SIRT5在脓毒症肺血管内皮细胞损伤中的作用及机制研究

doi:10.3969/j.issn.1674-8115.2025.09.004

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (9): 1116-1125.doi: 10.3969/j.issn.1674-8115.2025.09.004

SIRT5在脓毒症肺血管内皮细胞损伤中的作用及机制研究

赵善志, 郑相涛, 王晓峰, 陈尔真, 龚方晨, 陈影()

上海交通大学医学院附属瑞金医院急诊科，上海 200025

收稿日期:2025-05-05 接受日期:2025-08-07 出版日期:2025-09-28 发布日期:2025-09-30
通讯作者: 陈　影，主任医师，博士；电子信箱：bichatlion@163.com。
作者简介:第一联系人：为共同第一作者（co-first authors）。
基金资助:
国家重点研发计划(2024YFC3044600);国家自然科学基金(82300100);上海交通大学医学院“双百人”项目(20240804)

Role and mechanisms of SIRT5 in pulmonary microvascular endothelial cell injury in sepsis

ZHAO Shanzhi, ZHENG Xiangtao, WANG Xiaofeng, CHEN Erzhen, GONG Fangchen, CHEN Ying()

Department of Emergency, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2025-05-05 Accepted:2025-08-07 Online:2025-09-28 Published:2025-09-30
Contact: CHEN Ying, E-mail: bichatlion@163.com.
Supported by:
National Key Research and Development Program of China(2024YFC3044600);National Natural Science Foundation of China(82300100);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20240804)

摘要/Abstract

摘要：

目的·探讨沉默信息调节因子2相关酶5（sirtuin 5，SIRT5）在脓毒症肺血管内皮细胞损伤中的作用及机制。方法·对野生型（wild-type， WT）和Sirt5基因敲除的C57BL/6雄性小鼠行盲肠结扎穿孔术（cecal ligation and puncture，CLP），对小鼠安乐死后进行肺组织取材。苏木精-伊红（hematoxylin and eosin staining，H-E）染色评估肺组织炎症水平；伊文思蓝（Evans blue，EB）染色评估血管渗漏；肺组织免疫荧光染色评估小鼠凝血功能改变情况；采用免疫组化染色检测血管细胞黏附分子-1（vascular cell adhesion molecule-1，VCAM-1）蛋白表达，评估CLP小鼠内皮细胞炎症水平。采用基因编辑技术对人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）SIRT5基因进行敲减或者过表达，并使用脂多糖（lipopolysaccharide，LPS）刺激诱导内皮细胞炎症。采用蛋白质印迹法检测VCAM-1、组织因子（tissue factor，TF）等内皮细胞损伤标志物，通过实时荧光定量聚合酶链反应（quantitative real-time PCR，qPCR）检测白细胞介素-6（interleukin 6，IL-6）、IL-1β等炎症指标。对SIRT5过表达的HUVEC进行转录组测序，并通过qPCR验证关键基因如F2R样凝血酶或胰蛋白酶受体3（F2R like thrombin or trypsin receptor 3，F2RL3）或serpin家族成员3（serpin family A member 3，SERPINA3）以及转化生长因子-β 2/3（transforming growth factor β 2/3，TGF-β2/3）的表达。结果·Sirt5基因敲除显著加重CLP小鼠肺损伤，使其生存率降低（P<0.001）；H-E染色显示小鼠肺组织炎症浸润增加，EB染色观察到血管渗漏增加（P<0.001），免疫荧光染色可见纤维蛋白原沉积升高。SIRT5基因敲减的HUVEC中VCAM-1、TF蛋白及IL-6、IL-1β、VCAM-1、E选择素（E-selectin）等炎症因子mRNA表达上调（均P<0.001），而SIRT5过表达可逆转此效应。转录组测序分析表明SIRT5通过抑制F2RL3/SERPINA3/TGF-β通路调控内皮炎症与凝血反应。结论·SIRT5通过负向调控F2RL3/SERPINA3/TGF-β信号轴减轻内皮炎症及促凝血反应，提示其在脓毒症肺损伤中的潜在保护作用。

关键词: 脓毒症, 急性肺损伤, 内皮细胞, 沉默信息调节因子2相关酶5

Abstract:

Objective ·To investigate the role and mechanism of sirtuin 5 (SIRT5) in pulmonary microvascular endothelial cell injury in sepsis. Methods ·Wild-type (WT) and Sirt5 gene knockout C57BL/6 male mice underwent cecal ligation and puncture (CLP) surgery. Following euthanasia, lung tissues were collected. Pulmonary inflammation was assessed using hematoxylin and eosin (H-E) staining; vascular leakage was evaluated by Evans blue (EB) staining; coagulation function in mice was analyzed via immunofluorescence staining of lung tissues. Immunohistochemical staining was employed to detect vascular cell adhesion molecule-1(VCAM-1) protein expression, thereby assessing endothelial inflammation in CLP-treated mice. By using gene editing technology, SIRT5 was knocked down or overexpressed in human umbilical vein endothelial cells (HUVECs), and the cells were subsequently stimulated with lipopolysaccharide (LPS) to induce endothelial inflammation. Protein expression levels of VCAM-1, tissue factor (TF), and other endothelial injury markers were detected by Western blotting, and inflammatory cytokines such as interleukin-6 (IL-6) and IL-1β, were detected by quantitative real-time PCR (qPCR). In addition, transcriptomic sequencing was performed on HUVECs overexpressing SIRT5, and key genes including F2R-like thrombin or trypsin receptor 3 (F2RL3), serpin family A member 3 (SERPINA3), and transforming growth factor β2/β3 (TGF-β2/3) were validated by qPCR. Results ·Sirt5 knockout significantly aggravated lung injury in CLP mice, reducing their survival rates (P<0.001). H-E staining showed increased inflammatory infiltration in the lung tissue of the mice, while EB staining indicated increased vascular leakage (P<0.001). Immunofluorescence revealed elevated fibrinogen deposition. In HUVECs with SIRT5 knockdown, the protein levels of VCAM-1 and TF, as well as the mRNA levels of inflammatory factors including IL-6, IL-1β, VCAM-1, and E-selectin, were significantly upregulated (all P<0.001), whereas overexpression of SIRT5 reversed these effects. Transcriptome sequencing analysis indicated that SIRT5 regulated endothelial inflammation and coagulation responses by inhibiting the F2RL3/SERPINA3/TGF‑β pathway. Conclusion ·SIRT5 negatively regulates the F2RL3/SERPINA3/TGF‑β signaling axis, thereby alleviating endothelial inflammation and promoting coagulation responses, suggesting its potential protective role in sepsis-induced lung injury.

Key words: sepsis, acute lung injury, endothelial cells, sirtuin 5 (SIRT5)

中图分类号:

R364.5

赵善志, 郑相涛, 王晓峰, 陈尔真, 龚方晨, 陈影. SIRT5在脓毒症肺血管内皮细胞损伤中的作用及机制研究[J]. 上海交通大学学报（医学版）, 2025, 45(9): 1116-1125.

ZHAO Shanzhi, ZHENG Xiangtao, WANG Xiaofeng, CHEN Erzhen, GONG Fangchen, CHEN Ying. Role and mechanisms of SIRT5 in pulmonary microvascular endothelial cell injury in sepsis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(9): 1116-1125.

图/表 6

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Gene	Sequence (5′→3′)
SIRT5
Forward	GCCATAGCCGAGTGTGAGAC
Reverse	CAACTCCACAAGAGGTACATCG
IL-6
Forward	GAAGAGCGCCGCTGAGAAT
Reverse	GTGCAGAGGGTTTAATGTCAACT
IL-1β
Forward	ATGATGGCTTATTACAGTGGCAA
Reverse	GTCGGAGATTCGTAGCTGGA
VCAM-1
Forward	GGGAAGATGGTCGTGATCCTT
Reverse	TCTGGGGTGGTCTCGATTTTA
E-selectin
Forward	CGCGGGGACATTCACTCTC
Reverse	TGGAACTTGGATAGTCTCAGACA
F2RL3
Forward	GCTGCTGCATTACTCGGAC
Reverse	ACGTAGGCACCATAGAGGTTG
SERPINA3
Forward	CCTGAAGGCCCCTGATAAGAA
Reverse	GCTGGACTGATTGAGGGTGC
TGF-β2
Forward	CAGCACACTCGATATGGACCA
Reverse	CCTCGGGCTCAGGATAGTCT
TGF-β3
Forward	ACTTGCACCACCTTGGACTTC
Reverse	GGTCATCACCGTTGGCTCA

图1 Sirt5 基因敲除小鼠7 d生存率和肺组织炎症水平Note： A. 7-d survival curves of wild-type (WT) and Sirt5 knockout (Sirt5 KO) mice following CLP surgery (n=10, P<0.001). B. Representative H-E-stained images of lung tissues from WT-Sham group, WT-CLP group, KO-Sham group, and KO-CLP group (n=3).

Fig 1 7 d survival and lung inflammation levels in Sirt5 gene knockout mice

图2 Sirt5 基因敲除改变脓毒症小鼠肺组织血管渗漏情况Note： A. Gross images of lung tissues from each group 24 h post CLP surgery, 30 min after intravenous injection of 200 μL Evans blue (EB) via the tail vein, demonstrating EB dye extravasation (indicating vascular leakage). B. Quantitative analysis of EB dye extravasation in lung tissues (n=3. P<0.001). C. Representative immunofluorescence staining images of lung tissues 24 h post CLP surgery. CD31 (green, endothelial cell marker), fibrinogen (red), and nuclei (DAPI, blue) are shown (n=3).

Fig 2 Effect of Sirt5 gene knockout on pulmonary vascular permeability in septic mice

图3 小鼠肺组织和LPS刺激的HUVEC中炎症相关基因表达情况Note： A. Representative immunohistochemical staining images for VCAM-1 in lung tissues 24 h post-CLP (brown indicates positive staining; n=3). B. Western blotting analysis and quantification of VCAM-1 and TF protein levels in HUVECs transduced with empty vector (EV) or SIRT5 over-expression vector (OV), under unstimulated condition or stimulated condition (1 μg/mL LPS +2 μg/mL CD14) for 4 h (n=3). C‒F. qPCR analysis of mRNA expression levels of inflammatory cytokines [IL-6 (C), IL-1β (D), VCAM-1 (E) and E-selectin (F)] in HUVECs transduced with EV or OV, under unstimulated or stimulated conditions for 4 h (n=3).

Fig 3 Expression of inflammation-related genes in mouse lung tissue and HUVECs after LPS stimulation

图4 LPS刺激的HUVEC的转录组测序结果Note： A. Volcano plot of differentially expressed genes (DEGs) in EV-LPS or OV-LPS transduced HUVECs under LPS stimulation (1 μg/mL) for 4 h. Red dots represent upregulated genes (log2 fold change>1, P<0.05); blue dots represent downregulated genes (log2 fold change<-1, P<0.05); gray dots represent non-significant (Nosig) genes. Key DEGs (e.g., F2RL3, SERPINA3, TGF-β2 and TGF-β3) are labeled. B. Gene Ontology (GO) enrichment analysis (biological process) of DEGs in HUVECs transduced with EV-LPS versus OV-LPS upon LPS stimulation for 4 h. Significantly enriched pathways (P<0.05) are displayed at the top. Bubble size represents the number of genes; color intensity represents enrichment significance [-log10 (P value)].

Fig 4 Transcriptome sequencing results of LPS-stimulated HUVECs

图5 SIRT5 敲低或过表达对HUVEC中炎症/凝血相关基因表达的影响Note： A‒D. qPCR analysis of mRNA expression levels for inflammation/coagulation-related genes [TGF-β2 (A), TGF-β3 (B), SERPINA3 (C) and F2RL3 (D)] in HUVECs transduced with control shRNA (shCtrl) or SIRT5 shRNA (shSIRT5), under unstimulated or LPS-stimulated conditions for 4 h (n=3). E‒H. qPCR analysis of mRNA expression levels for inflammation/coagulation-related genes [TGF-β2 (E), TGF-β3 (F), SERPINA3 (G) and F2RL3 (H)] in HUVECs transduced with EV or SIRT5 OV, under unstimulated or LPS-stimulated conditions for 4 h (n=3).

Fig 5 Effects of SIRT5 knockdown or overexpression on the expression of inflammation/coagulation-related genes in HUVECs

参考文献 19

[1]	SINGER M, DEUTSCHMAN C S, SEYMOUR C W, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3)[J]. JAMA, 2016, 315(8): 801-810.
[2]	RUDD K E, JOHNSON S C, AGESA K M, et al. Global, regional, and national sepsis incidence and mortality, 1990‒2017: analysis for the Global Burden of Disease Study[J]. Lancet, 2020, 395(10219): 200-211.
[3]	TOMAZINI B M, MAIA I S, CAVALCANTI A B, et al. Effect of dexamethasone on days alive and ventilator-free in patients with moderate or severe acute respiratory distress syndrome and COVID-19: the CoDEX randomized clinical trial[J]. JAMA, 2020, 324(13): 1307-1316.
[4]	MEYER N J, GATTINONI L, CALFEE C S. Acute respiratory distress syndrome[J]. Lancet, 2021, 398(10300): 622-637.
[5]	GORMAN E A, O' KANE C M, MCAULEY D F. Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management[J]. Lancet, 2022, 400(10358): 1157-1170.
[6]	QIAO X Y, YIN J H, ZHENG Z H, et al. Endothelial cell dynamics in sepsis-induced acute lung injury and acute respiratory distress syndrome: pathogenesis and therapeutic implications[J]. Cell Commun Signal, 2024, 22(1): 241.
[7]	IBA T, CONNORS J M, NAGAOKA I, et al. Recent advances in the research and management of sepsis-associated DIC[J]. Int J Hematol, 2021, 113(1): 24-33.
[8]	ARORA J, MENDELSON A A, FOX-ROBICHAUD A. Sepsis: network pathophysiology and implications for early diagnosis[J]. Am J Physiol Regul Integr Comp Physiol, 2023, 324(5): R613-R624.
[9]	KE Z M, SHEN K K, WANG L, et al. Emerging roles of mitochondrial sirtuin SIRT5 in succinylation modification and cancer development[J]. Front Immunol, 2025, 16: 1531246.
[10]	WU Q J, ZHANG T N, CHEN H H, et al. The sirtuin family in health and disease[J]. Signal Transduct Target Ther, 2022, 7(1): 402.
[11]	GORBUNOVA V, SELUANOV A. SIRT5 slows skeletal muscle ageing by alleviating inflammation[J]. Nat Metab, 2025, 7(3): 447-449.
[12]	YU Q W, ZHANG J K, LI J Y, et al. Sirtuin 5-mediated desuccinylation of ALDH2 alleviates mitochondrial oxidative stress following acetaminophen-induced acute liver injury[J]. Adv Sci (Weinh), 2024, 11(39): e2402710.
[13]	HONG J Y, WANG X Y, MEI C G, et al. Competitive regulation by transcription factors and DNA methylation in the bovine SIRT5 promoter: roles of E2F4 and KLF6[J]. Gene, 2019, 684: 39-46.
[14]	WANG C H, HE D H, SHI C P. SIRT5 reduces the inflammatory response and barrier dysfunction in IL-17A-induced epidermal keratinocytes[J]. Allergol Immunopathol (Madr), 2023, 51(1): 30-36.
[15]	TESFAMARIAM B, DEFELICE A F. Endothelial injury in the initiation and progression of vascular disorders[J]. Vascul Pharmacol, 2007, 46(4): 229-237.
[16]	ZHANG X D, LING C X, XIONG Z Y, et al. Desuccinylation of TBK1 by SIRT5 regulates inflammatory response of macrophages in sepsis[J]. Cell Rep, 2024, 43(12): 115060.
[17]	WANG C H, HE D H, SHI C P. SIRT5 reduces the inflammatory response and barrier dysfunction in IL-17A-induced epidermal keratinocytes[J]. Allergol Immunopathol (Madr), 2023, 51(1): 30-36.
[18]	YAO P, CHEN T, JIANG P, et al. Functional skewing of TRIM21-SIRT5 interplay dictates IL-1β production in DSS-induced colitis[J]. EMBO Rep, 2022, 23(9): e54391.
[19]	LI R R, MENG M, CHEN Y, et al. ATP-citrate lyase controls endothelial gluco-lipogenic metabolism and vascular inflammation in sepsis-associated organ injury[J]. Cell Death Dis, 2023, 14(7): 401.

SIRT5在脓毒症肺血管内皮细胞损伤中的作用及机制研究

Role and mechanisms of SIRT5 in pulmonary microvascular endothelial cell injury in sepsis

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