丁苯酞对Aβ_(1-42)诱导人脐静脉内皮细胞凋亡的保护作用及机制研究
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摘要
目的:研究丁苯酞对β-淀粉样蛋白1-42(Aβ_(1-42))诱导人脐静脉内皮细胞(HUVEC)凋亡的保护作用及其机制。方法:将HUVEC分为正常对照组、Aβ_(1-42)组、TAK242组(10 nmol/L)、二甲基亚砜(DMSO)组(1‰DMSO)和丁苯酞低、中、高浓度组(40、80、160μg/L),除正常对照组和DMSO组外,其余各组细胞均加入50μmol/L Aβ1_(1-42)培养HUVEC 24 h,同时TAK242组、DMSO组和丁苯酞低、中、高浓度组细胞还加入相应浓度的药物作用30 min,每个浓度3个复孔。CCK-8法测定细胞活力,Hochest 33342/PI双染法观察细胞凋亡情况,膜联蛋白(Annexin)Ⅴ-异硫氰酸荧光素(FITC)流式细胞仪检测细胞凋亡率,Western blot法检测细胞中果蝇样受体4(TLR4)、环氧合酶2(COX-2)蛋白表达,ELISA法检测细胞中白细胞介素1(IL-1)、肿瘤坏死因子α(TNF-α)含量。结果:与正常对照组比较,Aβ_(1-42)组细胞活力减少、凋亡率增加,TLR4、COX-2蛋白表达和IL-1、TNF-α含量增加;与Aβ_(1-42)组比较,TAK242组和丁苯酞低、中、高浓度组细胞活力增加、凋亡率减少,TLR4、COX-2蛋白表达和IL-1、TNF-α含量减少,差异均有统计学意义(P<0.05或P<0.01)。结论:丁苯酞可改善Aβ_(1-42)所致的HUVEC凋亡,其机制可能与抑制TLR4、COX-2和炎症因子表达有关。
OBJECTIVE:To study the protective effect of butylphthalide on the apoptosis of human umbilical vein endothelial cells(HUVECs) induced by Aβ_(1-42) and its mechanism. METHODS:HUVECs were divided into normal control group,Aβ_(1-42) group,TAK242 group(10 nmol/L),DMSO group(1‰DMSO)and butylphthalide low-concentration,medium-concentration and high- concentration groups(40,80,160 μg/L). Except for normal control group and DMSO group,other groups were given 50μmol/L Aβ_(1-42) to culture HUVECs for 24 h. TAK242 group,DMSO group and butylphthalide low-concentration,medium-concentration and high-concentration groups were given relevant concentration of drugs for 30 min,with 3 holes for each concentration. The cell viability was determined by CCK-8 assay;cell apoptosis was observed by Hochest 33342/PI double staining;the cell apoptotic rate was detected by Annexin Ⅴ- fluorescein isothiocyanate(FITC) flow cytometry;the protein expression of TLR- 4 and COX- 2were determined by Western blot assay;the contents of IL-1 and TNF-α were detected by ELISA. RESULTS:Compared with normal control group,cell viability of HUVECs were decreased in Aβ_(1-42)group;while apoptotic rate,protein expression of TLR4 and COX-2,the contents of IL-1 and TNF-α were increased. Compared with Aβ_(1-42)group,cell viability of HUVECs were increased in TAK242 group and butylphthalide low- concentration,medium- concentration and high- concentration groups;while apoptotic rate,protein expression of TLR4 and COX-2,the contents of IL-1 and TNF-α were decreased,with statistical significance(P<0.05 or P<0.01). CONCLUSIONS:Butylphthalide can reduce HUVECs apoptosis induced by Aβ_(1-42),which may be related with inhibiting the expression of TLR4,COX-2 and inflammatory factors.
OBJECTIVE:To study the protective effect of butylphthalide on the apoptosis of human umbilical vein endothelial cells(HUVECs) induced by Aβ_(1-42) and its mechanism. METHODS:HUVECs were divided into normal control group,Aβ_(1-42) group,TAK242 group(10 nmol/L),DMSO group(1‰DMSO)and butylphthalide low-concentration,medium-concentration and high- concentration groups(40,80,160 μg/L). Except for normal control group and DMSO group,other groups were given 50μmol/L Aβ_(1-42) to culture HUVECs for 24 h. TAK242 group,DMSO group and butylphthalide low-concentration,medium-concentration and high-concentration groups were given relevant concentration of drugs for 30 min,with 3 holes for each concentration. The cell viability was determined by CCK-8 assay;cell apoptosis was observed by Hochest 33342/PI double staining;the cell apoptotic rate was detected by Annexin Ⅴ- fluorescein isothiocyanate(FITC) flow cytometry;the protein expression of TLR- 4 and COX- 2were determined by Western blot assay;the contents of IL-1 and TNF-α were detected by ELISA. RESULTS:Compared with normal control group,cell viability of HUVECs were decreased in Aβ_(1-42)group;while apoptotic rate,protein expression of TLR4 and COX-2,the contents of IL-1 and TNF-α were increased. Compared with Aβ_(1-42)group,cell viability of HUVECs were increased in TAK242 group and butylphthalide low- concentration,medium- concentration and high- concentration groups;while apoptotic rate,protein expression of TLR4 and COX-2,the contents of IL-1 and TNF-α were decreased,with statistical significance(P<0.05 or P<0.01). CONCLUSIONS:Butylphthalide can reduce HUVECs apoptosis induced by Aβ_(1-42),which may be related with inhibiting the expression of TLR4,COX-2 and inflammatory factors.
引文
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[2]Hwang EH,Kim TH,Oh SM,et al.Toll/IL-1 domain containing adaptor inducing IFN-β(TRIF)mediates in nate immune responses in murine peritoneal mesothelia cells through TLR3 and TLR4 stimulation[J].Cytokine,2016,doi:10.1016/j.cyto.2015.11.010.
[3]Yu H,Yang M,Wang Y,et al.p75NTR is mainly respon sible for Aβtoxicity but not for its internalization:a pri mary study[J].Neurol Sci,2012,33(5):1043-1050.
[4]张晓璇,邱海鹏,赵淑敏.丁苯酞对脑缺血再灌注大鼠HSP70及TLR4的影响研究[J].中国免疫学杂志,2014,30(10):1401-1403.
[5]Ma JF,Wang HM,Li QY,et al.Starvation triggers Abe ta42 generation from human umbilical vascular endotheli al cells[J].FEBS Lett,2010,584(14):3101-3106.
[6]Zhang W,Wang LZ,Yu JT,et al.Increased expression of TLR2 and TLR4 on peripheral blood mononuclear cell from patients with Alzheimer’s disease[J].J Neurol Sci,2012,315(1/2):67-71.
[7]Gambuzza ME,Sofo V,Salmeri FM,Soraci L,et al.Toll like receptors in Alzheimer’s disease:a therapeutic per spective[J].CNS Neurol Disord Drug Targets,2014,13(9):1542-1558.
[8]Shi S,Liang D,Chen Y,et al.Gx-50 reducesβ-amy loid-induced TNF-α,IL-1β,NO,and PGE2 expression and inhibits NF-κB signaling in a mouse model of Al zheimer’s disease[J].Eur J Immunol,2016,46(3):665-676.
[9]Wu D,Zhang X,Zhao M,et al.The role of the TLR4NF-κB signaling pathway in Aβaccumulation in primary hippocampal neurons[J].Acta physiologica Sinica,2015,67(3):319-328.
[10]Chen L,Bai Y,Zhao M,et al.TLR4 inhibitor attenuate amyloid-β-induced angiogenic and inflammatory factor in ARPE-19 cells:Implications for age-related macula degeneration[J].Mol Med Rep,2016,13(4):3249-3256.
[11]齐凡星,胡莹,卢军栋,等.丁苯酞治疗阿尔茨海默病的临床观察[J].中国药房,2016,27(17):2412-2414.