带电多囊体蛋白5在调控血管内皮细胞焦亡中的作用研究

doi:10.16138/j.1673-6087.2024.03.02

摘要/Abstract

摘要：

目的：探究带电多囊体蛋白5(charged multivesicular body protein 5, CHMP5)在人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）焦亡中的表达以及敲低CHMP5对HUVEC焦亡的影响。方法：采用聚肌胞苷酸（polyinosinic-polycytidylic acid，Poly I:C）刺激HUVEC建立病毒性脓毒症中双链RNA（double-stranded RNA，dsRNA）诱导血管内皮细胞损伤的体外模型。将HUVEC随机分为对照组、Lipo组、Poly I:C组、siNC组和siCHMP5+Poly I:C组，通过实时荧光定量PCR（real-time fluorescent quantitative PCR，qRT-PCR）检测各组CHMP5的表达。采用乳酸脱氢酶（lactate dehydrogenase，LDH）细胞毒性检测试剂盒检测LDH释放率、ELISA 检测白介素-1β（interleukin-1β，IL-1β）分泌及透射电子显微镜（transmission electron microscopy，TEM）检测HUVEC的超微结构以观察细胞膜的完整性，蛋白质印迹（Western blotting）检测相关蛋白胱天蛋白酶-3活性剪切体（cleaved caspase-3，活化Casp-3）、gasdermin E蛋白N端（gasdermin E N-terminal，GSDME-N）的表达水平，免疫荧光染色检测各组细胞相关蛋白的表达和定位。结果：与对照组相比，Poly I:C组HUVEC细胞肿胀，多处细胞膜破裂，同时活化Casp-3、GSDME-N和CHMP5蛋白表达上调（P<0.05）。RNAi技术敲低CHMP5后，与对照组和siNC组比较，siCHMP5+Poly I:C组LDH和IL-1β释放增加，焦亡相关蛋白表达水平上调以及GSDME蛋白募集分布改变，内皮细胞焦亡明显加重（均P<0.05）。结论：CHMP5在HUVEC细胞焦亡中呈现高表达。敲低CHMP5可增强HUVEC细胞焦亡，可能与抑制膜修复促进GSDME和GSDMD蛋白剪切引起的继发性细胞焦亡有关。

关键词: 细胞焦亡, 带电多囊体蛋白5, 血管内皮细胞, GSDME

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

中图分类号:

R631

孙俊楠, 张姣姣, 王海嵘. 带电多囊体蛋白5在调控血管内皮细胞焦亡中的作用研究[J]. 内科理论与实践, 2024, 19(03): 159-166.

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.

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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