论文部分内容阅读
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 investigate the effects of iso-1 [(S, R) -3- (4-hydroxyphenyl) -4,5-dihydro-5-isoxazoleacetic acid, a specific inhibitor of macrophage migration inhibitory factor (MIF) ] Can affect the activity of human umbilical vein endothelial cells (HUVECs) through the Toll-like Receptor 4 (TLR4) pathway, and lay a foundation for the targeted therapy of TLR4-related diseases with ISO-1. Methods The cultured HUVECs were divided into three groups: lipopolysaccharide (LPS) group, ISO-1 pretreatment group, LPS + ISO-1 group and culture medium control group. The expression of HUVECs was detected by RT-PCR, immunofluorescence, radioimmunoassay and chemical reaction The effect of ISO-1 on the expression of TLR4 and the production of tumor necrosis factor α (TNFα) and nitric oxide synthase (NOS) in HUVECs were detected. Results The pretreatment of HUVEC with high concentration of ISO-1 significantly inhibited the expression of TLR4 mRNA, especially in the groups of 25 and 50 μmol / L (P <0.05). The pretreatment of HUVEC with 50 μmol / L ISO-1 pretreated HUVEC 0.5, , 3h, can significantly reduce the expression of TLR4mRNA, 1h group the role of the most obvious (P <0.05). Immunofluorescence staining showed that HUVEC could express low levels of TLR4 and LPS (10ng / mL), which could promote the expression of TLR4. Pretreatment with 50μmol / L of ISO-1 for 1h reduced the expression of TLR4 and LPS-induced TNFα secretion and NOS (P <0.05). ISO-1 pretreatment inhibited the production of TNFα and NOS in HUVECs in a concentration-dependent manner. ISO-1 action time is short, pretreatment 1h group the best effect (P <0.05), then gradually weakened. Conclusion ISO-1 can affect the function of HUVEC by down-regulating TLR4, which can provide experimental evidence for the treatment of TLR4-related diseases such as atherosclerosis and inflammation by ISO-1.