Huang Ren, Guan Yan, Huang Wenjuan, Shang Yan, Xu Yanhong, Li Shuqi, Wan Rongwen
Department of Intensive Care Medicine, Hunan Provincial Hospital of Integrated Traditional Chinese and Western Medicine, Changsha, China.
Shock. 2025 Jul 1;64(1):97-105. doi: 10.1097/SHK.0000000000002588.
Background: Sepsis-acute kidney injury (AKI) is a common complication in critically ill patients with a very high mortality rate. Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is crucial in lipid metabolism; however, its role in the pathogenesis of sepsis-AKI remains unclear. Methods: Human renal tubular epithelial (HK2) cells stimulated with lipopolysaccharide (LPS) were used to establish sepsis-AKI cell models. Various assays, including cell counting kit 8, 5-ethynyl-2'-deoxyuridine staining, flow cytometry, and enzyme linked immunosorbent assay were employed to analyze the effects of LPS on HK2 cells. The levels of Fe 2+ , reactive oxygen species fluorescence intensity, and glutathione were measured to assess the impact of LPS on oxidative stress in HK2 cells. The expression of relevant genes was assessed by quantitative reverse transcription polymerase chain reaction and western blot. In terms of mechanism, the PROMO and JASPAR databases, chromatin immunoprecipitation assay, and dual luciferase reporter assay were engaged to predict and validate the transcriptional binding between upstream transcription factor 2 (USF2) and LPCAT3. In vivo experiments involved injecting adenovirus carrying Ad-sh-LPCAT3 via the tail vein to investigate the functional role of LPCAT3 in mice subjected to cecal ligation puncture-induced sepsis-AKI. Histological analyses were performed using hematoxylin and eosin staining, MASSON staining, and immunohistochemistry. Results: LPS inhibited the proliferation of HK2 cells while inducing apoptosis, inflammatory responses, and ferroptosis. LPCAT3 expression was upregulated in sepsis-AKI tissues and cells. Moreover, LPCAT3 knockdown weakened the sepsis-AKI in HK2 cells. Mechanistically, LPCAT3 was transcriptionally regulated by USF2, and LPCAT3 reversed the effects of si-USF2 in sepsis-AKI cell models via the nuclear factor erythroid 2-related factor 2/heme oxygenase-1/glutathione peroxidase 4 (NRF2/HO-1/GPX4) pathway. Mouse experiments demonstrated that LPCAT3 intensified sepsis-AKI through the same molecular mechanism in vivo . Conclusion: USF2 knockdown resulted in the downregulation of LPCAT3, thereby modulating the NRF2/HO-1/GPX4 pathway and aggravating sepsis-AKI and ferroptosis.
脓毒症相关性急性肾损伤（AKI）是危重症患者常见的并发症，死亡率极高。溶血磷脂酰胆碱酰基转移酶3（LPCAT3）在脂质代谢中起关键作用；然而，其在脓毒症相关性AKI发病机制中的作用尚不清楚。方法：采用脂多糖（LPS）刺激人肾小管上皮（HK2）细胞建立脓毒症相关性AKI细胞模型。运用多种检测方法，包括细胞计数试剂盒8、5-乙炔基-2'-脱氧尿苷染色、流式细胞术和酶联免疫吸附测定，分析LPS对HK2细胞的影响。检测Fe2+水平、活性氧荧光强度和谷胱甘肽水平，以评估LPS对HK2细胞氧化应激的影响。通过定量逆转录聚合酶链反应和蛋白质免疫印迹法评估相关基因的表达。在机制方面，利用PROMO和JASPAR数据库、染色质免疫沉淀测定和双荧光素酶报告基因测定，预测并验证上游转录因子2（USF2）与LPCAT3之间的转录结合。体内实验通过尾静脉注射携带Ad-sh-LPCAT3的腺病毒，研究LPCAT3在盲肠结扎穿刺诱导的脓毒症相关性AKI小鼠中的功能作用。采用苏木精-伊红染色、MASSON染色和免疫组织化学进行组织学分析。结果：LPS抑制HK2细胞增殖，同时诱导细胞凋亡、炎症反应和铁死亡。脓毒症相关性AKI组织和细胞中LPCAT3表达上调。此外，敲低LPCAT3可减轻HK2细胞中的脓毒症相关性AKI。机制上，LPCAT3受USF2转录调控，LPCAT3通过核因子红细胞2相关因子2/血红素加氧酶-1/谷胱甘肽过氧化物酶4（NRF2/HO-1/GPX4）途径逆转si-USF2在脓毒症相关性AKI细胞模型中的作用。小鼠实验表明，LPCAT3在体内通过相同的分子机制加重脓毒症相关性AKI。结论：敲低USF2导致LPCAT3下调，从而调节NRF2/HO-1/GPX4途径，加重脓毒症相关性AKI和铁死亡。
