| Background and purposed:It has been demonstrated that FA may be involved in protein-misfolding in brain and endothelium damage in cardiovascular system,and therefore may contribute to the occurrence of neurodegenerative diseases or vascular complications in diabetes.Hydralazine（HYD）is a hypotensive drug,because of its structure containing hydrazine,hydrazine can react with a variety of reactive carbonyl compounds to reduce the formation of glycation.Therefore,it has been used as an inhibitor of AGEs for many years.However,it is unclear whether HYD can remove FA,and the changes of FA level in plasma during the development of diabetes have not been reported.Therefore,this study is to explore the effect and possible mechanism of HYD intervention on the injury of Human Umbilical Vein Endothelial Cells（HUVECs）induced by FA through cell culture in vitro,and through the intervention of HYD on FA crosslink to protein in vitro and the effect of FA level in plasma of diabetic rats after HYD intervention,whether HYD can eliminate FA to inhibit the formation of AGEs,as a therapeutic drug to reduce the complications of diabetes to provide the basic experimental basis.Method:（1）MTT method was used to detect the effect of HYD and FA on the viability of HUVECs.（2）The mechanism of HUVECs injury induced by 200μM FA was studied by 80μM HYD.The cells viability,ROS,MDA,NO and SOD were detected,and the morphological changes were observed.（3）The HYD（0,3.13,6.25,12.5,25,50,100m M）and FA（0.5,5,50m M）combined with5mg·m L-1 BSA in 0.2M sodium phosphate buffer solution,at 37℃,p H7.4 for 0,24,48,72,96h.The combination of FA and BSA were determined by HPLC.（4）After one week of adaptive feeding,47 SD rats were randomly divided into two groups:Control group（n=15）and Diabetic model group（n=32）.Type 1 diabetic rats were prepared by intraperitoneal injection of 60 mg·kg-1 STZ.Their weekly body weight,daily food intake,weekly fasting blood glucose and FA concentration in plasma were recorded.When the fasting blood glucose of diabetic rats was more than 16.7mmol·L-1 for 4 weeks,the Diabetic model group were randomly divided into two groups:Diabetic model control group（n=15）,HYD group（n=15）.The HYD group was given an intraperitoneal injection of 20 mg·kg-1 HYD at 3:00 p.m.daily,while the other two groups were given an equal dose of saline.For four weeks,weekly body weight（adjusted dose）,daily food intake,weekly fasting blood glucose,weekly FA concentration in plasma were recorded.Result:（1）When 50,100,200,500μM FA and HUVECs were incubated for 24,48h,the viability of HUVECs decreased in a dose-and time-dependent（p<0.05）.20,40,80,120μM HYD and HUVECs incubated for 24,48h had no effect on the viability of HUVECs.（2）The viability of HUVECs was increased after 80μM HYD treated with 200μM FA（p<0.05）.The content of ROS,MDA and NO in HUVECs treated with 200μM FA for 24h was significantly higher than that in the control group（p<0.05）,but the viability of SOD was not changed.After 80μM HYD intervention,the content of ROS and MDA decreased（p<0.05）,but the content of NO,the viability of SOD remained unchanged.Inverted phase contrast microscope showed that in the control group,the cells adhered to the wall,the cytoplasm was plump,the boundary was clear,the shape was short fusiform or flat,and the monolayer of cells was typical cobblestone mosaic arrangement.In the 200μM FA group,the number of cells decreased significantly,the intercellular space became larger,the boundary was fuzzy,the cell deformation was obvious,and some cells fell off and died.After HYD intervention,the number of adherent cells increased significantly.（3）There was no significant difference in the binding content of 0,0.5,5m M FA and BSA in 0h without HYD co-incubation.The binding content of BSA and FA increased significantly with 50m M FA（p<0.05）.There was no difference in binding content of 0,0.5m M FA and BSA at24,48,72 and 96h（p>0.05）,but the binding content of 5,50m M FA and BSA increased gradually with time（p<0.05）.At 96h,the binding content of 50 m M FA and BSA was lower than that at 72h（p<0.05）.The binding content of FA and BSA decreased gradually and was dose-dependent under the co-incubation of HYD（3.13,6.25,12.5,25,50,100m M）.However,when HYD（3.13,6.25,12.5,25m M）was co-incubated with 50m M FA and BSA for 96h,the binding content of FA and BSA was lower than that of 72h.The co-incubation of 0.5 and 5m M FA and BSA was not combined with HYD.（4）After the establishment of 1 type diabetes model,the weight of rats in the Diabetic model group decreased.Compared with Diabetic model group,the weight of SD rats in Control group increased with time,and there was no significant difference in the weight of SD rats in HYD group after HYD intervention compared with Diabetic model control group（p>0.05）.The daily food intake of Control group rats was basically unchanged.After the establishment of 1 type diabetes model,the food intake of diabetic model group increased in the first week,and remained basically unchanged from the second week.At the 4th week of the intervention,compared with the Diabetic model control group,there was no significant difference in food intake in the HYD group.The fasting blood glucose of Control group rats was normal.The fasting blood glucose of rats in the diabetic model group increased at 1 week after streptozotocin injection and was greater than 16.7mmol·L-1.At the 4th week after the intervention,compared with the Diabetic model control group,there was no significant difference in the fasting blood glucose values of the rats in the HYD group（p>0.05）.Before STZ intraperitoneal injection,there was no significant difference in plasma FA content between the two groups（p>0.05）;at 1,2 and 3 weeks after STZ injection,there was no significant difference between the Control group and Diabetic model group,and the plasma FA content in the Diabetic model group was higher than that in the Control group for 4th week（p<0.05）.After intervention with HYD for 4 weeks,there was no significant difference in plasma FA content between the HYD group and the Diabetic model control group（p>0.05）;in Control group,there was no significant change in the plasma FA levels at 0th,4th,8th week.STZ induction but untreated with HYD of SD rats at 4th,8th compared with before STZ induction SD rats,the content of plasma FA was increased.Conclusion:HYD may inhibit the damage of HUVECs induced by FA and reduce the cross-linking between FA and protein by scavenging FA.It is suggested that the increase of FA in plasma may be related to the long-term development of diabetes. |
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