脂多糖诱导奶牛子宫内膜上皮细胞焦亡的分析

doi:10.11843/j.issn.0366-6964.2025.10.042

摘要/Abstract

摘要：

旨在分析脂多糖(LPS)诱导奶牛子宫内膜上皮细胞的焦亡现象。用0.1 μmol·L^-1的LPS处理奶牛子宫内膜上皮细胞，通过实时荧光定量PCR (qPCR)检测相关炎性因子(IL-1β、Nod-1、TNF-α、IL-18和IL-6)的mRNA转录水平，采用酶联免疫吸附试验(ELISA)检测相关炎性因子(IL-1β、IL-6和TNF-α)蛋白水平的变化，进一步用扫描电镜观察奶牛子宫内膜上皮细胞的形态变化，以及通过免疫印迹(Western blot)检测细胞焦亡相关蛋白(NLRP3、IL-18、IL-1β、GSDMD、Cleaved caspase-1和Cleaved GSDMD)的表达水平。结果显示，LPS刺激12 h后，奶牛子宫内膜上皮细胞炎性因子(IL-1β、Nod-1、TNF-α、IL-18和IL-6)的mRNA转录水平增加；细胞发生肿胀破裂；炎性因子IL-1β、IL-6和TNF-α的表达水平升高；焦亡与炎性相关蛋白(NLRP3、IL-18、IL-1β、GSDMD、Cleaved caspase-1和Cleaved GSDMD)的表达显著增强(P＜0.05)。LPS刺激可诱导奶牛子宫内膜上皮细胞发生细胞焦亡。

关键词: 细胞损伤, 奶牛子宫内膜上皮细胞, 细胞焦亡

Abstract:

This study aimed to analyze the pyroptosis of endometrial epithelial cells induced by lipopolysaccharide (LPS) in dairy cows. Endometrial epithelial cells of dairy cows were treated with 0.1 μmol·L^-1 LPS. The mRNA transcription levels of related inflammatory factors (IL-1β, Nod-1, TNF-α, IL-18 and IL-6) were detected by real-time fluorescence quantitative PCR (qPCR). The protein levels of related inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The morphological changes of endometrial epithelial cells of dairy cows were further observed by scanning electron microscopy. The expression levels of pyroptosis-related proteins (NLRP3, IL-18, IL-1β, GSDMD, Cleaved caspase-1 and Cleaved GSDMD) were detected by Western blot. The results showed that the mRNA transcription levels of inflammatory factors (IL-1β, Nod-1, TNF-α, IL-18 and IL-6) in endometrial epithelial cells of dairy cows increased after LPS stimulation for 12 h. Cell swelling and rupture; the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α were increased. The expression of pyroptosis and inflammatory-related proteins (NLRP3, IL-18, IL-1β, GSDMD, Cleaved caspase-1 and Cleaved GSDMD) was significantly enhanced (P < 0.05). LPS stimulation can induce pyroptosis in bovine endometrial epithelial cells.

Key words: cell damage, endometrial epithelial cells of dairy cows, pyroptosis

中图分类号:

S857.23

王峥钰, 付春宇, 黄子轩, 高滢, 于泳, 鲁文赓. 脂多糖诱导奶牛子宫内膜上皮细胞焦亡的分析[J]. 畜牧兽医学报, 2025, 56(10): 5232-5238.

WANG Zhengyu, FU Chunyu, HUANG Zixuan, GAO Ying, YU Yong, LU Wengeng. Analysis of LPS-induced Pyroptosis of Endometrial Epithelial Cells in Dairy Cows[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5232-5238.

图/表 5

表 1

图 1

图 2

图 3

图 4

参考文献 22

1	KELLY P , BARRY-REIDY A , BREWER A , et al. Improved filtration method to isolate pure populations of primary bovine endometrial epithelial and stromal cells for immunological studies[J]. Vet Res Commun, 2020, 44 (1): 29- 39. doi: 10.1007/s11259-020-09770-3
2	MADOZ L V , GIULIODORI M J , MIGLIORISI A L , et al. Endometrial cytology, biopsy, and bacteriology for the diagnosis of subclinical endometritis in grazing dairy cows[J]. J Dairy Sci, 2014, 97 (1): 195- 201. doi: 10.3168/jds.2013-6836
3	曹玥, 宋世豪, 倪婉仪, 等. 我国奶牛子宫内膜炎发生率、病原谱和主要病原菌耐药特征[J]. 中国兽医杂志, 2024, 60 (10): 88- 97.
	CAO Y , SONG S H , NI W Y , et al. The incidence, pathogenic spectrum and drug resistance characteristics of endometritis in dairy cows in China[J]. Chinese Journal of Veterinary Medicine, 2024, 60 (10): 88- 97.
4	郭建华. 奶牛子宫内膜炎病因及综合防治措施[J]. 中国乳业, 2022 (5): 60- 63.
	GUO J H . Causes and comprehensive prevention and treatment of endometritis in dairy cows[J]. China Dairy Industry, 2022 (5): 60- 63.
5	LIANG D , ARNOLD L M , STOWE C J , et al. Estimating US dairy clinical disease costs with a stochastic simulation model[J]. J Dairy Sci, 2017, 100 (2): 1472- 1486. doi: 10.3168/jds.2016-11565
6	CARNEIRO L C , CRONIN J G , SHELDON I M . Mechanisms linking bacterial infections of the bovine endometrium to disease and infertility[J]. Reprod Biol, 2016, 16 (1): 1- 7. doi: 10.1016/j.repbio.2015.12.002
7	JHAMAT N , NIAZI A , GUO Y , et al. LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function[J]. BMC Genomics, 2020, 21 (1): 385. doi: 10.1186/s12864-020-06777-7
8	TANG Y , WANG X , LI Z , et al. Heparin prevents caspase-11-dependent septic lethality independent of anticoagulant properties[J]. Immunity, 2021, 54 (3): 454- 467. doi: 10.1016/j.immuni.2021.01.007
9	AYERS M , LUNCEFORD J , NEBOZHYN M , et al. IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade[J]. J Clin Invest, 2017, 127 (8): 2930- 2940. doi: 10.1172/JCI91190
10	ZHAO Q , BI Y , GUO J , et al. Pristimerin protects against inflammation and metabolic disorder in mice through inhibition of NLRP3 inflammasome activation[J]. Acta Pharmacol Sin, 2021, 42 (6): 975- 986. doi: 10.1038/s41401-020-00527-x
11	鲁文赓, 谢何昕, 田春雨, 等. bAD-MSCs调控Caspase-3通路抑制LPS诱导的BEECs焦亡[J]. 中国兽医学报, 2023, 43 (6): 1228-1233, 1303.
	LU W G , XIE H X , TIAN C Y , et al. bAD-MSCs inhibit LPS-induced BEECs pyroptosis by regulating Caspase-3 pathway[J]. Chinese Journal of Veterinary Medicine, 2023, 43 (6): 1228-1233, 1303.
12	SHELDON I M , CRONIN J G , POSPIECH M , et al. Symposium review: Mechanisms linking metabolic stress with innate immunity in the endometrium[J]. J Dairy Sci, 2018, 101 (4): 3655- 3664. doi: 10.3168/jds.2017-13135
13	KOVACS S B , MIAO E A . Gasdermins: Effectors of Pyroptosis[J]. Trend Cell Biol, 2017, 27 (9): 673- 684. doi: 10.1016/j.tcb.2017.05.005
14	SHELDON I M , CRONIN J G , BROMFIELD J J . Tolerance and innate immunity shape the development of postpartum uterine disease and the impact of endometritis in dairy cattle[J]. Ann Rev Anim Biosci, 2019, 7, 361- 384. doi: 10.1146/annurev-animal-020518-115227
15	FERRARI E , CAPUCCIATI A , PRADA I , et al. Synthesis, structure characterization, and evaluation in microglia cultures of neuromelanin analogues suitable for modeling Parkinson's disease[J]. ACS Chem Neurosci, 2017, 8 (3): 501- 512. doi: 10.1021/acschemneuro.6b00231
16	ESPOSITO E , CUZZOCREA S . TNF-alpha as a therapeutic target in inflammatory diseases, ischemia-reperfusion injury and trauma[J]. Curr Med Chem, 2009, 16 (24): 3152- 3167. doi: 10.2174/092986709788803024
17	KIM J A , KO J H , KO A Y , et al. TSG-6 protects corneal endothelium from transcorneal cryoinjury in rabbits[J]. Invest Ophthalmol Vis Sci, 2014, 55 (8): 4905- 4912. doi: 10.1167/iovs.14-14538
18	WU D , PAN P , SU X , et al. Interferon regulatory factor-1 mediates alveolar macrophage pyroptosis during LPS-induced acute lung injury in mice[J]. Shock, 2016, 46 (3): 329- 338. doi: 10.1097/SHK.0000000000000595
19	LIU Y H , CHANG Y C , CHEN L K , et al. The ATP-P2X7 signaling axis is an essential sentinel for intracellular clostridium difficile pathogen-induced inflammasome activation[J]. Front Cell Infect Microbiol, 2018, 8, 84. doi: 10.3389/fcimb.2018.00084
20	CHEN X , HE W T , HU L , et al. Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis[J]. Cell Res, 2016, 26 (9): 1007- 1020. doi: 10.1038/cr.2016.100
21	RUAN J , XIA S , LIU X , et al. Cryo-EM structure of the gasdermin A3 membrane pore[J]. Nature, 2018, 557 (7703): 62- 67. doi: 10.1038/s41586-018-0058-6
22	MA X , LI Y , SHEN W , et al. LPS mediates bovine endometrial epithelial cell pyroptosis directly through both NLRP3 classical and non-classical inflammasome pathways[J]. Front Immunol, 2021, 12, 676088. doi: 10.3389/fimmu.2021.676088

基因Gene	引物Primer	序列(5′→3′) Sequence
β-Actin	Forward	TCCTGCGGCATTCACGAAACTAC
	Reverse	GTGTTGGCGTAGAGGTCCTTGC
IL-1β	Forward	ATGAAGAGCTGCATCCAACA
	Reverse	ATGGAAGACATGTGCGTAGG
TNF-α	Forward	TGACGGGCTTTACCTCATCT
	Reverse	TGATGGCAGACAGGATGTTG
IL-6	Forward	AACGAGTGGGTAAAGAACGC
	Reverse	CTGACCAGAGGAGGGAATGC
IL-18	Forward	CAGATAATGCACCCCAGACCA
	Reverse	TGGTTACGGCCAGACCTCTA
GSDMD	Forward	TGGTGTTGTCCTCCGGAATG
	Reverse	GCGTTTCACTCAGCATGGTC
NLRP3	Forward	ATCTGAAGCTCTGGTTGGTC
	Reverse	GAGTCTCCCAAGGCATTCTCC
Caspase-1	Forward	GTTCCAGGGTGAAGATAATGTTT
	Reverse	TGAAAATCGAACCTTGCGGA

[1]	刘佳金, 温小庆, 罗春海, 贾红豆, 王薇, 李丹阳, 付世新. FoxO1对高NEFA诱导的奶牛子宫内膜上皮细胞凋亡因子表达的影响[J]. 畜牧兽医学报, 2025, 56(9): 4708-4717.
[2]	王志浩, 郭龙, 王培莉, 李建基, 王亨. 细胞焦亡及其在角膜炎疾病中的作用机制研究进展[J]. 畜牧兽医学报, 2025, 56(3): 1089-1099.
[3]	王靖, 关淑文, 赵小博, 王琳玮, 郭刚, 蒋林树. 竹叶黄酮对H₂O₂诱导奶牛乳腺上皮细胞焦亡的保护作用[J]. 畜牧兽医学报, 2025, 56(1): 281-294.
[4]	李美艳, 邢晓莹, 李娜, 张椰莉, 乔宏萍, 武晓英. 甘草多糖对LPS诱导小鼠睾丸炎的调节作用[J]. 畜牧兽医学报, 2024, 55(6): 2741-2750.
[5]	苏依曼, 叶嘉莉, 邱文粤, 章心婷, 庞晓玥, 王荣梅, 唐兆新, 苏荣胜. 积雪草酸通过抑制HMGB1/TLR4/NF-κB通路减轻脂多糖诱导肉鸡肾细胞焦亡[J]. 畜牧兽医学报, 2024, 55(4): 1777-1786.
[6]	何婷, 刘思颖, 全锦雯, 苏伟谦, 黄江利, 李玙萌, 刘忠华, 余文兰. 高硼暴露通过诱导细胞焦亡加剧鸡肝细胞损伤[J]. 畜牧兽医学报, 2024, 55(10): 4747-4759.
[7]	马亚军, 焦智慧, 刘笑凝, 陆翔羽, 刘涛, 王月, 朴晨曦, 王洪斌. 脂肪间充质干细胞对小型猪肝缺血再灌注合并肝切除组织细胞焦亡的影响[J]. 畜牧兽医学报, 2024, 55(1): 355-364.
[8]	梁文清, 刘忠华, 常晓月, 何婷, 陈嘉, 马晓莉, 唐兆新, 余文兰. 长期高铜暴露通过影响线粒体自噬和细胞焦亡诱导大鼠肝组织损伤[J]. 畜牧兽医学报, 2022, 53(12): 4490-4500.
[9]	石玉华, 傅心雨, 郑梦洁, 吕倩, 师福山. 非经典细胞焦亡体外模型的构建[J]. 畜牧兽医学报, 2021, 52(8): 2284-2290.
[10]	聂雪伊, 刘蕾, 郑雪迪, 杨易, 徐金瑞, 王玉炯. BCG感染巨噬细胞后内质网应激对细胞焦亡的调控作用[J]. 畜牧兽医学报, 2021, 52(10): 2842-2851.
[11]	高泽乾，朱学亮，张志东，窦永喜. 病毒感染激活炎症小体的分子机制[J]. 畜牧兽医学报, 2016, 47(11): 2167-2174.
[12]	刘腾飞，田静，耿智霞，李会敏，余祖功. 抗菌药物对LPS诱导鸡肝细胞损伤的影响及复方甘草酸单胺的修复效应[J]. 畜牧兽医学报, 2015, 46(2): 309-316.