The Experimental Study On The Barrier Effect Of Modified Mimeng Flower Decoction On The Human Umbilical Vascular Endothelial Cells

Objective:This study was performed to investigate the effect of Modified Mimeng Flower decoction on the permeability of endothelial cells induced by advanced glycation end products modified human serum albumin(AGE-BSA),and then reserach the mechanism of Modified Mimeng Flower decoction to diabetic macular edema(DME),in order to provide experimental basis for Modified Mimeng Flower decoction used in clinic to preventing and curing DME.Methods:Human umbilical vein endothelial cells (EC304) were cultured in vitro.Used 100mg/L AGE-BSA to set up the diabetic microangiopathy model,in the same time 5mg/ml,10mg/ml,20mg/ml extract were added to the EC304 separately,the following studies were made after 24 hours incubation.1. Through MTT method to check up the inhibiton effoct of Modified Mimeng Flower decoction on the proliferation of EC304.2. Through immunofluorescence method to detect the protein expression of ZO-1,occludin regulated by Modified Mimeng Flower decoction.3. The changes of ZO-l,occludin expression of EC304 regulated by Modified Mimeng Flower decoction were measured with flow cytometry(FCM).4. Through immunofluorescence method to assayed the changes of F-actin regulated by Modified Mimeng Flower decoction.5. Through Western blot method to detect the protein expression of VEGF, Flt-1,Flk/KDR in EC304 regulated by Modified Mimeng Flower decoction.Results:1. The result of MTT test show that there are not many differences between normal group and low concentration AGE-BSA group (p>0.05),but the 100mg/L and high concentration of AGE-BSA group can reduce the optical density (OD)values compared with normal group (p<0.01),and these effects were dose dependent.;different concentration of Modified Mimeng Flower decoction with 100mg/L AGE-BSA groups can reduce the OD values compared with compared with normal group and show does dependent (p< 0.01)2. The result of immunofluorescence test show that the expression of ZO-1,occludin reduced in 100mg/L AGE-BSA group compared with normal group (p<0.01), and led to disruption of cortical ZO-1,occludin distribution while more intracellular ZO-1,occludin was observed. Modified Mimeng Flower decoction could up regulate the expression of ZO-1 and occludin (p <0.01),and show less disruption of cortical ZO-1,occludin distribution.3. The result of FCM test show that the expression of ZO-1,occludin reduced in 1 OOmg/L AGE-BSA group compared with normal group(p<0.01); Modified Mimeng Flower decoction could up regulate the expression of ZO-1 and occludin (p<0.01);the highest expression of ZO-1 was in the middle dose of Modified Mimeng Flower decoction,and the expression of occludin was does dependent in Modified Mimeng Flower decoction groups.4. The result of immunofluorescence test show that the fibers of F-actin in the cells of the normal group concentrated along the cell membrane and a little dense fibers were observed in the cytoplasm. With concentration intreased of AGE-BSA most of the peripheral fibers disappeared and more and more dense stress fibers were observed in the cytoplasm during the late stage. Modified Mimeng Flower decoction can reduced the redistribution of F-actin fibers and down regulate the expression of dense stress fibers in cytoplasm.5. The result of Western blot test show that the expression of VEGF,Flt-1,Flk/KDR increased in AGE-BSA group(p<0.01), Modified Mimeng Flower decoction can down regulate the expression of VEGF and Flk/KDR compared with the AGE-BSA group (p< 0.01),but could not regulate the expression of Flt-1.Conclusions:1. Modified Mimeng Flower decoction can inhibit the proliferation of EC304 and show dose dependent.2. Modified Mimeng Flower decoction can improve the expression of ZO-1and occluding,and reduced the redistribution of them.These may play a significant role in protecting the tight junction of EC304.3. Modified Mimeng Flower decoction can inhibit the redistribution of F-actin, maintain the normal shape of cytoskeleton to reduce the permeability of EC304,4. Reduced the disruption of blood barrier by Modified Mimeng Flower decoction may contributed to reducing the expression of VEGF and Flk/KDR.