PCSK9抑制剂依洛尤单抗对缺氧诱导的血管内皮细胞损伤的影响及其机制OA

Objective To investigate the effects and mechanism of PCSK9 inhibitor Evolocumab(evc)on hypoxia-induced human umbilical vein endothelial cells(HUVECs)injury.Methods HUVECs were cultured in vitro and divided into seven groups:control group,control+evc group,hypoxia group,hypoxia+evc group,hypoxia+evc+LPS(a TLR4 activator)group,hypoxia+evc+KLA(a TLR4 specific agonist)group,and hypoxia+evc+TAK-242(a TLR4 inhibitor)group.The cells were given no treatment in control group;the cells were cultured under hypoxia for 4 h in hypoxia group;the cells in hypoxia+evc group were treated with Evolocumab at final concentration of 10 μg/mL for 1 h and then hypoxia for 4 h;the cells in hypoxia+evc+LPS group,hypoxia+evc+KLA group,and hypoxia+evc+TAK-242 group were treated with 10 μg/mL Evolocumab combined with 0.5 μg/mL LPS,10 μg/mL KLA,or 100 μg/L TAK-242 for 1 h,and then hypoxia for 4 h,respectively.Protein immunoblotting was used to detect the expressions of PCSK9,TLR4,NF-κB-p65,and p-NF-κB-p65.Flow cytometry was employed to assess the apoptosis rate,and cell scratch assay was used to evaluate the cell migration ability.Protein immunoblotting or enzyme-linked immunosorbent assay(ELISA)was performed to detect the protein levels of inflammatory factors IL-6 and TNF-α.Transmission electron microscopy was used to observe mitochondrial ultrastruc-ture(membrane,cristae,matrix).Results Compared with control group,PCSK9 protein expression,apoptosis rate,IL-6 protein and TNF-α protein levels,and protein expressions of TLR4,NF-κB-p65,and p-NF-κB-p65 increased in hypoxia group,while the migration rate decreased(all P<0.05),and more severe mitochondrial ultrastructural damage was found.Compared with hypoxia group,PCSK9 protein expression,the apoptosis rate,IL-6 and TNF-α protein levels,and protein expressions of TLR4,NF-κB-p65,and p-NF-κB-p65 increased in hypoxia+evc group,while the migration rate increased(all P<0.05),and the mitochondrial ultrastruc-tural damage was improved.Compared with hypoxia+evc group,the protein expressions of TLR4,NF-κB-p65,and p-NF-κB-p65,the apoptosis rate,and IL-6 and TNF-α protein levels increased in hypoxia+evc+LPS group and hypoxia+evc+KLA group,while the migration rate decreased(all P<0.05).Compared with hypoxia+evc group,more severe mitochondrial ultrastructural damage was observed in hypoxia+evc+KLA group.Compared with hypoxia+evc group,the expressions of TLR4,NF-κB-p65,and p-NF-κB-p65 proteins,the apoptosis rate,and IL-6 and TNF-α protein levels decreased in hypoxia+evc+TAK-242 group,while the migration rate increased(all P<0.05).Conclusion Evolocumab can down-regulate PCSK9 protein expression,apoptosis and inflammatory factors in hypoxia-induced HUVECs,enhance the cell migration ability and improve the mitochondrial ultrastructural damage by blocking the TLR4/NF-κB pathway.