内质网应激预适应对LPS诱导的山羊子宫内膜上皮细胞炎性反应的保护作用

doi:10.11843/j.issn.0366-6964.2023.08.038

摘要/Abstract

摘要： 探究内质网应激（endoplasmic reticulum stress，ERS）在脂多糖（lipopolysaccharide，LPS）诱导的山羊子宫内膜上皮细胞（goat endometrial epithelial cells，gEECs）炎性反应中的作用，为阐明ERS在反刍动物子宫内膜炎中的作用机制提供前期基础。本研究使用ERS激活剂衣霉素（tunicamycin，TM）和抑制剂4-苯基丁酸（4-phenylbutyric acid，4-PBA）分别单独处理gEECs激活或抑制ERS，应用RT-qPCR和Western blot技术检测ERS对炎症相关基因表达的影响；接着，使用TM和4-PBA分别预处理gEECs，再利用大肠杆菌LPS处理诱导gEECs炎性反应，通过RT-qPCR和Western blot检测ERS对LPS诱导的gEECs炎性反应的影响及作用机制。结果显示，TM处理激活ERS显著抑制gEECs中炎性细胞因子IL-6和TNF-α mRNA的表达（P<0.05），显著抑制经典炎症通路相关基因TLR4、NF-κB P65和NLRP3 mRNA的表达（P<0.01）；同时，显著抑制NF-κB P65蛋白的磷酸化以及NLRP3蛋白的表达（P<0.05）。4-PBA处理则显著促进gEECs中炎症相关基因（IL-6、TNF-α、TLR4、NF-κB P65和NLRP3）的表达（P<0.05）。进一步研究发现，TM预处理gEECs显著抑制LPS诱导的炎性细胞因子IL-6 mRNA的表达（P<0.05）；显著抑制LPS诱导的TLR4、NF-κB P65和NLRP3 mRNA的表达以及NF-κB P65蛋白的磷酸化和NLRP3蛋白的表达（P<0.05）。4-PBA预处理gEECs则显著促进LPS诱导的gEECs炎性反应（P<0.05）。综上表明，ERS通过抑制NF-κB通路和NLRP3炎性小体通路的激活减轻LPS诱导的gEECs炎性反应，为进一步阐明山羊子宫内膜炎的发生发展提供了前期理论基础。

关键词: 内质网应激, 子宫内膜炎, 脂多糖, NF-κB通路, NLRP3炎性小体

Abstract: We attempted to investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced inflammatory responses in goat endometrial epithelial cells (gEECs) and provide a preliminary basis for elucidating the mechanism of ERS in ruminant endometritis. In this study, gEECs were treated with ERS activator tunicamycin (TM) and inhibitor 4-phenylbutyric acid (4-PBA) separately to activate and inhibit ERS, and RT-qPCR and Western blot techniques were used to detect the effect of ERS on inflammation-related genes expression. Next, gEECs were pretreated with TM and 4-PBA, respectively, and then treated with E.coli LPS to induce inflammatory responses in gEECs, the effects and mechanisms of ERS on the inflammatory responses induced by LPS in gEECs were detected by RT-qPCR and Western blot. The results showed that activation of ERS by TM treatment significantly inhibited the expression of inflammatory cytokines IL-6 and TNF-α mRNA in gEECs (P<0.05). The mRNA expression levels of classical inflammatory pathway-related genes TLR4, NF-κB P65, and NLRP3 was also significantly suppressed (P<0.01). Meanwhile, it also significantly inhibited the phosphorylation of NF-κB P65 protein and the expression of NLRP3 protein (P<0.05). However, 4-PBA treatment significantly promoted the expression of inflammation-related genes (IL-6, TNF-α, TLR4, NF-κB P65, and NLRP3) in gEECs (P<0.05). Further study revealed that TM pretreatment of gEECs significantly inhibited the expression of LPS-induced inflammatory cytokine IL-6 mRNA (P<0.05). It also significantly inhibited LPS-induced TLR4, NF-κB P65, and NLRP3 mRNA expression as well as phosphorylated NF-κB P65 protein and NLRP3 protein (P<0.05). 4-PBA pretreatment of gEECs then significantly promoted the LPS-induced inflammatory response in gEECs (P<0.05). The above results suggest that ERS alleviates LPS-induced inflammatory response in gEECs by inhibiting the activation of NF-κB pathway and NLRP3 inflammatory vesicle pathway. These results provide a pre-theoretical basis for further elucidation of the development of goat endometritis.

Key words: endoplasmic reticulum stress, endometritis, lipopolysaccharide, NF-κB pathway, NLRP3 inflammatory vesicles

中图分类号:

S857.2

郜康康, 扆妍妍, 赵一腾, 林鹏飞, 陈华涛, 靳亚平. 内质网应激预适应对LPS诱导的山羊子宫内膜上皮细胞炎性反应的保护作用[J]. 畜牧兽医学报, 2023, 54(8): 3546-3556.

GAO Kangkang, YI Yanyan, ZHAO Yiteng, LIN Pengfei, CHEN Huatao, JIN Yaping. Protective Effect of Endoplasmic Reticulum Stress Preadaptation on LPS-Induced Inflammatory Response in Goat Endometrial Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3546-3556.

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