蓬子菜总黄酮对人脐静脉内皮细胞氧化损伤的保护作用及其机制

Effects and related mechanism of flavone from Galium verum L on peroxide induced oxidative injuryin human umbilical vein endothelial cells

目的探讨蓬子菜总黄酮（FGVL）对过氧化氢所致人脐静脉内皮细胞氧化损伤的作用及其机制。方法将人脐静脉内皮细胞分为5组，包括对照组（加入1640完全培养基）、损伤组（加入100ixmo]／L过氧化氢培养4h）、FGVL1组（12．5mg／LFGVL培养24h后，加入100Ixmo]／L过氧化氢培养4h）、FGVL2组（25．0mg／LFGVL培养24h后，加入100μmol／L过氧化氢培养4h）和FGVL3组（50．0mg／LFGVL培养24h后，加入100μmo]／L过氧化氢培养4h）。采用硝酸还原酶法检测细胞上清液中的一氧化氮含量，ELISA检测细胞上清液中的6-酮-前列腺素1a（6-keto—PGF1a）、血栓烷素B：（TXB2）、白细胞介素-6和白细胞介素-22的分泌水平，RT—PCR检测细胞中磷脂酰肌醇3-激酶（PBK）、蛋白激酶B（Akt）及内皮型一氧化氮合酶（eNOS）的mRNA表达水平，Westernbolt检测磷酸化Akt（ser473）和磷酸化eNOS（ser）的蛋白表达水平，并观察细胞凋亡情况。结果（1）损伤组细胞上清液中的一氧化氮含量为（34．11±1．78）μmo]／L，低于对照组的（74．81±2．93）μmo]／L（P〈0．05）；FGVL2组和FGVL3组细胞上清液中的一氧化氮含量分别为（41．86±2．32）μmol／L和（62．79±1．16）μmol／L，均高于损伤组（P均〈0．05）。（2）损伤组细胞上清液中的6-keto—PGF1a含量为（44．84±3．87）ng／L，低于对照组的（82．38±3．98）ng／L（P〈0．05）；FGVL1组、FGVL2组和FGVL3组细胞上清液中的6-keto—PGF1a．含量分别为（52．76±1．78）μg／L、（56．58±1．44）μg／L和（67．78±2．02）ng／L，均高于损伤组（P均〈0．05）。损伤组细胞上清液中的TXB，含量为（43．37±3．96）ng／L，高于对照组的（25．56±1．75）ng／L（P〈0．05）；FGVL2组和FGVL3组细胞上清液中的TXB2含量分别为（32．41±1．68）ng／L和（28．23±2．15）ng／L，均低于损伤组（P均〈0．05）。

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-Prostacyelin-F1a （6-keto-PGF1a ）, 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 （ ser473 ） and p-eNOS （ser1177） 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 〈 O. 05 ）, which was significantly increased in group F2 （ （41.86 ±2. 32） μmol/L） and group F3 （ （62.79 ± 1.16 ） Ixmol/L） compared with injured group （ both P 〈 0.05 ）. （ 2 ） The secretion level of 6-keto-PGF1a 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 〈 O. 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） rig/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 〈 O. 05 ）. The contents of IL-6 were significantly lower in group FI, F2 and 173 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 ± O. 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 〈 O. 05 ）. The mRNA expression of Akt was similar among groups （ P 〉 0. 05 ）. （ 5 ） The relative levels of p-Akt （ set473 ） and p-eNOS （ ser1177 ） in injured group （0. 48 ± 0. 05 and O. 23 ± 0.03, respectively） were significantly lower compared to control group （0.71 ± 0. 12 and 0.66 ± 0.05, respectively,both P〈O. 05）, which was up-regulated in group F1, F2 and F3 groups compared to injured group（all P 〈 O. 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 ）. Conclusion The FGVL can reduce hydrogen peroxide induced oxidative injury in HUVEC by increasing the level of nitric oxide through PI3K/Akt/eNOS pathway.