[Role of ORAI1 in the balloon injury-induced rat carotid artery neointimal formation and the relationship between ORAI1 and sodium and calcium exchange 1].

Objective: To investigate the role of ORAI1 in the balloon injury-induced rat carotid artery neointimal formation and the relationship between ORAI1 and sodium and calcium exchanger(NCX)1. Methods: According to condition of carotid artery balloon injury and observation time, Sprague Dawley rats were divided into 3 groups including sham group, 7 days injury group, and 14 day injury group(n=3 each). According to virus of transfection, Sprague Dawley rats were divided into 2 groups including negative control group (negative lentiviruses particles transfection after establishment of carotid artery balloon injury model, n=3) and siORAI1 group(siORAI1 transfection after operation, n=3). Carotid artery neointimal formation was analyzed after HE staining. The ORAI mRNA expression level was detected using real-time PCR. The proliferating cell nuclear antigen (PCNA), ORAI and NCX1 protein expression level were measured by Western blot. Results: (1) Carotid artery intima was significantly thicker in 7 days and 14 days injury group than in sham group((0.54±0.11)µm2 and (0.89±0.12)µm2 vs.(0.11±0.08)µm2, both P<0.05). Relative expression level of PCNA protein was significantly higher in 7 days and 14 days injury group than in sham group(1.43±0.16 and 1.95±0.16 vs. 1, both P<0.05). Relative mRNA expression level of ORAI1 (1.39±0.14 and 1.78±0.21 vs. 0.56±0.09, both P<0.05) and protein expression level (1.42±0.19 and 1.78±0.22 vs. 1, both P<0.05) were significantly higher in 7 days and 14 days injury group than in sham group.(2) After 14 days, relative expression level of ORAI1 protein was significantly lower in siORAI1 group than in negative control group (0.21±0.161, P<0.05). Carotid artery intima thickness was significantly reduced in siORAI1 group compared to negative control group((0.19±0.14)µm2 vs.(0.91±0.23)µm2,P<0.05). Relative expression level of NCX1 protein was also significantly lower in siORAI1 group than in negative control group(0.53±0.131, P<0.05). Conclusions: ORAI1 may play a key role in the balloon injury-induced neointimal formation and vascular smooth muscle cells proliferation in rat carotid artery.ORAI1 knockdown could down regulate the NCX1 expression and attenuate the balloon injury-induced neointimal formation and vascular smooth muscle cells proliferation in rat carotid artery.

[Effects and related mechanism of 5-aza-2'-deoxycytidine on endothelial function in rats with hyperhomocysteinemia].

OBJECTIVE: To investigate the effects and related mechanism of 5-aza-2'-deoxycytidine(Aza) on endothelial function in Hyperhomocysteinemia rats. METHODS: Adult male SD rats were divided into 3 groups (n=7 each): control group, hyperhomocysteinemia (HHcy) group and Aza group according to the random number table. Control group rats were fed with normal diet. HHcy group rats were fed with diet adding 3% L-methionine. Aza group rats were fed with diet adding 3% L-methionine and Aza (0.5 mg/kg) injection for consecutive three days per week for 8 weeks. After 8 weeks, content of rat plasma homocysteine (Hcy) was detected by enzyme linked immunosorbent assay (ELISA). The rat mesenteric artery endothelium-dependent diastolic function was detected. The nitric oxide synthase (eNOS) activity and asymmetric dimethyl fine ammonia acid (ADMA) content were detected by ELISA, and the content of nitric oxide was detected by nitrate reductase method in the mesenteric arteries. The mRNA expression of DNA methyl transferase 1 (DNMT1) and dimethyl arginine acid dimethylamine hydrolase 2 (DDAH2) in the mesenteric arteries were detected by real-time fluorescence quantitative PCR, and the protein expressions of DNMT1 and DDAH2 in the mesenteric arteries were detected by Western blot. The DDAH2 promoter methylation level in the mesenteric arteries was detected by nested methylation specific PCR. RESULTS: (1) The content of plasma Hcy was significantly higher in the HHcy group and Aza group compared to the control group ((29.00±0.94) µmol/L and (26.43±0.47) µmol/L vs.(10.34±0.63) µmol/L, both P<0.01), which was significantly reduced in the Aza group compared with the HHcy group (P<0.05). (2) Acetylcholine-mediated relaxation at various concentrations was significantly lower in the HHcy group and the Aza group compared with the control group (both P<0.05), which was significantly increased in Aza group compared with HHcy group (P<0.05). SNP-mediated relaxation at various concentrations was similar among the three groups(all P>0.05). (3) Compared with the control group, the content of nitric oxide in the HHcy group was significantly decreased ((0.52±0.01) µmol/g vs.(0.42±0.00) µmol/g, P<0.01), which could be increased by Aza((0.49±0.01) µmol/g, P<0.05); the eNOS activity in the HHcy group was significantly decreased ((0.74±0.01) U/mg vs. (0.57±0.00) U/mg, P<0.01), which could be significantly increased by Aza ((0.65±0.01) U/mg, P<0.01); the content of ADMA in the HHcy group was significantly increased ((0.34±0.01) µmol/g vs. (0.37±0.00) µmol/g, P<0.05), which could be significantly decreased by Aza ((0.32±0.01) µmol/g, P<0.05). (4) Compared with the control group, the relative expression of DDHA2 mRNA in the HHcy group was significantly decreased (0.15±0.01 vs.0.12±0.01, P<0.01), which could be significantly increased by Aza (0.13±0.01, P<0.05); the relative expression of DDHA2 protein in the HHcy group was significantly decreased (0.31±0.02 vs. 0.24±0.01, P<0.01), which could be significantly increased by Aza (0.28±0.01, P<0.01). Compared with the control group, the relative expression of DNMT1 mRNA in the HHcy group was significantly increased (0.23±0.01 vs.0.43±0.01, P<0.01), which could be significantly decreased by Aza (0.39±0.01, P<0.05); the relative expression of DNMT1 protein in the HHcy group was significantly increased (0.35±0.01 vs. 0.50±0.01, P<0.01), which could be significantly decreased by Aza (0.47±0.01, P<0.05). (5) Compared with the control group, the methylated/non methylated ratio of DDHA2 promoter in the HHcy group was significantly increased (1.04±0.03 vs. 1.26±0.03, P<0.01), which could be significantly decreased by Aza (0.80±0.03, P<0.01). CONCLUSION: Aza can inhibit the activity of DNMT1, reduce DDAH2 promoter methylation level, increase the expression of DDAH2, decrease the content of ADMA, increase eNOS activity and content of nitric oxide, thus lead to the improvement of endothelial dysfunction in mesenteric artery of Hyperhomocysteinemia rats.

The serum level of C-reactive protein in patients undergoing GnRH agonist protocols for in vitro fertilization cycle.

BACKGROUND: The synchronization of the uterus and mature eggs at the molecular level is the key factor in embryo transfer, and the regulation of synchronization depends on a variety of cytokines. C-reactive protein (CRP), as the first acute phase reaction protein, is involved in the entire process of embryo transfer. The study is designed to investigate the correlation among CRP, sex hormone, controlled ovarian hyperstimulation (COH) cycle, and pregnancy outcome. MATERIALS AND METHODS: Ninety-two patients who accepted in vitro fertilization (IVF) treatment cycles because of tubal factor were included in the study. Seventy treated cases were included to complete final analysis with the full set of results. Respectively on the second day of the menstruation (Day-2) in gonadotropin-releasing hormone agonist (GnRH-a) short program treatment, on the morning in human chorionic gonadotropin (hCG) treatment (Day-hCG) and the embryo transplant day (Day-ET), plasma CRP level was tested by enzyme-linked immunosorbent assay (ELISA). The correlativity among CRP level, sex hormone, COH, and pregnancy outcome was analyzed by statistical methods. RESULTS: In the short program GnRH-a of 70 cases, there was no relationship between serum CRP level and the infertility age, gonadotropin (Gn) dosage, number of oocytes retrieved, the number of normal fertilization, and sex hormone. In the short program of GnRH-a, the change of serum CRP levels in Day-2, Day-hCG, Day-ET: serum CRP in Day-2 < Day-hCG < Day-ET and the level of serum CRP gradually increased in Day-2, Day-hCG, and Day-ET in both the pregnant group and non-pregnant group. In non-pregnant group, the ratio of hCG/D2 and ET/hCG-day were significantly higher than the pregnant group. The area under receiver operating characteristic (ROC) curve was 0.806, indicating the accuracy of diagnostic tests is medium, the authors chose the point which presents the ratio of CRP in Day-ET to Day-hCG which was less than 1.752 as a predictor of treatment outcome, the sensitivity of the experiment was 77.8%, and the specificity 75%. CONCLUSION: CRP as a sensitive inflammatory marker, CRP ratio of Day-ET/Day-hCG could be a predictor of treatment outcome by ROC curve analysis in COH program.