混合游离脂肪酸对大鼠肺微血管内皮细胞Toll样受体4表达的影响及其介导的炎症反应机制

目的研究混合游离脂肪酸（FFA）对大鼠肺微血管内皮细胞（RRPMVECs）Toll样受体4（TLR4）的激活作用及其介导的链式炎症反应机制。方法以不同浓度的FFA处理体外培养的RPMVECs，Westernblotting检测TLR4蛋白的表达。用干扰小RNA（siRNA）转染体外培养的RPMVECs（TLR4 siRNA组和杂序siRNA组），设立阴性对照组。分别用0．1mmol／LFFA、10ng／mL脂多糖（LPS）和5ng／mL肿瘤坏死因子-α（TNF-α）处理各组细胞，以未加药细胞作为相应的空白组。Real-TimePCR检测TLR4mRNA和NF-κB抑制蛋白激酶β（IKKβ）mRNA的表达，Westernblotting检测磷酸化核因子κB抑制因子（p-IκBα）和核因子κB（NF-κB）的表达，ELISA法检测细胞培养上清液中炎症因子白介素-1β（IL-1β）的表达。结果RPMVECs的TLR4蛋白相对表达量在0．1mmol／LFFA处理后显著增高（P〈0．05），并随FFA浓度的升高而增加。在杂序siRNA和阴性对照组，TLR4mRNA的相对表达量在LPS和FFA处理后显著升高（P〈0．05），TNF-α处理无明显变化；而在TLR4干扰组，3种处理后TLR4mRNA的表达均无显著变化。在杂序siRNA和阴性对照组，3种方式处理后RPMVECs的IKKβmRNA、p-IκBα及IL-1β的相对表达量均显著升高（P〈0．05），而TLR4siRNA干扰抑制了其在FFA和LPS处理组的表达，但对TNF-α组无抑制作用。结论FFA通过TLR4／IKKβ／NF-κB信号通路介导RPMVECs的炎症反应。

Effects of free fatty acid mixture on toll-like receptor-4 of pulmonary microvascular endothelial cells of rats and mechanism of FFA-induced inflammation response

Objective To explore the activating effect of the free fatty acid （FFA） mixture on the toll-like receptor-4 （TLR4） of rat pulmonary microvascular endothelial cells （RRPMVECs） and the mechanism of FFA- induced inflammation response. Methods RRPMVECs cultured in vitro were prepared by FFA of different concentrations. The expression of TLR4 protein was detected by the Western blotting. RRPMVECs were then divided into the TLR4 siRNA group （transfected by TLR4 siRNA）, scramble siRNA group （transfected by scramble siRNA）, and negative control group. All groups were prepared by FFA of 0.1 mmol/L, LPS of 10 ng/ mL, TNF-α of 5 ng/mL, and blank medium, respectively. The expressions of TLR4 mRNA and IKKβ mRNA were detected by the Real-Time PCR. The expressions of P-IκBα and NF-κB were measured by the Western blotting. The expression of inflammatory factor IL-1β in the cell culture supernatant was detected by the ELISA. Results The relative expression of TLR4 protein in RPMVECs increased significantly after being prepared by FFA of 0.1 mmol/L （P〈0.05） and increased with the concentration of FFA. The relative expressions of TLR4 mRNA of the scramble siRNA group and negative control group increased significantly after being prepared byLPS and FFA （P〈0.05） and did not significantly changed after being prepared by TNF-α. The expression of TLR4 mRNA of the TLR4 siRNA group did not significantly changed after being prepared by LPS, FFA, and TNF-α. The relative expressions of IKKβmRNA, p-IκBα, and IL-1β in RPMVECs of the scramble siRNA group and negative control group increased significandy after being prepared by LPS, FFA, and TNF-α （P〈0.05）. The interference of TLR4 siRNA could suppress the expressions of groups prepared by FFA and LPS, but did not suppress the expressions of groups prepared by TNF-α. Conclusion FFA induces the inflammation response of RPMVECs through the TLR4/IKKβ/NF-κB signaling pathway.