Relationships between endothelial nitric oxide synthase gene polymorphisms and osteoporosis in postmenopausal women

Objective:To investigate the relationships between endothelial nitric oxide synthases(eNOS) G894T and 27 bpvariable number tandem repeat(VNTR) gene polymorphisms and osteoporosis in the postmenopausal women of Chinese Han nationality.Methods:In the present study,281 postmenopausal women from Xi'an urban area in West China were recruited,and divided into osteoporosis,osteopenia,and normal groups according to the diagnostic criteria of osteoporosis proposed by World Health Organization(WHO).The bone mineral density(BMD) values of lumbar vertebrae and left hips were determined by QDR-2000 dual energy X-ray absorptiometry.Blood samples were tested for plasma biochemical indicators including testosterone,estradiol,calcitonin,osteocalcin,and procollagen type I amino-terminal propeptide by enzyme-linked immunosorbent assay(ELISA),tartrate-resistant acid phosphatase by spectrophotometric method,and the content of nitric oxide by Griess method.Genome DNA was extracted from whole blood,and G894T polymorphism of eNOS gene was analyzed by using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method and 27 bp-VNTR polymorphism of eNOS gene was genotyped by PCR method.Then the relationships between genotypes and biochemical indicators,genotypes and osteoporosis,and haplotypes and osteoporosis were analyzed.Results:The average BMD values of the femoral neck,ward's triangle and lumbar vertebrae 1～4(L1～L4) in the subjects with T/T genotype in eNOS G894T locus were significantly higher than those in the subjects with G/T and G/G genotypes(P<0.05).The average BMD of the femoral neck in the subjects with a/a genotype of eNOS 27 bp-VNTR locus was evidently higher than that in the subjects with b/b genotype(P<0.05).The plasma testosterone and os-teocalcin concentrations in the subjects of eNOS G894T G/T genotype were evidently higher than those in the subjects of other genotypes(P<0.05);the plasma estradiol concentration in the subjects of eNOS 27 bp-VNTR a/a genotype was obviously higher than that in the subjects of b/b genotype(P<0.01).eNOS G/G homozygous frequencies in osteoporosis women,osteopenia women,and normal women were 85.37%,76.38%,and 83.87%,respectively(P>0.05).0% osteoporosis woman,0.79% osteopenia women,and 3.23% normal women were eNOS a/a homozygous(P<0.05).The frequencies of eNOS 27 bp-VNTR a allele were 5.33% in the osteoporosis group,10.24% in the osteopenia group,and 16.13% in the normal group(P<0.05,odds ratio(OR)=0.29,95% confidence interval(CI)=0.11～0.77),suggesting that a/a genotype and a allele might have protective effects on osteoporosis.The haplotype analysis showed that G-b was 87.7%(214/244) in the osteoporosis group(P<0.05,OR=2.48,95% CI=1.18～5.18).G-a was 5.3%(13/244) in the osteoporosis group(P<0.05,OR=0.29,95% CI=0.11～0.77).G-b was a risk factor for osteoporosis,and G-a a protective factor.Conclusion:eNOS G894T G/T genotype influenced the plasma testosterone and osteocalcin concentrations,and T/T genotype influenced BMD.eNOS 27 bp-VNTR a/a genotype increased plasma estradiol concentration to have a protective effect on osteoporosis.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions

Objective: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats.Methods: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane,and then treated with MTX or BSO.Arthritis severity was evaluated macroscopically and microscopically.Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR).Results: The clinical arthritis severity was decreased after MTX treatment,while the BSO groups did not show significant changes compared with the disease group.The plasma NO level of the MTX group was significantly decreased compared with the disease group,but the BSO groups showed no difference from the disease group in plasma NO levels.The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group,but only showed a declining trend in the BSO groups compared with the disease group.Neither MTX nor BSO had an effect on the mRNA expressions of IL-4,transforming growth factor β (TGF-β),and tumor necrosis factor-α (TNF-α) in the spleen.Conclusions: MTX,but not BSO,can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.