血红蛋白β亚基通过调控肺血管内皮糖酵解减轻脓毒症急性肺损伤

Hemoglobin β‐subunit attenuates sepsis‐induced acute lung injury by modulating glycolysis in pulmonary vascular endothelium

摘要

目的：探究血红蛋白β亚基（HBB）在脓毒症急性肺损伤中的作用及机制。方法：4周龄雄性C57BL/6小鼠随机分成假手术（Sham）组、盲肠结扎穿刺（CLP）组和HBB过表达（HBB‐OE）+CLP组。通过尾静脉注射腺相关病毒（AAV）构建小鼠肺内皮HBB过表达模型，3周后行Sham和CLP处理。术后记录小鼠生存率；取肺组织进行HE染色并计算Smith评分；测量肺湿干质量比（W/D）；ELISA检测小鼠血清肿瘤坏死因子‐α（TNF‐α）、白细胞介素‐1β（IL‐1β）水平；Western Blot、qRT‐PCR与免疫荧光检测肺组织中HBB的表达水平。用慢病毒感染人脐静脉内皮细胞（HUVECs），成功后给予磷酸盐缓冲液（PBS）和脂多糖（LPS）处理。Western Blot与qRT‐PCR检测HBB、糖酵解关键酶（LDHA、HK2和PKM2）表达水平，比色法检测乳酸含量。结果：与Sham组相比，CLP组死亡率升高，肺组织损伤加重，W/D升高，血清TNF‐α、IL‐1β升高，HBB表达降低；HBB过表达后上述指标均有所下降（P<0.05）。细胞实验表明，HBB对LPS诱导的糖酵解关键酶表达升高和乳酸蓄积有显著抑制作用（P<0.05）。结论：HBB可能通过调控肺血管内皮糖酵解减轻脓毒症急性肺损伤。

Abstract

Objective: To investigate the role and mechanism of hemoglobin β‐subunit (HBB) in sepsis‐induced acute lung injury. Methods: Four‐week‐old male C57BL/6 mice were randomly assigned to three groups: a Sham group, a CLP group, and an HBB‐OE+CLP group. A pulmonary endothelial HBB overexpression model was established in mice via tail vein injection of an adeno‐associated virus. Three weeks post‐injection, Sham or CLP surgery was performed on the mice. Post‐procedure, survival rates were recorded. Lung tissues were harvested for HE staining and calculation of the Smith score, as well as for determining the lung W/D ratio. Serum levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) were quantified using ELISA. The expression of HBB in lung tissue was assessed by Western Blot, qRT‐PCR, and immunofluorescence. For in vitro experiments, human umbilical vein endothelial cells (HUVECs) were transduced with a lentivirus to modulate HBB expression. Following successful transduction, the cells were treated with either phosphate‐buffered saline (PBS) or lipopolysaccharide (LPS). The expression levels of HBB and key glycolytic enzymes (LDHA, HK2, and PKM2) were measured by Western Blot and qRT‐PCR. Lactate content was determined using a colorimetric assay. Results: Compared with Sham group, mice in the CLP group exhibited increased mortality, more severe lung tissue damage, an elevated lung W/D ratio, and higher serum levels of IL‐1β and TNF‐α. Notably, HBB expression was significantly decreased in the CLP group. Overexpression of HBB attenuated these CLP‐induced changes (P<0.05).In vitro, HBB overexpression significantly suppressed the LPS‐induced upregulation of key glycolytic enzymes and the subsequent lactate accumulation (P<0.05). Conclusion: HBB may alleviate sepsis‐induced ALI by regulating glycolysis in pulmonary vascular endothelial cells.

关键词

脓毒症 / 急性肺损伤 / 急性呼吸窘迫综合征 / 血红蛋白β亚基 / 糖酵解 / 细胞代谢

Key words

Sepsis / Acute Lung Injury / Acute Respiratory Distress Syndrome / Hemoglobin β‐Subunit / Glycolysis / Cellular Metabolism

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基金资助

国家重点研发计划(2023YFC2308404)

新发突发与重大传染病防控国家科技重大专项(2025ZD01902600)

国家自然科学基金面上项目(82272226)

国家自然科学基金面上项目(82572461)

国家自然科学基金青年科学基金项目（C类）(82502662)