Functional research of Arg 307>Gln locus genotype of P2X7R under high uric acid background

Acta Universitatis Medicinalis Anhui 2020 06 v.55 911-915     font:big middle small

Found programs:

Authors:Li Manyun; Tao Jinhui; Fang Xuan

Keywords:gouty arthritis;single nucleotide polymorphism;Arg 307>Gln;interleukin-1β

DOI:10.19405/j.cnki.issn1000-1492.2020.06.017

〔Abstract〕 Objective To analyze the functional changes of Arg 307>Gln mutant of purinergic receptor P2 X ligand-gated ion channel 7(P2 X7 R) in high uric acid background by establishing a human leukemic monocyte(THP-1) derived monocyte cell line in wild type or different mutants to express P2 X7 R stably. Methods THP-1 cells were transfected by lentivirus to establish a THP-1 cell line that stably expressed wild type, Arg 307>Gln mutantand empty virus. Then three types of wild type,Arg 307>Gln mutant and empty virus were set up separately,and each type was randomized into three groups: monosodium urate( MSU)( Group M),MSU and adenosine triphosphate( ATP)( Group MA) and unstimulated control group( Group C). Three groups of cells were stimulated by Phorbol-12-myristate-13-acetate( PMA),then PMA was removed by centrifugation. Group M and MA were added with MSU incubation,then ATP was added to MA group. Three groups of serum and cells were collected. Interleukin-1β( IL-1β) protein expression level in serum was detected by ELISA while mRNA expression of IL-1β,oligomerization domain( NOD)-like receptor family,pyrin domain containing 3( NLRP3),apoptosis-associated speck-like protein containing a caspase-1 recruitment domain( ASC) and Caspase-1 in transfected THP-1 cells was detected by qRT-PCR. Results There was no significant difference between wild type,Arg 307>Gln mutant and empty virus after adding MSU crystals alone( P>0. 05). However after incubation with ATP,the levels of IL-1βand NLRP3 in the wild type were significantly higher than those in the Arg 307>Gln mutant,and the difference was statistically significant( P<0. 05). The mRNA levels of ASC and Caspase-1 in the ATP-P2 X7 R-IL-1β signaling pathway were not significantly different between wild type and Arg 307>Gln mutant( P>0. 05). Conclusion Arg 307>Gln mutant significantly down-regulated the function of P2 X7 R and reduced the expression levels of IL-1β and NLRP3. Caspase-1 and ASC may be degraded during the activation pathway under high uric acid background.