灭活大肠杆菌与脂多糖诱导猪肠黏膜上皮细胞表达RegⅢγ的机理研究

doi:10.16431/j.cnki.1671-7236.2021.03.008

摘要/Abstract

摘要： 试验旨在探索革兰氏阴性菌大肠杆菌（Escherichia coli，E.coli）及其表面分子脂多糖（LPS）诱导胰腺再生蛋白Ⅲγ（RegⅢγ）表达调控的机制。首先，用不同浓度灭活E.coli（10⁹、10⁸、10⁷、10⁶、10⁵、10⁴ CFU/mL）和LPS （0.01、0.1、1、5、10、20、40、80 μg/mL）诱导猪肠黏膜上皮细胞（IPEC-JⅡ），用MTT法测D_{490 nm}值，检测E.coli和LPS对IPEC-JⅡ细胞活力的影响；其次，用不同浓度灭活E.coli（10⁷、10⁶、10⁵ CFU/mL）和LPS （0.01、0.1、1、5 μg/mL）处理IPEC-JⅡ细胞24 h，用实时荧光定量PCR和Western blotting检测RegⅢγ mRNA和蛋白的表达；最后，用1 μg/mL LPS处理IPEC-JⅡ细胞24 h，用实时荧光定量PCR和Western blotting检测p65、p38、JNK、ERK mRNA和蛋白表达及磷酸化水平。结果显示，除0.01 μg/mL LPS不抑制IPEC-JⅡ细胞活力外，其他浓度的灭活E.coli和LPS均可抑制IPEC-JⅡ细胞活力，且10⁹、10⁸ CFU/mL E.coli和10、20、40、80 μg/mL LPS组细胞活力极显著下降（P<0.01）；与对照组相比，10⁷、10⁶和10⁵ CFU/mL E.coli均能诱导RegⅢγ表达增加，且10⁵ CFU/mL E.coli组RegⅢγ mRNA表达量极显著高于对照组（P<0.01），蛋白表达量显著高于对照组（P<0.05）；0.01、0.1、1和10 μg/mL LPS均能诱导RegⅢγ表达增加，且0.1和1 μg/mL LPS组RegⅢγ mRNA表达量极显著高于对照组（P<0.01），RegⅢγ蛋白表达虽有增加趋势，但差异不显著（P>0.05）；与对照组相比，1 μg/mL LPS组p65、p38 mRNA表达量极显著增加（P<0.01），JNK、ERK mRNA表达量显著增加（P<0.05）；同时，p38、JNK蛋白表达量和磷酸化水平均极显著增加（P<0.01），p65蛋白磷酸化水平显著增加（P<0.05），ERK蛋白和磷酸化水平均增加，但差异不显著（P>0.05）。以上结果表明，灭活E.coli和LPS均可诱导RegⅢγ表达，1 μg/mL LPS可增加p65、p38和JNK蛋白的磷酸化水平。

关键词: RegⅢγ; 猪肠黏膜上皮细胞; 大肠杆菌; LPS; NF-κB; MAPK

Abstract: This experiment was intended to explore the regulation mechanism of Gram-negative bacterium Escherichia coli (E.coli) and its surface molecules lipopolysaccharide (LPS) induced the pancreas regeneration protein Ⅲ gamma (RegⅢγ) expression.Firstly,different concentration E. coli (10⁹,10⁸,10⁷,10⁶,10⁵ and 10⁴ CFU/mL) and LPS (0.01,0.1,1,5,10,20,40 and 80 μg/mL) were used to induce intestinal epithelia cells of porcine (IPEC-JⅡ),and D_{490 nm}value was determined by MTT method to judge the effect of E. coli and LPS on IPEC-JⅡ cell viability.Then,different concentration E. coli (10⁷,10⁶ and 10⁵ CFU/mL) and LPS (0.01,0.1,1 and 5 μg/mL) were used to induce IPEC-JⅡ for 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of RegⅢγ.Finally,1 μg/mL LPS was used to treat IPEC-JⅡ for 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression and phosphorylation level of p65,p38,JNK and ERK.The results showed that except 0.01 μg/mL LPS did not inhibit IPEC-JⅡ cell viability,all the other concentration of E. coli and LPS inhibited IPEC-JⅡ cell viability,moreover,the cell viability of 10⁹ and 10⁸ CFU/mL E. coli and 10,20,40,80 μg/mL LPS group were extremely significantly decreased (P<0.01).Compared with control group,10⁷,10⁶ and 10⁵ CFU/mL E. coli could increase the expression of RegⅢγ in IPEC-JⅡ cells,and the RegⅢγ mRNA expression of 10⁵ CFU/mL E. coli group was extremely significantly higher than that of control group (P<0.01),the quantity of protein expression was significantly higher than control group (P<0.05).0.01,0.1,1 and 10 μg/mL LPS could increase the expression of RegⅢγ in IPEC-JⅡ cells,moreover,the RegⅢγ mRNA expression of 0.1 and 1 μg/mL LPS group was extremely significantly higher than that of control group (P<0.01),although RegⅢγ protein expression had increasing trend,but had no significant difference (P>0.05).Compared with control group,the mRNA expression of p65 and p38 of 1 μg/mL LPS group were extremely significantly increased (P<0.01),and the mRNA expressions of JNK and ERK were significantly increased (P<0.05).Meanwhile,the protein expression levels of p38 and JNK and the phosphorylation level of p65 were significantly increased (P<0.01), and the phosphorylation level of p65 was significantly increased (P<0.05).Both ERK protein and phosphorylation levels increased,but the difference were not significant (P>0.05).The above results showed that E. coli and LPS could induce RegⅢγ expression,1 μg/mL LPS increased the phosphorylation levels of p65,p38 and JNK proteins.

Key words: RegⅢγ; IPEC-JⅡ; Escherichia coli; LPS; NF-κB; MAPK

中图分类号:

S852.6

孟江海, 向琼昊, 宋云云, 曹斌, 陈韬. 灭活大肠杆菌与脂多糖诱导猪肠黏膜上皮细胞表达RegⅢγ的机理研究[J]. 中国畜牧兽医, 2021, 48(3): 846-854.

MENG Jianghai, XIANG Qionghao, SONG Yunyun, CAO Bin, CHEN Tao. Mechanism Study on Inactivated E. coli and Lipopolysaccharide Co-induced IPEC-JⅡ Cells Expressing RegⅢγ[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 846-854.

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