| Background and purposeAtherosclerosis(atherosclerosis,AS)refers to a disease characterized by arterial stenosis,thickening of arterial walls,hardening and decreased elasticity,and the formation of atherosclerotic plaques on the intima of the artery.It is characterized by lipid accumulation,local inflammation,smooth muscle cell proliferation,apoptosis,necrosis and fibrosis,and involves chronic inflammatory responses caused by endothelial damage and activation of inflammatory cells.At present,the cause of the AS has not been completely determined,and may be related to factors such as age,gender,dyslipidemia,hypertension,smoking,diabetes,obesity,and infection.Vascular endothelial cell damage and dysfunction is the initial stage of AS,and it is also an important stage in AS.High cholesterol,especially low-density lipoprotein cholesterol,is a major risk factor for AS.Recent studies have shown that chemically modified lipoproteins,especially oxidatively modified LDL(ox-LDL),have a greater effect on AS than LDL.Ox-LDL can play a key role in the development of atherosclerosis by accelerating lipid peroxidation injury,accelerating foam cell formation,promoting vascular smooth muscle cell proliferation,inducing platelet adhesion and aggregation,and endothelial dysfunction.At present,the detection of ox-LDL in circulating plasma can mark the acute coronary syndrome and reflect its condition and course.It has been reported that ox-LDL in the circulating plasma is a recognized and effective index for the diagnosis and prediction of coronary heart disease.Histamine is a self-active substance.Under normal physiological conditions,histamine exists in inactive form in the granules of mast cells and basophils.When the body contacts the allergen again,the mast cells and basophils release histamine through degranulation and participate in allergic and inflammatory reactions by binding to histamine receptors,which can contract smooth muscle vasodilation of arterioles,capillaries and increase of vascular permeability.H1 R is one of the histamine receptors(HRs).It is mainly distributed on the surface of vascular endothelial cells,smooth muscle cells,neurons and immune cells of the skin and mucosa,and regulates vasodilation and bronchoconstriction.It was found that H1 R is expressed in mammalian vascular aorta and microvascular endothelial cells.In addition,atherosclerotic endothelial cells,macrophages and smooth muscle cells can express HR under the activation of histamine,and H1 R is the predominant one.Therefore,we speculate that H1 R may be involved in endothelial dysfunction of AS,and H1 R antagonist loratadine may provide a new treatment for atherosclerosis.Loratadine has been widely used in the treatment of allergic rhinitis,but its role in atherosclerosis remains to be explored.Studying the role of H1 R in AR endothelial dysfunction can provide new directions for the treatment of AR.In this study,we took human aortic endothelial cells HAECs as the research object.Firstly,JAR cells were used as a positive control to detect the expression of H1 R m RNA and protein in HAECs cells.Subsequently,the effects of ox-LDL on H1 R m RNA and protein expression in HAECs were examined by RT-q PCR and western blot.To study the effects of loratadine on ox-LDL-induced endothelial dysfunction,we treated HAECs cells with ox-LDL and different concentrations of loratadine,and then examined changes in cell adhesion,oxidative stress,and inflammation,including adhesion of monocytes to endothelial cells,expression of adhesion molecules VCAM-1 and E-selectin,expression of TNF-?,IL-6 and IL-8,and production of ROS.Then in the second part,in order to study the molecular mechanism of loratadine improving endothelial dysfunction induced by ox-LDL,we analyzed the effect of loratadine on the activation of AP1 signaling pathway and NF-?B signaling pathway in endothelial cells induced by ox-LDL.We treated HAECs cells with ox-LDL and different concentrations of loratadine,and then detected the phosphorylation of JNK and the phosphorylation of I?B?in HAECs cells,and the expression of c-Jun,c-Fos and p65 in the nucleus by western blotting.The activities of AP1 and NF-?B promoters were analyzed by dual-luciferase experiment.In this study,we explained the effects of loratadine on ox-LDL-induced endothelial dysfunction,and analyzed the molecular mechanism of its biological function.Therefore,this study not only revealed the biological function of H1 R in ox-LDL-induced endothelial dysfunction,but also provided new ideas for further clarifying the pathogenesis of AS and the diagnosis and treatment of AS.This research mainly includes the following two parts:Part 1 Effect of loratadine on ox-LDL-induced endothelial cell adhesion,inflammation and oxidative stress Purpose:The purpose os this part was to detect the expression of H1 R m RNA and protein in human aortic endothelial cells(HAECs),study the effect of ox-LDL on the expression of H1 R m RNA and protein in HAECs,and study the effects of loratadine on ox-LDL-induced endothelial dysfunction include cell adhesion,oxidative stress and inflammation by detecting the adhesion of monocytes to endothelial cells,the expression of adhesion molecules VCAM-1 and E-selectin,the expression of cytokines TNF-?,IL-6 and IL-8,and the production of ROS.Method:1.Total RNA and protein were extracted from HAECs cells and JAR cells which was used as control.The expression of H1 R m RNA and protein in endothelial cells was detected by semi-quantitative RT-PCR and western blot,respectively.2.Add 5 ?g/ml,10 ?g/ml and 20 ?g/ml of freshly prepared ox-LDL solution to HAECs cell culture medium to stimulate cells for 24 h.After collecting the cells,the effect of ox-LDL on the expression of H1 R m RNA and protein in endothelial cells was detected by RT-q PCR and western blot,respectively.3.Divide the cultured HAECs cells into four groups: a.blank control group;b.10?g/ml ox-LDL for 24 h;c.10 ?g/ml ox-LDL + 100 ?M with loratadine for 24 h;d.10 ?g/ml ox-LDL + 250 ?M loratadine for 24 h.Then the following experiments were carried out:(1)Calcein AM labeled mononuclear cells THP-1 were inoculated into HAECs.Two hours later,the non-adherent monocytes were washed.After fixation,THP-1adhered to HAECs were observed under a fluorescent microscope.(2)Collect the culture medium of HAECs cells in each group and detect the expression of cytokines TNF-?,IL-6 and IL-8 by ELISA.(3)The total RNA and protein of HAECs cells were extracted,the m RNA expression of VCAM,E-selectin,TNF-?,IL-6 and IL-8 were detected by RT-q PCR,and the protein expression of VCAM and E-selectin were detected by western blotting.(4)The HAECs cells were labeled with DCF-DA,then the green fluorescence signal was detected at the maximum excitation wavelength of 480 nm and the maximum emission wavelength of 525 nm by fluorescence microscope,and the fluorescence intensity was quantified by Image J software.Result:1.The expression of H1 R m RNA and protein in HAECs is comparable to that of JAR cells.2.In HAECs,compared with the control group without ox-LDL treatment,ox-LDL can significantly increase the expression of H1 R m RNA and protein in a dose-dependent manner.3.Upon ox-LDL treatment,there was a 3.5-fold increase in binding of monocytes to endothelial cells.However,two different doses of loratadine significantly attenuated this interaction,with 100 and 250 ?M lortadine causing 40% and 60%reductions in binding of monocytes to endothelial cells,respectively.4.Compared with basal conditions,ox-LDL treatment resulted in a 6-fold increase in the expression of VCAM-1,while the presence of 100 and 250 ?M loratadine ameliorated this effect by 25% and 50%,respectively.Meanwhile,loratadine had an equivalent inhibition on E-selectin,another vascular adhesion molecule.Compared to basal levels,ox-LDL treatment resulted in a considerable increase in the expression of VCAM-1 and ICAM-1,while treatment with loratadine at 100 and 250 ?M suppressed expression of these molecules by 28% and 70%,respectively.5.Ox-LDL treatment resulted in a 5 to 7-fold increase in the induction of TNF-?,IL-6and IL-8 m RNA.The addition of 100 and 250 ?M loratadine resulted in 30% and60% inhibition of ox-LDL action in a dose-dependent manner.Furthermore,loratadine exerted a very similar inhibitory effect on protein expression of TNF?,IL-6 and IL-8 induced by ox-LDL6.Compared to basal levels,ox-LDL increased the level of ROS signalling by3.9-fold,while treatment with 100 and 250 ?M loratadine suppressed ox-LDL-induced production of ROS by 30% and 54%,respectively.Conclusion:1.The expression abundance of H1 R in HAECs was high,and ox-LDL can stimulate the induction of H1 R expression in endothelial cells,which indicates that H1 R is related to the effect of ox-LDL.2.In HAECs,ox-LDL can induce monocyte adhesion,the expression of adhesion molecules VCAM-1 and E-selectin,the expression of cytokines TNF-?,IL-6 and IL-8,and the production of ROS.And loratadine can inhibit the above-mentioned cell adhesion,oxidative stress and inflammation induced by ox-LDL,that is,loratadine can ameliorate endothelial dysfunction induced by ox-LDL.Part 2 Study on the molecular mechanism of the biological function of loratadinePurpose:The purpose of this part was to study the effect of loratadine on activation of JNK,AP1 and NF-? B signaling pathway induced by ox-LDL in endothelial cells.By analyzing the molecular mechanism of loratadine in improving ox-LDL-induced endothelial dysfunction,we provided a theoretical basis for further elucidating the pathogenesis,diagnosis and treatment of AR.Method:1.Divide the cultured HAECs cells into four groups: a.blank control group;b.10?g/ml ox-LDL for 24 h;c.10 ?g/ml ox-LDL + 100 ?M with loratadine for 24 h;d.10 ?g/ml ox-LDL + 250 ?M loratadine for 24 h.Then the following experiments were carried out:(1)The total protein of cells was obtained by RIPA lysate.The expression of JNK,p-JNK,I?B? and P-I?B? in HAECs cells was detected by western blot.Then p-JNK/JNK and P-I?B?/ I?B?were used to indicate the activation of JNK and I?B?by ox-LDL,respectively.(2)The cells were collected and lysed with hypotonic buffer to remove the cytoplasmic components and obtain the nuclear protein.The expression of c-fos,c-jun and p65 protein in each group of HAECs cells was detected by western blot.2.Detection of AP1 and NF-?B promoter activity: firefly luciferase reporter gene plasmids containing NF-? B or AP-1 promoters and marine kidney luciferase reporter gene plasmids(as internal reference)were co transfected into HAECs cells with Lipofectamine 2000 transfection reagent;after 24 hours of transfection,the cells were divided into four groups: a.blank control group;b.10 ?g/ml ox-LDL;c.10 ?g/ml ox-LDL + 100 ?M with loratadine;d.10 ?g/ml ox-LDL +250 ?M loratadine.Cells were collected and lysed after 24 h,and the fluorescence of firefly luciferase and renilla luciferase were measured in turn.The relative luciferase activity was calculated using the firefly luciferase activity/renilla luciferase activity,which represents the promoter activities of NF-?B and AP-1,respectively.Result:1.Ox-LDL resulted in a 3-fold increase in the ratio of p-JNK/JNK on average.However,the presence of 100 and 250 ?M loratadine suppressed JNK activity by40% and 60%.2.There was a 3 to 4-fold increase in the induction of the two subunits of AP-1,c-fos and c-Jun,upon ox-LDL treatment.Notably,the presence of 100 ?M loratadine blocked the induction of c-Jun and c-Fos by about half,and 250 ?M loratadine completely abolished the influence of ox-LDL on c-Jun and c-Fos.3.When c-Jun/c-Fos containing AP1 promoter was transfected into cells,there was a38% and 72% reduction in ox-LDL-induced promoter induction as compared with basal conditions4.Compared with basal levels,ox-LDL induced a 3.5-fold increase in the ratio of p-I?B? to I?B?,while the presence of 100 and 250 ?M loratadine abolished this effect by 40% and 60%,respectively.5.ox-LDL caused a 3-fold increase in the accumulation of nuclear p65 as comapred to basal conditions,which was abolished by 30% and 50% in the presence of 100 and 250 ?M loratadine,respectively.6.When NF-?B promoter was introduced into cultured endothelial cells,ox-LDL activated a 45-fold increase in promoter activity.However,the presence of 100 and 250 ?M loratadine abolished this effect by 50% and 75%.Conclusion:1.Ox-LDL can activate the AP1 signaling pathway by enhancing JNK activation,AP1 promoter activity and nuclear accumulation of AP1 in endothelial cells.Loratadine can inhibit the activation of AP1 signaling pathway induced by ox LDL in a dose-dependent manner.2.Ox-LDL can activate the NF-?B signaling pathway by enhancing the activation of I?B?,the activation of NF-?B promoter and the nuclear accumulation of p65.Loratadine can inhibit the activation of NF-?B signaling pathway induced by ox LDL in a dose-dependent manner.3.Therefore,loratadine can improve ox-LDL-induced endothelial dysfunction by inhibiting ox-LDL-activated endothelial cell AP1 and NF-?B signaling pathways.Full text conclusion H1R antagonist loratadine can improve ox-LDL-induced endothelial cell adhesion,oxidative stress and inflammation.The molecular mechanism of H1 R involved inhibition of ox-LDL-activated AP1 and NF-?B signaling pathways in endothelial cell.Thus the mechanism of H1 R involvement in AS endothelial dysfunction was elucidated in the present study. |
PDF Full Text Request