Trans-Cinnamaldehyde Regulates Arginase 2 Through Deacetylation To Improve Endothelial Cell Damage Caused By Diabetes

ObjectiveDiabetic vascular lesion is one of the main pathologicalmanifestations of diabetic vascular complications.High glucose-induced vascular endothelial cell injury is the key to diabetic vascular injury.This experiment takes Human Umbilical VeinEndothelial Cells(HUVEC)as the research object,using high glucose-induced diabetic endothelial cell damage model to explore the improvement of diabetic endothelium by Trans-cinnamaldehyde(TCA)through deacetylation pathway The mechanism of cell damage.Method:1.High glucose(30 mM)model was used to induce HUVEC damage model.TCA was administered at 1,3,10,30 μM for 24 hours to detect cell viabilityto determine the concentration of TCA administered,and the endothelial cells were detected by nitrate reductase method.Nitric oxide(Nitric Oxide,NO)production level.2.Target Hunter software combined with CVD Platform to simulate the target molecular binding based on the similar structure of the compound,and predicted the protein target of TCA in vivo.3.In order to explore the possible targets of TCA in vivo,the experiment set up three groups:control group(NG),high glucose group(HG),TCA administration group(HG+TCA),using Histone deacetylase(HDAC)activity fluorescence The kit was used to detect the effect of TCA on the HD AC activity of endothelial cells induced by high glucose.4.In order to explore the effect of TCA on the HDAC pathway,various HDAC inhibitors were used to verify the high glucose-induced NOproduction of endothelial cells.5.To further explore the specific target of TCA,HDAC6 activity fluorescent kit was used to detect the activation level of HDAC6 activity by TCA.6.Detect the arginase(ARG)activity of different concentrations of TCA on high glucose-induced endothelial cells by the amount of urea produced by the ARG hydrolysis substrate L-arginine.7.To explore the effect of TCA on HD AC(Histone deacetylase,HD AC)pathway,various HDA C inhibitors were used to verify the detection of TCA on high glucose-induced endothelial cell ARG activity.8.Use qRT-PCR to detect the levels of ARG2 mRNA in the three groups of NG,HG,and HG+TCA;and use Western blot to detect the protein expression.9.In order to further explore the regulatory mechanism of TCA on ARG,immunoprecipitation method was used to detect the ARG2 acetylation level of NG,HG,HG+TCA.Result:1.Compared with the NG group,there was no significant difference in cellviabilityrate in the HG group(P>0.05),and there was no significant difference in endothelial cellviabilityrate in the TCA group at 1μM,3μM,and 10μM concentrations(P>0.05),at 30μM concentration.The cell viabilityrate decreased significantly(P<0.05),indicating that TCA at 10 μM concentration had no significant effect on cell viabilityrate;compared with the NG group,the endothelial cell NO production in the HG group was significantly reduced(P<0.05),which could be reversed after TCA The reduction of NO production in endothelial cells caused by high glucose indicates that TCA can significantly improve the level of NO production in endothelial cells induced by high glucose.2.According to the 3D structure of TCA small molecules,Target Hunter software combined with CVD Platform to predict the protein target of TCA in vivo,and select the top 10 prediction results based on p-value,three of which predict that TCA may deacetylate histones The enzyme has a strong binding effect.3.Compared with the NG group,the HDAC activity in the HG group was significantly reduced.After TCA administration,the HDA C activity of the endothelial cells in the HG group was significantly increased(P<0.05),indicating that TCA significantly increased the HDAC activity by stimulating the HDAC.Sugar-induced HDAC activity in endothelial cells.4.Compared with the NG group,high glucose incubation reduced NO production(P<0.05),TCA could reverse the reduction of NO production by endothelial cells caused by high glucose,and high glucose induced recovery of NO production by endothelial cells after TCA administration To the same level as the control group.Except for VB3,other HDAC inhibitors can block the damage of TCA on high glucose-induced endothelial cell NO production,indicating that TCA affects the endothelial cell NO production by agonizing the deacetylase.5.Compared with the NG group,when the TCA concentration was 10 nM,30 nM and 100 nM,the activity of HDAC6 was significantly increased.The TCA concentration of 30 nM had the strongest agonistic effect on HDAC6(P<0.05);Within a certain concentration range,HDAC6 activity can be directly stimulated,especially when the TCA concentration a is 30 nM,which has the most significant effect on HDAC6 activity.6.Compared with the NG group,the ARG activity of endothelial cells in the HG group was significantly increased(P<0.05).After administration of TCA at 1μM,3μM,and 10μM concentrations,the ARG activity of the endothelial cells in the HG group was significantly reduced(P<0.05).,Indicating that TCA can significantly improve the level of ARG activity in endothelial cells induced by high glucose.7.Compared with the NG group,high glucose incubation increased ARG activity(P<0.05),TCA could reverse the increase in ARG activity of endothelial cells caused by high glucose,and high glucose-induced endothelial cell ARG activity recovery after TCA administration To the same level as the control group.Except for VB3,other HD AC inhibitors can block the damage of TCA on high glucose-induced endothelial cell ARG activity,indicating that TCA affects the ARG activity of endothelial cells by agonizing the deacetylase.8.Compared with the NG group,there was no significant difference in ARG2 mRNA levels between the HG group and the TCA administration group(*P>0.05);compared with the NG group,the ARG2 protein expression in the HG group was significantly increased;High glucose-induced endogenous ARG2 protein expression in human umbilical vein endothelial cells decreased(*P<0.05),the results show that TCA can down-regulate the increase in protein content of endothelial cells induced by high glucose.9.Compared with the NG group,the high glucose-induced endothelial cell ARG2 acetylation level was significantly increased.After TCA administration,the high glucose-induced endothelial cell ARG2 acetylation level was significantly reduced(P<0.05).It shows that TCA can improve the endothelial cell damage caused by high glucose by directly regulating the acetylation level of endothelial cell ARG2.conclusion:(1)TCA has a protective effect on endothelial damage induced by high glucose.TCA protects endothelial cell damage by improving high glucose-induced NO production and abnormal levels of ARG activity in human umbilical vein endothelial cells.(2)The prediction and experimental results on the Target Hunter website show that TCA has an up-regulation effect on the HD AC activity of endothelial cells induced by high glucose.Screening with various HDA C inhibitors found that this effect is through TCA combined with Class Ⅰ and/or Class Ⅱ HD AC Achieved,and further reduced the activity of ARG and increased NO production.(3)In vitro experiments prove that TCA activates HDAC6 within a certain range,indicating that TCA may bind to HDAC6 in vivo,thereby deregulating the expression of ARG2 protein through deacetylation to regulate the NO pathway,improve diabetic vascular endothelial injury,and finally treat diabetic vascular disease.