TLR4沉默对高糖诱导胰岛微血管内皮细胞损伤的干预作用及机制(1)
【摘要】 目的 探討Toll样受体-4(TLR4)沉默对高糖诱导胰岛微血管内皮细胞损伤的干预作用及机制。方法 利用 siRNA 沉默MS-1细胞中TLR4的表达，实时荧光定量聚合酶链反应(qRT-PCR)检测TLR4 mRNA的表达，CCK-8 法检测细胞增殖，比色法测定一氧化氮水平，ELISA检测内皮素-1水平，羟胺法检测超氧化物歧化酶(SOD)活力，硫代巴比妥酸(TBA)法检测丙二醛水平，蛋白免疫印迹法检测TLR4、髓样分化因子88(MyD88)和核转录因子(NF)-κBp65蛋白的表达。将高糖处理的细胞分为高糖组及siRNA处理组(再分为siRNA-TLR4组及NC-TLR4组)进行上述指标的比较。结果 与高糖组比较，siRNA-TLR4组TLR4 mRNA的相对表达水平降低，细胞的增殖能力受抑制(P均<0.05)。此外，与高糖组比较，siRNA-TLR4组一氧化氮水平、SOD活力升高，内皮素-1、丙二醛水平下降(P均< 0.05)。与高糖组比较，siRNA-TLR4组TLR4、MyD88和NF-κBp65蛋白减少(P均< 0.05)。结论 沉默TLR4在MS-1细胞中的表达水平，减弱TLR4/NF-κB信号通路传递的炎症水平，间接降低氧化应激水平，从而改善高糖诱导的胰岛微血管内皮细胞损伤。
, http://www.100md.com
【关键词】 Toll样受体-4;高糖;胰岛;微血管内皮细胞
【Abstract】 Objective To evaluate the effect and mechanism of TLR4 silencing on high glucose-induced islet microvascular endothelial cell injury. Methods The expression of TLR4 in the MS-1 cells was silenced by siRNA. The expression of TLR4 mRNA was detected by qRT-PCR. The cell proliferation was measured by CCK-8 assay. The nitric oxode(NO) level was determined by colorimetry. The endothelin (ET)-1 level was measured by ELISA. The superoxide dismutase (SOD) activity was detected by hydroxylamine method. The malondialdehyde (MDA) level was detected by thiobarbituric acid (TBA) method. The expression levels of TLR4， MyD88 and NF-κBp65 proteins were detected by Western blot. The cells treated with high glucose were divided into the high glucose and isRNA silencing groups (further divided into siRNA-TLR4 and NC-TLR4 groups). Relevant parameters were statistically compared among different groups. Results Compared with the high glucose group， the relative expression level of TLR4 mRNA was significantly down-regulated， and the proliferation ability of the cells was inhibited in the siRNA-TLR4 group (both P < 0.05). In addition， compared with the high glucose group， the NO level and SOD activity were significantly increased， whereas the ET-1 and MDA levels were significantly decreased in the siRNA-TLR4 group (all P < 0.05). Compared with the high glucose group， the expression levels of TLR4， MyD88 and NF-κBp65 proteins were significantly down-regulated in the siRNA-TLR4 group (all P < 0.05). Conclusions Silencing the expression of TLR4 in the MS-1 cells attenuates the inflammatory levels of the TLR4/NF-κB signaling pathways and indirectly reduces the oxidative stress levels， thereby mitigating the high glucose-induced islet microvascular endothelial cell damage.
【Key words】 Toll-like receptor-4;High glucose;Islet;Microvascular endothelial cell, http://www.100md.com(林雪波)
, http://www.100md.com
【关键词】 Toll样受体-4;高糖;胰岛;微血管内皮细胞
【Abstract】 Objective To evaluate the effect and mechanism of TLR4 silencing on high glucose-induced islet microvascular endothelial cell injury. Methods The expression of TLR4 in the MS-1 cells was silenced by siRNA. The expression of TLR4 mRNA was detected by qRT-PCR. The cell proliferation was measured by CCK-8 assay. The nitric oxode(NO) level was determined by colorimetry. The endothelin (ET)-1 level was measured by ELISA. The superoxide dismutase (SOD) activity was detected by hydroxylamine method. The malondialdehyde (MDA) level was detected by thiobarbituric acid (TBA) method. The expression levels of TLR4， MyD88 and NF-κBp65 proteins were detected by Western blot. The cells treated with high glucose were divided into the high glucose and isRNA silencing groups (further divided into siRNA-TLR4 and NC-TLR4 groups). Relevant parameters were statistically compared among different groups. Results Compared with the high glucose group， the relative expression level of TLR4 mRNA was significantly down-regulated， and the proliferation ability of the cells was inhibited in the siRNA-TLR4 group (both P < 0.05). In addition， compared with the high glucose group， the NO level and SOD activity were significantly increased， whereas the ET-1 and MDA levels were significantly decreased in the siRNA-TLR4 group (all P < 0.05). Compared with the high glucose group， the expression levels of TLR4， MyD88 and NF-κBp65 proteins were significantly down-regulated in the siRNA-TLR4 group (all P < 0.05). Conclusions Silencing the expression of TLR4 in the MS-1 cells attenuates the inflammatory levels of the TLR4/NF-κB signaling pathways and indirectly reduces the oxidative stress levels， thereby mitigating the high glucose-induced islet microvascular endothelial cell damage.
【Key words】 Toll-like receptor-4;High glucose;Islet;Microvascular endothelial cell, http://www.100md.com(林雪波)