DEAD-box解旋酶3X-连锁（DDX3X）在免疫介导性肝损伤小鼠模型和HBV相关肝损伤患者中的表达分析

潘桢桢; 徐玲; 朱先茹; 范子豪; 曹亚玲; 莫胤康; 闫赛; 任锋

doi:10.12449/JCH260116

DEAD-box解旋酶3X-连锁（DDX3X）在免疫介导性肝损伤小鼠模型和HBV相关肝损伤患者中的表达分析

DOI: 10.12449/JCH260116

首都医科大学附属北京佑安医院北京肝病研究所，北京 100069

基金项目:

国家自然科学基金 (81770611);

国家自然科学基金 (82002243);

北京自然科学基金和北京市教委联合资助重点项目 (KZ202010025035);

首都卫生发展科研专项重点攻关项目 (SF2020-1-1151);

北京市科技计划“首都临床诊疗技术研究及示范应用”专项课题 (Z191100006619096);

北京市科技计划“首都临床诊疗技术研究及示范应用”专项课题 (Z191100006619097);

北京市医院管理中心“青苗”计划专项 (QML20201702);

北京市医管局“登峰”人才计划 (DFL20221503);

高层次公共卫生技术人才建设项目 (Subject leaders-02-13)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：潘桢桢负责设计实验，分析、解释数据，起草文章，统计分析；徐玲负责实施研究，审阅文章，统计分析；朱先茹负责实施研究，分析、解释数据，统计分析；范子豪负责设计实验，统计分析；曹亚玲负责审阅文章；莫胤康负责分析、解释数据；闫赛负责统计分析；任锋负责审阅文章，获取研究经费，技术及材料支持。

详细信息

通信作者:
任锋， renfeng7512@ccmu.edu.cn （ORCID： 0000-0001-7736-8637）

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出版历程
- 收稿日期: 2025-06-22
- 录用日期: 2025-09-18
- 出版日期: 2026-01-25

Role of endoplasmic reticulum stress-mediated DEAD-box helicase 3 X-linked in a mouse model of concanavalin A-induced immune-mediated liver injury

Institute of Hepatology，Beijing YouAn Hospital，Capital Medical University，Beijing 100069，China

Research funding:

National Natural Science Foundation of China (81770611);

National Natural Science Foundation of China (82002243);

Key Projects of the Beijing Municipal Education Commission’s Science and Technology Plan (KZ202010025035);

Special Key Research Project of Capital Health Development Scientific Research (SF2020-1-1151);

The Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing (Z191100006619096);

The Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing (Z191100006619097);

Beijing Hospitals Authority Youth Programme (QML20201702);

Talent Cultivation plan of “Climbing the Peak” of Beijing Municipal Hospital Administration (DFL20221503);

High-level Public Health Technical Personnel Construction Project (Subject leaders-02-13)

More Information

Corresponding author: REN Feng， renfeng7512@ccmu.edu.cn （ORCID： 0000-0001-7736-8637）

摘要

摘要: 目的探讨DEAD-box解旋酶3X-连锁（DDX3X）在免疫介导性肝损伤（ILI）中的作用，并阐明其通过调控内质网应激（ERS）依赖性凋亡通路的作用机制及其与乙型肝炎临床进展的相关性。方法采用刀豆蛋白A（ConA）尾静脉注射建立小鼠ILI模型；对肝细胞特异性DDX3X敲除小鼠（DDX3X^ΔHep）和DDX3X-flox小鼠（DDX3X^fl/fl）分别尾静脉注射PBS（对照组）和不同浓度的ConA，通过Log-rank生存分析、血清天冬氨酸氨基转移酶（AST）及丙氨酸氨基转移酶（ALT）检测、肝组织苏木精-伊红（HE）染色评估肝损伤严重程度；采用实时荧光定量PCR（qRT-PCR）和蛋白质印迹法（Western Blot）检测肝组织中葡萄糖调节蛋白78（GRP78）、CCAAT/增强子结合蛋白同源蛋白（CHOP）及DDX3X的表达；通过腹腔注射4-苯基丁酸（4-PBA，100 mg/kg）抑制ERS；收集北京佑安医院2019—2023年健康对照、慢性乙型肝炎及HBV相关肝衰竭（HBV-LF）患者血清样本各30例，肝组织样本各6例，通过酶联免疫吸附分析检测血清DDX3X水平，qRT-PCR/Western Blot分析肝组织靶标表达。计量资料多组间比较采用单因素方差分析，进一步两两比较使用LSD-t检验。采用Kaplan-Meier法绘制生存曲线。结果相较于正常对照组，ConA诱导肝损伤后小鼠肝脏DDX3X表达显著升高（P<0.05）。在致死剂量ConA处理后，DDX3X^ΔHep小鼠72 h生存率为55%，显著高于DDX3X^fl/fl对照组的20%（P<0.05），血清ALT和AST的水平亦同步显著降低（P值均<0.000 1），ERS标志物GRP78与CHOP表达同步下调（P<0.05）。在ConA诱导的小鼠肝损伤中，与PBS预处理组相比，4-PBA预处理抑制ERS不仅减轻了ConA诱导的肝损伤（ALT和AST降低，P值均<0.001），亦使肝脏DDX3X的mRNA与蛋白表达降低（P值均<0.01）。临床样本分析结果中，慢性乙型肝炎及HBV-LF患者肝组织DDX3X的mRNA和蛋白表达均高于健康对照（P值均<0.01），HBV-LF患者血清DDX3X水平显著升高（P<0.000 1）。结论 DDX3X通过调控ERS依赖性凋亡通路（GRP78/CHOP）加重ILI，其表达与乙型肝炎疾病进展相关，可作为潜在治疗靶点。
- 内质网应激 /
- 刀豆蛋白A /
- 免疫介导性肝损伤
Abstract: Objective To investigate the role of DEAD-box helicase 3 X-linked （DDX3X） in immune-mediated liver injury （ILI）， and to clarify its mechanism by regulating endoplasmic reticulum stress （ERS）-dependent apoptotic pathway and its association with the clinical progression of hepatitis B. Methods Mice were given injection of concanavalin A （ConA） via the caudal vein to establish a model of ILI， PBS （control group） and different concentrations of ConA were injected into the tail vein of hepatocyte-specific DDX3X-knockout mice （DDX3X^ΔHep and DDX3X-flox mice （DDX3X^fl/fl）， respectively.. The log-rank survival analysis， measurement of the serum levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）， and HE staining of liver tissue were performed to assess liver injury， and qRT-PCR and Western Blot were used to measure the mRNA and protein expression levels of glucose-regulated protein 78 （GRP78）， CCAAT/enhancer-binding protein homologous protein （CHOP）， and DDX3X in liver tissue. Intraperitoneal injection of 4-phenylbutyric acid （4-PBA， 100 mg/kg） was performed to inhibit ERS. Serum samples （n=30） and liver tissue samples （n=6） were collected from healthy controls， chronic hepatitis B （CHB） patients， and hepatitis B virus-associated liver failure （HBV-LF） patients； ELISA was used to measure the serum level of DDX3X， and qRT-PCR/Western Blot was used to analyze the expression of targets in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the control group of mice， the expression of DDX3X in the liver of mice induced by ConA was significantly increased after liver injury （P<0.05）， and hepatocyte-specific DDX3X knockout increased the 72-hour survival rate of mice by 55% （compared with 20% in the DDX3X^fl/fl group）， with significant reductions in the serum levels of ALT and AST （P<0.000 1） and the expression levels of the ERS markers GRP78 and CHOP （P<0.05）. After ERS was inhibited by 4-PBA， there was alleviation of liver injury （with reductions in ALT and AST， P <0.001） and a reduction in DDX3X expression （P<0.01）. The analysis of clinical samples showed that the mRNA and protein expression levels of liver DDX3X in CHB patients and HBV-LF patients were significantly higher than those in healthy controls （all P<0.01）， and there was a significant increase in the serum level of DDX3X in HBV-LF patients （P<0.000 1）. Conclusion DDX3X exacerbates ILI by regulating the ERS-dependent apoptotic pathway （GRP78/CHOP）， and its expression is associated with the progression of hepatitis B. Therefore， it can be used as a potential therapeutic target.
- Endoplasmic Reticulum Stress /
- Concanavalin A /
- Immune-Mediated Liver Injury

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注： ConA，刀豆蛋白A；DDX3X，DEAD-box解旋酶3X-连锁；ALT，丙氨酸氨基转移酶；AST，天冬氨酸氨基转移酶；ERS，内质网应激；PBS，磷酸盐缓冲溶液；4-PBA，4-苯基丁酸；CHOP，CCAAT增强子结合蛋白同源蛋白；GRP78，葡萄糖调节蛋白78；CHB，慢性乙型肝炎；HBV-LF，乙型肝炎病毒相关肝衰竭。

图 1 ERS介导DDX3X促进ConA诱导的小鼠ILI的作用研究流程图

Figure 1. Study flowchart of the role of ERS in mediating DDX3X-promoted immune-mediated liver injury induced by ConA in mice

下载: 全尺寸图片幻灯片

注： a，qRT-PCR检测DDX3X的mRNA水平；b，通过ImageJ测定Western Blot图像灰度值，并进行定量；c，Western Blot检测DDX3X的蛋白表达水平；d，肝组织病理学结果（HE染色，×10），黑色箭头处为损伤区域。*P<0.05，**P<0.01，****P<0.000 1。DDX3X，DEAD-box解旋酶3X-连锁；HPRT，次黄嘌呤磷酸核糖基转移酶；ConA，刀豆蛋白A；β-actin，β-肌动蛋白。

图 2 DDX3X在ConA诱导的肝损伤期间的表达情况

Figure 2. Expression of DDX3X in the liver during liver injury induced by ConA

下载: 全尺寸图片幻灯片

注： a，DDX3X^fl/fl和DDX3X^ΔHep小鼠用ConA（40 mg/kg）处理72 h生存曲线；b，DDX3X^fl/fl和DDX3X^ΔHep小鼠用ConA（25 mg/kg）处理8 h后ALT、AST水平；c，DDX3X^fl/fl和DDX3X^ΔHep小鼠用ConA（40 mg/kg）处理8 h后ALT、AST水平；d，肝组织病理学结果（HE染色，×10），黑色箭头处为损伤区域。****P<0.000 1。ALT，丙氨酸氨基转移酶；AST，天冬氨酸氨基转移酶；DDX3X，DEAD-box解旋酶3X-连锁；DDX3X^fl/fl，DDX3X基因的野生型等位基因型小鼠；DDX3X^ΔHep，肝细胞特异性DDX3X敲除小鼠；ConA，刀豆蛋白A。

图 3 肝细胞特异性敲除DDX3X可改善ConA诱导的小鼠肝损伤

Figure 3. Hepatocellular specific knockout of DDX3X can improve liver injury induced by ConA in mice

下载: 全尺寸图片幻灯片

注： a，GRP78和CHOP的mRNA水平；b，Western Blot检测结果；c，GRP78和CHOP的蛋白表达水平。*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。GRP78，葡萄糖调节蛋白78；HPRT，次黄嘌呤磷酸核糖基转移酶；DDX3X，DEAD-box解旋酶3X-连锁；CHOP，CCAAT增强子结合蛋白同源蛋白；β-actin，β-肌动蛋白；ConA，刀豆蛋白；DDX3X^fl/fl，DDX3X基因的野生型等位基因型小鼠；DDX3X^ΔHep，肝细胞特异性DDX3X敲除小鼠。

图 4 肝细胞特异性敲除DDX3X减少ERS相关凋亡标志物的表达

Figure 4. Hepatocellular specific knockout of DDX3X reduces the expression of ERS-related apoptotic markers

下载: 全尺寸图片幻灯片

注： a，血清ALT和AST水平；b，肝组织病理学结果（HE染色，×10），黑色箭头处为损伤区域；c，GRP78、CHOP、DDX3X的mRNA水平；d，Western Blot检测结果；e，GRP78、CHOP、DDX3X的蛋白表达水平。**P<0.01，***P<0.001，****P<0.000 1。ALT，丙氨酸氨基转移酶；AST，天冬氨酸氨基转移酶；4-PBA，4-苯基丁酸；PBS，磷酸盐缓冲溶液；ConA，刀豆蛋白A；GRP78，葡萄糖调节蛋白78；HPRT，次黄嘌呤磷酸核糖基转移酶；CHOP，CCAAT增强子结合蛋白同源蛋白；DDX3X，DEAD-box解旋酶3X-连锁；β-actin，β-肌动蛋白。

图 5 抑制ERS可下调ConA诱导的小鼠肝损伤中DDX3X的表达

Figure 5. Inhibition of ERS significantly reduced the expression of DDX3X in ConA-induced liver injury in mice

下载: 全尺寸图片幻灯片

注： a，GRP78、CHOP和DDX3X的mRNA水平；b，Western Blot检测结果；c，GRP78、CHOP和DDX3X的蛋白表达水平；d，血清DDX3X水平。**P<0.01，***P<0.001，****P<0.000 1。GRP78，葡萄糖调节蛋白78；CHOP，CCAAT增强子结合蛋白同源蛋白；DDX3X，DEAD-box解旋酶3X-连锁。

图 6 DDX3X在ILI患者中的表达情况

Figure 6. Expression of DDX3X in patients with immune-mediated liver injury

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因	正向（5′-3′）	反向（5′-3′）
HPRT（小鼠）	AGTCCCAGCGTCGTGATTAG	GCCTCCCATCTCCTTCATGA
DDX3X（小鼠）	GGCCGTGGAGATAGAAGTGG	TGCACTGCCAATTCTCTCGT
GRP78（小鼠）	CTTTGATCAGCGGGTCATGG	AGCTCTTCAAATTTGGCCCG
CHOP（小鼠）	TCACTACTCTTGACCCTGCG	ACTGACCACTCTGTTTCCGT
HPRT（人）	TTCCTCCTCCTGAGCAGTCA	ATCCAACACTTCGTGGGGTC
DDX3X（人）	GGGTGTCAAACTTCAACCGC	TCCAGTCACCGGCATAGAGA
GRP78（人）	TAGCGTATGGTGCTGCTGTC	TTTGTCAGGGGTCTTTCACC
CHOP（人）	TGGAAGCCTGGTATGAGGAC	TGTGACCTCTGCTGGTTCTG
注：HPRT，次黄嘌呤磷酸核糖基转移酶；DDX3X，DEAD-box解旋酶3X-连锁；GRP78，葡萄糖调节蛋白78；CHOP，CCAAT增强子结合蛋白同源蛋白。

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