AcSDKP体外抑制脂多糖诱导的小鼠RAW264.7巨噬细胞促炎功能研究*

doi:10.3969/j.issn.1672-5069.2017.02.005

摘要/Abstract

摘要： 目的探讨N-乙酰-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）对小鼠RAW264.7巨噬细胞促炎功能的影响。方法取小鼠RAW264.7巨噬细胞体外培养，分为对照组、脂多糖（LPS）组和LPS加不同浓度的AcSDKP（1 nmol/L、10 nmol/L和100 nmol/L）处理组。采用荧光定量PCR法检测细胞肿瘤坏死因子（TNF）-α、白细胞素（IL）-1β、IL-6和诱导型一氧化氮合成酶（iNOS）mRNA相对水平；使用Transwell小室检测RAW264.7细胞的趋化能力；采用Western blot法检测细胞iNOS、磷酸化的蛋白激酶复合体抑制物(p-IKK)、磷酸化的κB抑制蛋白(p-IκB)和磷酸化的P65（p-P65）蛋白表达水平。结果 LPS处理组TNF-α、IL-1β和IL-6 mRNA水平显著高于对照组【分别高出（41±2.1）倍、（1073±80.8）倍和（1726±110.2）倍，P<0.01】；AcSDKP（10 nmol/L和100 nmol/L）处理组TNF-α水平显著低于LPS处理组【分别降低19.0% 和39.3%，P<0.01】；AcSDKP（1 nmol/L、10 nmol/L和100 nmol/L）处理组IL-1β水平显著低于LPS处理组【分别降低22.08%、35.8%和38.2%，P<0.01】；AcSDKP（1 nmol/L、10 nmol/L和100 nmol/L）处理组IL-6水平显著低于LPS处理组【分别降低15.8%、35.7%和43.3%，P<0.01】；LPS处理组穿过小室的细胞数为（136±5.3）个/HP，显著高于对照组【（64±5.3）个/HP，P<0.01】，而AcSDKP处理组穿过小室的细胞数【分别为（105±4.8）、（85.3±5.0）和（73.7±5.6）个/HP】，显著低于LPS组【（136±5.3）个/HP，P<0.05】；LPS处理组iNOS mRNA和蛋白表达水平显著高于对照组【分别为（21±2.5）倍和（5.9±0.4）倍，P<0.01】，而AcSDKP处理组iNOS mRNA显著低于LPS处理组【分别降低37.8%、23.3%和33.1%，P<0.05】；在10 nmol/L和100 nmol/L浓度AcSDKP处理组，两种蛋白水平显著低于LPS处理组【分别降低27.0%和40.2%，P<0.05】；LPS处理组p-IKK、p-IκB和p-P65表达量均显著高于对照组【分别为（25.9±4.8）倍、（9.5±0.6）倍和（2.1±0.2）倍，P<0.01】，而AcSDKP（10 nmol/L）处理组三者水平较LPS处理组显著下降【分别为42.5%、17.8%和29.7%，P<0.05】。结论 AcSDKP可抑制LPS诱导的小鼠RAW264.7巨噬细胞的炎症反应，这一效应可能与阻断IKK/ IκB/NF-κB通路有关。

关键词: RAW264.7巨噬细胞, N-乙酰-丝氨酰-天门冬酰-赖氨酰-脯氨酸, 炎症因子, NF-κB, 体外

Abstract: Objective To investigate the effect of N-acetyl-seryl-aspartyl-lysyl-proline（AcSDKP） on mouse RAW264.7 macrophage cell line.Methods RAW264.7 mouse macrophage cells were cultured in vitro. Cells were divided into five groups，namely the control group （vehicle only），lipopolysaccharides （LPS） group （LPS only），and AcSDKP intervention groups （LPS plus AcSDKP at 1 nmol/L，10 nmol/L and 100 nmol/L）. The mRNA levels of tumor necrosis factor-α （TNF-α），interleukin （IL）-1β，IL-6 and inducible nitric oxide synthase（iNOS） were measured by real-time RT-PCR. The chemotactic motility of RAW264.7 cells was detected by using Transwell assay. Western blot was performed to determine the iNOS，phosphorylated inhibitor of κB protein kinase complex （p-IKK），phosphorylated inhibitor of κB （p-IκB） and phosphorylated P65（p-P65） expression.Results The mRNA levels of TNF-α，IL-1β and IL-6 in LPS group were increased by（41±2.1），（1073±80.8） and（1726±110.2） folds compared with in control group， respectively（P<0.01）；the TNF-α mRNA levels in AcSDKP （1 nmol/L，10 nmol/L and 100 nmol/L） treatment groups were decreased by 8.1% （P>0.05），19.0% （P<0.01） and 39.3% （P<0.01），respectively as compared with in LPS group，IL-1β were decreased by 22.0%，35.8% and 38.2%，respectively，（P<0.01），and IL-6 were decreased by 15.8%，35.7% and 43.3%， respectively （P<0.01）；The migrated cell counts across the chamber membrane in LPS group （136±5.3）/HP were significantly higher than those in control group [（64±5.3），P<0.01]，however，the cell counts in AcSDKP treatment groups [（105±4.8） /HP，（85.3±5.0）/HP and （73.7±5.6）/HP，P<0.05] were significantly lesser than those in LPS group （P<0.05）；The mRNA and protein levels of iNOS were increased by （21±2.5） and （5.9±0.4） folds compared with those in the control group，However，the mRNA levels of iNOS in AcSDKP treatment groups were decreased by 37.8%，23.3% and 33.1%，respectively，and their protein levels were decreased by 27.0% and 40.2%（P<0.01）at the concentration of 10 nmol/L and 100 nmol/L of AcSDKP intervention；Relative levels of p-IKK，p-IκB and p-P65 protein in LPS group were increased by （25.9±4.8），（9.5±0.6） and （2.1±0.2） folds，respectively，as compared with those in control group；After AcSDKP （10 nmol/L） treatment，these protein levels were decreased by 42.5%（P<0.01），17.8%（P<0.05） and 29.7%（P<0.05），respectively，than those in cells treated with LPS alone.Conclusions AcSDKP exhibits a significant inhibitory effect on the proinflammatory profiles induced by LPS in mouse Raw264.7 macrophages in vitro，and this effect may be related to the blockage of the IKK/IκB/NF-κB signaling pathway.

Key words: RAW264.7 macrophages, N-acetyl-seryl-aspartyl-lysyl-proline, Inflammatory cytokines, Nuclear factor-κB, In vitro

李兵航，李梦婷，何玲楠，周达，白洁，丁永年，陈源文，范建高. AcSDKP体外抑制脂多糖诱导的小鼠RAW264.7巨噬细胞促炎功能研究*[J]. 实用肝脏病杂志, 2017, 20(2): 142-147.

Li Binghang，Li Mengting，He Lingnan，et al.. Inhibitory effect of AcSDKP on the proinflammatory profiles induced by lipopolysaccharides in a mouse RAW264.8 macrophage cell line in vitro[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2017, 20(2): 142-147.

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