Protective Effects And Mechanisms Of Diplacone On Vascular Endothelial Cells Against Homocysteine-induced Damage

Objective:Atherosclerosis(AS)is an arteriosclerotic disease characterized by thickening,hardening and elastic degeneration of the arterial wall.It is thought that the formation process of AS is a chronic inflammatory reaction.The inflammatory injury of vascular endothelial cells is the initiating link of AS.Both epidemiological and basic studies have shown that hyperhomocysteinemia(Hyperhomocysteine,HHCy)is a risk factor for vascular endothelial injury,and the incidence of AS in HHCy patients is often accompanied by the occurrence of AS.Therefore,protection of vascular endothelial cells has important clinical significance for preventing and treating AS and related diseases.Diplacone is a natural flavone flavone derived from the fruit of Paulownia Paulownia.It has good anti-inflammatory,antiseptic,antioxidant activity and anti-tumor effect.Through the establishment of HCy induced human umbilical vein endothelial cell(HUVEC)inflammatory damage model,the protective effect of Diplacone on vascular endothelial cells was studied,and the mechanism of Diplacone intervention was further explored.Methods 1.HUVEC cultureHUVEC were cultivated in DMEM medium supplemented with10% Fetal Bovine Serum in 37?,5%CO2 constant temperature and constant humidity incubator,when the cells grew to 80%-90% and was harvested by 0.25% trypsinization with EDTA to carry out conventional passages.2.Establishing inflammatory damage model of vascular endothelial cell.(1)The determination of the optimum concentration of HCy damage.The cultured HUVEC was divided into normal control group(no HCy),HCy model group and matrix blank group(no cell zero control group),and the HCy model group was divided into 0.25 mmol/L HCy group,1 mmol/L HCy group,2 mmol/L HCy group,4 mmol/L HCy group and 6 group.HCy was added to the treated model group cells respectively,and 24 h was incubated with the normal control group,and the survival rate of the cells was detected by adding MTT,and the optimum damage concentration was obtained by the plot analysis.(2)The determination of the best time for HCy damage.The cultured HUVEC was divided into normal control group(no HCy),HCy model group and matrix blank group(no cell zero control group),and the HCy model group was divided into 36 h group,24 h group,12 h group,6 h group and 2 h group.2 mmol/L HCy(the optimum damage concentration)were added to the treated model cells at different time respectively.The 36 h,24 h,12 h,6 h and 2 h were co cultured with the normal control group.Then MTT was added to detect the cell survival rate,and the optimal time for the HCy induced HUVEC to produce the damage was obtained by the graph analysis.3.Experimental groupingThe experiment was divided into 6 groups: control group,2 mmol/L HCy model group and drug group.The drug group was further divided into 0.1 ?mol/L Diplacone +2 mmol/L HCy group,1 ?mol/L Diplacone+2 mmol/L HCy group,10 ?mol/L Diplacone +2 mmol/L HCy group and 100 ?mol/L Diplacone+2 mmol/L HCy group.4.Protective effect of Diplacone on vascular endothelial injury induced by homocysteine.(1)Activity changes were detected by MTT,and Changes of cell morphologic were observed under an inverted microscope;(2)Morphological changes of nuclear apoptosis were detected by Hoechst33342 nucleic acid staining;(3)Changes of cells apoptosis rate cells cycle were detected by flow cytometry.5.Protective mechanisms of Diplacone on vascular endothelial injury induced by homocysteine.(1)ELISA method were used to detect the release of VCAM-1,ICAM-1,P-selectin and E-selectin adhesion factors in cell culture fluid.(2)HUVEC ROS cell ratio,MDA content,SOD and GSH-Px activity were detected by different kits.(3)Western Bloting was used to detect protein expression levels of apoptotic genes Bax,Bcl2,Casepase-9,and adhesion molecules VCAM-1,ICAM-1,P-selectin,E-selectin,and nuclear factor NF-?B.(4)qRT-PCR was used to detect the mRNA level of apoptotic genes Bax,Bcl2,Casepase-3,P53 and adhesion molecules VCAM-1,ICAM-1,P-selectin,E-selectin,and NF-?B.Results 1.Observation of cell statusThe resuscitated cells were observed under an inverted microscope,the cells were circular,transparent individuals and suspended in the cell culture medium.The cells were cultured for 24 h,which basically adhered to the wall,cells shape was short fusiform,with transparent color.The number of cells continued to increase with the increase of incubation time,HUVECs' shape was fusiformor polygon,they were close together to the wall and showed paving stone mosaic-like arrangement.2.The establishment of the optimum concentration and time of HCy damageHUVEC was pretreated with different concentrations of homocysteine for 24 h,the optimal damage concentration and time were obtained that was 2 mmol/L and 12 h.3.Protective effect of Diplacone on homocysteine-induced vascular endothelial cell(1)The effect on cell survivalCompared with the normal control group,the survival rate of the HCy model group was significantly reduced(P<0.01).Compared with the HCy model group,there was no significant difference in the cell survival rate of the 0.1 ?mol/L Diplacone group,but the cell survival rate of the 1?mol/L and 10 ?mol/L Diplacone groups increased significantly(P<0.01),and there was no significant change in the cell survival rate in the 100 ?mol/L Diplacone group.That is,0.1 ?mol/L~10 ?mol/L Diplacone can inhibit HCy damage to HUVEC and increase cell survival rate in a concentration dependent manner.(2)The effect on cell morphologyThe cell morphology was observed under the microscope.Compared with the normal control group,the cell volume of the HCy model group crinkled,the shape tended to be slender,the shape was irregular,and part of the cells fell off.The cell morphology of the 0.1 mol/L to 10 ?mol/L Diplacone group was gradually improved,gradually becoming full,and the number of shedding decreased gradually.(3)The effect on the morphology of nuclear apoptosisCompared with the normal control group,the cell nuclei of the HCy model group showed obvious morphological characteristics of apoptosis,and most of the nuclei were bright blue fluorescence,chromosome agglutination and edge aggregation.The nuclei of 0.1 ?mol/L ~ 10 ?mol/L Diplacone groups showed a significant decrease in the concentration dependence of the apoptotic cells.The number of nuclei with uniform blue fluorescence increased gradually,and the degree of chromosome agglutination and the degree of agglutination decreased.(4)The effect on the rate of apoptosisCompared with the normal control group,the apoptosis rate in the HCy model group increased significantly(P<0.05).Compared with the HCy model group,there was no obvious change in the cell apoptosis rate in the 0.1 ?mol/L Diplacone group,but the apoptosis rate of the 1?mol/L and 10 ?mol/L Diplacone groups decreased significantly(P<0.05),and there was no significant change in the apoptosis rate of the 100 ?mol/L Diplacone group.That is,0.1~10 ?mol/L Diplacone can reduce HCy induced apoptosis in a concentration dependent manner.(5)The effect on cell cycleCompared with the normal control group,the cell proportion of G0/G1 in HCy model group increased significantly(P<0.05),and the proportion of cells in S phase decreased significantly(P<0.05).Compared with the HCy model group,there was no obvious change in the cell cycle of the 0.1 ?mol/L Diplacone group and the 1 ?mol/L Diplacone group,but the specific gravity of the cells in G0/G1 decreased,the specific gravity of the S phase increased,and the specific gravity of the G0/G1 period of the 10 ?mol/L Diplacon group was significantly lower(P<0.05),and the specific gravity of the cells in the S stage increased significantly.0.05)there was no obvious change in the cell cycle of the 100 ?mol/L Diplacone group,that is,0.1 ?mol/L ~10 ?mol/L Diplacone could have a concentration dependent effect on the removal of HCy blocking cycle.4.Protective effect mechanism of Diplacone on homocysteine-induced vascular endothelial cell(1)The influence of Diplacone on injured-HCy VCAM-1,ICAM-1,P-selectin and E-selectin content,mRNA and protein expression level in HUVECELISA,Western Bloting and qRT-PCR results show,compared with the normal control group,the levels of VCAM-1,ICAM-1,P-selectin,E-selectin and the expression of protein and mRNA in the HCy model group were significantly increased(P<0.05).Compared with the HCy model group,the content of VCAM-1,ICAM-1 and P-selectin in the 10 ?mol/L Diplacone group decreased significantly(P<0.05),and there was no significant change in the 1?mol/L and 100 ?mol/L Diplacone groups;The gene and protein expression levels of 1 ?mol/L and 10 ?mol/L Diplacone groups were significantly decreased(P<0.05),and there was no significant change in the gene and protein expression levels in the 0.1 ?mol/L and 100 ?mol/L Diplacone groups.(2)The influence of Diplacone on HCy oxidative stress index ROS,SOD,GSH-Px and MDA induced by HUVEC were detected by different kits.Kits results show,compared with the normal control group,the ratio of ROS cells and the content of MDA in the HCy model group increased significantly(P<0.05),and the activity of SOD and GSH-Px was significantly decreased(P<0.05).Compared with the HCy model group,the ratio of ROS cells in 1,10 and 100 ?mol/L Diplacone groups decreased significantly(P<0.05),and GSH-Px activity increased significantly(P<0.05).There was no significant change in the ratio and activity of ROS cells in 0.1 mu mol/L Diplacone group,and the activity of 0.1 ?mol/L was significant Increased(P<0.05),MDA content decreased significantly(P<0.05),SOD activity and MDA content in 100 ?mol/L Diplacone group did not change significantly.(3)The effects of Diplacone on HCy induced HUVEC Diplacone and HCy induced HUVEC apoptosis gene Bax,Bcl2 and Casepase-9 protein expression levelWestern Bloting results show,Compared with the normal model group,the expression level of Bax and Casepase-9 protein in the HCy model group increased significantly,and the protein expression level of the inhibitor of apoptosis index Bcl2 was significantly decreased(P<0.05).Compared with the HCy model group,the expression level of Bax protein in 1 ?mol/L and 10 ?mol/L Diplacone groups decreased significantly(P<0.05),and the expression level of Bcl2 protein increased significantly(P<0.05).There was no significant change in the expression level of Bax and protein in 0.1 ?mol/L and 100 ?mol/L Diplacone groups.10 ?mol/L.The level of expression decreased significantly(P<0.05),but there was no significant change in Casepase-9 protein expression level in 0.1,1,100 and 100 ?mol/L Diplacone group,mol/L.(4)The effects of Diplacone on HCy induced HUVEC Diplacone and HCy induced HUVEC apoptosis gene Bax,Bcl2,P53 and Casepase-3 Mrna expression levelQRT-PCR results show,Compared with the normal model group,the expression level of Bax and Casepase-9 protein in the HCy model group increased significantly,and the protein expression level of the inhibitor of apoptosis index Bcl2 was significantly decreased(P<0.05).Compared with the HCy model group,the expression level of Bax protein in 1 ?mol/L and 10 ?mol/L Diplacone groups decreased significantly(P<0.05),and the expression level of Bcl2 protein increased significantly(P<0.05).There was no significant change in the expression level of Bax and protein in 0.1 ?mol/L and 100 ?mol/L Diplacone groups.10 ?mol/L.The level of expression decreased significantly(P<0.05),but there was no significant change in Casepase-9 protein expression level in 0.1,1,100 and 100 ?mol/L Diplacone group,mol/L.(5)The effect of Diplacone on the expression of HUVEC NF-?B protein and mRNA induced by HCyWestern Bloting and qRT-PCR results show,Compared with the normal control group,the expression level of NF-?B and mRNA in HCy group increased significantly(P<0.05).Compared with the HCy model group,the expression level of NF-?B protein in 1 ?mol/L and 10 ?mol/L Diplacone groups decreased significantly(P<0.05).The expression level of NF-?B protein in 0.1 ?mol/L and 100 ?mol/L Diplacone groups had no significant changes,and the expression level of the 10 mu group was significantly lower than that of 0.1 ?mol/L and 1 ?mol/L.There was no significant change in NF-?B mRNA expression level in 100 ?mol/L Diplacone group.Conclusion 1.Under the experimental conditions,the most suitable damage concentration of HCy induced HUVEC damage is 2 mmol/L,and the optimal damage time is 12 h.It can induce endothelial cell injury and reduce cell survival rate.2.1~10 ?mol/L has protective effect on HUVEC damage induced by 2 mmol/L HCy,which can improve the morphology of cell and cell nucleus apoptosis,inhibit cell apoptosis and cycle arrest.3.Diplacone decreased the secretion of VCAM-1,ICAM-1 and P-selectin and increased the expression of gene and protein in HUVEC induced by 2 mmol/L HCy.4.Diplacone can reduce the ratio of HUVEC ROS and MDA level induced by 2 mmol/L HCy,and also increase the activity of SOD and GSH-Px of antioxidant index.5.Diplacone can reduce the increase of mRNA or protein expression of Bax,Casepase-9,Casepase-3 and P53 in the HUVEC induced apoptosis index of HUVEC induced by 2 mmol/L HCy,and increase the mRNA and protein expression of the apoptosis inhibition index Bcl2.6.Diplacone can interfere with the increase of NF-?B gene and protein expression in cultured HUVEC induced by 2 mmol/L HCy.