Abstract
Background:Previous studies[1] have shown that ATP acts on the sputum receptor P2X ligand-gated ion channel 7 (P2X7R) as a second signal to induce gouty arthritis.Objectives:In this study, the functional changes of three SNP genotypes, Ala348 to Thr, Glu 496 to Ala and Arg307 to Gln, in P2X7R were analyzed with high uric acid.Methods:After transfection of HEK-293T cells by lentivirus, observing the uptake ability of HEK-293T cells to ethidium bromide. The effect of three different mutants on the P2X7 receptor was thus observed on the P2X7 channel. In addition, THP-1 cells were also transfected, stable expression of a THP-1 cell line that has been transfected with a wild-type or different mutants and thus established. Then three types were set up separately, and each type was randomized into three groups: MSU(labeled M), MSU+ATP (labeled MA), and unstimulated control group (labeled C).Detection of IL-1β protein expression level in serum by ELISA and NLRP3, ASC and Caspase-1 mRNA levels in transfected THP-1 cells by qRT-PCR.Results:1.These three variants have different effects on the uptake function of ATP-induced ethidium+bromide in transfection of HEK-293T cells by lentivirus. Ala348to Thr increased P2X7-dependent ethidium+bromide uptake (145% of wild-type P2X7response, P<0.001). In contrast, Absent or very reduced P2X7function was found in Glu496to Ala and Arg307to Gln subjects, appeared to abolish P2X7-dependent dye uptake (38% and 32% of wild-type P2X7responses, P<0.001,), who were compared with wild-type.2.Compared the IL-1β levels of the three variants with the wide-type and empty virus in THP-1 cells, the Ala348to Thr mutation significantly up-regulated the serum levels of IL-1β compared with the wide-type and empty virus in group MA with high uric acid (P=0.0007;P=0.013, respectively). Moreover, similar results have also been shown in IL-1β mRNA expressions (P=0.0334; P=0.0307, respectively). The Glu496to Ala and Arg307to Gln mutations down-regulated the serum levels of IL-1β compared with the wide-type in group MA (P=0.0189;P=0.0164, respectively).3.NLRP3 mRNA was significantly increased in the Ala348to Thr mutation compared with the wide-type and empty virus in group MA (p =0.0003;P=0.0001, respectively). However, NLRP3 mRNA was significantly reduced in the Glu496to Ala and Arg307to Gln mutations compared with the wide-type in group MA (p =0.0294;P=0.0279, respectively).4.Wild-type was signifcantly higher than empty virus in the ASC gene expression in group MA(P=0.0022). Morever, the Ala348to Thr mutation was higher than empty virus while Arg307to Gln mutation was lower than that in group MA (P=0.0138;P=0.0283, respectively).5.Unlike NLRP3 gene expression, the data showed that the expression of caspase-1 mRNA in group C, M and MA all with no statistical significance, respectively (P>0.05).Conclusion:Our data revealed that Ala348to Thr up-regulate the functional status of P2X7R and Glu496to Ala and Gln460to Arg down-regulate the functional status of P2X7R, which resulted in a significant increase or decrease in IL-1β and NLRP3 expression levels with high uric acid.
Published Version
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