The Effects Of Diisopropylamine Dichloroacetate, Pioglitazone And Adrenomedullin On The Endothelial Cell Functions Of Human Umbilical Vein In Vitro

Objective l.This experiment aims at researching whether diisopropylamine dichloroacetate (DIPA), pioglitazone (PIO) and adrenomedullin (AM) have effects on human umbilical vein endothelial cells (HUVECs). 2. To evaluate the possible protective effects on HUVECs mechanisms of DIPA, PIO and AM involved.Methods The HUVECs were selected to be a model and the following aspects were studied: 1. Effects of the drugs on the endothelial dysfunction induced by high glucose: (1) Apoptosis peaks were assessed by flow cytometry. (2)The levels of NO in supernatant were measured with chemistry method by spectro-photometer. (3) The levels of sICAM-1 in culture medium were assayed by ELISA.2. The role of protein kinase C (PKC) in the endothelial dysfunction induced by high glucose and the effects of the drugs:(l) To investigate the role of PKC8 or PKCa in endothelial dysfunction in HUVECs induced by high glucose and the effects of drugs, the translocation of PKC § or PKCa in a single HUVECs after high glucose or the drugs exposure was observed by Laser-Scanning Confocal Microscope.(2) To investigate the role of PKC δ orPKCa in endothelial dysfunction in HUVECs induced by high glucose and the effects of the drugs, PKC8 and PKCa's expressions analysis was conducted quantificationally by western blotting. 3. The levels of pyruvate dehydrogenase (PDH) in HUVECs cultured with high glucose or DIPA were assayed with chemistry method by ELISA Reader. HUVECs were cultured and divided into 5 groups according to the drugs in culture media, each group had been cultured for 48h : (1) Matched control group :with no drugs;(2)High glucose group: 30mM glucose; (3) DIPA(10'5M, W4M, 10"3M) + high glucose group ;(4) PIO (10'9M> 10"7]VL 10"5M) +high glucose group; (5) AM (10-9M. 1(T8M, 10"7M) + high glucose group;Results l.High glucose could induce HUVECs apoptosis. The concentration of NO in culture supernatants of high glucose group was reduced significantly than that of control group. The level of sICAM-1 of high glucose group increased than that of control group.2. By Laser-Scanning Confocal Microscope after high glucose or the drugs exposure, it was observed that high glucose could induce the translocation of PKCa from plasm to nucleus in HUVECs. And high glucose could induce the translocation of PKC5 from nucleus to plasm and membran in HUVECs.3. The Western Blotting results showed that high glucose could increase the PKC8 expressions in HUVECs.While the expressions of PKCa in high glucose group was not different with that of the control.4.DIPA, PIO and AM could inhibit the increasing apoptosis peaks induced by high glucose. 5. DIPA, PIO and AM could reverse the concentration of NO in culture supernatants to normal, and could inhibit the increasing levels of sICAM-1 induced by high glucose.6. By Laser-Scanning Confocal Microscope, it was observed that DIPA, PIO and AM could inhibit the translocation of PKCa from plasm to nucleus in HUVECs induced by high glucose,they also could inhibitthe translocation of PKC8 from nucleus to plasm and membran in HUVECs.7. The Western Blotting results showed that DIPA, PIO and AM could significantly inhibit the increasing expressions of PKC8 in HUVECs induced by high glucose. The expressions of PKCa in the drug groups were not different from that of the high glucose group.8.DIPA could increase the activities of PDH in a concentration- dependent manner. The PDH levels of DIPA groups were higher than that of control group and high glucose group. Conclusion High glucose could induce HUVECs apoptosis. The concentration of NO in high glucose group was reduced and the level of sICAM-1 increased significantly. Endothelial cell dysfunctions induced by high glucose were associated with the activation of PKCa and PKC8. High glucose could induce the translocation and increase the expressions of PKC5 in HUVECs, and high glucose could induce the translocation of PKCa .DIPA, PIO and AM could correct the endothelial cell dysfunctions induced by high glucose.The inhibition of PKCa and PKC8 play some roles during the process.