摘要
目的探讨巨噬细胞移动抑制因子(MIF)特异抑制剂ISO-1[(S,R)-3-(4-羟苯基)-4,5-二氢-5-异噁唑乙酸,甲酯]是否通过Toll样受体4(TLR4)途径影响人脐静脉内皮细胞(HUVEC)活性,从而为ISO-1用于TLR4相关疾病的靶向治疗奠定基础。方法将传代培养的HUVEC分为脂多糖(LPS)组、ISO-1预处理组、LPS+ISO-1组和培养液对照组,通过RT-PCR、免疫荧光染色、放射免疫分析和化学反应法,检测ISO-1对HUVEC TLR4表达和肿瘤坏死因子α(TNFα)、一氧化氮合酶(NOS)产生的影响。结果高浓度ISO-1预处理HUVEC,可明显抑制TLR4mRNA表达,其中以25和50μmol/L浓度组最为明显(P<0.05);用50μmol/L ISO-1分别预处理HUVEC0.5、1、2、3h,均能明显降低TLR4mRNA表达,其中1h组的作用最为明显(P<0.05)。免疫荧光染色结果显示,HUVEC可表达低水平TLR4,LPS(10ng/mL)可促进TLR4表达,而50μmol/L的ISO-1预处理1h,可明显降低TLR4表达和LPS诱导的HUVEC TNFα分泌和NOS的产生(P<0.05)。ISO-1预处理对HUVEC产生TNFα和NOS的抑制呈现浓度依赖性。ISO-1作用时间短暂,预处理1h组的作用效果最好(P<0.05),此后逐渐减弱。结论 ISO-1可通过下调TLR4影响HUVEC功能,该结果可为ISO-1用于动脉粥样硬化和炎症等TLR4相关疾病的治疗提供实验依据。
Objective To study the effect of ISO-1 [（S,R）-3-（4-hydroxyphenyl）-4,5-dihydro-5-isoxazole acetic acid methyl ester],a specific antagonist of MIF（Macrophage migration inhibition factor）,on TLR4（Toll-like receptor） activities in HUVEC（Human umbilical vein endothelial cells）,and to provide experimental evidence for the treatment of TLR4-related diseases in which ISO-1 is targeted.Methods HUVECs were cultured and divided into 4 groups： the LPS（lipid polysaccharide） group,the ISO-1 pretreatment group,the LPS＋ISO-1 group and the Medium control group.Effects of ISO-1 on TLR4 expression,TNFα（tumor necrosis factor） and NOS（nitric oxide synthase） secretion were investigated with RT-PCR,immunofluorescense staining,radioimmunoassay and enzyme-chemical reactions.Results Expression of TLR4 in HUVEC was obviously inhibited by pretreatment with high concentration of ISO-1,especially in the 25 and 50 μmol/L groups（P0.05）.Low expression of TLR4 was also detected at 0.5,1,2 and 3 h after pretreatment with 50 μmol/L ISO-1,in which the lowest appeared at 1h（P0.05）.Immunofluorescense showed a low level TLR4 in normal HUVEC,however,expression could be enhanced by LPS（10 ng/mL）.TLR4 expression,TLR4-induced TNFα and NOS secretion were markedly suppressed 1h after the pretreatment with 50 μmol/L ISO-1,and such inhibition was dose dependent（P0.05）.The effective time of ISO-1 was short and the maximal was at 1 h.Conclusion ISO-1 can exert inhibitory effects through down-regulating the TLR4 activities of HUVEC,which suggests that ISO-1 may be a potential target-agent in the treatment of TLR4-related diseases,such as atherosclerosis and inflammation.
出处
《山东大学学报（医学版）》
CAS
北大核心
2010年第8期65-69,共5页
Journal of Shandong University:Health Sciences
基金
山东大学2009大学生创新课题(2009282)
关键词
脂多糖
TOLL样受体4
巨噬细胞移动抑制因子
脐静脉内皮细胞
Lipid polysaccharide
Macrophage migration inhibition factor 4
Toll-like receptor
Human umbilical vein endothelial cells
