| Background: Atherosclerosis(AS),a kind of chronic pathological manifestation,is characterized by abnormal lipid accumulation in arterial wall.The cardiovascular and cerebrovascular diseases caused by AS are threatening people’s health and lives.Among them,endothelial dysfunction(ED)has been recognized as one of initial processes in the progression of AS.Therefore,improvement and prevention of ED is the research hotspot and difficult points in treatment and prevention of AS.SelS,as a single transmembrane selenoprotein,is located on the endoplasmic reticulum membrane.Sel S is characteristically expressed in various organs including liver,adipose tissue,skeletal muscle,kidney,islet and blood vessel.The biological functions of SelS include reduction of endoplasmic reticulum stress,resistance to oxidative stress,regulation of inflammation,and roles in glycolipid metabolism.Among inflammatory factors,TNF-α is a one of the key factors which induce ED in metabolic and inflammatory diseases.At present,the effect of SelS on vascular endothelial inflammatory injury remains unknown.Thus,in this study,we explored the effect and relevant mechanism of SelS on TNF-α induced ED.Methods:The low-density lipoprotein receptor knockout(LDLR KO)mice were fed with high fat diet(HFD)for 16 weeks.The level of EGF-like module-containing mucin-like hormone receptor-like 1(EMR1,also named as F4/80),tumor necrosis factor receptor-1(TNFR-1),phosphorylated nuclear factor-κB p65(p-NF-κB p65)and SelS were tested in the thoracic aorta in mice using immunohistochemistry.Human umbilical vein endothelial cells(HUVECs)were treated with exogenous recombinant human TNF-α and related pathway inhibitors.TNF-α-induced ED and inflammatory injury were observed and the molecular mechanism of TNF-α-induced endothelial injury was determined via series of methods,including real-time quantitative polymerase chain reaction and western blot.In order to regulate SelS expression in HUVECs,the pcDNA3.1-SelS recombinant plasmid was constructed by recombinant DNA technology and SelS specific small-interference RNAs were constructed by siRNA interference technology.Then,the effect and mechanism of SelS on TNF-α-induced endothelial injury were examined.Results: The expression of inflammatory indicators and SelS were increased in arotic AS plaques of LDLR KO mice fed with HFD: compared with LDLR KO mice fed with regular chow diet,F4/80 expression was increased(0.11±0.01 vs 0.06±0.01,p<0.01),TNFR-1 expression was increased(0.010±0.004 vs 0.003±0.003,p<0.01),p-NF-κB p65 expression was increased(0.010±0.004 vs 0.003±0.002,p<0.01),and SelS expression were up-regulated(0.06±0.02 vs 0.010±0.004,p<0.01)in the aortic AS plaques of LDLR KO mice fed with HFD.TNF-α induced vascular endothelial dysfunction: compared to control group,the cell viability was decreased [(53.93±11.20)(%)vs(93.42±7.63)(%),p<0.01],nitric oxide(NO)content was decreased(1.07±0.22 mmol/ml vs 2.12±0.28 mmol/ml,p<0.01),endothelial nitric oxide synthase(eNOS)mRNA level were reduced(0.56± 0.11 vs 1.01 ± 0.16,p < 0.05),and eNOS protein expression was reduced(0.55±0.17 vs 2.17±0.23,p<0.01)in TNF-α group.Whereas,the endothelin-1(ET-1)mRNA level was increased(6.45±1.06 vs 0.93±0.64,p<0.01),and the release of reactive oxygen species(ROS)were increased(220.36±40.95 vs 117.66±16.27,p<0.01)in TNF-α group compared to control group.TNF-α induced vascular endothelial inflammatory injury: compared with control group,the amount of THP-1 adhesion was increased(554±111.45 vs 110±13.45,p<0.01),the level of supernatant intercellular adhesion molecule(s ICAM-1)was increased(56.97±8.12 pg/ml vs 12.23±4.43 pg/ml,p<0.01),supernatant vascular cell adhesion molecule-1(sVCAM-1)level was increased(150.13±22.30 pg/ml vs 17.56±9.08 pg/ml,p<0.01),ICAM-1 mRNA expression was elevated(12.31±2.01 vs 1.03±0.66,p<0.01),VCAM-1 mRNA expression was elevated(25.74±4.54 vs 0.90±0.63,p<0.01),interlukin-6(IL-6)mRNA expression was elevated(4.76±1.06 vs 1.00±0.49,p<0.01),interlukin-1β(IL-1β)mRNA expression was elevated(3.00±0.45 vs 1.00±0.45,p<0.01),interlukin-8(IL-8)mRNA expression was elevated(3.39±0.53 vs 1.07±0.22,p<0.01),and monocyte chemoattractant protein-1(MCP-1)m RNA expression was elevated(5.61±1.18 vs 0.97±0.21,p<0.01)in HUVECs in TNF-α group.The relationship between TNF-α induced vascular endothelial injury and the activation of p38 mitogen-activated protein kinase(p38 MAPK)and nuclear factor-κB(NF-κB)signaling pathways:compared with control group,p-p38 MAPK protein expression was increased(1.23±0.27 vs 0.29±0.12,p<0.01),p-c-jun protein expression was increased(0.85±0.15 vs 0.11±0.07,p < 0.01),phosphorylated inhibitory kappa B α(p-IκBα)protein expression was increased(0.87±0.14 vs 0.28±0.17,p<0.01),phosphorylated IκB kinase β(p-IKKβ)protein expression was increased(0.61±0.13 vs 0.17±0.22,p<0.05),cytoplasmic NF-κB p65 was reduced(0.39±0.17 vs 1.22±0.24,p<0.01),and the expression of nuclear NF-κB p65 was increased(1.32±0.24 vs 0.69±0.16,p<0.01)in HUVECs in TNF-α group.Morever,compared with TNF-α group,ICAM-1 mRNA level was reduced(0.10±0.09 vs 0.95±0.17,p<0.01),VCAM-1 mRNA level was reduced(0.59±0.24 vs 2.56±0.35,p<0.01),IL-6 mRNA level was reduced(2.66±0.74 vs 5.24±0.96,p<0.01),IL-1β mRNA level was reduced(1.47±0.69 vs 3.29±0.69,p<0.01),MCP-1 mRNA level was reduced(2.33±0.83 vs 7.73±0.10,p<0.01),and IL-8 mRNA level was reduced(1.30±0.23 vs 3.94±0.72,p<0.01)in HUVECs treated with p38 MAPK pathway inhibitor(SB203580).Compared with TNF-α group,ICAM-1 mRNA level was decreased(0.10±0.09 vs 0.95±0.17,p<0.01),VCAM-1 mRNA level was decreased(0.08±0.09 vs 2.56±0.35,p<0.01),IL-6 mRNA level was decreased(1.81±0.76 vs 5.24±0.96,p<0.01),IL-1β mRNA level was decreased(1.19±0.66 vs 3.29±0.69,p<0.01),MCP-1 mRNA level was decreased(1.85±1.23 vs 7.73±0.10,p<0.01),and IL-8 mRNA level was decreased(1.32±0.32 vs 3.94±0.72,p<0.01)in HUVECs treated with NF-κB pathway inhibitor(S2882).The effect and mechanism of SelS overexpression on TNF-α induced vascular endothelial injury: compared with TNF-α+E-Vector group,the cell viability was increased [(81.76±1.47)(%)vs(51.37±11.30)(%),p<0.01],NO content was increased(1.71±0.31 mmol/ml vs 1.08±0.26 mmol/ml,p<0.01),eNOS mRNA level was elevated(0.81±0.10 vs 0.53±0.18,p<0.05),and eNOS protein level was elevated(1.30±0.22 vs 0.50±1.06,p<0.01)in HUVECs in TNF-α+pc-SelS group.Whereas,the ET-1 mRNA level was reduced(3.64±0.99 vs 6.65±1.05,p<0.01),and the release of ROS was reduced(126.20±31.72 vs 221.63±40.27,p<0.01)in TNF-α+pc-SelS group compared to TNF-α+E-Vector group.Compared with TNF-α+E-Vector group,the amout of THP-1 adhesion was decreased(297±105.87 vs 554±111.45,p<0.01),s ICAM-1 level was decreased(32.11±10.17 pg/ml vs 57.66±17.61 pg/ml,p<0.01),sVCAM-1 level was decreased(100.79±17.65 pg/ml vs 153.66±25.37 pg/ml,p<0.01),ICAM-1 mRNA level was decreased(5.34±2.40 vs 12.31±2.42,p<0.01),VCAM-1 mRNA level was decreased(14.60±4.61 vs 26.41±4.56,p<0.01),IL-6 mRNA level was decreased(2.14±0.60 vs 4.83±0.98,p<0.01),IL-1β m RNA level was decreased(1.62±0.41 vs 2.98±0.42,p < 0.01),MCP-1 mRNA level was decreased(2.18±0.75 vs 5.62±1.08,p<0.01),IL-8 mRNA level was decreased(1.34±0.50 vs 3.27±0.82,p<0.01),p-p38 MAPK protein expression was reduced(0.59±0.17 vs 3.27±0.82,p < 0.01),p-c-jun protein expression was reduced(0.33±0.11 vs 0.86±0.15,p < 0.01),p-IKKβ protein expression was reduced(0.10±0.01 vs 0.61±0.13,p < 0.01),p-IκBα protein expression was reduced(0.42±0.11 vs 0.87±0.14,p<0.01),cytoplasmic NF-κB p65 expression was increased(0.90±0.12 vs 0.25±0.15,p<0.01),nuclear NF-κB p65 expression was declined(0.38±0.20 vs 1.17±0.18,p<0.01)in HUVECs in TNF-α+pc-SelS group.The effect and mechanism of SelS knockdown on TNF-α induced vascular endothelial injury: compared with TNF-α+Neg.RNA group,the cell viability was decreased [(26.57±7.62)(%)vs(52.27±13.17)(%),p<0.01],NO content was decreased(0.46±0.15 mmol/ml vs 1.02±0.23 mmol/ml,p<0.01),eNOS mRNA level was reduced(0.18±0.12 vs 0.52±0.15,p<0.01),and eNOS protein level was decreased(0.08±0.07 vs 0.63±0.22,p<0.01)in HUVECs in TNF-α+SelS siRNA group.Whereas,compared with TNF-α+Neg.RNA group,the ET-1 mRNA level was increased(9.35±1.03 vs 6.73±1.30,p<0.01),and the release of ROS was increased(352.20±31.82 vs 211.84±41.46,p<0.01)in TNF-α+SelS siRNA group compared to TNF-α+Neg.RNA group.Meanwhile,compared with TNF-α+Neg.RNA group,the amout of THP-1 adhesion was increased(1371±98.51 vs 554±102.69,p<0.01),s ICAM-1 level was increased(93.10±10.14 pg/ml vs 60.52±10.51 pg/ml,p<0.01),sVCAM-1 level was increased(276.73±20.06 pg/ml vs 164.00±18.62 pg/ml,p<0.01),ICAM-1 mRNA level was increased(25.75±5.49 vs 12.43±4.73,p<0.01),VCAM-1 mRNA level was increased(46.33±5.93 vs 25.42±7.74,p<0.01),IL-6 m RNA level was increased(8.71±1.44 vs 4.92±0.93,p<0.01),IL-1β mRNA level was increased(6.26±0.87 vs 3.10±1.03,p<0.01),MCP-1 mRNA level was increased(9.14±1.35 vs 5.32±1.57,p<0.01),IL-8 mRNA level was increased(5.25±1.21 vs 3.03±0.80,p<0.01),p-p38 MAPK protein expression was increased(1.87±0.23 vs 1.14±0.35,p<0.01),p-c-jun protein expression was increased(1.76±0.18 vs 1.15±0.30,p<0.01),p-IKKβ protein expression was increased(0.68±0.09 vs 0.20±0.05,p<0.01),p-IκBα protein expression was increased(1.20±0.27 vs 0.82±0.08,p<0.05),cytoplasmic NF-κB p65 expression was reduced(0.08±0.04 vs 0.63±0.20,p<0.01),and the expression of nuclear NF-κB p65 was increased(1.42±0.17 vs 0.78±0.22,p<0.01)in HUVECs in TNF-α+SelS siRNA group.Conclusions:The expression of SelS was increased in arotic AS plaques of LDLR KO mice fed with HFD,suggesting that SelS may participate in the AS process.TNF-α treatment induced vascular endothelial cell dysfunction and activation of p38 MAPK and NF-κB signaling pathways.TNF-α induced endothelial inflammatory injury was reduced by using p38 MAPK and NF-κB inhibitors.The results indicate that TNF-α induced endothelial injury through activation of p38 MAPK and NF-κB signaling pathways.TNF-α induced endothelial injury and the activation of p38 MAPK and NF-κB pathways was inhibited by SelS overexpression,and TNF-α induced endothelial injury and the activation of p38 MAPK and NF-κB pathways was enhanced by SelS knockdown.It has suggested that SelS could protect endothelial cells from TNF-α induced injury.Hence,Sel S may be a novel target for the prevention of vascular endothelial injury in AS,which is characterized by persistent existence of inflammation and endothelial dysfunction as its initial process. |
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