升降散对LPS诱导大鼠肺泡巨噬细胞NF-κB信号的影响
Effect of Shengjiang San on NF-κB Activity in LPS-activated Rat Alveolar Marcrophage Cell Line
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摘要: 目的 研究升降散对LPS诱导大鼠肺泡巨噬细胞(NR8383)TLR4表达及其下游NF-κB信号通路的影响。方法 用不同浓度LPS（0、2、10 μg/mL）诱导NR8383细胞，采用Real-time PCR和Western Blotting分别检测TLR4的mRNA和蛋白表达水平。用带有NF-κB的质粒转染NR8383细胞，双荧光素酶报告基因检测NF-κB活性，Western Blotting检测磷酸化p65表达水平，Real-time PCR检测细胞因子TNF-α、A20、IL-6和IL-1β的mRNA表达水平，ELISA检测细胞上清液中TNF-α、IL-6和IL-1β的含量。将升降散作用于LPS成功诱导NF-κB上调的NR8383细胞，Real-time PCR、Western Blotting、双荧光报告基因检测和ELISA实验检测升降散在NR8383细胞中对LPS诱导NF-κB信号通路的影响。结果 LPS成功诱导NR8383细胞中NF-κB上调，在蛋白表达和mRNA表达水平均证实，LPS能梯度诱导TLR4高表达，且与LPS浓度呈正相关；同时，LPS能够诱导TLR4下游NF-κB的激活，增加下游靶基因编码细胞因子的合成与分泌，且与LPS浓度呈正相关。10 μg/mL升降散作用NR8383细胞后，能抑制LPS诱导TLR4的高表达和下游NF-κB的激活。结论 LPS能梯度诱导NR8383细胞中TLR4的高表达及其下游NF-κB的激活，升降散能够显著抑制该诱导作用。Abstract: OBJECTIVE To investigate the effect of Shengjiang San on expression of TLR4 and NF-κB in LPS-induced rat pulmonary alveolar macrophage cell. METHODS NF-κB signal pathway in NR8383 cells was stimulated with LPS. The expression level of TLR4 was measured by QRT-PCR and Western Blotting separately. NF-κB activity was detected by Dual Luciferase Reporter Gene Assay kit. The protein expression level of p-p65 was detected by Western Blotting. The mRNA level of TNF-α，A20，IL-6， IL-1β was measured by QRT-PCR， and the production of TNF-α， IL-6， IL-1β in the supernatant of NR8383 was determined with ELISA. Shengjiang San treated NR8383 cells， which were induced NF-κB signal pathway by LPS. NF-κB activity was detected by Dual Luciferase Reporter Gene Assay kit， qRT-PCR， Western Blotting and ELISA. RESULTS LPS induced TLR overpression in both mRNA and protein levels. Moreover， NF-κB signaling pathway and downstream target cytokines were increased with dose-dependent. 10 μg/mL Shengjiang San treatment abrogated the induction of LPS to TLR4 and NF-κB signaling pathway. CONCLUSION Shengjiang San significantly reduced LPS-activated NF-κB signaling pathway in NR8383.
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