低氧环境下NLRP3信号通路促进非酒精性脂肪性肝炎小鼠的肝细胞焦亡

doi:10.12122/j.issn.1673-4254.2025.09.22

摘要/Abstract

摘要：

目的探讨低氧环境下NLRP3信号通路在非酒精性脂肪性肝炎（NASH）肝细胞焦亡中的作用。方法 24只6周龄的雄性C57BL/6小鼠，随机分为低氧对照组（A组）、低氧NASH模型组（B组）、低氧NASH模型+NLRP3抑制剂组（C组）、低氧NASH模型+Caspase-1抑制剂组（D组），6只/组。小鼠在模拟海拔5000 m的低压氧舱中饲养6周。A组小鼠喂食基础饲料，B、C、D组小鼠喂食蛋氨酸胆碱缺乏（MCD）饲料，其中C、D组小鼠分别每隔1 d腹腔注射NLRP3抑制剂（20 mg/kg）、Caspase-1抑制剂（25 mg/kg）。实验终点时处死小鼠，收集小鼠血清和肝脏组织，检测小鼠血清空腹血糖（FBG）、总胆固醇（TC）、甘油三酯（TG）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平；HE染色、Masoon染色及透射电镜观察小鼠肝脏组织病理变化；ELISA法检测小鼠血清IL-1β、IL-18水平；免疫组化和Western blotting法检测小鼠肝脏组织NLRP3、Caspase-1、IL-1β、消皮素D（GSDMD）蛋白表达水平。结果与A组相比，B组小鼠血清FBG、TC、TG、ALT、AST水平均增加（P<0.05）；肝脏脂质含量、炎性细胞浸润及胶原纤维沉积均增加；肝脏组织NLRP3、Caspase-1、IL-1β、GSDMD蛋白表达水平均增加（P<0.05）；肝组织线粒体肿胀严重，嵴断裂，基质局部溶解、空泡化，部分线粒体外膜破裂，胞浆中可见部分核糖体丢失，核膜模糊，部分粗面内质网扩张成囊状。与B组相比，C、D组小鼠血清TC、TG、ALT、AST水平明显降低（P<0.05），FBG差异无统计学意义（P>0.05）；肝脏脂质含量、炎性细胞浸润及胶原纤维沉积均显著减少；肝脏组织NLRP3、Caspase-1、GSDMD、IL-1β蛋白表达水平均降低（P<0.05）；肝组织线粒体肿胀、嵴断裂、空泡化程度减轻，部分线粒体外膜破裂，胞浆中可见部分核糖体丢失，粗面内质网形态结构正常。结论低氧环境下，NLRP3信号通路在促进NASH肝细胞焦亡中起关键作用，而通过抑制该通路可以有效减轻肝脏炎症，为NASH的治疗提供了潜在的治疗靶点。

关键词: 低氧, 非酒精性脂肪性肝炎, 肝细胞焦亡, NLRP3炎症通路

Abstract:

Objective To investigate the regulatory role of the NLRP3 signaling pathway in hepatocyte pyroptosis in nonalcoholic steatohepatitis (NASH) under hypoxia. Methods Twenty-four male C57BL/6 mice were randomized equally into hypoxic control (A), hypoxic NASH model (B), hypoxic NASH+NLRP3 inhibitor (C), and hypoxic NASH+caspase-1 inhibitor (D) groups. In groups B-D, the mice were fed a methionine choline-deficient (MCD) diet under hypoxic conditions (to simulate a 5000 m altitude) for 6 weeks; the mice in groups C and D received intraperitoneal injections of the respective inhibitors every other day. Results Compared with those in group A, the mice in group B showed significantly elevated serum levels of FBG, TC, TG, ALT and AST, increased liver lipid content, inflammatory cell infiltration and collagen fiber deposition, and enhanced hepatic expressions of NLRP3, caspase-1, IL-1β and GSDMD proteins, with obvious swelling, cristae breakage, vacuolization, and outer membrane disruption of the mitochondria, ribosome loss in the cytoplasm, destruction of the nuclear membrane, and pathological changes of the rough endoplasmic reticulum. Treatment with NLRP3 inhibitor and caspase-1 inhibitor both significantly lowered serum levels of TC, TG, ALT and AST (but without significantly affecting FBG) in the mouse models, and reduced liver lipid content, inflammatory cell infiltration, collagen deposition, and expression levels of NLRP3, caspase-1, GSDMD and IL-1β. The treatments also significantly improved pathological changes in the mitochondria, ribosomes and endoplasmic reticulum in liver tissues of the mice. Conclusion NLRP3 signaling pathway plays a key role in promoting hepatocyte pyroptosis in NASH mice under hypoxic condition, and inhibiting this pathway can effectively reduce liver inflammation, suggesting its potential as a therapeutic target for NASH treatment.

Key words: hypoxia, nonalcoholic steatohepatitis, hepatocyte pyroptosis, NLRP3 inflammatory pathway

罗善玉, 朱强, 闫玉翡, 纪宗红, 邹华杰, 张瑞霞, 巴应贵. 低氧环境下NLRP3信号通路促进非酒精性脂肪性肝炎小鼠的肝细胞焦亡[J]. 南方医科大学学报, 2025, 45(9): 2026-2033.

Shanyu LUO, Qiang ZHU, Yufei YAN, Zonghong JI, Huajie ZOU, Ruixia ZHANG, Yinggui BA. NLRP3 signaling pathway promotes hepatocyte pyroptosis in mice with nonalcoholic steatohepatitis in hypoxic environment[J]. Journal of Southern Medical University, 2025, 45(9): 2026-2033.

图/表 9

表1 各组小鼠生化指标水平

Tab.1 Levels of biochemical indicators of the mice from each group (Mean±SD)

Indicator	Group A	Group B	Group C	Group D	F	P
FBG (mmol/L)	4.33±0.99	16.57±2.41*	14.98±2.40	15.70±2.22	45.37	<0.001
TC (mmol/L)	1.15±0.30	2.43±0.65*	1.76±0.26^#	1.78±0.71^#	6.02	0.004
TG (mmol/L)	0.59±0.06	2.42±0.36*	1.83±0.22^#	1.64±0.27^#	54.57	<0.001
AST (U/L)	141.50±21.22	241.90±31.35*	193.32±12.09^#	201.98±12.54^#	23.56	<0.001
ALT (U/L)	33.77±5.08	52.37±7.74*	39.60±7.77^#	42.82±7.57^#	7.15	0.002

表2 各组小鼠血清L-1β、IL-18水平

Tab.2 Serum levels of L-1β and IL-18 of the mice from each group(Mean±SD, pg/mL)

Groups	IL-1β	IL-18
Group A	21.66±6.67	43.99±11.79
Group B	48.31±11.65*	168.48±28.93*
Group C	36.84±6.53^#	92.21±15.47^#
Group D	32.56±8.26^#	97.02±19.98^#
F	10.01	39.13
P	<0.001	<0.001

图1 各组小鼠肝组织HE染色结果

Fig.1 HE staining of the liver tissues in each group (Original magnification: ×400).

图2 各组小鼠肝组织Masson染色结果

Fig.2 Masson staining of the liver tissue in each group (×400).

图3 各组小鼠肝组织免疫组化染色结果

Fig.3 Masson staining of the liver tissue in each group (×400).

图4 各组小鼠肝组织NLRP3、Caspase-1、GSDMD、IL-1β阳性表达面积

Fig.4 Comparison of relative expression levels of NLRP3, caspase-1, GSDMD, and IL-1β in the liver tissues among the groups. ****P<0.0001vs Group A, ####P<0.0001 vs Group B, ###P<0.001 vs Group B.

图5 各组小鼠肝组织蛋白免疫印迹条带图

Fig.5 Protein bands in Western blotting in each group.

图6 各组小鼠NLRP3、caspase-1、GSDMD、IL-1β蛋白表达结果

Fig.6 Relative protein expression levels of NLRP3, caspase-, GSDMD, and IL-1β in each group.****P<0.0001 vs Group A,***P<0.001 vs Group A,**P<0.01 vs Group A,####P<0.0001 vs Group B,###P<0.001 vs Group B, ##P<0.01, #P<0.05 vs Group B.

图7 各组小鼠肝组织透射电镜结果

Fig.7 Transmission electron microscopy of the liver tissue in each group.↑: Rough endoplasmic reticulum (RER);↑: Mitochondria; ↑: Vacuolization (×25 000).

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