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目的探讨二十碳五烯酸(eicosapentaenoic acid,EPA)对脂多糖(lipopolysaccharide,LPS)诱导的人单个核细胞核转录因子-κB(nuclear factor-kappa B,NF-κB)活性、VEGF、IL-1α和IL-6分泌的影响。方法分离人外周血单个核细胞,将其分为空白对照组、LPS组、0.030g.L-1EPA处理组、0.050g.L-1EPA处理组。LPS组仅加入LPS进行培养(LPS终浓度为10mg.L-1),EPA处理组先加入2种浓度的EPA(EPA终浓度分别为0.030g.L-1和0.050g.L-1)培养1h,再加入LPS进行培养。LPS刺激6h、12h、24h后,收集各组上清液,ELISA检测上清液中VEGF、IL-1α和IL-6的分泌量;LPS刺激24h后的沉淀细胞用Western blot法检测人单个核细胞NF-κB活性。结果 LPS刺激6h后,空白对照组、LPS组、0.030g.L-1EPA处理组、0.050g.L-1EPA处理组VEGF含量(ng.L-1)分别为:22.57±8.86、66.49±4.21、46.18±2.35、45.49±6.61;12h后分别为:18.05±3.18、107.30±5.70、61.29±2.86、54.34±7.41;24h后分别为:20.49±5.92、157.63±5.95、59.54±4.20、53.13±11.42。LPS刺激6h后IL-1α含量(ng.L-1)分别为:15.63±2.98、75.41±4.12、53.60±4.71、31.03±8.49;12h后分别为:40.37±4.51、408.00±47.93、142.80±14.65、99.17±5.86;24h后分别为:63.37±1.99、929.73±27.97、322.03±101.80、161.23±14.59。LPS刺激6h后IL-6含量(ng.L-1)分别为:34.94±2.71、117.60±7.89、82.25±14.56、60.66±2.12;12h后分别为:51.00±6.65、183.60±8.64、127.37±11.48、71.61±8.16;24h后分别为:68.04±21.53、297.50±25.72、132.37±20.87、102.45±21.46。与空白对照组相比,LPS刺激后人单个核细胞NF-κB p65表达和VEGF、IL-1α、IL-6分泌明显升高。EPA抑制LPS诱导的人单个核细胞NF-κB p65活性;下调其VEGF、IL-1α和IL-6分泌,与LPS组比较差异均有统计学意义(均为P<0.05)。结论 LPS激活人单个核细胞NF-κB,促进其VEGF及细胞因子表达。EPA抑制LPS诱导的人单个核细胞NF-κB活性,下调其VEGF及细胞因子表达。这为EPA应用于各种新生血管性疾病和炎症性疾病的防治提供了理论依据。
Objective To investigate the effect of eicosapentaenoic acid (EPA) on the activity of nuclear factor-kappa B (NF-κB) induced by lipopolysaccharide (LPS) in human mononuclear cells And IL-6 secretion. Methods Human peripheral blood mononuclear cells were isolated and divided into blank control group, LPS group, 0.030g.L-1EPA treatment group and 0.050g.L-1EPA treatment group. LPS group was cultured with only LPS (final concentration of LPS was 10 mg.L-1). EPA group was added with two concentrations of EPA (final concentration of EPA: 0.030gL-1 and 0.050gL-1) LPS training. The supernatant of each group was collected after LPS stimulation for 6h, 12h and 24h. The secretion of VEGF, IL-1alpha and IL-6 in the supernatant was detected by ELISA. The precipitated cells after stimulation with LPS for 24h were detected by Western blot Cell NF-κB activity. Results After 6h stimulation with LPS, the levels of VEGF (ng.L-1) in the blank control group, LPS group, 0.030gL-1EPA treatment group and 0.050gL-1EPA treatment group were 22.57 ± 8.86,66.49 ± 4.21,46.18 ± 2.35, 45.49 ± 6.61; after 12h, they were: 18.05 ± 3.18,107.30 ± 5.70,61.29 ± 2.86,54.34 ± 7.41 respectively; after 24h were 20.49 ± 5.92,157.63 ± 5.95,59.54 ± 4.20,53.13 ± 11.42. The levels of IL-1α (ng.L-1) after LPS stimulation for 6h were: 15.63 ± 2.98, 75.41 ± 4.12, 53.60 ± 4.71, 31.03 ± 8.49, respectively; 40.37 ± 4.51,408.00 ± 47.93,142.80 ± 14.65 , 99.17 ± 5.86; respectively, after 24h: 63.37 ± 1.99,929.73 ± 27.97,322.03 ± 101.80,161.23 ± 14.59. The levels of IL-6 (ng.L-1) after LPS stimulation for 6h were: 34.94 ± 2.71,117.60 ± 7.89,82.25 ± 14.56,60.66 ± 2.12, respectively; 51.00 ± 6.65,183.60 ± 8.64,127.37 ± 11.48 , 71.61 ± 8.16; respectively, after 24 hours: 68.04 ± 21.53,297.50 ± 25.72,132.37 ± 20.87,102.45 ± 21.46. Compared with the blank control group, the expression of NF-κB p65 and the secretion of VEGF, IL-1α and IL-6 in LPS-stimulated human mononuclear cells were significantly increased. EPA inhibited the LPS-induced NF-κB p65 activity in human mononuclear cells; down-regulated the secretion of VEGF, IL-1α and IL-6, which was significantly different from LPS group (all P <0.05). Conclusion LPS can activate NF-κB in human mononuclear cells and promote the expression of VEGF and cytokines. EPA inhibits LPS-induced NF-κB activity in human mononuclear cells and down-regulates its expression of VEGF and cytokines. This provides a theoretical basis for the EPA to be used in the prevention and treatment of various neovascular diseases and inflammatory diseases.