The Function Changes On Mutation Of Different SNPs In The P2X7 Receptor With High Uric Acid

Objective Previous studies have shown that ATP acts on the sputum receptor P2 X ligand-gated ion channel 7(P2X7R)as a second signal to induce gouty arthritis.In this study,the functional changes of three SNP genotypes,Ala348 to Thr,Glu 496 to Ala and Arg307 to Gln,in P2X7 R were analyzed in a high uric acid background by establishing a THP-1 derived monocyte cell line in wild type or different mutants to express P2X7 R stably.Methods After transfection of HEK-293 T cells by lentivirus,observing the uptake ability of HEK-293 T 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 by lentivirus,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 of wild type,mutant types or empty virus 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-1? protein expression level in supernatants by ELISA and Caspase-1 and NLRP3 m RNA expression levels in transfected THP-1 cells by q RT-PCR.SPSS software was used to analyze the data,and the difference of P<0.05 was statistically significant.Results These three variants have different effects on the uptake function of ATP-induced ethidium+ bromide in transfection of HEK-293 T cells by lentivirus.Gain-of-function variant,Ala348 to Thr,increased P2X7-dependent ethidium+ bromide uptake(145% of wild-type P2X7 response),at all functional polymorphic positions showed brisk uptake of ethidium+ bromide which was more than the wild-type value.In contrast,Absent or very reduced P2X7 function was found in Glu496 to Ala and Arg 307 to Gln subjects,appeared to abolish P2X7-dependent dye uptake(38% and 32% of wild-type P2X7 responses),who were compared with wild-type.Compared the level of IL-1?in the three variants with the wide-type and empty virus in THP-1 cells,the Ala348 to Thr mutation significantly up-regulated the supernatant levels of IL-1 ?compared with the wide-type and empty virus in group MA under high uric acid background(P=0.0007;P=0.013,respectively).The wide-type had higher levels of IL-1? than the empty virus,but with no statistical significance.However,there were no significant differences in group C and M,respectively.Moreover,the m RNA expressions of IL-1 ? in Ala348 to Thr mutation showed a higher level than the wide-type and empty virus in group MA under high uric acid background(P=0.0334;P=0.0307,respectively).The Glu496 to Ala and Arg307 to Gln mutations down-regulated the supernatant levels of IL-1? compared with the wide-type in group MA(P=0.0189;P=0.0164,respectively).In addition,the two variants had lower levels than the empty virus while the wide-type had higher levels of IL-1 ? than that with no statistical significance in group MA,respectively.Furthermore,there were no significant differences in group C and M,respectively.Likewise,the m RNA expressions of IL-1?in Glu496 to Ala and Arg307 to Gln mutations showed a lower level than the wide-type in group MA under high uric acid background(P=0.0037;P<0.001,respectively).The m RNA level of NLRP3 was significantly increased in the Ala348 to Thr mutation compared with the wide-type and empty virus in group MA(p =0.0003;P=0.0001,respectively).However,The m RNA level of NLRP3 was significantly reduced in the Glu496 to Ala and Arg307 to Gln mutations compared with the wide-type in group MA(p=0.0294;P=0.0279,respectively).Whereas compared the m RNA level of NLRP3 in the wild-type had no significant change with the empty virus.In addition,there were also no significant difference in two variants compared with the empty virus,respectively.Moreover,the expression of NLRP3 m RNA in group C and M with no statistical significance,respectively.Wild-type was signifcantly higher than empty virus in the ASC gene expression in group MA(P=0.0022).Morever,the Ala348 to Thr mutation was higher than empty virus while Arg307 to Gln mutation was lower than that in group MA(P=0.0138;P=0.0283,respectively).However,there was no statistical significance in the Glu496 to Ala mutation compared with empty virus.Meanwhile,compared the m RNA expression of ASC in three SNPs with wild-type were with no statistical significance,respectively.Furthermore,there was no signifcant difference in the expression of ASC m RNA in groups C and M,respectively.Unlike NLRP3 gene expression,the data showed that the m RNA level of Caspase-1 in group C?M and MA all with no statistical significance,respectively.Conclusion Our data revealed that Ala348 to Thr up-regulate the functional status of P2X7 R and Glu496 to Ala and Arg307 to Gln down-regulate the functional status of P2X7 R,which resulted in a significant increase or decrease in IL-1? and NLRP3 expression levels in high uric acid background.