Expression And Significance Of ELOVL6 And Related NLRP3 Inflammatory Bodies In Patients With Gout Stimulated By Uric Acid

Objective:To investigate the expression and clinical significance of Elongation of very long chain fatty acids protein 6(ELOVL6)and NLRP3 inflammatory bodiesin inflammatory response of gout arthritis(GA)under uric acid stimulation.Methods:(1)33GA(AGA:15,NAGA:18)and healthy controls(HC:20)were enrolled into our study.The peripheral venous heparin anticoagulant blood was divided into 2ml/ tube and plasma was separated.The mononuclear cells were extracted and divided into MSU(100μg/ml)stimulated and non-stimulated groups.The mononuclear cells were incubated in a 37 cell ℃incubator containing 5%CO2 for 0h,12 h,24h,and the mononuclear cells were collected.The expression levels of ELOVL6,NLRP3,Caspase-1 and IL-1β m RNA in both the AGA、NAGA group and the HC group PBMCs were detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR);Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of IL-1β in the supernatant.In addition,the general clinical data and laboratory data were collected for single-factor analysis,Pearson correlation analysis.(2)The anti-inflammatory effect of sodium urate crystal(MSU)on human peripheral blood mononuclear cell line THP-1 was analyzed.THP-1 cells were stimulated with 0,25,50,100 and 200μg/ml MSU crystals for 0h,12 h,and 24 h,respectively.The effect of MSU crystals at different concentrations and time on the expression of ELOVL6 and Nod like receptor protein3(NLRP3),cysteine-specificaspartate proteolytic enzyme(caspase-1),proinflammatory factor interleukin-1β(IL-1β)m RNA was investigated;Western Blot was used to test the expression of ELOVL6,NLRP3,Caspase-1protein in THP-1 cells stimulated by MSU.(3)SPSS Statistics17.0 statistical software was used for analysis and Graph Pad Prism7.0 software was used for drawing.Results:The results were as follows:(1)After MSU crystal stimulated PBMCs for 0h,12 h and 24 h,the expression level of ELOVL6 m RNA reached a peak in the urate stimulated 12 h NAGA group,which was higher than that in the stimulated 12 h AGA group(P<0.05),and the difference was statistically significant.(2)The expression level of ELOVL6 m RNA was higher in the NAGA group after 12 h and 24 h of stimulation than in the HC group after 12 h and 24 h of stimulation,and the expression level was higher in the AGA group and NAGA group after 12 h and 24 h of stimulation than in the non-stimulation group(P<0.05),the difference was statistically significant.(3)The expression level of NLRP3 m RNA in the AGA group and NAGA group stimulated by MSU was lower than that in the AGA group and NAGA group after 12 h and 24 h,while the expression level in the AGA group and NAGA group without stimulation was higher than that in the HC group after 24 h,and the difference was statistically significant(P<0.05).(4)The content of IL-1β in the supernatant of PBMCs cells reached its peak at 24 h after stimulation in AGA group,which was significantly higher than that in 24 h group stimulated by NAGA(P<0.05),and the difference was significant.(5)The expression levels of ELOVL6 m RNA,IL-1βm RNA and IL-1β,IL-18 in the supernatant of THP-1 stimulated by 200μg/ml MSU crystal for 24 h were significantly increased,NLRP3 and Caspase-1 m RNA were stimulated by 200μg/ml MSU crystal for 12h(P<0.05),and the difference was statistically significant.(6)The expression level of THP-1 protein increased significantly at the concentration of 200μg/ml for 12 h,and reached the peak at 24 h after stimulation with 200μg/ml(P<0.05),and the expression level of ELOVL6 reached a peak at 24 h after stimulation with 200μg/ml(P <0.05).Conclusion:(1)In this study,it was found that after overexpression of ELOVL6 in PBMC of gout patients and thp-1 cells stimulated by uric acid,NLRP3 gene and protein level decreased and caspase-1 gene and protein level increased,which confirmed that the expression of ELOVL6 was increased in high uric acid environment,NLRP3 was expressed negatively.(2)Through this study,we found that ELOVL6 can be recognized by MSU crystals,and its expression varies with the change of MSU stimulation.Moreover,after the concentration and time dependent MSU crystals activate the NLRP3 inflammatory body,ELOVL6 may negatively regulate the NLRP3 inflammatory signal pathway to participate in the inflammatory process of gout,and is more likely to play a role in non-acute gout.