Investigation of the impact of dibutyl phthalate on oxygen-glucose deprivation-induced human brain micro-vascular endothelial cell injury based on the TLR4/MyD88/NF-KB pathway
Objective To investigate the impact of dibutyl phthalate on oxygen-glucose deprivation-induced human brain microvascular endothelial cell injury based on the TLR4/MyD88/NF-κB pathway.Methods Human brain microvascular endothelial cells(HBMECs)were randomly divided into control group,oxygen-glucose depriva-tion group(OGD group),dibutyl phthalate low,medium,high dose groups(NBP-L,M,H groups),and dibutyl phthalate high dose+pcDNA3-TLR4 group(NBP-H+pcDNA3-TLR4 group).Cell proliferation,invasion,and migra-tion abilities were assessed using CCK-8 assay,Transwell assay,and scratch assay,respectively.The levels of oxi-dative stress markers(SOD,MDA)and inflammatory cytokines(IL-6,IL-8,TNF-a)in the cells were measured by ELISA.Protein expression of TLR4,MyD88,p-NF-KB,and NF-κB in the cells was detected using Western blot-ting.Results Compared with the control group,the OGD group showed significantly reduced cell survival rate(41.26％±3.78％),invasive cell count(95.36±7.81),scratch healing rate(41.16％±3.21％),and SOD activity(8.71±0.69),while malondialdehyde(MDA)content(65.42±5.69),IL-6(89.41±7.52),IL-8(83.91±7.86),and tumor necrosis factor-alpha(TNF-α)(95.67±8.62)levels were significantly increased(P＜0.05).Compared with the OGD group,the NBP-L,M,and H groups showed significantly increased cell survival rate,invasive cell count,scratch healing rate,and SOD activity,as well as significantly decreased MDA content,IL-6,IL-8,and TNF-α lev-els,and these effects were dose-dependent(P＜0.05).Compared with the NBP-H group,the NBP-H+pcDNA3-TLR4 group showed significantly reduced cell survival rate(46.34％±3.89％),invasive cell count(101.43±8.02),scratch healing rate(45.33％±3.46％),and SOD activity(9.40±0.73),while MDA content(59.16±5.43),IL-6(80.89±7.41),IL-8(80.51±7.46),and TNF-α levels(90.81±7.40)were significantly increased(P＜0.05).Compared with the Control group,the OGD group showed significantly increased expression of Toll-like receptor 4(TLR4)(1.45±0.14),myeloid differentiation primary response 88(MyD88)protein(1.32±0.14),and phosphorylated nuclear fac-tor kappa B(p-NF-κB)/NF-κB ratio(1.18±0.12)(P＜0.05).Compared with the OGD group,the NBP-L,M,and H groups showed significantly reduced expression of TLR4 and MyD88 protein and p-NF-κB/NF-κB ratio,and these effects were dose-dependent(P＜0.05).The NBP-H+pcDNA3-TLR4 group showed significantly higher expression of TLR4,MyD88 protein,and p-NF-κB/NF-κB ratio compared to the NBP-H group(P＜0.05).Conclusion NBP can improve the injury of human brain microvascular endothelial cells induced by oxygen-glucose deprivation,and its mechanism of action may be related to the regulation of the TLR4/MyD88/NF-κB signaling pathway.
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