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

doi:10.3969/j.issn.1006-7795.2021.05.001

Abstract

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

CLC Number:

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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URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2021.05.001

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2021/V42/I5/691

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