Effect of soluble uric acid on the gene expression of inflammatory markers and also the viability of human peripheral blood mononuclear cells.
,1,* Alireza bahiraee
,2 Solaleh emamgholipour
1. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2. Department of Medical Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
3. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Overproduction of uric acid as the final compound of purines catabolism, can play emerging roles in human diseases. here, we aimed to explore the effects of soluble uric acid induction on the activation of basic proliferative pathways using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt) assay and also the gene expression of inflammatory markers in human peripheral blood mononuclear cells (pbmcs).
The proliferation rate of pbmcs separated from 10 ml heparinized peripheral blood samples obtained from 4 healthy adult volunteers, were measured using mtt assay. pbmcs were seeded in rpmi 1640 supplement with 5% (v/v) fetal bovine serum and 100 iu/ml penicillin. uric acid was solved with the prewarmed media (37 °c), and then passed through sterile 0.20-μm filters. thus, the mtt assay was performed with various concentrations of soluble uric acid (0, 3, 5, 6, 10, 12, 18 and 20 mg/dl) on the pbmcs after 24 hours incubation. all results were stated as percentage alterations in absorbance compared with pbmcs incubated with plain culture medium. next, we treated pbmcs with different concentration of soluble uric acid (0, 5, 12 and 20 mg/dl) for 24 hours and total rna was isolated using geneall rna extraction kit. therefore, cdna was synthesized by the quantitect reverse transcription kit. to investigate the gene expression of interleukin (il)-6, il-1β and tnf-α as inflammatory markers, real-time pcr were performed.
This study showed that soluble uric acid (0-20 mg/dl) induced a concentration-dependent proliferative effect in pbmcs from healthy people after cells incubation for 24 hours which was performed by using mtt colorimetric assay (p < 0.001). moreover, to investigate the role of uric acid on inflammatory pathways, pbmcs were exposed to soluble uric acid at different concentrations or were left untreated for 24 hours. different concentration of soluble uric acid did not induce detectable expression of il-6, il-1β, and tnf-α mrnas even at the highest concentration (20 mg/dl) after 24 hours incubation.
The results showed that the soluble uric acid can rise the proliferation of pbmcs in a concentration-dependent manner which can be inversely associated with disease severity. although, different concentrations of soluble uric acid did not change the gene expression of inflammatory markers, which may suggest the possible contribution of other causes among hyperuricemia in activating inflammatory pathways.
Uric acid, mtt assay, pbmc, interleukin