乌司他丁通过抑制NOD样受体热蛋白结构域相关蛋白3炎症小体激活保护脓毒症相关肺损伤

doi:10.3877/cma.j.issn.1674-6880.2025.03.003

中华危重症医学杂志(电子版) ›› 2025, Vol. 18 ›› Issue (03) : 189 -196. doi: 10.3877/cma.j.issn.1674-6880.2025.03.003

论著

乌司他丁通过抑制NOD样受体热蛋白结构域相关蛋白3炎症小体激活保护脓毒症相关肺损伤

龙飞宇¹^,², 祝鑫蕊¹, 伍佳莉¹, 晏丕军³, 王茂华¹^,^†(

)

¹646000　四川泸州，西南医科大学附属医院麻醉科
²610000　成都，四川大学华西医院麻醉科
³646000　四川泸州，四川省肾脏疾病临床医学研究中心

收稿日期:2024-11-18 出版日期:2025-06-30
通信作者: 王茂华
基金资助:
四川省科技计划资助项目(2022YFS0612)

Ulinastatin protects against sepsis-associated lung injury by inhibiting the activation of NOD-like receptor thermal protein domain associated protein 3 inflammasome

Feiyu Long¹^,², Xinrui Zhu¹, Jiali Wu¹, Pijun Yan³, Maohua Wang¹^,^†()

¹Department of Anesthesiology, TheAffiliated Hospital of Southwest Medical University, Luzhou 646000, China
²Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu 610000, China
³Clinical Research Center for Kidney Diseases of Sichuan Province, Luzhou 646000, China

Received:2024-11-18 Published:2025-06-30
Corresponding author: Maohua Wang

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引用本文：

龙飞宇, 祝鑫蕊, 伍佳莉, 晏丕军, 王茂华. 乌司他丁通过抑制NOD样受体热蛋白结构域相关蛋白3炎症小体激活保护脓毒症相关肺损伤[J/OL]. 中华危重症医学杂志(电子版), 2025, 18(03): 189-196.

Feiyu Long, Xinrui Zhu, Jiali Wu, Pijun Yan, Maohua Wang. Ulinastatin protects against sepsis-associated lung injury by inhibiting the activation of NOD-like receptor thermal protein domain associated protein 3 inflammasome[J/OL]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2025, 18(03): 189-196.

目的

探讨不同剂量的乌司他丁对脓毒症大鼠肺损伤的保护作用，以及对NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎症小体激活的影响。

方法

将72只Sprague Dawley大鼠分为假手术组、脓毒症组、低剂量组和高剂量组，每组各18只。脓毒症组、低剂量组和高剂量组采用盲肠结扎穿刺法（CLP）建立脓毒症大鼠模型，假手术组开腹后游离盲肠后不结扎。建模完成后低剂量组大鼠腹腔注射乌司他丁50 000 U/kg，高剂量组腹腔注射乌司他丁100 000 U/kg，假手术组和脓毒症组腹腔注射等体积等渗NaCl溶液。造模后每组取10只大鼠观察其7 d生存率；造模后24 h，将每组另外8只大鼠处死并采集大鼠腹主动脉血进行动脉血氧分压测量，采集大鼠肺组织进行苏木素-伊红（HE）染色观察肺部损伤情况并进行评分；采用酶联免疫吸附测定（ELISA）法检测肺组织中白细胞介素1β（IL-1β）、IL-6及肿瘤坏死因子α（TNF-α）的水平；western-blotting法检测肺组织中NLRP3、caspase-1及凋亡相关斑点样蛋白（ASC）蛋白表达；免疫荧光观察肺组织NLRP3阳性细胞百分比。

结果

4组大鼠7 d生存情况比较，差异有统计学意义（Log-rank检验：χ² = 7.727，P = 0.005）；且脓毒症组大鼠7 d生存情况明显低于低剂量组和高剂量组（P均< 0.05）。4组大鼠肺损伤评分、动脉血氧分压，肺组织中IL-1β、IL-6、TNF-α水平以及NLRP3、caspase-1、ASC蛋白表达水平和NLRP3阳性细胞百分比比较，差异均有统计学意义（F = 30.691、11.787、13.042、6.669、18.953、52.539、46.265、61.609、39.339，P均< 0.05）。进一步两两比较发现，与假手术组相比，脓毒症组大鼠的动脉血氧分压明显降低，肺组织损伤评分、IL-1β、IL-6、TNF-α水平及NLRP3、caspase-1、ASC蛋白表达水平和NLRP3阳性细胞百分比均明显升高（P均< 0.05）；与脓毒症组相比，给予乌司他丁后，低剂量组及高剂量组大鼠的动脉血氧分压均明显升高，且高剂量组升高更为明显，而肺组织损伤评分、IL-1β、IL-6、TNF-α水平以及NLRP3、caspase-1、ASC蛋白表达水平和NLRP3阳性细胞百分比均明显降低，且高剂量组降低更为明显（P均< 0.05）。

结论

乌司他丁可能通过抑制NLRP3炎症小体的激活改善脓毒症大鼠的生存情况以及肺部损伤。

关键词: 乌司他丁, 脓毒症, 肺损伤, NOD样受体热蛋白结构域相关蛋白3炎症小体

Objective

To investigate the protective effects of different doses of ulinastatin on sepsis-induced lung injury in rats and their influence on the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome.

Methods

Seventy-two Sprague Dawley rats were divided into a sham group, a sepsis group, a low-dose group, and a high-dose group, with 18 rats in each group. The sepsis, low-dose, and high-dose groups underwent cecal ligation and puncture (CLP) to establish a sepsis model, while the sham group underwent laparotomy without ligation. After modeling, rats in the low-dose and high-dose groups received intraperitoneal injections of ulinastatin (50 000 or 100 000 U/kg, respectively), whereas rats in the sham and sepsis groups received an equal volume of isotonic NaCl solution. Ten rats in each group were monitored for 7-day survival status. At 24 h post-modeling, the remaining eight rats in each group were euthanized for sample collection. Arterial blood was drawn from the abdominal aorta to measure the partial pressure of oxygen. Lung tissue was collected for histopathological examination (hematoxylin-eosin staining and injury scoring), for enzyme-linked immunosorbent assay (ELISA) to quantify interleukin-1 beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α) levels, for western-blotting to assess NLRP3, caspase-1 and apoptosis-associated speck-like protein (ASC) expression, and for immunofluorescence to determine the percentage of NLRP3 positive cells.

Results

There was a statistically significant difference in the 7-day survival status of rats among the four groups (Log-rank test: χ² = 7.727, P = 0.005). The 7-day survival status of the sepsis group was significantly worse than that of the low-dose and high-dose groups (both P < 0.05). The lung injury scores, arterial partial pressure of oxygen, levels of IL-1β, IL-6 and TNF-α in lung tissue, expression of NLRP3, caspase-1 and ASC proteins, and percentage of NLRP3 positive cells were compared among the four groups, and the differences were statistically significant (F = 30.691, 11.787, 13.042, 6.669, 18.953, 52.539, 46.265, 61.609, 39.339; all P < 0.05). Further pairwise comparisons revealed that compared with the sham group, the arterial partial pressure of oxygen in the sepsis group was significantly reduced, while the lung tissue injury score, levels of IL-1β, IL-6 and TNF-α, expression of NLRP3, caspase-1 and ASC proteins, and percentage of NLRP3 positive cells were significantly increased (all P < 0.05). Compared with the sepsis group, the arterial partial pressure of oxygen in the low-dose and high-dose groups increased significantly after administration of ulinastain, and the increase was more pronounced in the high-dose group (all P < 0.05). The lung tissue injury score, levels of IL-1β, IL-6 and TNF-α, expression of NLRP3, caspase-1 and ASC proteins, and percentage of NLRP3 positive cells were significantly lower in the low-dose and high-dose groups than in the sepsis group, with the high-dose group showing a more significant decrease (all P < 0.05).

Conclusion

Ustutidine may improve survival status and lung injury in septic rats by inhibiting the activation of NLRP3 inflammasome.

Key words: Ulinastain, Sepsis, Lung injury, NOD-like receptor thermal protein domain associated protein 3 inflammasome

图1 各组大鼠7 d生存曲线图（每组n = 10）

图2 各组大鼠肺组织病理图（HE染色　× 200）注：HE.苏木素-伊红；a~d图分别为假手术组、脓毒症组、低剂量组、高剂量组大鼠肺组织病理结果；a图显示假手术组大鼠肺组织无明显炎症细胞浸润，肺间隔正常，肺泡间质无明显出血和水肿；b图显示脓毒症组大鼠肺泡发生损伤，肺间隔增厚，肺泡上皮细胞肿胀，肺泡融合，肺泡间质可见明显的炎症细胞浸润；c图显示，与脓毒症组相比，乌司他丁处理后大鼠中性粒细胞浸润减少，充血减少，肺间隔增厚有所改善；d图显示高剂量组大鼠肺损伤程度改善更加明显

表1 各组大鼠肺损伤评分及PaO₂比较（

± s）

组别	只数	肺损伤评分（分）	PaO₂（mmHg）
假手术组	8	4.5 ± 1.6	86 ± 10
脓毒症组	6	12.7 ± 1.9^a	60 ± 9^a
低剂量组	7	11.4 ± 2.2^b	71 ± 8^b
高剂量组	7	9.1 ± 1.3^bc	79 ± 6^bc
F值		30.691	11.787
P值		<0.001	<0.001

表1 各组大鼠肺损伤评分及PaO₂比较（

± s）

组别	只数	肺损伤评分（分）	PaO₂（mmHg）
假手术组	8	4.5 ± 1.6	86 ± 10
脓毒症组	6	12.7 ± 1.9^a	60 ± 9^a
低剂量组	7	11.4 ± 2.2^b	71 ± 8^b
高剂量组	7	9.1 ± 1.3^bc	79 ± 6^bc
F值		30.691	11.787
P值		<0.001	<0.001

表2 各组大鼠肺组织炎症因子水平比较（ng/L，

± s）

组别	只数	IL-1β	IL-6	TNF-α
假手术组	8	26 ± 10	43 ± 19	228 ± 52
脓毒症组	6	84 ± 18^a	107 ± 34^a	597 ± 102^a
低剂量组	7	72 ± 26^b	90 ± 28^b	511 ± 122^b
高剂量组	7	60 ± 19^bc	81 ± 31^bc	462 ± 109^bc
F值		13.042	6.669	18.953
P值		<0.001	0.002	<0.001

表2 各组大鼠肺组织炎症因子水平比较（ng/L，

± s）

组别	只数	IL-1β	IL-6	TNF-α
假手术组	8	26 ± 10	43 ± 19	228 ± 52
脓毒症组	6	84 ± 18^a	107 ± 34^a	597 ± 102^a
低剂量组	7	72 ± 26^b	90 ± 28^b	511 ± 122^b
高剂量组	7	60 ± 19^bc	81 ± 31^bc	462 ± 109^bc
F值		13.042	6.669	18.953
P值		<0.001	0.002	<0.001

图3 各组大鼠肺组织NLRP3、caspase-1及ASC蛋白表达水平注：NLRP3. NOD样受体热蛋白结构域相关蛋白3；ASC.凋亡相关斑点样蛋白

表3 各组大鼠肺组织NLRP3、caspase-1及ASC蛋白相对表达量比较（

± s）

组别	只数	NLRP3	caspase-1	ASC
假手术组	6	0.25 ± 0.09	0.35 ± 0.07	0.58 ± 0.06
脓毒症组	6	0.90 ± 0.12^a	0.93 ± 0.13^a	2.17 ± 0.34^a
低剂量组	6	0.72 ± 0.07^b	0.84 ± 0.09^b	1.68 ± 0.31^b
高剂量组	6	0.61 ± 0.09^bc	0.61 ± 0.08^bc	0.76 ± 0.08^bc
F值		52.539	46.265	61.609
P值		<0.001	<0.001	<0.001

表3 各组大鼠肺组织NLRP3、caspase-1及ASC蛋白相对表达量比较（

± s）

组别	只数	NLRP3	caspase-1	ASC
假手术组	6	0.25 ± 0.09	0.35 ± 0.07	0.58 ± 0.06
脓毒症组	6	0.90 ± 0.12^a	0.93 ± 0.13^a	2.17 ± 0.34^a
低剂量组	6	0.72 ± 0.07^b	0.84 ± 0.09^b	1.68 ± 0.31^b
高剂量组	6	0.61 ± 0.09^bc	0.61 ± 0.08^bc	0.76 ± 0.08^bc
F值		52.539	46.265	61.609
P值		<0.001	<0.001	<0.001

图4 各组大鼠肺组织NLRP3表达免疫荧光图（ × 200）注：NLRP3. NOD样受体热蛋白结构域相关蛋白3；DAPI. 4',6-二脒基-2-苯基吲哚；假手术组大鼠肺组织NLRP3荧光染色在细胞质中呈弱染表现，NLRP3阳性细胞表达率较低；脓毒症组胞质中NLRP3染色荧光强度明显高于假手术组，阳性表达明显增高；给予低剂量乌司他丁后，胞质中NLRP3染色阳性细胞表达率较脓毒症组降低；而高剂量组大鼠NLRP3阳性细胞表达率较低剂量组进一步降低

1	Martin-Loeches I, Singer M, Leone M. Sepsis: key insights, future directions, and immediate goals. A review and expert opinion[J]. Intensive Care Med, 2024, 50 (12): 2043-2049.
2	Liu D, Huang SY, Sun JH, et al. Sepsis-induced immunosuppression: mechanisms, diagnosis and current treatment options[J]. Mil Med Res, 2022, 9 (1): 56.
3	Kumar V. Pulmonary innate immune response determines the outcome of inflammation during pneumonia and sepsis-associated acute lung injury[J]. Front Immunol, 2020, 11: 1722.
4	Kelley N, Jeltema D, Duan Y, et al. The NLRP3 inflammasome: an overview of mechanisms of activation and regulation[J]. Int J Mol Sci, 2019, 20 (13): 3328.
5	Mangan MSJ, Olhava EJ, Roush WR, et al. Targeting the NLRP3 inflammasome in inflammatory diseases[J]. Nat Rev Drug Discov, 2018, 17 (8): 588-606.
6	邵义明，张良清，邓烈华，等. 乌司他丁对全身炎症反应综合征的治疗作用[J]. 中国危重病急救医学，2005，17（4）：228-230.
7	Xu CE, Zhang MY, Zou CW, et al. Evaluation of the pharmacological function of ulinastatin in experimental animals[J]. Molecules, 2012, 17 (8): 9070-9080.
8	Karnad DR, Bhadade R, Verma PK, et al. Intravenous administration of ulinastatin (human urinary trypsin inhibitor) in severe sepsis: a multicenter randomized controlled study[J]. Intensive Care Med, 2014, 40 (6): 830-838.
9	Capcha JMC, Moreira RS, Rodrigues CE, et al. Using the cecal ligation and puncture model of sepsis to induce rats to multiple organ dysfunction[J]. Bio Protoc, 2021, 11 (7): e3979.
10	Fang S, Li P, Zhu C, et al. Research progress of ulinastatin in the treatment of liver diseases[J]. Int J Clin Exp Pathol, 2020, 13 (11): 2720-2726.
11	Wang H, Liu B, Tang Y, et al. Improvement of sepsis prognosis by ulinastatin: a systematic review and meta-analysis of randomized controlled trials[J]. Front Pharmacol, 2020, 10: 1697.
12	Ju M, He H, Chen S, et al. Ulinastatin ameliorates LPS-induced pulmonary inflammation and injury by blocking the MAPK/NF-κB signaling pathways in rats[J]. Mol Med Rep, 2019, 20 (4): 3347-3354.
13	Jiang YX, Huang ZW. Ulinastatin alleviates pulmonary edema by reducing pulmonary permeability and stimulating alveolar fluid clearance in a rat model of acute lung injury[J]. Iran J Basic Med Sci, 2022, 25 (8): 1002-1008.
14	Cao C, Yin C, Shou S, et al. Ulinastatin protects against LPS-induced acute lung injury by attenuating TLR4/NF-κB pathway activation and reducing inflammatory mediators[J]. Shock, 2018, 50 (5): 595-605.
15	杨如江，张光成. 乌司他丁对新生大鼠脓毒症肺损伤缓解作用的研究[J]. 中国临床药理学杂志，2024，40（15）：2207-2211.
16	Danielski LG, Giustina AD, Bonfante S, et al. The NLRP3 inflammasome and its role in sepsis development[J]. Inflammation, 2020, 43 (1): 24-31.
17	Fu J, Wu H. Structural mechanisms of NLRP3 inflammasome assembly and activation[J]. Annu Rev Immunol, 2023, 41: 301-316.
18	Gong T, Yang Y, Jin T, et al. Orchestration of NLRP3 inflammasome activation by ion fluxes[J]. Trends Immunol, 2018, 39 (5): 393-406.
19	Blevins HM, Xu Y, Biby S, et al. The NLRP3 inflammasome pathway: a review of mechanisms and inhibitors for the treatment of inflammatory diseases[J]. Front Aging Neurosci, 2022, 14: 879021.
20	沈沛，何国丽，徐娜娜，等. 脓毒症相关性急性肺损伤信号通路的研究进展[J/OL]. 中华危重症医学杂志（电子版），2024，17（4）：326-330.
21	Chen G, Hou Y, Li X, et al. Sepsis-induced acute lung injury in young rats is relieved by calycosin through inactivating the HMGB1/MyD88/NF-κB pathway and NLRP3 inflammasome[J]. Int Immunopharmacol, 2021, 96: 107623.
22	张建楠，刘文，昌广平，等. 核苷酸结合寡聚化结构域样受体蛋白3炎性小体在脓毒症急性肾损伤大鼠肾脏组织的表达及其影响[J/OL]. 中华危重症医学杂志（电子版），2020，13（1）：55-59.
23	Qiu J, Xiao X, Gao X, et al. Ulinastatin protects against sepsis-induced myocardial injury by inhibiting NLRP3 inflammasome activation[J]. Mol Med Rep, 2021, 24 (4): 730.
24	Huang W, Zhang H, Wang L, et al. Ulinastatin attenuates renal ischemia-reperfusion injury by inhibiting NLRP3 inflammasome-triggered pyroptosis[J]. Int Immunopharmacol, 2024, 143 (Pt 1): 113306.

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Ulinastatin protects against sepsis-associated lung injury by inhibiting the activation of NOD-like receptor thermal protein domain associated protein 3 inflammasome

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Ulinastatin protects against sepsis-associated lung injury by inhibiting the activation of NOD-like receptor thermal protein domain associated protein 3 inflammasome