Vitro Study Of Ginkgo Biloba Extract (gbe50) Prevention Of Endothelial Dysfunction

PARTⅠProtective effects of GBE50 and flavonoids on hypoxia induced ROS production and apoptosis of HUVECsObjective To investigate the protective effect of GBE50 and flavonoids on hypoixa induced ROS production and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods HUVECs were obtained by digestion of human umbilical veins and cultured conventionally. It was divided into 4 groups. The N group was normal control; the H group was exposed to hypoxia for 24h; the HG group was administrated with GBE50 (12．5, 25, 50μg/ml respectively) for 4h before being exposed to hypoxia for 24 h; the HF group was administrated with flavonoids (12．5, 25, 50μg/ml respectively) for 4h before being exposed to hypoxia for 24h. ROS levels and apoptosis rate (by Annexin V and TUNEL) was measured in all groups.Results 1．A statistically significant difference (P<0．05) regarding the ROS level was found between the H group (29．27±2．21) % and the N group (9．88±0．97) %. ROS level of the HG group decreased compared to that of the H group, but no statistically significant difference (P>0．05) was found in the N group and the H group. The ROS level of all the HF groups were significantly (P<0．05) lowered compared to that of the H group whose ROS level reached the lowest level (7．42±1．81) % at a flavonoids concentration of 50μg/ml. 2．The positive rate of AnnexinV of the H group (18．47±2．53) % increased significantly (P＜0．05) compared to that of the N group (9．00±1．00) %, and it was significantly (P＜0．05) decreased in the HG group when the concentration of GBE50 was 25μg/ml (10．69±2．08) %. The positive rate of Annexin V of HF group decreased at all concentrations, but no significant difference was found between the HF group and the H group. The protective function of GBE was most prominent at a flavonoids concentration of 50μg/ml. Under this condition, the positive rate of Annexin V is (14．25±2．22) %. 3．The positive rate of TUNEL of the H group (8．59±1．10) % increased significantly (P＜0．05) compared to that of N group (1．28±0．65) %; it decreased significantly (P＜0．05) compared to H group on different concentration of GBE50 (3．58±0．28) % or flavonoids (3．06±0．40) % (25μg/ml and 50μg/ml respectively).Conclusion The results clearly show that hypoxia can induce HUVECs' apoptosis with a higher level of ROS, while GBE50 and flavonoids can attenuate or significantly recover endothelial dysfunction induced by hypoxia. Therefore, GBE50 and flavonoids may be beneficial agents in protecting against endothelial dysfunction induced by hypoxia.PARTⅡPreliminary research on the protective mechanisms of GBE50 on hypoxia-induced endothelial dysfunctionObjective To investigate the molecular mechanisms of GBE50 protective effects against endothelial dysfunction induced by hypoxia.Methods HUVECs were obtained by digestion of human umbilical veins and cultured conventionally. It was divided into 4 groups. The N group was normal control; the NG group was administrated with GBE50(25μg/ml) for 4h; the H group was exposed to hypoxia for 24h; the HG group was administrated with GBE50(25μg/ml) for 4h, then exposed to hypoxia for 24h. RT-PCR and Western-Blot were applied to detect the changes of expression of vWF, ET-1, PPARγand eNOS in all groups.Results 1．There was a statistically significant difference(P<0．05) of the mRNA expression of vWF between N group(0．55±0．05) % and H group(0．84±0．14) %; the mRNA expression of vWF of HG group(0．58±0．24) % decreased significantly (P<0．05) compared to that of H group. 2．There was a statistically significant difference(P<0．05) of the mRNA expression of ET-1 between N group(0．14±0．03) % and H group(0．31+0．03) %; the mRNA expression of ET-1 of HG group(0．26±0．02) % decreased significantly (P<0．05) compared to that of H group. 3．There was a statistically significant difference(P<0．05) of the protein expression of eNOS between N group(0．30±0．04) % and H group(0．59±0．08) %; the protein expression of eNOS of HG group(0．43±0．08) % was partially decreased (P>0．05) compared to that of H group. 4．The mRNA expressions of PPARγin above groups were as follows: (0．40±0．02) % in N group, (0．42±0．01) % in H group, (0．42±0．01) % in HG group. There was no significant difference among the above groups. 5．The protein expressions of PPARγin the above groups were as follows: (0．10±0．02) % in N group, (0．13±0．05) % in H group, (0．13±0．04) % in HG group. There was no significant difference among above groups.Conclusion Hypoxia increased the expression of vWF, ET-1, and eNOS. However, mRNA expressions of vWF and ET-1 were reduced markedly with a partially decreased eNOS after the HUVECs was pretreated with GBE50 25μg/ml for 4 hours before hypoxia. GBE50 could attenuate endothelial dysfunction induced by hypoxia. But neither the mRNA expression nor the protein expression of PPARγwere changed in all groups. The results indicate that PPARγmay not be involved in the regulation of HUVECs' function on hypoxia or treatment of GBE 50．PARTⅢPreliminary research on anti-apoptosis function of GBE50 on hypoxia-induced endothelial dysfunctionObjective To explore the anti-apoptosis function of GBE50 on endothelialdysfunction induced by hypoxia.Methods Apoptotic gene expression profiles of HUVECs were detected before andafter hypoxia or GBE50 treatment using Human Cell apoptotic cDNA array. Severaldifferentially expressed genes were assessed by RT-PCR.Results 1．Compared with the normal control, HUVECs showed a significantup-regulation of 15 genes after hypoxia treatment; while treatment of GBE50 could significantly up-regulate the expression of 38 genes and down-regulate 69 genes. 2．Some of these genes were selected to be further assessed by RT-PCR. In comparison with the normal control, the expression of ATM and TNFSF10 were boosted by hypoxia: (0．47±0．09) % in H group vs. (0．40±0．01) % in N group; and (0．20±0．13) % in H group vs. (0．05±0．01) % in N group respectively, P<0．05．The down-regulation of these two genes were obvious after treatment of GBE50 (25μg/ml, 4h) before hypoxia, the expression of ATM in HG group was (0．23±0．11) %, and that of TNFSF10 in HG group was (0．01±0．01) %, P<0．05．Conclusion The changes in expression of ATM and TNFSF10 is closely related to hypoxia and GBE50 treatment. These two genes may play an important role in endothelial cell apoptosis induced by hypoxia and anti-apoptosis of GBE50．...