丁苯酞对糖氧剥夺条件下人脑微血管内皮细胞炎症反应的调节作用

Regulate Effect of Butylphthalide on the Inflammatory Response of HBMEC under Glucose and Oxygen Deprivation

目的研究丁苯酞调节糖氧剥夺(oxygen-glucosedeprivation,OGD)条件下人脑微血管内皮细胞(humanbrain microvascularendothelialcells,HBMEC)炎症反应的机制。方法采用OGD方法处理HBMEC,将细胞分为对照组、模型组和丁苯酞低、中、高剂量组。其中对照组为正常培养的HBMEC,模型组为OGD处理的细胞,低剂量组为OGD+5μmol·L^(-1)的丁苯酞,中剂量组为OGD+10μmol·L^(-1)的丁苯酞,高剂量组为OGD+20μmol·L^(-1)的丁苯酞。采用流式细胞术检测细胞凋亡水平,CCK-8法检测细胞活力的变化,Western-Blot法检测细胞中HMGB1、TLR4、NF-κB(p-P65)、caspase-1和procaspase-1的表达水平,免疫酶联法检测上清中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α),白细胞介素6(IL-6)的分泌水平。实时荧光定量PCR检测HMGB1、TLR4、NF-κB(P65)、caspase-1、IL-1β的mRNA表达。结果 OGD处理可以诱导HBMEC活力下调和HBMEC细胞凋亡,且具有时间依赖性,相比对照组具有显著性差异(P<0.05);而丁苯酞干预后,HBMEC细胞活力比模型组显著上调(P<0.05),细胞凋亡率比模型组显著下降(P<0.05)。OGD处理可以导致HBMEC中HMGB1-TLR4-NF-κB信号的激活,诱导炎症因子caspase-1、IL-1β的表达上调,相比对照组具有显著性差异(P<0.05);而丁苯酞干预可以显著下调HMGB1-TLR4-NF-κB信号的表达,下调caspase-1、IL-1β的表达。结论丁苯酞可能通过抑制HMGB1-TLR4-NF-κB信号的表达调节OGD下HBMEC细胞炎症反应。

OBJECTIVE To study the effect of butylphthalide(NBP)on the inflammatory response of human brain microvascular endothelial cells(HBMEC)under the condition of glucose and oxygen deprivation(OGD).METHODS HBMEC cells were treated by OGD.The cells were divided into control group,model group,low,middle high dose of NBP group.The control group was normal cultured,the model group were OGD treated,the low dose group was OGD+5 mol·L-1 NBP,the middle dose group was OGD+10 mol·L-1 NBP,and the high dose group was OGD+20 mol·L-1 NBP.The cell apoptosis was detected by flow cytometry.The changes of cell vitality were detected by CCK-8.The expression of HMGB1,TLR4,NF-κB(p-P65),caspase-1 and procaspase-1 were detected by Western-Blot.The secretion level of interleukin 1β(IL-1β),tumor necrosis factorα(TNF-α)and interleukin 6(IL-6)in supernatant were detected by immunoenzyme linked immunosorbent assay.The mRNA expression of HMGB1,TLR4,NF-κB(P65),Caspase-1 and IL-1βwere detected by real time fluorescence quantitative PCR.RESULTS OGD treatment could induce HBMEC activity and apoptosis of HBMEC to be down regulated and time-dependent,which was significantly different from that of the control group(P<0.05).After NBP treatment,the activity of HBMEC was significantly higher than that of the model group(P<0.05),and the apoptotic rate was decreased significantly compared with the model group(P<0.05).OGD treatment could lead to the activation of HMGB1-TLR4-NF-κB signal in HBMEC,and the expression of inflammatory factors caspase-1 and IL-1βwas up-regulated.Compared with the control group,there was a significant difference(P<0.05).NBP could reduce the expression of HMGB1-TLR4-NF-NF-κB signal and down regulate the expression of caspase-1 and IL-1β.CONCLUSION NBP may regulate the inflammatory response of HBMEC cells under OGD by inhibiting the expression of HMGB1-TLR4-NF-κB signaling.