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目的:探讨副干酪乳酸杆菌(Lactobacillus paracacei,L.para)预刺激抑制脂多糖(Lipopolysacchride,LPS)诱导的炎性细胞因子释放的调节机制。方法:用佛波酯诱导THP-1细胞分化为巨噬细胞样细胞;以L.para预刺激为实验组,小剂量LPS(10ng/ml)预刺激和TLR2激动剂Pam3CSK4预刺激为对照组,预刺激24 h后,再用LPS大剂量(1μg/ml)刺激经分化的THP-1细胞;检测预刺激对TLR信号通路负调控因子A20、SOCS1、SOCS3、IRAK3的表达水平,对TLR4和CD14的膜表达水平和mRNA水平,以及对大剂量LPS诱导的炎性细胞因子IL-1β、TNF-α水平的影响。各目的蛋白mRNA水平检测采用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)法,TLR4和CD14的膜表达检测采用流式细胞术。结果:L.para等预刺激能够降低LPS诱导的IL-1β、TNF-α表达水平;并能够上调细胞TLR信号途径负调控因子A20、SOCS1、SOCS3、IRAK3的mRNA水平,但不影响细胞膜的TLR4和CD14阳性百分率;IRAK1/4抑制剂能够减弱预刺激诱导的负调控因子表达水平,并减弱预刺激对LPS诱导炎症因子释放的抑制作用。结论:副干酪乳酸杆菌预刺激能够通过上调TLR信号通路负调控因子A20、SOCS1、SOCS3、IRAK3等的表达抑制单核巨噬细胞对LPS刺激的炎性细胞因子的释放。
AIM: To investigate the regulatory mechanism of Lactobacillus paracacei (L. para) pre-stimulation on the inhibition of inflammatory cytokine release induced by lipopolysacchride (LPS). Methods: THP-1 cells were induced to differentiate into macrophage-like cells by phorbol ester. Pretreatment with L. parara as experimental group, pretreatment with low dose LPS (10ng / ml) and pretreatment with TLR2 agonist Pam3CSK4 as control group, After pre-stimulation for 24 h, THP-1 cells were stimulated with high dose of LPS (1 μg / ml), and the expression of negative regulatory factors A20, SOCS1, SOCS3 and IRAK3 of TLR signaling pathway was detected by pre-stimulation. Membrane and mRNA levels, as well as the effects of high-dose LPS on inflammatory cytokines IL-1β and TNF-α. The mRNA level of each target protein was detected by real-time quantitative polymerase chain reaction (RT-qPCR) method, and the membrane expression of TLR4 and CD14 was detected by flow cytometry. Results: Pretreatment with L.para could decrease the expression of IL-1β and TNF-α induced by LPS, and upregulate the mRNA levels of A20, SOCS1, SOCS3 and IRAK3, but not TLR4 And the positive percentage of CD14. IRAK1 / 4 inhibitor decreased the expression of negative regulation factor induced by pre-stimulation and attenuated the inhibitory effect of pre-stimulation on LPS-induced inflammatory factor release. Conclusion: Lactobacillus paracasei pretreatment can inhibit the release of inflammatory cytokines stimulated by LPS by up-regulating the expression of negative regulatory factors TLR signaling pathway such as A20, SOCS1, SOCS3 and IRAK3.