竹叶黄酮对H2O2诱导奶牛乳腺上皮细胞焦亡的保护作用

doi:10.11843/j.issn.0366-6964.2025.01.026

摘要/Abstract

摘要：

旨在以竹叶黄酮(BLF)为添加物，探究BLF对过氧化氢(H₂O₂)诱导奶牛乳腺上皮细胞(BMECs)焦亡的保护作用。本研究以BMECs为研究对象，分为对照组、80 μg·mL^-1 BLF处理组、800 μmol·L^-1 H₂O₂处理组和80 μg·mL^-1 BLF+800 μmol·L^-1 H₂O₂处理组。利用RT-qPCR、Western blot、免疫荧光、流式细胞术等技术，检测细胞焦亡相关基因和蛋白表达以及凋亡相关微粒样蛋白(ASC)荧光强度。结果表明，与对照组相比，800 μmol·L^-1 H₂O₂处理BMECs 8 h显著增加了奶牛乳腺上皮细胞的焦亡(P＜0.05)；H₂O₂处理后，NLRP3、ASC、GSDMD、IL-18、IL-1β等焦亡相关基因的相对表达量均显著升高(P＜0.05)，尤其是NLRP3，说明H₂O₂显著增加BMECs焦亡。与H₂O₂处理组相比，BLF可以降低正常细胞焦亡水平，显著降低NLRP3、Nek7、ASC、GSDMD、IL-18、IL-1β基因mRNA上调的趋势，显著缓解了H₂O₂导致的NLRP3、ASC、Nek7、pro-caspase1、caspase1 p20、GSDMD-N、IL-18和IL-1β胞内焦亡相关蛋白表达的升高(P＜0.05)，ASC的荧光强度显著降低(P＜0.05)。综上所述，H₂O₂显著上调NLRP3、ASC、Caspase-1、GSDMD、IL-1β、IL-18焦亡基因和蛋白表达水平，诱导奶牛乳腺上皮细胞焦亡。BLF通过降低NLRP3炎症小体通路蛋白和基因表达，降低了炎症因子水平，从而缓解奶牛乳腺上皮细胞焦亡。

关键词: 竹叶黄酮, 奶牛乳腺上皮细胞, 细胞焦亡

Abstract:

In this study, bamboo leaf flavonoids (BLF) was used as an additive to explore its protective effect on hydrogen peroxide (H₂O₂)-induced pyroptosis of bovine mammary epithelial cells (BMECs). In this experiment, the concentration of BLF was 80 μg·mL^-1 and the concentration of H₂O₂ was 800 μmol·L^-1. The BMECs without BLF treatment were set as the control group, and the treatment groups were BLF treatment group, H₂O₂ treatment group, BLF and H₂O₂ co-treatment group. The expression of pyroptosis-related genes and related proteins and the fluorescence intensity of apoptosis-related microparticle-like protein(ASC) were detected by RT-qPCR, Western blot, immunofluorescence and flow cytometry. The results showed that compared with the control group, 800 μmol·L^-1 H₂O₂ treatment of BMECs for 8 h significantly increased the pyroptosis of bovine mammary epithelial cells (P < 0.05). After H₂O₂ treatment, the relative expression levels of NLRP3, ASC, GSDMD, IL-18, IL-1β genes which related to pyroptosis genes were significantly increased (P < 0.05), especially the expression of NLRP3, indicating that H₂O₂ significantly increased the pyroptosis of BMECs. Compared with the H₂O₂ treatment group, BLF could reduce the pyroptosis level of normal cells, significantly reduce the mRNA up-regulation trend of NLRP3, Nek7, ASC, GSDMD, IL-18, IL-1β, and significantly alleviate the increase of intracellular pyroptosis protein expression of NLRP3, ASC, Nek7, pro-caspase1, caspase1 p20, GSDMD-N, IL-18 and IL-1β induced by H₂O₂ (P < 0.05), and the fluorescence intensity of ASC was significantly reduced (P < 0.05). In summary, H₂O₂ significantly up-regulated the expression levels of pyroptosis-related genes and proteins such as NLRP3, ASC, Caspase-1, GSDMD, IL-1β, and IL-18, and induced pyroptosis in bovine mammary epithelial cells. In conclusion, BLF reduced the level of inflammatory factors by reducing the expression of NLRP3 inflammasome pathway proteins and genes, thereby alleviating the pyroptosis of dairy cow mammary epithelial cells.

Key words: bamboo leaf flavonoids, dairy cow mammary epithelial cells, pyroptosis of cells

中图分类号:

S823.91

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.

WANG Jing, GUAN Shuwen, ZHAO Xiaobo, WANG Linwei, GUO Gang, JIANG Linshu. The Protective Effect of Bamboo Leaf Flavonoids on H₂O₂-induced Pyroptosis in Bovine Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 281-294.

图/表 11

表 1

试剂与耗材"

试剂 Reagent	来源 Source
H₂O₂ Hydrogen peroxide	天津光复，中国
DMEM/F12、FBS、双抗DMEM/F12，FBS，Penicillin-Streptomycin	Gibco，美国
0.25%胰蛋白酶-EDTA 0.25% trypsin-EDTA	Gibco，美国
PBS	Wissen，中国
细胞培养皿、细胞培养板、离心管、枪头 Cell culture dish，Cell culture plate，Centrifuge tube，Gun tip	Corning，美国
BCA试剂盒BCA kit	Beyotime，中国
二甲基亚砜(DMSO) Dimethyl sulfoxide(DMSO)	Solarbio，中国
蛋白酶抑制剂Protease inhibitor	Beyotime，中国
RIPA、PMSF	Beyotime，中国
TBST	Solarbio，中国
聚丙烯酰胺电泳凝胶试剂盒Polyacrylamide electrophoresis gel kit	Beyotime，中国
Hoechst 33342/PI双染试剂盒Hoechst 33342/PI double dye kit	Solarbio，中国
SDS-PAGE缓冲液SDS-PAGE buffer	Beyotime，中国
预染蛋白Marker Prestain protein Marker	LABLEAD，中国
PVDF膜PVDF membrane	Beyotime，中国
脱脂奶粉Skim milk powder	Beyotime，中国
Tris	Solarbio，中国
SDS	Solarbio，中国
甘氨酸Glycine	Solarbio，中国
甲醇Carbinol	Solarbio，中国
cDNA反转录试剂盒cDNA reverse transcription kit	Erik，中国
SYB^RTM Premix ExTaq Ⅱ	Erik，中国
TRIzol	Beyotime，中国
氯仿、异丙醇、无水乙醇Chloroform，isopropyl alcohol，anhydrous ethanol	北京化工厂，中国
ECL化学发光试剂盒ECL chemiluminescence kit	Beyotime，中国
羊抗兔IgG、羊抗鼠IgG Sheep Anti-Rabbit IgG, Sheep Anti-Mouse IgG	LABLEAD，中国
SIRT1、p65、NLRP3、ASC、IL-1β、Nek7、Lamin B1抗体 SIRT1, p65, NLRP3, ASC, IL-1β, Nek7, Lamin B1 antibody	Santa，中国
Caspase-1、GSDMD-N抗体Caspase-1, GSDMD-N antibody	Abcam，美国
IL-18抗体IL-18 antibody	Bioss，中国
β-actin抗体β-actin antibody	CST，美国
抗荧光淬灭封片液(含DAPI) Anti-fluorescence quenched sealing solution (DAPI included)	Beyotime，中国

表 1

表 2

表 3

图 1

图 2

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图 8

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试验仪器 Test instrument	来源 Source
荧光倒置显微镜Inverted fluorescence microscope	Motic，中国
CO₂细胞培养箱CO₂ cell incubator	Thermo，美国
双功能气浴恒温振荡器Dual-function gas bath thermostatic oscillator	常州峥嵘，中国
恒温恒湿箱Constant temperature and humidity box	上海一恒，中国
荧光定量PCR仪Fluorescent quantitative PCR instrument	Roche，瑞士
垂直电泳转印系统Vertical electrophoretic transfer system	BIO-RAD，美国
天能化学发光成像仪Celestial chemiluminescence imager	上海天能，中国
Gene Max基因扩增仪Gene Max Gene amplification instrument	杭州博日，中国
Cyto FLEX流式细胞仪Cyto FLEX flow cytometry	BECKMAN COULTER，中国

基因 Gene	引物序列(5′→3′) Primer sequences(5′→3′)
含有一个吡啶结构域3的Nod样受体 NLRP3	F: AGATCATGTTGGACTGGGCA R: CATCAAAGCCGTCCATGAGG
凋亡相关斑点样蛋白 ASC	F: AGCCAGGCCTGCACTTTATA R: CATCTTGCTTGGGTTGGTGG
半肌氨酸天冬氨酸蛋白酶1 Caspase 1	F: ATGGGATCTGCGGGACTATG R: CCAGGTTCTCGGATGACTGT
哺乳动物相关激酶7 Nek7	F: TTCAGCTCCAAAACCACAGC R: ACACAGCGAGTACAGGTTCA
白细胞介素-1β IL-1β	F: AAACTCCAGGACAGAGAGCAAAA R: CTCTCCTTGCACAAAGCTCATG
白细胞介素-18 IL-18	F: TGGCTGCAGAACAAGTAGAAG R: TGGTCTGGGGTGCATTATCT
消皮素D GSDMD	F: TTTGTAGTGACCGAGGTGCT R: ATGTCCCAGTCAGAGCCAAT
β-肌动蛋白 ACTB	F: CAAGTACCCCATTGAGCACG R: GTCATCTTCTCACGGTTGGC
甘油醛-3-磷酸脱氢酶 GAPDH	F: TGGAAAGGCCATCACCATCT R: CCCACTTGATGTTGGCAG

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竹叶黄酮对H₂O₂诱导奶牛乳腺上皮细胞焦亡的保护作用

The Protective Effect of Bamboo Leaf Flavonoids on H₂O₂-induced Pyroptosis in Bovine Mammary Epithelial Cells

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