Study On The Effect Of Zuoguijiangtangling On Vascular Endothelial Cells Injured By Glucose, Insulin And Low-Density Lipoprotein

OBJECTIVE: To establish an injured model of endothelial cell, which might be used to research diabetes vascular complications in vitro, well-growing subcultured human umbilical vein endothelial cell line ECV-304 was exposed to different concentration of glucose, insulin and oxidized low-density lipoprotein (ox-LDL); To investigate the effect of Zuoguijiangtangling (ZGJTL) on vascular endothelial cells injured by glucose, insulin and low-density lipoprotein.METHODS: There are two parts in this paper. Part 1,ECV-304 cells were randomized into 9 groups, and each group was exposed to different concentration lever of glucose, insulin and ox-LDL for 24 hours according to the principle of orthogonal design. The common morphology changes of the cells were observed by optics microscope and the hyper-microstructure changes of cells were surveyed by electron microscope. Cell viability was measured by MTT test, and gel electrophoresis was carried out for assaying DNA fragmentation. Through comprehensive analysis, we got the injured model of endothelial cell. Part 2, the cells having been incubated in the model conditioned fluid (MCF): 20 mmol/L glucose, 30 mU/L insulin and 200 mg/L ox-LDL were randomly divided into 5 groups: model group, lowdosage of ZGJTL group, middle dosage of ZGJTL group, high dosage ofZGJTL group, Gliclazide group, compared with a normal blank group. Thecells in each group were cultured for additional 48 hours. In the same way, cell viability, common morphology changes and hyper-microstructure of cells were examined. The supernatants were used to determine tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) contents. Several reagent kits were used to measure the contents of nitric oxide(NO), endothelin-l(ET-l) and intercellular adhesion molecule-1 (ICAM-1) respectively.RESULTS:(1)Compared with the other group, the group of cells incubated with a concentration of MCF:20 mmol/L glucose, 30 mU/L insulin and 200 mg/L ox-LDL were showed to pyknocyte, wide intercellular space and abnormal granule in cytoplasm, on the other hand, the few were showed to fibroblast-shape ECV-304 cells, and ZGJTL with a final concentration of 20mg/ml could obviously antagonize the changes of the cells in MCF group(P<0.05). High dose and low dose of ZGJTL could markedly decrease the apoptotic or necrotic changes induced by MCF(P <0.05). (2)The viability (measured by MTT test) of the cells in MCF decreased markedly <0.01 vs blank group). Middle dose and high dose of ZGJTL, Gliclazide could inhibit the effect (P <0.05), but the three groups didn't show any marked difference.(3)MCF could promote the secretion of PAI, ET-1 and ICAM-1(P <0.01, P <0.05, P <0.05 respectively), but decrease the release of NO(P <0.05). And tPA secreted by the group of cells in MCF didn't show any statistics difference compared with any other group(P >0.05). However, different dose of ZGJTL could obviously antagonize the changes of PAI(P <0.01), so did Gliclazide(P <0.01), whose function is inferior to that of the high, middle and low dose of ZGJTL(P <0.05, P <0.01, P <0.01respectively). The contents of NO in Gliclazide group, high dose and middle dose of ZGJTL are notably enhanced compared with that of the model group (all P <0.01), and there is marked difference among the three groups. Different dose of ZGJTL and Gliclazide could also inhibit the enhance of ET-1 in the model group. Moreover, the effects of middle dose and low dose of ZGJTL were notabler than Gliclazide (P <0.05), but the high dose of ZGJTL wasn't vs Gliclazide (P>0.05). Middle dose of ZGJTL and Gliclazide could also significantly inhibit the increase of ICAM-1 induced by MCF (P <0.01), with the function of Gliclazide inferior to that of middle doseofZGJTL(P<0.01).CONCLUSION: All the results being mentioned above suggested that MCF may play the greatest role in alleviating ECV-304 cells' injury induced by glucose, insulin and ox-LDL, it not only could induce cell apoptosis or necrosis , but also could facilitate blood coagulation, inhibit fibrinolysis a...