目的:观察葛根黄酮(FGVL)对过氧化氢诱导的人脐静脉内皮细胞(HUVEC)氧化损伤的影响。并探讨相关机制。
方法:HUVEC分为5组:对照组(1640完全培养基),损伤组(HUVEC用100μmol/L过氧化氢处理4h),FGVL组(HUVEC用12.5mg/LFGVL治疗(F1组),25.0mg/L(F2组),50.0mg/L(F3组),在过氧化氢之前24小时)。用硝酸还原酶法测定一氧化氮含量。6-酮-前列环素-F1α(6-酮-PGF1α),血栓烷B2(TXB2),ELISA法测定白细胞介素(IL)-6和IL-22。磷脂酰肌醇3-激酶(PI3K)mRNA表达,RT-PCR检测蛋白激酶B(Akt)和内皮型一氧化氮合酶(eNOS)。通过蛋白质印迹测定p-Akt(ser(473))和p-eNOS(ser(1177))的蛋白表达。Hoechst33258染色后用荧光显微镜观察细胞凋亡。
结果:(1)损伤组一氧化氮含量明显低于对照组((34.11±1.78)μmol/Lvs.(74.81±2.93)μmol/L,P<0.05),与损伤组相比,F2组((41.86±2.32)μmol/L)和F3组((62.79±1.16)μmol/L)明显升高(均P<0.05)。(2)损伤组的6-酮-PGF1α分泌水平((44.84±3.87)ng/L)明显低于对照组((82.38±3.98)ng/L,P<0.05),F1组显著升高((52.76±1.78)ng/L),FGVL2组为(56.58±1.44)ng/L,FGVL3组为(67.78±2.02)ng/L,均高于损伤组(均P<0.05)。损伤组TXB2的分泌水平((43.37±3.96)ng/L)明显高于对照组((25.56±1.75)ng/L,P<0.05),F2组((32.41±1.68)ng/L)和F3组(28.23±2.15)ng/L较损伤组显著降低(均P<0.05)。(3)损伤组IL-6和IL-22含量显著高于(539.74±11.63)ng/L和(23.70±3.05)ng/L,分别)比对照组((288.67±19.52)ng/L)和((23.70±3.05)ng/L,分别,均P<0.05)。F1、F2、F3组IL-6含量均显著低于损伤组(均P<0.05)。F2、F3组IL-22含量显著低于损伤组(P均<0.05)。(4)损伤组PI3KmRNA和eNOSmRNA的相对水平(分别为0.68±0.09和0.22±0.03)明显低于对照组(分别为0.81±0.12和0.63±0.11,两者P<0.05),F2组PI3KmRNA(0.76±0.03),F3组PI3KmRNA(0.83±0.06),F1组eNOSmRNA(0.37±0.08),F2(0.53±0.04)和F3(0.56±0.09)高于损伤组(均P<0.05)。各组AktmRNA表达水平相似(P>0.05)。(5)损伤组p-Akt(ser(473))和p-eNOS(ser(1177))的相对水平(分别为0.48±0.05和0.23±0.03)明显低于对照组(分别为0.71±0.12和0.66±0.05,两者P<0.05),与损伤组相比,F1,F2和F3组上调(均P<0.05)。(6)损伤组细胞凋亡率明显高于对照组((63.67±11.37)%vs.(4.67±1.15)%,P<0.05),F1组显著降低((43.33±4.16)%),F2((18.33±4.93)%)和F3(15.67±2.08)%(均P<0.05).
结论:FGVL可通过PI3K/Akt/eNOS途径增加一氧化氮水平,从而减轻过氧化氢诱导的HUVEC氧化损伤。
OBJECTIVE: To investigate the effects of flavone from
Galium verum L (FGVL) on hydrogen peroxide induced oxidative injury in human umbilical vein endothelial cells (HUVEC), and explore related mechanisms.
METHODS: HUVEC were divided into five groups: control group (1640 complete medium), injured group (HUVEC treated with 100 μmol/L hydrogen peroxide for 4 h), FGVL group (HUVEC treated with 12.5 mg/L FGVL (group F1), 25.0 mg/L (group F2), 50.0 mg/L (group F3) for 24 h before hydrogen peroxide). The nitric oxide content was measured by nitric acid reductase method. The 6-keto-Prostacyclin-F1α (6-keto-PGF1α), thromboxane B2 (TXB2), interleukin(IL)-6 and IL-22 were determined by ELISA.mRNA expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) was detected by RT-PCR.Protein expression of p-Akt (ser(473)) and p-eNOS (ser(1177)) was determined by Western blot.Cell apoptosis was observed with fluorescence microscope after Hoechst33258 staining.
RESULTS: (1) The contents of nitric oxide were significantly lower in the injured group than in the control group ((34.11±1.78) μmol/L vs. (74.81±2.93) μmol/L, P<0.05), which was significantly increased in group F2 ((41.86±2.32) μmol/L) and group F3 ((62.79±1.16) μmol/L) compared with injured group (both P<0.05). (2)The secretion level of 6-keto-PGF1α was significantly lower in the injured group ((44.84±3.87) ng/L) than in the control group ((82.38±3.98) ng/L, P<0.05), which was significantly increased in group F1 ((52.76±1.78) ng/L), FGVL 2 group which was(56.58±1.44) ng/L and FGVL 3 group which was(67.78±2.02) ng/L than that of injured group(all P<0.05). The secretion level of TXB2 was significantly higher in the injured group((43.37±3.96) ng/L) than in the control group ((25.56±1.75) ng/L, P<0.05), which was significantly reduced group F2 group ((32.41±1.68) ng/L) and group F3 ((28.23±2.15) ng/L) than that of injured group(both P<0.05). (3) The contents of IL-6 and IL-22 were significantly higher in the injured group ((539.74±11.63) ng/L) and ((23.70±3.05) ng/L, respectively) than in the control group ((288.67±19.52) ng/L) and ((23.70±3.05) ng/L, respectively, both P<0.05). The contents of IL-6 were significantly lower in group F1, F2 and F3 compared to that of injured group(all P<0.05). The contents of IL-22 were significantly lower in group F2 and F3 than that of injured group(both P<0.05). (4)The relative levels of PI3K mRNA and eNOS mRNA in injured group (0.68±0.09 and 0.22±0.03, respectively) were significantly lower compared to control group(0.81±0.12 and 0.63±0.11, respectively, both P<0.05), PI3K mRNA in group F2 (0.76±0.03) and group F3 (PI3K mRNA 0.83±0.06) as well as eNOS mRNA in group F1 (0.37±0.08), F2 (0.53±0.04) and F3 (0.56±0.09) than those of injured group(all P<0.05). The mRNA expression of Akt was similar among groups (P>0.05). (5) The relative levels of p-Akt (ser(473)) and p-eNOS (ser(1177)) in injured group (0.48±0.05 and 0.23±0.03, respectively) were significantly lower compared to control group (0.71±0.12 and 0.66±0.05, respectively, both P<0.05), which was up-regulated in group F1, F2 and F3 groups compared to injured group(all P<0.05). (6) The cell apoptosis rate in injured groups was significantly higher compared to control group which ((63.67±11.37)% vs. (4.67±1.15)%, P<0.05) which was significantly reduced in group F1((43.33±4.16)%), F2((18.33±4.93)%) and F3((15.67±2.08)%) compared to injured group(all P<0.05).
CONCLUSIONS: The FGVL can reduce hydrogen peroxide induced oxidative injury in HUVEC by increasing the level of nitric oxide through PI3K/Akt/eNOS pathway.