基于TLR4/MyD88/NF-κB通路探讨丁苯酞对氧糖剥夺诱导的人脑微血管内皮细胞损伤的影响

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⁃κB pathway

目的 基于TLR4/MyD88/NF-κB通路探讨丁苯酞对氧糖剥夺诱导的人脑微血管内皮细胞(HBMEC)损伤的影响。方法 采用随机数字表法,将HBMEC分为对照组(Control组)、糖氧剥夺组(OGD组)、丁苯酞低、中、高剂量组(NBP-L、M、H组)和丁苯酞高剂量+pcDNA3-TLR4组(NBP-H+pcDNA3-TLR4组)。采用CCK-8、Transwell法和划痕实验分别检测细胞增殖、侵袭和迁移能力;ELISA检测细胞中氧化应激指标(SOD、MDA)和炎症因子水平(IL-6、IL-8、TNF-α);蛋白质印迹法检测细胞中TLR4、MyD88、p-NF-κB、NF-κB蛋白表达。结果 和Control组相比,OGD组细胞存活率(41.26%±3.78%)、侵袭细胞数(95.36±7.81)、划痕愈合率(41.16%±3.21%)、细胞中SOD活性(8.71±0.69)均明显降低,细胞中MDA含量(65.42±5.69)、IL-6(89.41±7.52)、IL-8(83.91±7.86)、TNF-α(95.67±8.62)含量明显升高(P<0.05);和OGD组相比,NBP-L、M、H组细胞存活率、侵袭细胞数、划痕愈合率、细胞中SOD活性均明显增加,细胞中MDA含量、IL-6、IL-8、TNF-α含量明显降低,且呈剂量依赖(P<0.05);和NBP-H组相比,NBP-H+pcDNA3-TLR4组细胞存活率(46.34%±3.89%)、侵袭细胞数(101.43±8.02)、划痕愈合率(45.33%±3.46%)、细胞中SOD活性(9.40±0.73)均明显降低,细胞中MDA含量(59.16±5.43)、IL-6(80.89±7.41)、IL-8(80.51±7.46)、TNF-α含量(90.81±7.40)明显升高(P<0.05)。和Control组相比,OGD组细胞中TLR4(1.45±0.14)、MyD88蛋白(1.32±0.14)表达及p-NF-κB/NF-κB比值(1.18±0.12)均明显升高(P<0.05);和OGD组相比,NBP-L、M、H组细胞中TLR4、MyD88蛋白表达及p-NF-κB/NF-κB比值均含量明显降低,且呈剂量依赖(P<0.05);NBP-H+pcDNA3-TLR4组细胞中TLR4(1.39±0.13)、MyD88蛋白(1.26±0.13)表达及p-NF-κB/NF-κB比值(1.02±0.11)明显高于NBP-H组(P<0.05)。结论 丁苯酞可改善氧糖剥夺诱导的HBMEC损伤,其作用机制可能和调控TLR4/MyD88/NF-κB信号通路有关。

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-α)in the cells were measured by ELISA.Protein expression of TLR4,MyD88,p-NF-κB,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-αlev-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-αlevels(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.