Study On Shh/NF-κB In Endothelial Injury And Atherosclerosis

Objective:Sonic hedgehog（Shh）,an evolutionally-highly conserved morphological factor whose maturation、transportation and function are closely related to cholesterol.Endothelial cell injury plays an important role in the development and progression of Atherosclerosis（AS）.However,the role of Shh in endothelial cell injury and atherosclerosis and its mechanism have not been fully elucidated.Here,gene chip used to analysis the Shh expression.In vivo,high fat feed Apolipoprotein E knockout(ApoE^-/-)mice and in vitro,the oxidized low density lipoprotein（ox-LDL）simulated human umbilical vein endothelial cells（HUVECs）construct models of atherosclerosis then observe the role of Shh in atherosclerosis and possible molecular mechanisms,provide new ideas for anti-atherosclerosis and explore the clinical application value of Shh.Methods:1.The expression of different Genes in aortic tissues of atherosclerotic mice was analyzed by Gene microarray.Kyoto Encyclopedia of Genes and Genomes（KEGG）and Gene Set Enrichment Analysis（GSEA）was used to observe the expression of Hedgehog signaling pathway.The ApoE^-/-mice treatment by high-fat diet and HUVECs induced by ox-LDL,then real-time quantitative polymerase chain reaction（RT-qPCR）and Western blot（WB）were used to verify the results of gene chips.2.In HUVECs,after plasmid encoding human Shh gene（phShh）,recombinant Shh protein（rShh）and Shh siRNA pretreatment,ox-LDL stimulated for 24 h,and the effects of Shh overexpression and knockout on endothelial cell injury were observed through the expression of reactive oxygen species（ROS）,endothelial nitric oxide synthase（eNOS）,nitric oxide（NO）,intercellular adhesion molecule 1（ICAM-1）and vascular cell adhesion molecule 1（VCAM-1）.Apoptotic cells were detected by Flow Cytometer,Caspase 3 activity,and Cleaved Caspase 3,Bcl-2 and Bax protein expression.3.The binding of Shh protein to NF-κB p65 was observed by protein immunoprecipitation.The effects of plasmid encoding human Shh gene（phShh）,recombinant Shh protein（rShh）on the phosphorylated proteins of IKKα/β、IκBαand NF-κB p65 were detected by WB,and the effects of recombinant Shh protein on the activity of NF-κB p65 were observed by nuclear protein.It suggested that Shh could play a protective role of endothelium by inhibiting the activation of NF-κB signaling pathway.4.Construction of ApoE^-/-mice atherosclerosis model and lentivirus intervention treatment,according to the experiment aim were randomly divided into the Chow group,High fat diet group（HFD group）,high fat diet+lentivirus empty carrier intervention group（NC group）,high fat diet+Shh lentivirus group（Lv-Shh group）and a high fat diet+Shh sh RNA lentivirus group（Sh-Shh group）,lentivirus was injected by tail vein,2×10⁷ TU/mouse,the effects of high fat diet and Shh intervention on weight,blood lipid,plaque size,eNOS,NO,lung dry and wet weight and ICAM-1,VCAM-1 were observed.Results:1.Gene chip analysis revealed the downregulation of Hedgehog signaling pathway in atherosclerosis.RT-qPCR and WB showed that mRNA and protein expressions of Shh were down-regulated in aortic tissues of the high fat diet group compared with the chow group.After the stimulation of HUVECs with ox-LDL,the mRNA and protein expressions of Shh were down-regulation gradually with the increase of ox-LDL concentration.Shh expression was down-regulated in atherosclerosis and ox-LDL-induced endothelial injury.2.After treatment with HUVECs by plasmid encoding human Shh gene（phShh）,recombinant Shh protein（rShh）and Shh siRNA,ox-LDL was stimulated for 24hours:（1）Compared with the ox-LDL group,ROS expression was down-regulated after phShh and rShh overexpression and Shh siRNA induced ROS expression up-regulated.（2）Compared with the ox-LDL group,phShh and rShh promoted the expression of NO and eNOS,and Shh siRNA inhibited the expression of NO and eNOS.（3）Compared with the ox-LDL group,ICAM-1 and VCAM-1 expression was down-regulated after phShh and rShh overexpression and Shh siRNA induced ICAM-1 and VCAM-1 expression up-regulated.（4）Overexpression of Shh by phShh and rShh,apoptotic cells were reduced,Caspase 3 activity and Cleaved caspase 3 expression were down-regulated compared with the ox-LDL treated group.（5）Ox-LDL induced down-regulation of Bcl-2,up-regulation of Bax,and down-regulation of Bcl-2/Bax ratio.Ph Shh and rShh inhibited ox-LDL-induced down-regulation of Bcl-2 and up-regulation of Bax.The above results preliminarily showed that Shh can reduce ox-LDL-induced endothelial oxidative stress,inflammatory response and mitochondria-mediated apoptosis,and improve endothelial cell injury.3.Relationship between Shh and NF-κB signaling pathway:（1）The interaction between Shh protein and NF-κB p65 protein was confirmed by immunoprecipitation.（2）Compared with the ox-LDL group,phShh and rShh inhibited the phosphorylation levels of IKKα/β,IκBαand NF-κB p65 proteins.（3）Compared with the ox-LDL group,Shh recombinant protein reduced the level of NF-κB p65 protein in the nucleus and inhibited the nuclear translocation of NF-κB p65.4.High fat feeding induced atherosclerosis in ApoE^-/-mice after lentivirus intervention:（1）Compared with the Chow group,the weight of the HFD group increased,the serum levels of T-CHO,TG and LDL-C were up-regulated,and HDL-C was down-regulated.However,in ApoE^-/-mice fed with high fat,there was no statistical difference in body weight and lipid level between the HFD group,NC group,Lv-Shh group and Sh-Shh group.（2）Oil red O staining showed that the plaque area of the HFD group increased significantly compared with the Chow group.Compared with the NC group,the aortic gross plaque area of mice after Lv-Shh intervention was significantly reduced,and the plaque area of mice after Sh-Shh lentivirus intervention was significantly increased.（3）Compared with the Chow group,the high fat diet reduced serum NO levels and inhibited eNOS mRNA and protein expression in aortic tissues.Compared with the NC group,Lv-Shh lentivirus intervention promoted eNOS and NO synthesis,and Sh-Shh lentivirus intervention inhibited eNOS and NO synthesis.（4）Compared with the Chow group,the high fat diet induced an increase in lung wet weight and an up-regulation of lung wet weight/lung dry weight ratio.Compared with the NC intervention group,the lung wet weight in Lv-Shh group was down-regulated and the lung wet weight/lung dry weight ratio was down-regulated,while the lung wet weight in Sh-Shh group was up-regulated and the lung wet weight/lung dry weight ratio was up-regulated.（5）Compared with the Chow group,the high fat diet induced ICAM-1 and VCAM-1mRNA expression in aortic tissues.Compared with the NC group,Lv-Shh lentivirus intervention inhibited ICAM-1 and VCAM-1 expression,and Sh-Shh lentivirus intervention promoted ICAM-1 and VCAM-1 expression.It has been preliminarily confirmed in vivo that Shh can regulate the vasoconstriction and relaxation function,inhibit vascular inflammation,and play an anti-atherosclerosis role.Conclusion:The results of this study confirmed that Shh expression was down-regulated in atherosclerotic aortic tissue and endothelial cell injury.Shh inhibited oxidative stress,inflammation and mitochondrial mediated apoptosis of endothelial cells significantly.The mechanism may be related to the inhibition of downstream endothelial cell injury and apoptosis regulated by NF-κB signaling pathway after activation of Shh,thereby reducing the formation of atherosclerosis.Therefore,the Shh/NF-κB pathway is likely to be a pathway target for the prevention and treatment of atherosclerotic diseases.