不同浓度丁酸钠抑制氧化型低密度脂蛋白诱导THP-1细胞炎症反应的研究

Different concentrations of sodium butyrate inhibit inflammatory response of THP-1 cells induced by oxidized low-density lipoprotein

目的探讨不同浓度的丁酸钠抑制氧化型低密度脂蛋白(Ox-LDL)诱发人单核巨噬THP-1细胞炎症反应能力及其机制。方法分别使用终浓度为50、100、200、400、800μmol/L的丁酸钠预处理THP-1细胞24 h,再以终浓度为50 mg/L的Ox-LDL刺激THP-1细胞24 h,采用实时荧光定量PCR法和ELISA法检测IL-1β、TNF-α、IL-10和TGF-β表达量,实时荧光定量PCR法和免疫印迹法检测NLRP3和Caspase-1的表达量,免疫印迹法检测自噬相关蛋白LC3-Ⅱ和Beclin-1的表达量,以及NF-κB p65在细胞核和胞浆中的分布情况。本研究同时设置正常对照组和无丁酸钠预处理的Ox-LDL组。结果与Ox-LDL组比较,分别经终浓度为200、400、800μmol/L丁酸钠预孵育后,THP-1细胞IL-10和TGF-β表达量显著升高(P<0.01);50、100、200、400μmol/L丁酸钠预处理组细胞TNF-α表达量明显下降(P<0.01);50和100μmol/L丁酸钠预处理组细胞IL-1β表达量明显下降(P<0.01);50、100、200、400、800μmol/L丁酸钠预处理组细胞Caspase-1表达量明显下降(P<0.01);仅400和800μmol/L丁酸钠预处理组细胞NLRP3蛋白表达量明显下降(P<0.01),其他浓度丁酸钠抑制效果不明显。Ox-LDL刺激后THP-1细胞NF-κB p65蛋白明显由细胞浆移位至细胞核内(P<0.01),而经50、100、200、400、800μmol/L丁酸钠预处理后,NF-κB p65蛋白核转位现象明显受到抑制(P<0.01)。与Ox-LDL组比较,200、400、800μmol/L丁酸钠可以显著上调THP-1细胞Beclin-1表达量(P<0.01),50、100、200、400、800μmol/L丁酸钠可以显著上调LC3-Ⅱ表达量(P<0.01)。结论丁酸钠可以抑制由Ox-LDL诱发的THP-1细胞炎症反应,其可能的机制是通过上调THP-1细胞自噬水平,抑制THP-1细胞NF-κB信号通路和NLRP3炎症复合体活化而产生抗炎效果。本结果为采用“肠道微生物组-免疫炎症轴”理论干预高血脂患者体内慢性非可控性炎症提供了实验依据。

Objective To investigate the ability of different concentrations of sodium butyrate inhibiting oxidized low-density lipoprotein(Ox-LDL)-induced inflammatory response in THP-1 cells and its mechanism.Methods THP-1 cells were pre-treated with sodium butyrate in final concentration of 50,100,200,400,800μmol/L for 24 h,and THP-1 cells were stimulated with Ox-LDL in final concentration of 50 mg/L for 24 h,the real-time fluorescent quantitative PCR was used to determine mRNA expression of IL-1β,TNF-α,IL-10 and TGF-β,and ELISA was used to determine concentrations of IL-1β,TNF-α,IL-10 and TGF-βin supernatant.The real-time fluorescent quantitative PCR and Western blotting assay were used to determine the expression of NLRP3 and Caspase-1.The expression of autophagy-related proteins LC3-Ⅱ,Beclin-1 and the distributions of NF-κB p65 in nucleus and cytoplasm were analysed by Western blotting assay.Results The THP-1 cells pre-incubated with sodium butyrate in final concentrations of 200,400,800μmol/L can significantly increase the expression of anti-inflammatory factors IL-10 and TGF-β stimulated by Ox-LDL(P<0.01).Pre-treatment of 50,100,200,400μmol/L sodium butyrate can significantly inhibit the expression of pro-inflammatory TNF-αthat stimulated by Ox-LDL(P<0.01).Pre-treatment of 50,100μmol/L sodium butyrate can significantly inhibit the expression of pro-inflammatory factor IL-1β stimulated by Ox-LDL(P<0.01).Pre-treatment of 50,100,200,400,800μmol/L sodium butyrate can significantly inhibit the expression of Caspase-1(P<0.01),but only 400 and 800μmol/L sodium butyrate can significantly inhibit the NLRP3 expression.There were no obvious inhibition effects in other concentrations of sodium butyrate.After treatment,Ox-LDL and NF-κB p65 of THP-1 cells was significantly shifted from cytoplasm to nucleus(P<0.01),and after pre-treatment with 50,100,200,400,800μmol/L sodium butyrate,the nuclear translocation of NF-κB p65 was obviously inhibited(P<0.01).The concentration of 200,400,800μmol/L sodium butyrate can significantly up-regulate the expression of Beclin-1 in THP-1 cells(P<0.01),and 50,100,200,400,800μmol/L sodium butyrate can significantly increase the expression of LC3-Ⅱ in THP-1 cells(P<0.01).Conclusion Sodium butyrate can be inhibit the inflammatory response of THP-1 cells induced by ox-LDL,which may be by up-regulating the level of autophagy of THP-1 cells and inhibit the activation of NF-κB signaling pathway and NLRP3 inflammatory complex in THP-1 cells.These results provide a theoretical basis for the intervention of chronic uncontrollable inflammation in hyperlipidemia patients via“gut microbiomeimmune inflammatory axis”.