| Objective The goal of this study was to detect the genotype frequency and allele frequency of aryl hydrocarbon receptor (AHR) and its repressor AHRR whether or not confer susceptibility to rheumatoid arthritis (RA) in Han Chinese population, and to analyze association between the polymorphisms (rs2066853, rs2292596) and the main clinical parameters in RA; To explore the possible reasons of the effects about smoking> Leflunomide on RA in vivo and vitro through detecting the mRNA levels of aryl AHR, its response gene cytochrome P4501A1 (CYP1A1)/AHRR and pro-inflammatory cytokines IL-17/FOXP3+ in peripheral blood mononuclear cells (PBMC) of both patients and healthy controls.MethodsPart 1 A total of 201 patients were recruited from Anhui Provincial Hospital, Anhui Medical University.All the patients fulfilled the 1987 revised criteria of the American College of Rheumatology for the classification of RA. During the time,200 healthy controls from Physical Examination Center of the hospital were included, all of that were excluded from other autoimmune diseases. Clinical data were recorded in detail. All the blood samples of donors were stored in anti-coagulated tubes with EDTA. The single nucleotide polymorphisms (SNP) of AHR rs2066853 and AHRR rs2292596 were detected by polymerase chain reaction (PCR). Statistical analysis was performed with SPSS 17.0 software. The probability level as<0.05 was considered statistically significant.Part 2 Real-time PCR was used to assessed the mRNA levels of AHR, AHRR, CYP1A1, IL-17, FOXP3 from 62 patients with RA and 30 healthy subjects, of whose PBMC was stimulated by2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),cirgarette smoke condensate(CSC), Leflunomide for 24 hours, then effects of smoking, Leflunomide on the expression of AHR, AHRR and CYP1A1 were analyzed. The nonparametric Mann-Whitney U tests were used to compare the median between different groups. Spearman’s rank correlation coefficient was used to analyze the association. Statistical analysis was performed with SPSS 17.0 software. The probability level as <0.05 was considered statistically significant.Results(1) No deviations from HWE were observed in two polymorphisms. (For SNP rs2066853, controls, χ2=0.067,P=0.795; cases, χ2=2.539, P=0.111; For SNP rs2292596, controls,χ2=0.611, P=0.410; cases, χ2=1.852, P=0.174).(2) No statistically significant different about the allele and genotype frequencies of rs2066853 were detected between cases and controls (P>0.05). Also we did not find the association of clinical features with rs2066853 in RA patients (P>0.05).(3) The allele and genotype frequencies of rs2292596 were statistically significant different between two groups. Also,a statistically significantly increased risk of RA was found to be associated with the G allele of rs2292596, the odds ratio was (G versus C χ2=29.570,p=0.000,OR:2.176,95%CI:1.642-2.886). However, we failed to find any association between clinical data and the polymorphism.(4) The expression of AHR, AHRR and CYP1A1 at mRNA level were significantly higher in patients who were smoking than those who were non-smokers. However, there were no statistically significant differences between smoking healthy subjects and nonsmoking controls (P>0.05)(5)Positive correlation was detected betweenCYPlAl mRNA and IL-17, but the association was not found between AHR/AHRR mRNA and IL-17,further more, we did not find the correlation between FOXP3 expression and AHR/AHRR/CYP1A1 mRNA expression levels.(6) The relative expression levels of AHR, AHRR, CYP1A1 mRNA in PBMCs from RA patients were significantly decreased after LEF treated for 3 months.(7) The expression of AHR, AHRR and CYP1A1 at mRNA level were significantly increased in PBMC of patients with RA who were stimulated with CSC (1mg/ml,10mg /ml) and TCDD (10 umol/ml) in vitro when compared with not stimulated. The expressions were all significantly decreased after LEF stimulated.However, there were no statistically significant differences in healthy controls (P>0.05).Conclusions(1) This study demonstrated the polymorphisms of rs2292596 was significantly with genetic susceptibility to RA patients, furthermore, it suggested the G allele of rs2292596 might be associated with a dangerous effect on RA in Han Chinese populations.(2) Smoking is involved in the pathogenesis of RA and then becomes a risk factor, of what may due to the activation of AHR and the differentiation of Th17 cells.(3) There may be some defects in AHR of PBMC in patients with RA, what make AHR activated and highly expressed with its liands. Or AHR is not activated for some inhibited pathways in PBMC of healthy cases.(4)AHR probably was activated in a dose-dependent fashion.(5)Different ligands of AHR may have divergent effects on RA. |
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