the siCHMP5+Poly I:C group. Conclusions The CHMP5 is highly expressed in HUVEC pyroptosis. Knockdown of CHMP5 can enhances HUVEC cellular pyroptosis, which may be related to the inhibition of membrane repair and promoting secondary cellular pyroptosis induced by cleavage of GSDME and GSDMD proteins.

Key words: Pyroptosis, Charged multivesicular body proteins 5, Human umbilical vein endothelial cell, Gasdermin E N-terminal

CLC Number:

R631

SUN Junnan, ZHANG Jiaojiao, WANG Hairong. Study on the role of charged multivesicular body proteins 5 in regulating pyroptosis of vascular endothelial cells[J]. Journal of Internal Medicine Concepts & Practice, 2024, 19(03): 159-166.

Figures/Tables 12

Table 1

Figure 1

Figure 2

Table 2

Figure 3

Figure 4

Table 3

Table 4

Figure 5

Figure 6

Table 5

Figure 7

References 26

[1]	Corman V M, Lienau J, Witzenrath M. Coronaviren als ursache respiratorischer infektionen[J]. Internist (Berl), 2019, 60(11):1136-1145. doi: 10.1007/s00108-019-00671-5 pmid: 31455974
[2]	张姣姣, 孙俊楠, 王海嵘. 磷酸氯喹对双链RNA诱导的血管内皮细胞焦亡的影响[J]. 上海交通大学学报(医学版), 2022, 42(10):1404-1412.
[3]	de Rivero Vaccari JC, Dietrich WD, Keane RW, et al. The inflammasome in times of COVID-19[J]. Front Immunol, 2020, 11:583373.
[4]	Ferreira AC, Soares VC, de Azevedo-Quintanilha IG, et al. SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes[J]. Cell Death Discov, 2021, 7(1):43. doi: 10.1038/s41420-021-00428-w pmid: 33649297
[5]	Han Y, Zhang H, Mu S, et al. Lactate dehydrogenase, an independent risk factor of severe COVID-19 patients: a retrospective and observational study[J]. Aging (Albany NY), 2020, 12(12):11245-11258.
[6]	Ding J, Wang K, Liu W, et al. Pore-forming activity and structural autoinhibition of the gasdermin family[J]. Nature, 2016, 535(7610):111-116.
[7]	Jorgensen I, Miao EA. Pyroptotic cell death defends against intracellular pathogens[J]. Immunol Rev, 2015, 265(1):130-142. doi: 10.1111/imr.12287 pmid: 25879289
[8]	Shi J, Zhao Y, Wang K, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death[J]. Nature, 2015, 526(7575):660-665.
[9]	Kuriakose T, Kanneganti TD. Pyroptosis in antiviral immunity[J]. Curr Top Microbiol Immunol, 2023, 442:65-83.
[10]	Li Y, Yuan Y, Huang ZX, et al. GSDME-mediated pyroptosis promotes inflammation and fibrosis in obstructive nephropathy[J]. Cell Death Differ, 2021, 28(8):2333-2350. doi: 10.1038/s41418-021-00755-6 pmid: 33664482
[11]	Lian LH, Milora KA, Manupipatpong KK, et al. The double-stranded RNA analogue polyinosinic-polycytidylic acid induces keratinocyte pyroptosis and release of IL-36γ[J]. J Invest Dermatol, 2012, 132(5):1346-1353. doi: 10.1038/jid.2011.482 pmid: 22318382
[12]	Rogers C, Fernandes-Alnemri T, Mayes L, et al. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death[J]. Nat Commun, 2017, 8:14128. doi: 10.1038/ncomms14128 pmid: 28045099
[13]	Orzalli MH, Prochera A, Payne L, et al. Virus-mediated inactivation of anti-apoptotic Bcl-2 family members promotes gasdermin-E-dependent pyroptosis in barrier epithelial cells[J]. Immunity, 2021, 54(7):1447-1462.e5. doi: 10.1016/j.immuni.2021.04.012 pmid: 33979579
[14]	朱莹莹, 王海嵘. 带电多囊体蛋白5的研究进展[J]. 中国细胞生物学学报, 2020, 42(07):1215-1220.
[15]	Karunakaran KB, Balakrishnan N, Ganapathiraju MK. Interactome of SARS-CoV-2 / nCoV19 modulated host proteins with computationally predicted PPIs[J]. Res Sq, 2020, rs.3.rs-28592.
[16]	Ward DM, Vaughn MB, Shiflett SL, et al. The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells[J]. J Biol Chem, 2005, 280(11):10548-10555. doi: 10.1074/jbc.M413734200 pmid: 15644320
[17]	Son F, Umphred-Wilson K, Shim JH, et al. Assessment of ESCRT protein CHMP5 activity on client protein ubiquitination by immunoprecipitation and Western blotting[J]. Methods Mol Biol, 2019, 1998:219-226. doi: 10.1007/978-1-4939-9492-2_16 pmid: 31250305
[18]	Liu Y, Wang Y, Liu J, et al. The circadian clock protects against ferroptosis-induced sterile inflammation[J]. Biochem Biophys Res Commun, 2020, 525(3):620-625.
[19]	Liu Y, Mei Y, Han X, et al. Membrane skeleton modulates erythroid proteome remodeling and organelle clearance[J]. Blood, 2021, 137(3):398-409. doi: 10.1182/blood.2020006673 pmid: 33036023
[20]	Zhang Y, Chen X, Gueydan C, et al. Plasma membrane changes during programmed cell deaths[J]. Cell Res, 2018, 28(1):9-21. doi: 10.1038/cr.2017.133 pmid: 29076500
[21]	Sarhan J, Liu BC, Muendlein HI, et al. Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection[J]. Proc Natl Acad Sci U S A, 2018, 115(46):E10888-E10897.
[22]	Dai E, Meng L, Kang R, et al. ESCRT-Ⅲ-dependent membrane repair blocks ferroptosis[J]. Biochem Biophys Res Commun, 2020, 522(2):415-421.
[23]	Greenblatt MB, Park KH, Oh H, et al. CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts[J]. J Exp Med, 2015, 212(8):1283-1301.
[24]	Rühl S, Shkarina K, Demarco B, et al. ESCRT-dependent membrane repair negatively regulates pyroptosis downstream of GSDMD activation[J]. Science, 2018, 362(6417):956-960. doi: 10.1126/science.aar7607 pmid: 30467171
[25]	Kovacs SB, Miao EA. Gasdermins: effectors of pyroptosis[J]. Trends Cell Biol, 2017, 27(9):673-684. doi: S0962-8924(17)30084-3 pmid: 28619472
[26]	Zhou B, Abbott DW. Gasdermin E permits interleukin-1 beta release in distinct sublytic and pyroptotic phases[J]. Cell Rep, 2021, 35(2):108998.

基因		引物序列 (5'to 3')
GAPDH	正向引物	GGAGCGAGATCCCTCCAAAAT
	反向引物	GGCTGTTGTCATACTTCTCATGG
CHMP5	正向引物	CAGAAAGCCTTGCGAGTT
	反向引物	ACCGTGGTCTTGGTGTCC

组别	对照	Lipo	Poly I:C	P
活化Casp-3	0.176±0.002	0.216±0.002	1.097±0.014^1）	<0.000 1
GSDME-N	0.293±0.022	0.358±0.027	0.985±0.075^1）	<0.000 1
CHMP5	0.925±0.017	0.973±0.060	1.123±0.007^1）	0.001 3

项目	对照	Lipo	siRNA NC-1	siRNA NC-2	si CHMP5-1	si CHMP5-2	si CHMP5-3	P
mRNA水平	1.020±0.007	0.968±0.019	0.898±0.021^1）	1.032±0.008	0.173±0.029^1）	0.220±0.003^1）	0.593±0.033^1）	<0.000 1
蛋白水平	1.060±0.006	1.186±0.008^1）	0.802±0.053	0.962±0.072	0.584±0.010^1）	1.140±0.013	1.153±0.065	0.091 2

项目	对照	Poly I:C	siNC	si CHMP5	P
LDH释放率(%)	0.244±0.036	0.470 ± 0.012^1）	0.469±0.020	0.610±0.001^2）	<0.000 1
IL-1β (pg/mL)	93.360±0.851	367.900±27.120^1）	360.900±29.290	531.400±39.180^2）	0.000 5

组别	对照	Poly I:C	siNC	si CHMP5	P
活化Casp-3	0.067±0.009	1.027±0.014^1）	0.707±0.204^2）	0.765±0.010	0.010 9
GSDME-N	0.368±0.005	0.900±0.011^1）	0.687±0.087^2）	1.124±0.066^2）	<0.000 1
活化Casp-1	0.817±0.026	24.270±0.702^1）	27.060±0.153	24.780±1.194^2）	<0.000 1
GSDMD-N	1.178±0.108	11.440±0.240^1）	9.920±0.916	18.630±0.102^2）	<0.000 1