Buddleja Side The Prevention And Treatment Of Diabetic Retinopathy Vegf Signal Transduction Mechanisms

Objective:To investigate the inhibition effect of Mimeng Flower decoction on the proliferation of Human Umbilical Vascular Endothelial Cell(HUVEC) induced by chronic hypoxia and to study the effect of Mimeng Flower decoction to diabetic retinopathy of rat,and further to study their mechanism of signal transduction,in order to provide experimental basis for Mimeng Flower decoction used in clinic to preventing and curing diabetic retinophty(DR).Methods:Animal model study:Used diabetic rats induced by streptozocin(STZ) as animal model. Used calcium dobesilate as positive control medicine.Mimeng Flower decoction had three different concentration which were low dosage,median dosage and high dosage.The following studies were made when the animals were administered for 4 months.1.To observe the general status of diabetic rats induced by STZ,such as food,urinary produc-tion,weight,et al.2.To test the blood item of diabetic rats induced by STZ,such as blood glucose, glycosylated hemoglobin(GHb),the whole blood viscosity,plasma viscosity, Triglyceride(TG),Cholestrerol(CHO)and so on.3.Pathological changes of pancreases of diabetic rats were observed with hematoxylin and eosin(HE) stained method.4.Opacity degree of lens of diabetic rats were observed with slip lamp and photo analyzed system.5.Pathological changes of retinals in diabetic rats were observed with hematoxylin and eosin(HE) stained method.6.Through immunohistochemistry method to detect the protein expression of ICAM-1,E-selectin in retinal of diabetic rats induced by STZ.7.The pathological changes of retinal capillary vessels were observed in stretched preparation of retinal vessels digested stained with periodic acid-Schiff(PAS).8.Through RT-PCR method to detect the expression of VEGFmRNA,Flt-1mRNA,Flk-1/KDRmRNA in retinal of diabetic rats induced by STZ.9.Through Western blot method to detect the phosphorylation level of VEGF,Flt-1,Flk-1/KDR in retinal of diabetic rats induced by STZ. Cell culture in vitro study:HUVEC were cultured in vitro.Used 100μmol/L Cocl₂ to set up the cell chemistry hypoxia model,in the same time,10,20,40mg/ml Mimeng Flower decoction extract were added to the HUVEC separately,the following studies were made after 24h incubation.1.Through WST-8 method to check up the inhibition effect of Mimeng Flower decoction on the proliferation of HUVEC induced by chronic hypoxia.2.The changes of cell cycle and proliferating cell nuclear antigen(PCNA) expression of HUVEC regulated by Mimeng Flower decoction were measured with flow cytometry (FCM).3.Through RT-PCR method to detect the expression of VEGFmRNA,Flt-1mRNA,Flk-1/KDRmRNA in HUVEC regulated by Mimeng Flower decoction.4.Through Western blot method to detect the phosphorylation level of VEGF,Flt-1,Flk-1/KDR in HUVEC regulated by Mimeng Flower decoction.5.Through immunohistochemistry method to detect the protein expression of ICAM-1,E-selectin,vWF regulated by Mimeng Flower decoction.ResultsAnimal model study(after 4 months):1.General status:STZ induced diabetic rats had lasting and stable hyperglycaemia, displayingwith the obvious signs of "polydipsia,polyphagia,hyperdiuresis and loss of weight".Median dosage of Mimeng Flower decoction can increase the body weight of rats compared with DM model group(p＜0.05).2.Blood glucose:Compared with normal group,the blood glucose of the rats increased mark-edly in DM model group(p＜0.01).Compared with model group,the blood glucose of the rats reduced in median and high dosage group after taking 4 month's medicine.3.GHb:Compared with normal group,GHb of the rats increased markedly in DM model group(p＜0.01).Compared with model group,GHb of the rats reduced in median and high dosage group(p＜0.01).4.Blood rheology:Compared with normal group,the whole blood viscosity and plasma viscosity of the rats increased markedly in DM model group(p＜0.01).Compared with model group,the whole blood viscosity and plasma viscosity of the rats reduced in median and high dosage group and Haochang group(p＜0.01).There was no remarkable difference between median,high dosage and Haochang group(p＞0.05).The whole blood viscosity reduced in low dosage group compared with model group(p＜0.01) while increased compared with Haochang group(p＜0.05).There was no remarkable difference in plasma viscosity between low dosage group and Haochang group(p＞0.05).5.Serum lipoids:Compared with normal group,TG and CHO of the rats increased markedly in DM model group(p＜0.01).Compared with model group,CHO reduced in Haochang group(p＜0.01) while there was no difference between model and Haochang group(p＞0.05).Compared with model group,TG and CHO reduced in Mimeng Flower groups (p＜0.01).There was no difference between median and high dosage group(p＞0.05). TG and CHO reduced in median and high dosage group compared with low dosage and Haochang group(p＜0.05).6.Pancreas:Remarkable pathological changes of pancreases of diabetic rats were found in DM model group.In median and high dosage group,the pathological changes of pancreases of diabetic rats reduced.7.Cataract:4 months after making model,compared with normalgroup,remarkable opacity of lens were observed in another five groups(p＜0.05),compared with model group,the degree of cataract relieved remarkablely in median dosage group(p＜0.05).8.Retinal:Remarkable pathological changes of retinal of diabetic rats were found in DM model group.In median dosage group,the pathological changes of retinal of diabetic rats reduced.9.The result of immunohistochemistry test show that the expression of ICAM-1,E-selectin in retinal increased in DM model group compared with blank group(p＜0.01).Mimeng Flower decoction groups and Haochang group could down regulate the expression of ICAM-1,E-selectin compared with model group(p＜0.01).In median dosage group the expression of ICAM-1,E-selectin reduced compared with Haochang group.10.The result of PAS stained show that remarkable pathological changes of retinal capillary vessels of diabetic rats were found in DM model group.In median dosage group,the pathological changes of retinal capillary vessels of diabetic rats reduced.11.The result of RT-PCR test show that the expression of VEGFmRNA,Flk-1/KDRmRNA increased in DM model group compared with normal group(p＜0.01).Mimeng Flower decoction groups could down regulate the expression of VEGFmRNA,Flk-1/KDRmRNA and up regulate the expression of Flt-1 mRNA compared with model group.12.The result of Western-blot test show that the phosphorylation level of VEGF,Flk-1/KDR increased while Flt-1 reduced in DM model group compared with normal group.Mimeng Flower decoction groups and Haochang group could down regulate the phosphorylation level of VEGF,Flk-1/KDR and up regulate the phosphorylation level of Flt-1 compared with DM model group and these effects were dose dependent.Cell culture in vitro study:Cells grew more rapidly in hypoxia group than in Mimeng Flower decoction groups.1.The results of WST-8 test show that the hypoxia group can increase the optical density (OD) values compared with blank group(p＜0.01);Mimeng Flower decoction groups can reduce the OD values obviously compared with hypoxia group(p＜0.01) and these effects were dose dependent;The high dose of Mimeng Flower decoction group's OD values were the lowest.2.The results of FCM test show that cells in G₀/G₁ phase reduced while cells in S phase increased in hypoxia group compared with blank group(p＜0.01).In Mimeng Flower decoction groups,cells in G₀/G₁ phase increased while cells in S phase reduced compared with hypoxia group(p＜0.01) and these effects were dose dependent.Further more, Mimeng Flower decoction can induce HUVEC apoptosis.3.The results of FCM test show that the expession of PCNA increased in hypoxia group compared with blank group(p＜0.01).Mimeng Flower decoction could down regulate the expression of PCNA significantly compared with hypoxia group and expression of PCNA showed dose-dependent in Mimeng Flower decoction groups(p＜0.01).4.The results of RT-PCR test show that the expression of VEGFmRNA,Flk-1/KDRmRNA increased while Flt-1mRNA reduced in hypoxia group compared with blank group(p＜0.01).Mimeng Flower decoction could down regulate the expression of VEGFmRNA,Flk-1/KDRmRNA and up regulate the expression of Flt-1mRNA compared with hypoxia group(p＜0.01) and these effects were dose dependent.5.The results of Western-blot test show that the phosphorylation level of VEGF,Flk-1/KDR increased while Flt-1 reduced in hypoxia group compared with blank group.Mimeng Flower decoction could down regulate the phosphorylation level of VEGF,Flk-1/KDR and up regulate the phosphorylation level of Flt-1 compared with hypoxia group. 6.The results of immunohistochemistry test show that the expression of ICAM-1,E-selectin,vWF increased in hypoxia group compared with blank group(p＜0.01). Mimeng Flower decoction could down regulate the expression of ICAM-1,E-selectin,vWF compared with hypoxia group(p＜0.01) and the expression of ICAM-1,E-selectin reduced in high dose compared with low dose group(p＜0.01).ConclusionAnimal model study:1.STZ induced diabetic rat is a simple,easy and reliable animal model.2.Mimeng Flower decoction has the function of improving the general status,reducing the level of blood glucose,GHb,Blood rheology and Serum lipoids and inhibiting the development of cataract of DM rats induced by STZ to a certain.3.Median dosage of Mimeng Flower decoction can reduce the the pathological changes of retinal and retinal capillary vessels in diabetic rats to a certain.4.The effect of improving the pathological changes of retinal and retinal capillary vessels may be contributed to downregulating ICAM-1,E-selectin expression in retinal.5.Downregulating the protein phosphorylation level and mRNA expression of VEGF,Flk-1/KDR mRNA and upregulating the protein phosphorylation level and mRNA expression of Flt-1 mRNA may play a significant role in improving the pathological changes of retinal and retinal capillary vessels of DM rats.Cell culture in vitro study:1.Mimeng Flower decoction can markly inhibit the HUVEC proliferation in hypoxia state.2.The effect of inhibiting proliferation by Mimeng Flower decoction may be contributed to inducing G₁/S transition arrest and increasing the apoptosis of HUVEC.3.The effect of inhibiting proliferation by Mimeng Flower decoction may be contributed to downregulating PCNA expression and hindering the DNA synthesis of HUVEC.4.Downregulating the protein phosphorylation level and mRNA expression of VEGF,Flk-1/KDR mRNA and upregulating the protein phosphorylation level and mRNA expression of Flt-1 mRNA may play a significant role in inhibiting proliferation of HUVEC by Mimeng Flower decoction.5.Mimeng Flower decoction can improve the cells' function by reducing the expression of ICAM-1,E-selectin,vWF in HUVEC.