| According to recently research,the Delta-Notch signaling pathways,especially the main component,Delta-like ligand 4(Dll4),is considered to be another important signaling pathway participating in angiogenesis.The interaction between VEGF signaling and Notch signaling pathways involves in many aspects.Researches of tumor vessels showed that the combined inhibition of VEGF and Notch signaling pathways could inhibit tumor growth.Notch signaling pathway is expected to be the new drug target for anti-neovascularization,it provide us a new direction or method for the treatment of diabetic retinopathy.We have achieved a huge progress in the field of prevention and treatment of complications related to diabetes with traditional Chinese medicine,either the monomer or compound effective component of traditional Chinese medicine.The experimental and clinical studies have revealed that traditional Chinese medicine has notable advantages.One of the traditional Chinese medicines,Qidengmingmu capsule,is made from three TCM extractions,which are puerarin,astragaloside,scutellarein.The preliminary pharmacodynamics of this capsule has demonstrated that Qidengmingmu capsule can impair the retinal microcapillary damage in diabetic rats,it can also improve the ocular electrical physiological functions,et al.According to the literatures of the researches of traditional Chinese medicine,the main purpose of those study were focused on oxidative stress reaction and free radicals,inflammation cytokines,glycosylation end products.As the diversity of the effective components of traditional Chinese medicine and the complexity of pathogenesis of DR,the drug targets for most of the Chinese traditional medicine extract is not yet entirely clear.This study is based on the Dll4/Notch-VEGF signaling pathways and is designed to find the potential drug targets of the effective components of Qidengmingmu capsule.The research results would help to optimize the drug design,improve the clinical effect,meanwhile,the results would be the guidelines for the other ocular diseases and the systemic vascular diseases,and expand the application of traditional Chinese medicine.First Part Cell modeling of human umbilical vein endothelial cells under high-glucose in vitroObjective: Due to the easier manipulation and the high repetition of cell models,the human umbilical vein endothelial cells under high-glucose stimulation were chose for diabetic retinopathy research.However,there have been few reports on how to select and establish endothelial cell models in high-glucose environment.How to establish the cell model that can simulate the optimal microenvironment of diabetic retinopathy is the purpose of this part of the experiment.Methods: Cell viability was determined by MTT method to check cell proliferation,at same time,cell metabolism was measured by the real-time glucose levels,which indirectly reflects the cell metabolic activity in high glucose cultivation environment.The optimal glucose concentration for cell model was chose based on the two results above.Results: Compared with normal control group,the cells activity cultured with 30mmol/L and 40mmol/L glucose began to decline from 24 h;the glucose concentration in the cell medium was decreased with time.The reduction of the glucose concentration in cells cultured with 30mmol/L and 40mmol/L glucose were significant different at 48 h and 72 h.However,40mmol/L high-glucose-stressed cells were suffered more damage.So,cells under 30mmol/L glucose stimulation can be chose as the DR cell model in vitro.Meanwhile,in order to eliminate the toxic effect of the metabolites on the cells,48 h is the best observation point for the drug intervention.Second Part The effect of the main components of Qidengmingmu capsule on cell viability and cell migrationObjective: Cell viability would decrease under high-glucose stimulation.Effective drugs can protect the cells from damagin or apoptosis,and reduce the incidence of neovascularization.Meanwhile,the endothelial cell’s migration ability would change under the environment with high glucose.Due to the migration of endothelial cells is the key step of angiogenesis,we evaluate the cell migration for highlighting the drug’s effect on anti-angiogenesis.Methods: We took two parallel intervention approaches on cells under high-glucose(HG)stimulation,namely the Notch signaling pathway inhibitor(gamma secretase inhibitors,DAPT)and the main components of Qidengmingmu capsule.The cell viability was observed for evaluating the protection on cells.And also,Transwell assay was taken for cell migration evaluation.Results: DAPT and the three effective components of Qidengmingmu capsule in certain concentration can protect HG-stressed cells and eliminate the damage due to toxic of hyperglycemia.Meanwhile,the migration of endothelial cells increased in high-glucose environment,but the three active components of Qidengmingmu capsule and DAPT were able to partially inhibit the migration of endothelial cells induced by high glucose.Third Part Effect of Qidengmingmu capsule on Dll4 / Notch –VEGF signaling pathway under HG stimulationObjective: The recent researches of Notch signaling pathway on neovascularization in DR is not enough to elucidate the mechanism of angiogenesis.Tip cells sprouting is the key step for angiogenesis,and Notch signaling pathway plays an important role on this step and crosstalk with VEGF signaling pathway.Thus inhibiting the Notch signaling pathway helps to depress neovascularization.The results in second parts have revealed that the Qidengmingmu capsule can depress the cell migration,but the mechanism is not clear yet.Whether the mechanism is related with Notch signaling need further research.So we use the effective components of Qidengmingmu capsule to co-culture with HG-stress cells,and analyze the main molecules on Dll4/Notch-VEGF signaling pathway,which are Notch1 and Notch4,VEGF and VEGFR2,VEGFR1,Ephrin B4,and Eph B4,and discuss the effect of Qidengmingmu capsule on anti-angiogenesis.Methods: Normal cell group,high-glucose(HG)group,DAPT+HG group,puerarin + HG group,astragaloside+ HG group,and scutellarein+HG group were set up in this part.Real-time PCR was taken to measure the expression of Dll4,Notch1 and Notch4,VEGF and VEGFR2,VEGFR1,Ephrin B4,Eph B4,and western blot was taken for validation the proteins of Dll4,Notch1,Notch4,and VEGF.Results: Compared with normal cells,the expression of Dll4,Notch1,VEGF,VEGFR1,VEGFR2,Eph B4,and Ephrin B2 were increased significantly,and Notch4 was decreased in HG group.With DAPT intervention,the expression of Dll4,Notch1,VEGF and VEGFR2 in cells was increased,and the expression of Notch4 was up regulated.With puerarin intervention,the expression of Dll4,Notch1 and VEGFR2 in cells decreased,and VEGF and Notch4 showed no significant change.With astragaloside intervention,the xpression of Dll4,Notch1 and VEGF in cells decreased,and the expression of Notch4 was increased,and VEGFR2 did not change significantly.With scutellarein intervention,the expression of VEGF and VEGFR2 were decreased.VEGFR1,Eph B4 and Ephrin B2 did not change significantly in each intervention group.Conclusion: 1.The optimal glucose concentration for the culture of human umbilical vein endothelial cells as the cell model of diabetic retinopathy in vitro was 30mmol/L;2.The three active ingredients of Qidengmingmu capsule can protect the cell viability under HG-stimulation;it can alos partially restrain the migration of endothelial cells induced by HG;3.The three active ingredients of Qidengmingmu capsule had certain effect on Dll4 / Notch-VEGF signaling pathway,but puerarin,astragaloside IV and scutellarein might have different drug targets.The puerarin might works mainly on Notch signaling pathway,and astragaloside IV might have targets on both Notch and VEGF signaling pathway,the effect of scutellarein might aims at VEGF signaling pathway. |
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