|
| 超级增强子促进PSAP表达介导急性肝衰竭的实验研究 |
| Experimental Study on Super-Enhancers-Mediated Acute Liver Failure by Promoting PSAP Expression |
| 投稿时间：2026-01-14 修订日期：2026-08-16 |
| DOI： |
| 中文关键词: 急性肝衰竭 表观遗传调控 超级增强子 前鞘脂激活蛋白 |
| 英文关键词: Acute liver failure Epigenetic regulation Super enhancers Prosaposin |
| 基金项目:无 |
|
| 摘要点击次数: 0 |
| 全文下载次数: 0 |
| 中文摘要: |
| 目的 探讨超级增强子（SEs）调控前鞘脂激活蛋白（PSAP）表达在急性肝衰竭（ALF）肝细胞损伤中的作用和机制。方法 通过整合3个独立的ALF转录组数据集，筛选肝特异性SEs相关的差异表达基因，采用蛋白互作网络及单细胞数据分析鉴定关键基因。使用脂多糖（LPS，1 μg/mL）处理人肝永生化细胞（THLE-2）在体外构建ALF细胞损伤模型。在LPS处理或磷酸盐缓冲液（PBS）处理THLE-2细胞后，通过CCK-8检测不同处理条件下的THLE-2细胞活性，试剂盒检测细胞上清中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和乳酸脱氢酶（LDH）的释放水平，实时荧光定量逆转录聚合酶链反应检测白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNFα）和PSAP的mRNA表达水平，蛋白质免疫印迹检测裂解型半胱氨酸天冬氨酸特异性蛋白酶-3（Caspase-3）和PSAP蛋白表达水平。通过肝组织的染色质免疫共沉淀测序（ChIP-seq）数据可视化分析PSAP的SEs区域。构建PSAP敲低以及靶向其SEs的THLE-2细胞模型并用LPS（1 μg/mL）处理，验证PSAP在ALF细胞损伤中的作用机制。结果 共筛选出74个在ALF中一致性上调且受SEs调控的基因，其中PSAP在肝细胞中表达最高。与LPS未处理组相比，LPS处理组细胞活性下降，ALT、AST和LDH释放水平升高，IL-1β和TNFα的mRNA水平升高，以及裂解型Caspase-3蛋白表达水平升高，提示体外ALF细胞损伤模型构建成功。此外，与LPS未处理组相比，PSAP在ALF细胞损伤模型中mRNA和蛋白水平均升高。与阴性对照组或空载体组相比，敲低PSAP或靶向SEs均可改善LPS诱导的细胞损伤，表现为细胞活性升高，ALT、AST和LDH释放水平下降，IL-1β和TNFα的mRNA水平下降，以及裂解型Caspase-3蛋白表达水平降低。ChIP-seq显示PSAP的SEs区域（chr10:71,817,502-71,871,378）中存在丰富且连续的组蛋白H3第27位赖氨酸乙酰化信号。结论 SEs可能通过驱动PSAP表达介导ALF肝细胞损伤，抑制PSAP或靶向其SEs区域可能改善肝细胞损伤。 |
| 英文摘要: |
| Objective To investigate the role and mechanism of super-enhancers (SEs)-mediated regulation of prosaposin (PSAP) expression in hepatocyte injury during acute liver failure (ALF). Methods Three independent ALF transcriptome datasets were integrated to screen for differentially expressed genes associated with liver-specific SEs. Protein-protein interaction networks and single-cell data analysis were used to identify key genes. An in vitro ALF cell injury model was established by treating human immortalized hepatocytes (THLE-2) with lipopolysaccharide (LPS, 1 μg/mL). After treatment of THLE-2 cells with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS), cell viability was measured by CCK-8 assay; the release levels of alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) in cell supernatants were detected using commercial kits; mRNA levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα) and PSAP were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR); protein levels of cleaved cysteine-aspartic acid protease-3 (Caspase-3) and PSAP were examined by western blotting. The SEs region of PSAP was visualized and analyzed using chromatin immunoprecipitation sequencing (ChIP-seq) data from liver tissues. THLE-2 cell models with PSAP knockdown and SEs targeting were constructed and treated with LPS (1 μg/mL) to verify the mechanism of PSAP in ALF cell injury. Results A total of 74 genes consistently upregulated in ALF and regulated by SEs were identified, among which PSAP showed the highest expression in hepatocytes. Compared with the untreated group, LPS treatment led to decreased cell viability, elevated release of ALT, AST and LDH, increased mRNA levels of IL-1β and TNFα, and upregulated protein expression of cleaved Caspase-3, confirming successful establishment of the in vitro ALF cell injury model. Moreover, mRNA and protein levels of PSAP were both increased in the ALF cell model compared with the untreated group. Compared with the negative control or empty vector groups, PSAP knockdown or SEs targeting ameliorated LPS-induced cell injury, as reflected by enhanced cell viability, reduced release of ALT, AST and LDH, decreased mRNA levels of IL-1β and TNFα, and downregulated expression of cleaved Caspase-3. ChIP-seq revealed abundant and continuous histone h3 lysine 27 acetylation signals in the SEs region of PSAP (chr10:71,817,502–71,871,378). Conclusion SEs mediate hepatocyte injury in ALF by driving PSAP expression. Inhibition of PSAP or targeting its SEs region can alleviate hepatocyte injury. |
|
View Fulltext
查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
