烟碱对LPS诱导的兔子宫内膜上皮细胞炎症因子及前列腺素分泌的影响

doi:10.16431/j.cnki.1671-7236.2021.11.040

摘要/Abstract

摘要： 试验旨在探索α7烟碱乙酰胆碱受体（α7 nicotinic acetylcholine receptor，α7nAChR）的高亲和力激动剂烟碱对脂多糖（lipopolysaccharides，LPS）诱导的兔子宫内膜上皮炎症的作用机制。分离发情后期兔的子宫内膜上皮细胞，用100 ng/mL LPS对细胞进行炎性刺激12 h。用CCK8法检测不同浓度烟碱（5、10和20 μg/mL）对细胞存活率的影响，筛选合适浓度的烟碱进行后续试验。将细胞分为对照组（CON）、LPS、LPS+烟碱、LPS+烟碱+甲基牛扁碱（MLA）（α7nAChR的特异性颉颃剂）组，通过ELISA法检测细胞培养上清液中白细胞介素-1β（interleukin 1β，IL-1β）、IL-6、IL-8、肿瘤坏死因子-α（tumor necrosis factor，TNF-α）、前列腺素E₂（prostaglandin E₂，PGE₂）和前列腺素F_2α（PGF_2α）的含量。试验成功分离兔子宫内膜上皮细胞，且传至第5代仍保持良好的生长状态。CCK8检测结果显示，20 μg/mL烟碱组细胞存活率显著降低（P<0.05），10 μg/mL烟碱对细胞存活率无显著影响（P>0.05），所以选择10 μg/mL烟碱进行后续试验。ELISA结果显示，与对照组相比，LPS组IL-1β、IL-6、IL-8、TNF-α、PGE₂和PGF_2α的含量显著增加（P<0.05）；与LPS组相比，LPS+烟碱组显著降低IL-1β、IL-6、IL-8、TNF-α、PGE₂和PGF_2α的含量（P<0.05），LPS+烟碱+MLA组炎性因子和前列腺素的含量差异不显著（P<0.05）。以上结果表明，烟碱对LPS诱导的兔子宫内膜上皮细胞分泌IL-1β、IL-6、IL-8、TNF-α、PGE₂和PGF_2α具有下调作用，推测烟碱通过α7nAChR介导炎性因子和前列腺素分泌下调而发挥抗炎作用，该结果可为研究α7nAChR作为子宫内膜炎治疗靶点的药物选择提供参考。

关键词: α7nAChR; 烟碱; 炎性细胞因子; 前列腺素

Abstract: The purpose of this study was to investigate the mechanism of nicotine which is the high affinity agonist of α7nAChR on the inflammation of rabbit endometrial epithelial cells induced by lipopolysaccharides (LPS). Epithelial cells were isolated from the endometrium of rabbits in late estrus and stimulated with 100 ng/mL LPS for 12 h. CCK8 method was used to detect the effects of nicotine in 5, 10 and 20 μg/mL on cell survival rates, and appropriate concentrations of nicotine were screened for subsequent tests. The cells were divided into control group (CON), LPS group, LPS+nicotine group, LPS+nicotine+methyllycaconitine citrate(MLA, specific antagonist of α7nAChR) group. The contents of interleukin-1β(IL-1β), IL-6, IL-8, tumor necrosis factor-α(TNF-α), prostaglandin E₂(PGE₂) and prostaglandin F_2α(PGF_2α) in the supernatant of cell culture were detected by ELISA. Rabbit endometrial epithelial cells were successfully isolated and maintained good growth until the fifth generation. CCK8 test results showed that the survival rate of 20 μg/mL nicotine group was significantly decreased (P<0.05), and 10 μg/mL nicotine had no significant effect on the survival rate of cells(P>0.05), so 10 μg/mL nicotine was selected for the following test. ELISA results showed that compared with control group, the content of IL-1β, IL-6, IL-8, TNF-α, PGE₂ and PGF_2α was significantly increased in LPS group (P<0.05). When compared with LPS group, the content of IL-1β, IL-6, IL-8, TNF-α, PGE₂ and PGF_2α was significantly decreased in LPS+nicotine group(P<0.05), and the content of inflammatory factors and prostaglandins in LPS+nicotine+MLA group had no significant difference (P>0.05). These results suggested that nicotine could down-regulate the secretion of IL-1β, IL-6, IL-8, TNF-α, PGE₂ and PGF_2α in LPS-induced rabbit endometrial epithelial cells. It was speculated that nicotine played an anti-inflammatory role by down-regulating inflammatory factors and prostaglandins mediated by α7nAChR. These results might provide a reference for the selection of α7nAChR as a therapeutic target for endometritis.

Key words: α7nAChR; nicotine; inflammatory cytokines; prostaglandin

中图分类号:

S852.2

魏泓毓, 鲍朝霞, 陈灰, 曹金山, 贺鹏飞. 烟碱对LPS诱导的兔子宫内膜上皮细胞炎症因子及前列腺素分泌的影响[J]. 中国畜牧兽医, 2021, 48(11): 4302-4310.

WEI Hongyu, BAO Zhaoxia, CHEN Hui, CAO Jinshan, HE Pengfei. Effects of Nicotine on LPS-induced Inflammatory Cytokines and Prostaglandin Secretion in Rabbit Endometrial Epithelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4302-4310.

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