EPHX2 rs751141变异位点对肾脏微炎症的影响及其分子机制研究

doi:10.3969/j.issn.1006-7795.2021.05.001

首都医科大学学报 ›› 2021, Vol. 42 ›› Issue (5): 691-697.doi: 10.3969/j.issn.1006-7795.2021.05.001

• 检验医学与临床 • 下一篇

EPHX2 rs751141变异位点对肾脏微炎症的影响及其分子机制研究

马亮¹, 赵海玲², 赵婷婷², 赵美美¹, 刘怡¹, 高芃¹, 李平², 曹永彤^1*

1.中日友好医院检验科,北京 100029;
2.中日友好医院临床医学研究所免疫炎性疾病北京市重点实验室,北京 100029

收稿日期:2021-08-24 发布日期:2021-10-29
通讯作者: 国家自然科学基金面上项目(82074221,81973627),北京临床重点专科项目(2020)。

Effect of EPHX2 rs751141 mutation on renal microinflammation and its molecular mechanism

Ma Liang¹, Zhao Hailing², Zhao Tingting², Zhao Meimei¹, Liu Yi¹, Gao Peng¹, Li Ping², Cao Yongtong^1*

1. Department of Clinical Laboratory, China-Japan Friendship Hospital,Beijing 100029, China;
2. Beijing Key Laboratory of Immune-Mediated Inflammatory Diseases, Institute of Clinical Medical Science, China-Japan Friendship Hospital, Beijing 100029,China

Received:2021-08-24 Published:2021-10-29
Contact: National Natural Science Foundation of China (82074221,81973627), Key Clinical Specialty Project of Beijing (2020).

摘要/Abstract

摘要： 目的研究EPHX2 rs751141基因多态性(G860A,R287Q)对可溶性环氧化物水解酶(soluble epoxide hydrolase, sEH)活性的影响及其参与肾脏微炎症的分子机制。方法以人的肾小管上皮细胞(HK2)cDNA为模板,利用p-UCT DNA连接试剂盒、点突变技术及亚克隆技术,分别构建包含EPHX2基因rs751141位点等位基因G和A的重组质粒pcDNA3.1/V5-His-G(野生型:WT)和pcDNA3.1/V5-His-A(突变型:R287Q)。以Lipofectamine 2000转染试剂分别将上述重组质粒转染至HK2细胞,给予足量的11,12-环氧二十碳三烯酸(11,12-epoxyeicosatrienoic acid,11,12-EET);共培养后,利用ELISA试剂盒检测11,12-二羟基二十碳三烯酸(11,12-dihydroxyeicosatrienoic acid,11,12-DHET),采用t检验分析R287Q突变对sEH水解酶活性的影响;利用流式细胞术检测细胞上清中炎症因子浓度白细胞介素-17A(interleukin-17A,IL-17A)和IL-1的浓度;RT-PCR检测IL-17A和IL-1β等mRNA表达水平;Western blotting方法检测PI3K/AKT信号通路相关蛋白表达。结果与WT组相比,R287Q突变型重组质粒组水解活性明显降低(P<0.01),细胞上清中IL-17A浓度以及IL-1等炎症因子浓度显著下降(P<0.05)。与单纯WT组相比,WT+EET组的炎症因子浓度显著降低,但仍明显高于R287Q+EET组(P<0.05)。RT-PCR结果显示R287Q突变组中IL-17A和IL-1β等的mRNA表达明显低于WT组;与单纯WT组相比,WT+EET组IL-17A以及IL-1β等炎症因子mRNA表达降低,但仍明显高于R287Q+EET组(P<0.05)。Western blotting结果表明R287Q组可能通过调控PI3K/AKT信号通路减轻肾小管上皮细胞炎症水平。结论 R287Q突变型重组质粒对11,12-EET的水解能力降低,炎症水平显著下降;外源性11,12-EET可降低WT组炎症水平。EPHX2 rs751141位点等位基因A的肾脏保护作用可能通过调控PI3K/AKT信号通路参与减缓炎症的分子机制。

关键词: EPHX2 rs751141基因多态性, 可溶性环氧化物水解酶活性, 肾脏微炎症, PI3K/AKT信号通路, 等位基因

Abstract: Objective To study the effect of EPHX2 rs751141 polymorphism (G860A, R287Q) on the activity of soluble epoxide hydrolase (sEH) and the molecular mechanism of its involvement in renal microinflammation. Methods With human renal tubular epithelial cells (HK2) cDNA used as a template, the recombinant plasmids pcDNA3.1/V5-His-G (wild type, WT) and pcDNA3.1/V5- His-A (mutant: R287Q) containing EPHX2 rs751141 alleles G and A were constructed using p-UCT DNA linkage kit, point mutation technique and subclone technique. The recombinant plasmids were transfected into HK2 cells with Lipofectamine2000 transfection reagent and co-cultured with 11,12-epoxyeicosatrienoic acid (11,12-EET). The level of 11,12-dihydroxyeicosatrienoic acid (11,12-DHET) was detected by ELISA kit and the effect of R287Q mutation on the activity of sEH was analyzed by T test. The levels of IL-17A and IL-1 in supernatants were measured by flow cytometry technique. The levels of IL-17A and IL-1β mRNA were detected by RT-PCR, and PI3K/AKT signal pathway related protein were detected by Western Blot. Results Compared with WT group, the hydrolytic activity of R287Q group was significantly decreased (P<0.01), and the levels of IL-17A and IL-1 in supernatant were significantly decreased (P<0.05). Compared with WT group, the level of inflammatory factors in WT+EET group was significantly lower, but still higher than that in R287Q+EET group (P<0.05). The mRNA expression of IL-17A and IL-1β in the R287Q mutation group was significantly lower than that in the WT group, and the mRNA expression of IL-17A and IL-1β in the WT+EET group was significantly lower than that in the WT group, but still higher than R287Q + EET group (P<0.05). Western blotting results indicated that R287Q group may modulate PI3K/AKT signaling pathway to reduce inflammation in renal tubular epithelial cells. Conclusions R287Q group decreased the hydrolytic ability of 11,12-EET and significantly reduce inflammation, while exogenous 11,12-EET reduce inflammation in WT group. The renal protection of EPHX2 rs751141 allele A may be involved in the molecular mechanism by regulating the PI3K/AKT signaling pathway.

Key words: EPHX2 rs751141 polymorphism, soluble epoxide hydrolase activity, renal microinflammation, PI3K/AKT signal pathway, allele

中图分类号:

马亮, 赵海玲, 赵婷婷, 赵美美, 刘怡, 高芃, 李平, 曹永彤. EPHX2 rs751141变异位点对肾脏微炎症的影响及其分子机制研究[J]. 首都医科大学学报, 2021, 42(5): 691-697.

Ma Liang, Zhao Hailing, Zhao Tingting, Zhao Meimei, Liu Yi, Gao Peng, Li Ping, Cao Yongtong. Effect of EPHX2 rs751141 mutation on renal microinflammation and its molecular mechanism[J]. Journal of Capital Medical University, 2021, 42(5): 691-697.

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EPHX2 rs751141变异位点对肾脏微炎症的影响及其分子机制研究

Effect of EPHX2 rs751141 mutation on renal microinflammation and its molecular mechanism

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