PAD4抑制剂GSK484通过抑制H3Cit表达减轻小鼠脓毒症肺损伤后内皮功能障碍的发生

doi:10.12122/j.issn.1673-4254.2024.12.16

摘要/Abstract

摘要：

目的探讨PAD4抑制剂（GSK484）对脓毒症后H3Cit表达的抑制作用，并探究其对脓毒症引起的内皮功能障碍的改善效果。方法 C57BL/6小鼠18只通过盲肠结扎穿孔术（CLP）构建小鼠脓毒症模型，实验分为假手术组，模型组和GSK484治疗组，6只/组，治疗组在术后第2天腹腔注射给予GSK484（4 mg/kg）。ELISA检测小鼠血清VEGF、ESM-1、IL-6、IL-1β水平，HE染色观察肺组织病理学变化，免疫荧光和Western blotting检测各组小鼠肺组织中F-actin、VE-cadherin、ZO-1蛋白表达，Western blotting检测肺组织VE-Cadherin和H3Cit蛋白表达情况。肺组织原代分离培养肺微血管内皮细胞，使用脂多糖（LPS，10 μg/mL）干预24 h构建脓毒症模型。检测细胞成管能力，CCK-8检测细胞增殖，流式检测细胞凋亡，ELISA检测细胞上清中VEGF、ESM-1、IL-6、IL-1β水平。结果与假手术组相比，脓毒症小鼠肺组织肺脏血管充血、肺泡断裂、肺泡腔及肺间质水肿、肺泡隔增厚、中性粒细胞浸润等，血清中炎症因子IL-6、IL-1β和内皮细胞因子VEGF、ESM-1含量均升高（P<0.05），肺组织中胞间连接蛋白F-actin、VE-cadherin、ZO-1表达降低，H3Cit蛋白表达升高（P<0.05），GSK484治疗可以有效改善这些变化。LPS诱导肺微血管内皮细胞上清中炎症因子表达量升高、内皮细胞因子VEGF、ESM-1含量均升高（P<0.05），细胞成管能力降低；2.5 μmol/L浓度GSK484处理可降低炎症因子和内皮细胞因子表达（P<0.05）。结论 GSK484的使用可有效抑制脓毒症后H3Cit表达，进一步改善脓毒症后内皮功能障碍的发生。

关键词: 脓毒症, 肺损伤, PAD4抑制剂, 小鼠肺微血管内皮细胞, 内皮功能障碍, GSK484

Abstract:

Objective To investigate the inhibitory effect of GSK484, a PAD4 inhibitor, on H3Cit expression following sepsis and its effects for improving sepsis-induced endothelial dysfunction. Methods Eighteen C57BL/6 mice were randomized into sham-operated group, sepsis model group and GSK484 treatment group (n=6), and in the latter two groups, models of sepsis were established by cecal ligation and puncture (CLP). The mice in GSK484 treatment group were given an intraperitoneal injection of GSK484 (4 mg/kg) on the second day following the surgery. Twenty-four hours after the injection, the mice were euthanized for measurement of serum levels of VEGF, ESM-1, IL-6 and IL-1β using ELISA. Lung tissue pathology was observed with HE staining, and pulmonary expressions of F-actin, VE-cadherin, ZO-1 and H3Cit proteins were detected using immunofluorescence staining and Western blotting. In primary cultured of mouse lung microvascular endothelial cells, the effect of stimulation with LPS (10 μg/mL) for 24 h on tube formation, proliferation, apoptosis and expressions of VEGF, ESM-1, IL-6 and IL-1β were assessed using CCK-8 assay, flow cytometry and ELISA. Results Compared to the sham-operated mice, the septic mice exhibited significant lung tissue pathologies characterized by vascular congestion, alveolar rupture, edema, and neutrophil infiltration. Serum levels of IL-6, IL-1β, VEGF, and ESM-1 were elevated, pulmonary expressions of F-actin, VE-cadherin, and ZO-1 were decreased, and H3Cit expression was increased significantly in the septic mice. GSK484 treatment effectively mitigated these changes in the septic mice. The LPS-stimulated endothelial cells showed increased productions of IL-6, IL-1β, VEGF and ESM-1, which were significantly reduced after treatment with 2.5 μmol/L GSK484. Conclusion GSK484 treatment effectively suppresses H3Cit expression in septic mice to ameliorate sepsis-induced endothelial dysfunction.

Key words: sepsis, lung injury, PAD4 inhibitor, mouse pulmonary microvascular endothelial cells, endothelial dysfunction, GSK484

苏晓飞, 李霖, 戴靖榕, 肖宝, 金子琦, 刘斌. PAD4抑制剂GSK484通过抑制H3Cit表达减轻小鼠脓毒症肺损伤后内皮功能障碍的发生[J]. 南方医科大学学报, 2024, 44(12): 2396-2403.

Xiaofei SU, Lin LI, Jingrong DAI, Bao XIAO, Ziqi JIN, Bin LIU. GSK484, a PAD4 inhibitor, improves endothelial dysfunction in mice with sepsis-induced lung injury by inhibiting H3Cit expression[J]. Journal of Southern Medical University, 2024, 44(12): 2396-2403.

图/表 9

表1 引物序列

Tab.1 Primer sequence

Primers	F (5'-3')	R (5'-3')
β-actin	AGGGAAATCGTGCGTGAC	CATACCCAAGAAGGAAGGCT
IL-6	TCCGGAGAGGAGACTTCACA	TTGCCATTGCACAACTCTTTTC
IL-1β	GAAATGCCACCTTTTGACAGTG	TGGATGCTCTCATCAGGACAG

图1 小鼠肺组织HE染色及病理评分

Fig. 1 HE staining (Original magnification: ×400) and pathological score (n=3) of mouse lung tissue. *P<0.05 vs Sham group; #P<0.05 vs cecal ligation and puncture (CLP) group.

图2 各组脓毒症小鼠血清中血管内皮相关因子和炎症因子变化

Fig.2 Alterations in serum levels of vascular endothelial-related and inflammatory factors in septic mice in different groups (n=6). *P<0.05 vs Sham group; #P<0.05 vs CLP group.

图3 免疫荧光检测各组脓毒症诱导的小鼠肺组织中F-actin、VE-cadherin和ZO-1的表达

Fig.3 Analysis of F-actin, VE-cadherin and ZO-1 expressions in mouse lung tissues in different groups using immunofluorescence staining (×400, n=3). *P<0.05 vs sham group; #P<0.05 vs CLP group.

图4 Western blotting检测各组脓毒症诱导的小鼠肺组织中VE-Cadherin和H3Cit表达比较

Fig.4 VE-Cadherin and H3Cit protein expressions in mouse lung tissues in different groups detected by Western blotting (n=3). *P<0.05 vs sham group; #P<0.05 vs CLP group.

图5 免疫荧光CD31鉴定原代小鼠肺为血管内皮细胞

Fig.5 Immunohistofluorescent detection of CD31 in primary mouse lung vascular endothelial cells (×400).

图6 细胞增殖率及IL-6和IL-1β mRNA表达水平

Fig.6 Cell proliferation rate and mRNA expression levels of IL-6 and IL-1β. A. The cell proliferation rate was assessed with CCK-8 assay after a 3-h treatment with GSK484 at different concentrations. B: mRNA expression levels of IL-6 and IL-1β measured by RT-qPCR after a 24-h exposure to LPS at different concentrations. *P<0.05 vs 0 μmol/L group.

图7 GSK484对LPS诱导脓毒症细胞模型成管能力影响

Fig.7 Effect of GSK484 on tube-forming ability of LPS-induced mouse lung vascular endothelial cells.*P<0.05 vs control group; #P<0.05 vs LPS group.

图8 GSK484预处理对LPS诱导脓毒症细胞上清中血管内皮相关因子和炎症因子水平影响

Fig.8 Effect of GSK484 on levels of vascular endothelial-related and inflammatory factors in the supernatants of LPS-induced mouse lung vascular endothelial cells. *P<0.05 vs control group; #P<0.05 vs LPS group.

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