车叶草苷调节NLRP3/Caspase-1/GSDMD信号通路对脓毒症大鼠肺组织细胞焦亡的影响

doi:10.11958/20221430

天津医药 ›› 2023, Vol. 51 ›› Issue (6): 607-612.doi: 10.11958/20221430

车叶草苷调节NLRP3/Caspase-1/GSDMD信号通路对脓毒症大鼠肺组织细胞焦亡的影响

孙立燕¹(), 刘泽茹², 苏永胜³, 艾宏亮¹

1 潍坊市中医院重症医学科（邮编261000）
2 济宁医学院附属医院呼吸内科
3 河南大学淮河医院呼吸内科

收稿日期:2022-09-06 修回日期:2022-12-22 出版日期:2023-06-15 发布日期:2023-06-20
作者简介:孙立燕（1990），女，主治医师，主要从事重症系统疾病诊治方面研究。E-mail：sunliyan1990@163.com
基金资助:
潍坊市中医药科研资助项目(2019-02-008)

Effect of asperuloside on pyroptosis of lung tissue in septic rats by regulating NLRP3/Caspase-1/GSDMD signaling pathway

SUN Liyan¹(), LIU Zeru², SU Yongsheng³, AI Hongliang¹

1 Department of Critical Medicine, Weifang Hospital of Traditional Chinese Medicine, Weifang 261000, China
2 Department of Respiratory Medicine, Affiliated Hospital of Jining Medical University
3 Department of Respiratory Medicine, Huaihe Hospital of Henan University

Received:2022-09-06 Revised:2022-12-22 Published:2023-06-15 Online:2023-06-20

孙立燕, 刘泽茹, 苏永胜, 艾宏亮. 车叶草苷调节NLRP3/Caspase-1/GSDMD信号通路对脓毒症大鼠肺组织细胞焦亡的影响[J]. 天津医药, 2023, 51(6): 607-612.

SUN Liyan, LIU Zeru, SU Yongsheng, AI Hongliang. Effect of asperuloside on pyroptosis of lung tissue in septic rats by regulating NLRP3/Caspase-1/GSDMD signaling pathway[J]. Tianjin Medical Journal, 2023, 51(6): 607-612.

摘要/Abstract

摘要：

目的探讨车叶草苷调节核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）/胱天蛋白酶1（Caspase-1）/消皮素D（GSDMD）信号通路对脓毒症大鼠肺组织细胞焦亡的影响。方法取50只SD大鼠腹腔内注射5 mg/kg脂多糖建立脓毒症肺损伤模型，随机数字表法分为模型（M）组、车叶草苷低剂量（AL）组（17.5 mg/kg）、车叶草苷中剂量（AM）组（35 mg/kg）、车叶草苷高剂量（AH）组（70 mg/kg）、车叶草苷高剂量（70 mg/kg）+尼日利亚霉素（NLRP3激活剂，1 mg/kg）组（AH+N组），每组10只；另取10只SD大鼠灌胃等剂量生理盐水作对照（C）组。分组干预后，检测各组大鼠肺功能指标[每分钟通气量（MV）、吸气阻力（Ri）]与动脉血氧分压[p（O₂）]；HE染色检测各组大鼠肺组织病理形态；瑞氏-姬姆萨染色分类计数各组大鼠肺泡灌洗液（BALF）中性粒细胞、巨噬细胞和淋巴细胞数；酶联免疫吸附试验（ELISA）检测各组大鼠血清炎性细胞因子白细胞介素（IL）-6、IL-8及IL-1β水平；免疫荧光染色检测各组大鼠肺组织细胞焦亡情况，比较NLRP3与GSDMD在肺组织中表达情况；Western blot检测各组大鼠肺组织NLRP3/Caspase-1/GSDMD信号通路相关蛋白表达。结果与C组比较，M组MV、p（O₂）降低，Ri及BALF中性粒细胞、巨噬细胞、淋巴细胞数升高，血清IL-6、IL-8及IL-1β水平升高，肺组织NLRP3、GSDMD相对荧光强度和NLRP3、Caspase-1、GSDMD蛋白表达升高（P<0.05）；与M组比较，AL组、AM组、AH组MV、p（O₂）均升高，Ri及BALF中性粒细胞、巨噬细胞、淋巴细胞数下降，血清IL-6、IL-8及IL-1β水平下降，肺组织NLRP3、GSDMD相对荧光强度和NLRP3、Caspase-1、GSDMD蛋白表达均降低（P<0.05）；与AH组比较，AH+N组MV、p（O₂）降低，Ri及BALF中性粒细胞、巨噬细胞、淋巴细胞数升高，血清IL-6、IL-8及IL-1β水平升高，肺组织NLRP3、GSDMD相对荧光强度和NLRP3、Caspase-1、GSDMD蛋白表达升高（P<0.05）。结论高剂量车叶草苷可通过下调NLRP3/Caspase-1/GSDMD通路活性，抑制脓毒症大鼠肺组织炎症，减弱肺组织炎性损伤及肺组织细胞焦亡，修复肺功能。

关键词: 脓毒症, 肺损伤, 细胞焦亡, 车叶草苷, 核苷酸结合寡聚化结构域样受体蛋白3, 胱天蛋白酶1, 消皮素D

Abstract:

Objective To investigate the influence of asperuloside on pyroptosis of lung tissue in septic rats by regulating NOD-like receptor protein 3 (NLRP3)/cysteine-containing aspartate-specific proteases 3 (Caspase-1)/gasdermin D (GSDMD) signaling pathway. Methods Fifty SD rats were injected intraperitoneally with 5 mg/kg lipopolysaccharide (LPS) to establish the septic lung injury model. Rats were randomly grouped into the model (M) group, the low-dose asperuloside (AL) group (17.5 mg/kg), the medium-dose asperuloside (AM) group (35 mg/kg), the asperuloside high-dose (AH) group (70 mg/kg) and the high-dose asperuloside (70 mg/kg) + nigericin (NLRP3 activator, 1 mg/kg) group (AH+N group), 10 rats in each group. Another 10 SD rats were intraperitoneally injected with the same dose of normal saline and used as the control (C) group. After rats in each group were intervened with terpenoside and nigericin, pulmonary function indexes {minute ventilation (MV), inspiratory resistance (Ri)} and arterial oxygen partial pressure [p(O₂)] were detected in each group. HE staining was applied to detect the pathological morphology of lung tissue of rats in each group. Wright-Giemsa staining was applied to classify and count numbers of neutrophils, macrophages and lymphocytes in bronchoalveolar lavage fluid (BALF) of rats in each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of inflammatory cytokines interleukin (IL)-6, IL-8 and IL-1β in each group of rats. Immunofluorescence staining was used to detect the pyroptosis of lung tissue in each group. Expression levels of NLRP3 and GSDMD in lung tissue were compared. Western blot assay was used to detect expression levels of NLRP3/Caspase-1/GSDMD signaling pathway-related proteins in lung tissue of rats in each group. Results Compared with the C group, MV and p(O₂) were obviously decreased in the M group, and Ri, counts of neutrophils and macrophages, lymphocyte in BALF, serum levels of IL-6, IL-8, IL-1β, relative fluorescence intensity of NLRP3 and GSDMD in lung tissue, and protein expressions of NLRP3, Caspase-1 and GSDMD were obviously increased (P<0.05). Compared with the M group, MV and p(O₂) were increased in the AL group, the AM group and the AH group. Ri, counts of neutrophils and macrophages, lymphocyte in BALF, serum levels of IL-6, IL-8, IL-1β, relative fluorescence intensity of NLRP3 and GSDMD in lung tissue, and protein expressions of NLRP3, Caspase-1 and GSDMD were all decreased in a dose-dependent manner (P<0.05). Compared with the AH group, MV and p(O₂) decreased in the AH+N group, Ri, counts of neutrophils and macrophages, lymphocyte in BALF, serum levels of IL-6, IL-8, IL-1β, relative fluorescence intensity of NLRP3 and GSDMD in lung tissue, and protein expressions of NLRP3, Caspase-1 and GSDMD increased (P<0.05). Conclusion Asperuloside can inhibit lung tissue inflammation in sepsis rats by down-regulating NLRP3/Caspase-1/GSDMD pathway, attenuate lung tissue inflammatory damage and lung tissue pyroptosis, and restore lung function.

Key words: sepsis, lung injury, pyroptosis, asperuloside, NLRP3, Caspase-1, GSDMD

中图分类号:

R563.9

图/表 8

参考文献 19

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组别	MV（mL）	Ri（kPa^-1·L^-1·s^-1）	p（O₂）（mmHg）
C组	7.69±0.86	24.97±4.65	98.82±10.61
M组	2.71±0.42^a	59.68±6.80^a	54.75±5.15^a
AL组	3.91±0.48^b	50.98±6.12^b	65.12±6.24^b
AM组	4.80±0.51^bc	42.24±5.16^bc	75.82±7.43^bc
AH组	7.13±0.73^bcd	26.43±4.52^bcd	95.93±9.52^bcd
AH+N组	2.93±0.38^e	55.32±6.03^e	58.12±5.36^e
F	129.083^＊＊	69.525^＊＊	61.205^＊＊

组别	MV（mL）	Ri（kPa^-1·L^-1·s^-1）	p（O₂）（mmHg）
C组	7.69±0.86	24.97±4.65	98.82±10.61
M组	2.71±0.42^a	59.68±6.80^a	54.75±5.15^a
AL组	3.91±0.48^b	50.98±6.12^b	65.12±6.24^b
AM组	4.80±0.51^bc	42.24±5.16^bc	75.82±7.43^bc
AH组	7.13±0.73^bcd	26.43±4.52^bcd	95.93±9.52^bcd
AH+N组	2.93±0.38^e	55.32±6.03^e	58.12±5.36^e
F	129.083^＊＊	69.525^＊＊	61.205^＊＊

组别	中性粒细胞	巨噬细胞	淋巴细胞
C组	2.68±0.65	22.76±2.43	5.09±0.67
M组	13.10±1.12^a	54.20±6.51^a	81.94±17.62^a
AL组	10.68±0.95^b	46.59±5.27^b	64.75±13.41^b
AM组	8.19±0.70^bc	39.84±4.25^bc	45.01±9.46^bc
AH组	3.15±0.57^bcd	25.90±2.56^bcd	6.13±0.81^bcd
AH+N组	12.38±1.31^e	53.32±6.03^e	80.61±15.73^e
F	242.906^＊＊	80.336^＊＊	88.312^＊＊

组别	中性粒细胞	巨噬细胞	淋巴细胞
C组	2.68±0.65	22.76±2.43	5.09±0.67
M组	13.10±1.12^a	54.20±6.51^a	81.94±17.62^a
AL组	10.68±0.95^b	46.59±5.27^b	64.75±13.41^b
AM组	8.19±0.70^bc	39.84±4.25^bc	45.01±9.46^bc
AH组	3.15±0.57^bcd	25.90±2.56^bcd	6.13±0.81^bcd
AH+N组	12.38±1.31^e	53.32±6.03^e	80.61±15.73^e
F	242.906^＊＊	80.336^＊＊	88.312^＊＊

组别	TNF-α	IL-8	IL-1β
C组	27.35±5.02	204.94±32.25	16.87±3.03
M组	183.46±21.53^a	968.40±92.30^a	242.64±28.12^a
AL组	136.59±17.84^b	761.25±77.62^b	193.45±20.51^b
AM组	107.86±13.46^bc	589.32±54.92^bc	134.45±12.51^bc
AH组	31.12±6.32^bcd	229.86±30.74^bcd	21.13±3.54^bcd
AH+N组	164.63±20.14^e	960.11±90.58^e	230.36±25.92^e
F	185.239^＊＊	251.860^＊＊	296.171^＊＊

车叶草苷调节NLRP3/Caspase-1/GSDMD信号通路对脓毒症大鼠肺组织细胞焦亡的影响

Effect of asperuloside on pyroptosis of lung tissue in septic rats by regulating NLRP3/Caspase-1/GSDMD signaling pathway

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组别	n	NLRP3	GSDMD
C组	10	1.00±0.00	1.00±0.00
M组	10	2.89±0.36^a	2.57±0.32^a
AL组	10	2.36±0.29^b	2.19±0.26^b
AM组	10	2.02±0.25^bc	1.84±0.29^bc
AH组	10	1.16±0.20^bcd	1.13±0.17^bcd
AH+N组	10	2.64±0.34^e	2.31±0.33^e
F		84.386^＊＊	63.873^＊＊

组别	n	NLRP3	Caspase-1	GSDMD
C组	10	0.19±0.04	0.10±0.02	0.21±0.04
M组	10	1.20±0.16^a	1.08±0.13^a	1.29±0.15^a
AL组	10	0.98±0.13^b	0.81±0.11^b	0.95±0.12^b
AM组	10	0.71±0.10^bc	0.59±0.07^bc	0.78±0.09^bc
AH组	10	0.22±0.05^bcd	0.13±0.03^bcd	0.25±0.06^bcd
AH+N组	10	1.16±0.11^e	1.04±0.14^e	1.26±0.16^e
F		178.049^＊＊	204.821^＊＊	178.037^＊＊

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