Protective Effects Of Schisandrin On Diastolic Dysfunction By High Glucose In Cultured Human Umbilical Vein Endothelial Cells

Objective:In this experiment,the high glucose damage model is established through culturing human umbilical vein endothelial cells in vitro.The cells are pretreated with Schisandrin of different concentrations before the stimulation of high glucose.Explore the possible mechanisms and effects of Schisandrin on the diastolic dysfunction of HUVECs induced by high glucose,according to observing the morphology of HUVECs and measuring the activity of RhoA and eNOS and the secretion of NO.Methods:The HUVECs were cultured and the cells which attached for 12 hours were randomized into five groups:1)normal group(NG):the final concentration of glucose is 5.5 mmol/L;2)high glucose group(HG):the final concentration of glucose is 30 mmol/L;3)high glucose with low doses of Schisandrin group(HG+5): Schisandrin concentration is 5?mol/L;4)high glucose with medium doses of Schisandrin group(HG+10):Schisandrin concentration is 10?mol/L;5)high glucose with high doses of Schisandrin group(HG+20):Schisandrin concentration is 20?mol/L.The cells were pretreated with Schisandrin for 24 hours before stimulated by high glucose.Cells and supernatant were collected 48 hours after treatment of high glucose.The morphology of HUVECs at various groups were observed under light microscope.Immunocytochemiacal stain was used to detect the level of p-MYPT1.The levels of RhoA activity were determined by RhoA Activation Assay Biochem kit.The levels of eNOS activity were examined by NOS Activation Assay Biochem kit.The levels of NO were measured by nitrate reduction test.Results:1 In this experiment,GTP-RhoA and p-MYPT1 represent the activation of RhoA/ROCK.p-MYPT1 was basically expressed in the cytoplasm of HUVECs by immunocytochemistry,after treated by high glucose for 48 hours,the cytoplasm was stained for brown,the expression of p-MYPT1 was increased significantly,and gradually decreased to normal level after pretreatment with Schisandrin of different concentrations.In the Western blotting results,we found slight GTP-RhoA protein expressed in normal control group,while the expression of GTP-RhoA increased compared to normal control group(P<0.01).There was no difference between pretreatment group with Schisandrin of 5?mol/L and high glucose group(P>0.05).Nevertheless pretreatment group with Schisandrin(10?mol/L and 20?mol/L)significantly suppressed the level of GTP-RhoA(P<0.01),the distinction was statistically significant(P<0.05),and the group of 20?mol/L has no difference with normal control group(P>0.05).It turned out that,high glucose can improve the activity of RhoA and increase the expression of p-MYPT1 in HUVECs.When cells are pretreated with Schisandrin in a certain concentration,the activity of RhoA can be reduced and the expression of p-MYPT1 decreased;2 Compared with normal control group,the production of NO and activity of eNOS in the high glucose group declined markedly,which are statistically significant(P<0.01).The level of NO and eNOS in pretreatment group with Schisandrin of 5?mol/L had no difference with the high glucose group(P>0.05),while intervention with concentration of 10?mol/L and 20?mol/L decreased evidently compared with high glucose group,which showed dose dependence.In addition,the pretreatment group of 20?mol/L had no difference with nomal control group on the level of NO and eNOS(P>0.05).The results showed us:high glucose can suppress the activation of eNOS in HUVECs and decreases the production of NO.After intervention with pretreatment with Schisandrin,the activity of eNOS induced by high glucose can be improved dose dependently within a certain range,which results in a rise in secretion of NO.Conclusions:1 Endothelial dysfunction which induced by high glucose may be associated with a rise of RhoA/Rho kinase activity,a drop of eNOS activity and the reduction of NO production.2 Schisandrin can improve the function of endothelial cells by lowering the activity of RhoA/Rho kinase,raising the activity of eNOS and the production of NO.And thus it can protect the diastolic dysfunction of HUVECs induced by high glucose.The protection on cells with a concentration of 20?mol/L is most obvious.