Protective Effect And Mechanism Of Pinoresinol Diglucoside On Oxidative Injury Of Vascular Endothelial Cells

BackgroundIn recent years,with the improvement of people’s living standards and the aging of the population,the incidence of atherosclerosis(AS)continues to increase.As a disease seriously harmful to human health,the pathogenesis of AS is extremely complex.Looking for its exact etiology and pathogenesis has been the direction of medical research workers.AS is a chronic disease whose initiation is endothelial cell damage and dysfunction.Despite many factors leading to endothelial injury,but the endothelial cell oxidative damage get more attention,and it is considered to be an important risk factor for AS.Studies have confirmed that under external stimulations,the antioxidant mechanism of vascular endothelial cells is damaged and the cell redox homeostasis is disturbed,which could cause accumulation of intracellular reactive oxygen species,namely oxidative stress.As one major production of oxidative stress,oxidized low-density lipoprotein(ox-LDL)is the primary causative factor for vascular endothelial cell dysfunction.Lectin-like oxidized low-density lipoprotein-1(LOX-1),one of the specific receptor of ox-LDL,is expressed mainly in endothelial cells and could be up-regulated by ox-LDL,vascular angiotensin II,endothelin and cytokines.The binding of LOX-1 to ox-LDL induces up-regulation of multiple genes,such as intercellular adhesion molecule-1(ICAM-1),Vascular Cell Adhesion Molecule 1(VCAM-1)and monocyte chemoattractant protein-1(MCP-1),so mononuclear cells adhere to the vascular endothelial surface and move into the endothelium to assimilate lipids.And inflammation in endothelial cells was triggered,damage and dysfunction of endothelial cells was caused,so that AS was triggered or aggravated eventually.The apoptosis of endothelial cells is closely associated with AS development.The intracellular signal transduction pathways are important mediums for the response of cells to external stimulations.Among them,p38MAPK/NF-κB is the important pathway that closely related to the oxidative damage.It has been verified that,the ox-LDL/LOX-1 system can activate the p38MAPK/NF-κB pathway,and ox-LDL induces the protein expressions of LOX-1 and IC AM-1 via regulation of the pathway.The disorder of lipid metabolism is also risk factor for AS.Therefore,many lipid-lowering drugs have been prescribed for AS intervention in clinical practice,such as statins.Pinoresinol diglucoside(PDG),extracted from Eucommia ulmoides Oliver,is one kind of bisepoxylignan that has functions on lowing blood pressure and blood lipid,inhibiting the tumor cells growth,and anti-bacteria or virus.Researches had shown that PDG have the protective function on vascular endothelial cell damage;However,whether the vascular protection of PDG is achieved by reducing oxidative damage of endothelial cells,and what is its protective mechanism?These problems have not been reported yet.This is the main content of this subject.We hope that this subject will provide experimental basis for finding new targets for anti-AS drugs.The study is divided into two parts:(1)We established a high-fat mouse model,and the mice were given PDG treatment.We used toorvastatin as a positive control,to study the protective effect of PDG on vascular endothelium,and whether it played the role through inhibiting endothelial cell oxidative damage?(2)We built a oxidative damage model in vitro by using human umbilical vein endothelial cell(HUVEC),to explore whether PDG inhibit endothelial cell oxidative damage induced by ox-LDL by influencing the p38MAPK/NF-κB signaling pathway?Methods1 Experiments in high-fat mouse model1.1 Establishment of mouse model and groupingThe 8-week aged Apoe mice that were fed with high fat diet were randomly divided into 4 groups(n = 5 in each group):high fat mouse model group;low PDG concentration group(intragastric administration every other day with the dosage of 5mg/kg/d,and successive administration for 4 weeks);high PDG concentration group(intragastric administration every other day with the dosage of 10mg/kg/d,and successive administration for 4 weeks);and the positive control group(atorvastatin administration every other day with the dosage of 10mg/kg/d,and successive administration for 4 weeks).Normal control were wild mice(n = 3)with normal diet for 4 weeks.All mice in the above 5 groups were sacrificed after 8 weeks cultivation.Then relative tissues were collected for the following experiments.1.2 Morphological examination of vascular endothelial cells in high-fat mouse modelTissues were fetched out from the thoracic aorta.The morphological changes of vascular endothelial cells were observed by HE staining.1.3 Detection methods of vascular endothelial oxidative damage indexTotal cholesterol(TC),serum ox-LDL content,serum NO content and MDA content were determined using the corresponding kit detection,respectively.Reverse transcription-polymerase chain reaction(RT-PCR)was used for mRNA expressions of LOX-1 and ICAM-1 in thoracic aorta tissue in mice.2 Experiments in vitro2.1 Establishment of oxidative damage model and groupingThe cell lines were human umbilical vein endothelial cells(HUVEC).The lowest concentration of ox-LDL(100 μg/mL)and the optimal detection time point(24h)were obtained by preliminary experiment.Meanwhile,the optimal concentration of PDG was set as 0.1 μM.SB203580(SB)is a specific inhibitor of the p38MAPK signaling pathway.In this vitro experiment,there were 6 groups as follows:Control group:HUVECs;Oxidative model group:ox-LDL-HUVECs;High PDG concentration group:ox-LDL-HUVECs + PDG(1 μm);Low PDG concentration group:ox-LDL-HUVECs + PDG(0.1μM);SB inhibitor group:ox-LDL-HUVECs +SB(10 μmol/L);PDG +SB inhibitor group:ox-LDL-HUVECs +PDG(1 μM)+ SB(10 μmol/L).2.2 Detection of apoptosis of HUVECsThe HUVECs in logarithmic growth phase were picked.Flow cytometry was applied to determine the cell apoptosis in each group.2.3 Detection methods of each indexFor ROS measurement,reactive oxygen was detected using fluorescent probe DCFH-DA.NO level,MDA content and SOD activity were determined by instructions of the corresponding reagent kits,respectively.The mRNA expressions of LOX-1,ICAM-1,NF-κB,eNOS and iNOS were determined by real-time RT-PCR.Protein measurement of LOX-1,ICAM-1,p38MAPK and NF-κB was conducted by western blot.Results1 In high-fat mouse model1.1 Morphological results of vascular endothelial cellsHE staining results show that in the high-fat mouse model,vascular were swelling in a round shape,endothelial injury was significant.Compared with the high-fat model group,the swelling of endothelial cell was significantly reduced in the positive control group and PDG group.1.2 Detection results of vascular endothelial oxidative damage indexIn comparison with control,mRNA expressions of LOX-1 and ICAM-1,and the contents of TC and ox-LDL in the high-fat mouse model were significantly increased;whereas,the content of NO was dramatically decreased(P<0.05).Compared with the high-fat mouse model,PDG and positive control treatment resulted in a lower mRNA expression of LOX-1 and ICAM-1,and content of TC and ox-LDL,but a higher NO content(P<0.05).PDG showed a concentration-dependent effect on these factors.2 In vitro experiment2.1 Detection results of apoptosis of HUVECsCompared with control,apoptosis rate in the oxidative model was obviously increased(P<0.05);however,PDG and SB inhibitor significantly inhibited the cell apoptosis(P<0.05).2.2 Measurement of each indicatorCompared with control,levels of ROS and content of MDA,mRNA expressions of LOX-1,ICAM-1,NF-κB and iNOS,and protein expressions of p38MAPK and NF-κB in the oxidative model were significantly increased;SOD activity,levels of NO and mRNA expression of eNOS were significantly decreased(P<0.05).PDG and SB treatments significantly inhibited these changes in the oxidative model group(P<0.05).Conclusions1 PDG could reduce the content of TC,ox-LDL and MDA,increase the level of NO,downregulate the mRNA expression of LOX-1 and ICAM-1 in endothelial cells of the high-fat mice model.PDG had a protective effect on vascular endothelial cells of high-fat mice.2 PDG could antagonize oxidative damage of HUVECs induced by ox-LDL,increase the activity of antioxidant enzymes SOD,increase the level of NO,up-regulate the mRNA expression of eNOS,down-regulate the mRNA expression of iNOS,reduce the levels of MDA and ROS,decrease the expression of LOX-1 and ICAM-1 in endothelial cells.PDG could improve the antioxidant capacity ofendothelial cells,and protect against endothelial injury caused by lipid peroxidation.3 PDG could reduce the apoptosis of HUVECs induced by ox-LDL through inhibiting the activity of p38MAPK/NF-κB pathway.PDG played a protective role on vascular endothelial cells.Innovation and significance1.It was the first time to confirm that PDG,the main component of Eucommia Ulmoides Oliver,had the protective effect on oxidative injury of endothelial cells induced by ox-LDL in the course of the onset of atherosclerosis.2.It was the first time to confirm that PDG could significantly inhibit the apoptosis of HUVECs induced by ox-LDL in vitro.PDG may play a protective role on endothelial cells by inhibiting the activity of p38MAPK/NF-κB pathway.