连翘酯苷A通过激活PPAR-γ抑制中性粒细胞胞外捕获网形成减轻脓毒症相关急性呼吸窘迫综合征

doi:10.3877/cma.j.issn.2095-9133.2025.03.010

中华卫生应急电子杂志 ›› 2025, Vol. 11 ›› Issue (03) : 180 -187. doi: 10.3877/cma.j.issn.2095-9133.2025.03.010

论著

连翘酯苷A通过激活PPAR-γ抑制中性粒细胞胞外捕获网形成减轻脓毒症相关急性呼吸窘迫综合征

白霖果¹, 秦康杰², 郑杰², 李俊杰², 梅鸿², 刘鑫鑫², 覃松², 冯帮海³, 余琨²^,^†(

)

¹550000　贵州贵阳，贵州中医药大学第二附属医院神经外科
²563000　贵州遵义，遵义医科大学附属医院重症医学科
³563000　贵州遵义，遵义市中医院重症医学科

收稿日期:2025-02-27 出版日期:2025-06-18
通信作者: 余琨
基金资助:
国家自然科学基金(82460373); 贵州省科技厅基础研究计划项目(黔科合基础-ZK-2024-299，ZK-2023-544); 遵义市科技与大数据局科学技术基金项目：(遵市科合HZ字（2023）221号，（2023）199号，（2023）366号); 贵州省中医药管理局(QZYY-2024-137); 贵州省卫健委(gzwkj2024-310)

Forsythiaside A activates PPAR-γ to inhibit neutrophil extracellular traps and alleviate sepsis-associated acute respiratory distress syndrome

Linguo Bai¹, Kangjie Qin², Jie Zheng², Junjie Li², Hong Mei², Xinxin Liu², Song Qin², Banghai Feng³, Kun Yu²^,^†()

¹Department of Neurosurgery, Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550000, China
²Intensive Care Unit, Zunyi Medical University Affiliated Hospital, Zunyi 563000, China
³Intensive Care Unit, Zunyi Hospital of Traditional Chinese Medicine, Zunyi 563000, China

Received:2025-02-27 Published:2025-06-18
Corresponding author: Kun Yu

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引用本文：

白霖果, 秦康杰, 郑杰, 李俊杰, 梅鸿, 刘鑫鑫, 覃松, 冯帮海, 余琨. 连翘酯苷A通过激活PPAR-γ抑制中性粒细胞胞外捕获网形成减轻脓毒症相关急性呼吸窘迫综合征[J/OL]. 中华卫生应急电子杂志, 2025, 11(03): 180-187.

Linguo Bai, Kangjie Qin, Jie Zheng, Junjie Li, Hong Mei, Xinxin Liu, Song Qin, Banghai Feng, Kun Yu. Forsythiaside A activates PPAR-γ to inhibit neutrophil extracellular traps and alleviate sepsis-associated acute respiratory distress syndrome[J/OL]. Chinese Journal of Hygiene Rescue(Electronic Edition), 2025, 11(03): 180-187.

目的

探究连翘酯苷A（FA）调控中性粒细胞胞外捕获网减轻脓毒症相关急性呼吸窘迫综合征（ARDS）的作用及可能机制。

方法

80只小鼠随机分为假手术（sham）组、模型（CLP）组、FA组及FA+过氧化物酶体增殖物激活受体γ（PPAR-γ）组，采用经典盲肠结扎穿孔术建立脓毒症肺损伤小鼠模型；sham组仅行开关腹手术；小干扰PPAR-γ（siPPAR-γ）以10 nmol/20 g的剂量经尾静脉注射（2次/周，2周），FA以80 mg/kg的剂量经腹腔注射（1次/d，3 d）。模型构建成功后，采用卡普兰-迈耶曲线（Kaplan-Meier）生存曲线分析每组10只小鼠7 d累积生存率；剩余小鼠每组随机选取6只，取肺组织采用酶联免疫吸附试验（ELISA）试剂盒检测炎症因子（TNF-α、IL-6、IL-1β）及中性粒细胞胞外捕获网标记物（NE-DNA、MPO-NDA）水平；可见分光光度法丙二醛（MDA）水平、光电比色法测定超氧化物歧化酶（SOD）水平、荧光分光光度法测定活性氧（ROS）水平；计算肺W/D质量比；行苏木素-伊红（HE）染色光镜下观察病理改变并行肺损伤病理学评分；免疫荧光检测肺组织H3-cit蛋白表达；蛋白质印迹（Western blotting）实验检测PPAR-γ、瓜氨酸化组蛋白H3（H3-cit）、髓过氧化物酶（MPO）及中性粒细胞弹性蛋白酶（NE）蛋白相对表达水平。

结果

Kaplan-Meier生存曲线分析显示，FA组7 d累积生存率均明显高于CLP组及FA+ PPAR-γ组（P <0.05）。HE染色CLP组可见部分肺泡破坏，肺泡间隔增厚、大量炎性细胞浸润间隔及肺泡、部分肺泡透明膜形成伴肺泡萎陷；FA组HE染色可见肺泡及间质炎性细胞浸润明显减少，肺间隔厚度及肺泡萎陷减少；与sham组比较，CLP组肺损伤病理评分和肺W/D质量比升高（P<0.05），炎症因子TNF-α、IL-6及IL-1β水平升高，（P<0.05），NE-DNA、MPO-NDA表达升高（P<0.05），MDA和ROS水平升高（P<0.05），SOD水平降低（P<0.05）；PPAR-γ蛋白表达下降（P<0.05），H3-cit、MPO及NE蛋白表达升高（P<0.05）；与CLP组比较，FA组肺损伤病理评分和肺W/D质量比降低（P<0.05），炎症因子TNF-α、IL-6及IL-1β水平降低（P<0.05），NE-DNA、MPO-NDA表达水平降低（P<0.05），MDA和ROS水平降低（P<0.05），SOD水平升高（P<0.05），PPAR-γ蛋白表达升高（P<0.05），H3-cit、MPO及NE蛋白降低（P<0.05）。

结论

FA通过激活PPAR-γ来抑制中性粒细胞胞外捕获网形成减轻脓毒症相关急性呼吸窘迫综合征。

关键词: 连翘酯苷A, 过氧化物酶体增殖物激活受体γ, 中性粒细胞胞外捕获网, 脓毒症相关急性呼吸窘迫综合征

Objective

To explore the role of forsythoside A (FA) in regulating neutrophil extracellular traps (NETs) and alleviating sepsis-related ARDS, as well as its possible mechanism.

Methods

A total of 80 mice were randomly divided into sham, model (CLP), FA, and FA+PPAR-γ groups. A sepsis-induced lung injury mice model was established using the classic cecal ligation and puncture (CLP) procedure. The sham group underwent only laparotomy. siPPAR-γ (10 nmol/20g) was administered via tail vein injection (twice a week for 2 weeks), while FA was administered via intraperitoneal injection (80 mg/kg, once daily for 3 days). After successful model establishment, Kaplan-Meier survival curves were used to analyze the cumulative 7-day survival rate of 10 mice per group. For the remaining mice, six per group were randomly selected to collect lung tissue. Inflammatory factors (TNF-α, IL-6, IL-1β) and NET markers (NE-DNA, MPO-NDA) were detected using ELISA kits. SOD, MDA, and ROS levels were measured by spectrophotometry. The lung wet/dry weight (W/D) ratio was calculated. Pathological changes were observed under light microscope after H&E staining, and lung injury histopathological scoring was conducted. Immunofluorescence was used to detect H3-cit protein expression in lung tissue. Western blotting was performed to measure the relative expression levels of PPAR-γ, H3-cit, MPO, and NE proteins.

Results

Kaplan-Meier survival curve analysis revealed that the 7-day cumulative survival rate in the FA group was significantly higher than in the CLP and FA+PPAR-γ groups (P<0.05). The CLP group showed partial alveolar destruction, thickened alveolar septa, extensive inflammatory cell infiltration, alveolar and partial alveolar hyaline membrane formation, and alveolar collapse. In the FA group, H&E staining showed significant reduction in inflammatory cell infiltration, thinner alveolar septa, and less alveolar collapse. Compared to the sham group, the CLP group exhibited higher lung injury histopathological scores and W/D ratios (P<0.05), increased levels of inflammation factors (TNF-α, IL-6, IL-1β, P<0.05), elevated NE-DNA and MPO-NDA expressions (P<0.05), increased MDA and ROS levels (P<0.05), and decreased SOD levels (P<0.05). PPAR-γ protein expression was reduced (P<0.05), while H3-cit, MPO, and NE protein expressions were elevated (P<0.05). Compared to the CLP group, the FA group showed lower lung injury histopathological scores and W/D ratios (P<0.05), decreased levels of inflammatory factors (TNF-α, IL-6, IL-1β, P<0.05), reduced NE-DNA and MPO-NDA expression (P<0.05), lower MDA and ROS levels (P<0.05), higher SOD levels (P<0.05), and increased PPAR-γ protein expression (P<0.05). H3-cit, MPO, and NE proteins were reduced (P<0.05).

Conclusion

Forsythoside A alleviates sepsis-related ARDS by activating PPAR-γ to inhibit the formation of neutrophil extracellular traps.

Key words: Forsythoside A, PPAR-γ, Neutrophil extracellular traps, Sepsis-related acute respiratory distress syndrome

图1 7 d累积生存率

图2 各组小鼠肺组织HE染色情况（×100）

表1 各组小鼠肺组织W_湿/D_干重质量比、肺损伤病理评分及氧化应激指标（MDA、SOD、ROS）表达比较（

±s）

分组	鼠数	肺损伤病理评分	肺W/D质量比	MDA（U/mg）	SOD（nmol/mg）	ROS（U/mg）
Sham组	6	0.21±0.03	3.50±0.48	11.93±2.84	66.06±7.31	58.44±4.88
CLP组	6	0.77±0.05^a	6.38±0.46^a	71.27±4.60^a	20.72±3.45^a	194.56±7.26^a
FA组	6	0.58±0.06^ab	5.40±0.63^ab	44.08±5.43^ab	45.12±6.54^ab	113.92±9.38^ab
FA+siPPAR-γ组	6	0.89±0.03^abc	7.81±0.43^abc	82.15±4.41^abc	12.63±2.43^abc	237.25±5.58^abc
F值		244.40	76.85	302.10	123.90	788.40
P值		<0.001	<0.001	<0.001	<0.001	<0.001

表1 各组小鼠肺组织W_湿/D_干重质量比、肺损伤病理评分及氧化应激指标（MDA、SOD、ROS）表达比较（

±s）

分组	鼠数	肺损伤病理评分	肺W/D质量比	MDA（U/mg）	SOD（nmol/mg）	ROS（U/mg）
Sham组	6	0.21±0.03	3.50±0.48	11.93±2.84	66.06±7.31	58.44±4.88
CLP组	6	0.77±0.05^a	6.38±0.46^a	71.27±4.60^a	20.72±3.45^a	194.56±7.26^a
FA组	6	0.58±0.06^ab	5.40±0.63^ab	44.08±5.43^ab	45.12±6.54^ab	113.92±9.38^ab
FA+siPPAR-γ组	6	0.89±0.03^abc	7.81±0.43^abc	82.15±4.41^abc	12.63±2.43^abc	237.25±5.58^abc
F值		244.40	76.85	302.10	123.90	788.40
P值		<0.001	<0.001	<0.001	<0.001	<0.001

表2 各组小鼠肺组织TNF-α、IL-6和IL-1β表达比较（

±s）

分组	鼠数	IL-1β（ng/mL）	TNF-α（ng/mL）	IL-6（ng/mL）
Sham组	6	30.04±4.29	16.74±4.40	26.06±4.66
CLP组	6	151.32±5.60^a	304.72±14.28^a	187.80±6.48^a
FA组	6	104.81±11.82^ab	166.61±8.11^ab	98.10±10.37^ab
FA+siPPAR-γ组	6	164.88±4.88^abc	343.87±5.56^abc	211.99±9.64^abc
F值		416.70	1 653.00	662.80
P值		<0.001	<0.001	<0.001

表2 各组小鼠肺组织TNF-α、IL-6和IL-1β表达比较（

±s）

分组	鼠数	IL-1β（ng/mL）	TNF-α（ng/mL）	IL-6（ng/mL）
Sham组	6	30.04±4.29	16.74±4.40	26.06±4.66
CLP组	6	151.32±5.60^a	304.72±14.28^a	187.80±6.48^a
FA组	6	104.81±11.82^ab	166.61±8.11^ab	98.10±10.37^ab
FA+siPPAR-γ组	6	164.88±4.88^abc	343.87±5.56^abc	211.99±9.64^abc
F值		416.70	1 653.00	662.80
P值		<0.001	<0.001	<0.001

图3 各组肺组织H3-cit免疫荧光结果（20μm）注：H3-cit为瓜氨酸化组蛋白H3（绿色），DAPI为4'，6-二脒基-2-苯基吲哚（蓝色染色显示细胞核），Merge为H3-cit与DAPI共染

表3 各组小鼠肺组织MPO-DNA、NE-DNA表达及H3-cit阳性细胞面积比较（

±s）

分组	鼠数	MPO-DNA（pg/mL）	NE-DNA（pg/mL）	H3-cit阳性细胞面积（%）
Sham组	6	112.27±9.47	79.61±4.60	0.37±0.11
CLP组	6	300.57±17.80^a	185.15±8.46^a	8.78±0.62^a
FA组	6	214.10±14.23^ab	148.32±7.85^ab	3.58±0.68^ab
FA+siPPAR-γ组	6	348.56±8.60^abc	222.71±7.32^abc	11.12±1.19^abc
F值		379.40	429.10	250.80
P值		<0.001	<0.001	<0.001

表3 各组小鼠肺组织MPO-DNA、NE-DNA表达及H3-cit阳性细胞面积比较（

±s）

分组	鼠数	MPO-DNA（pg/mL）	NE-DNA（pg/mL）	H3-cit阳性细胞面积（%）
Sham组	6	112.27±9.47	79.61±4.60	0.37±0.11
CLP组	6	300.57±17.80^a	185.15±8.46^a	8.78±0.62^a
FA组	6	214.10±14.23^ab	148.32±7.85^ab	3.58±0.68^ab
FA+siPPAR-γ组	6	348.56±8.60^abc	222.71±7.32^abc	11.12±1.19^abc
F值		379.40	429.10	250.80
P值		<0.001	<0.001	<0.001

图4 连翘酯苷A对PPAR-γ、H3-cit、MPO、NE蛋白表达的影响注：NE为中性粒细胞弹性蛋白酶，MPO为髓过氧化物酶，H3-cut为瓜氨酸化组蛋白H3，PPAR-γ为过氧化物酶体增殖物激活受体γ，β-actin为β-肌动蛋白

表4 各组小鼠肺组织PPAR-γ、H3-cit、MPO、NE蛋白表达水平比较（

±s)

分组	鼠数	PPAR-γ	H3-cit	MPO	NE
Sham组	6	1.05±0.05	0.43±0.03	0.40±0.06	0.38±0.026
CLP组	6	0.65±0.02^a	0.71±0.04^a	0.75±0.03^a	0.80±0.06^a
FA组	6	0.88±0.03^ab	0.59±0.06^ab	0.62±0.09^ab	0.54±0.03^ab
FA+siPPAR-γ组	6	0.60±0.05^abc	1.01±0.07^abc	0.97±0.06^abc	0.97±0.57^abc
F值		80.77	82.16	44.33	101.50
P值		<0.001	<0.001	<0.001	<0.001

表4 各组小鼠肺组织PPAR-γ、H3-cit、MPO、NE蛋白表达水平比较（

±s)

分组	鼠数	PPAR-γ	H3-cit	MPO	NE
Sham组	6	1.05±0.05	0.43±0.03	0.40±0.06	0.38±0.026
CLP组	6	0.65±0.02^a	0.71±0.04^a	0.75±0.03^a	0.80±0.06^a
FA组	6	0.88±0.03^ab	0.59±0.06^ab	0.62±0.09^ab	0.54±0.03^ab
FA+siPPAR-γ组	6	0.60±0.05^abc	1.01±0.07^abc	0.97±0.06^abc	0.97±0.57^abc
F值		80.77	82.16	44.33	101.50
P值		<0.001	<0.001	<0.001	<0.001

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[2]	张晨露, 蒋敏, 罗健英. 抗磷脂抗体和子痫前期的研究进展[J/OL]. 中华临床医师杂志(电子版), 2020, 14(02): 140-143.

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Forsythiaside A activates PPAR-γ to inhibit neutrophil extracellular traps and alleviate sepsis-associated acute respiratory distress syndrome

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Forsythiaside A activates PPAR-γ to inhibit neutrophil extracellular traps and alleviate sepsis-associated acute respiratory distress syndrome