阿魏酸钠通过NALP3炎性小体对实验性肺纤维化的影响

[The Effect of Sodium Ferulate in Experimental Pulmonary Fibrosis via NALP3 Inflammasome].

作者信息

Kuang Ju, Wei Xiao-Lin, Xie Min

机构信息

Department of Respiratory and Critical Care Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.

出版信息

Sichuan Da Xue Xue Bao Yi Xue Ban. 2017 Jul;48(4):503-508.

PMID:28752965

Abstract

OBJECTIVE

To investigate the activation of NALP3 inflammasome in the process of experimental pulmonary fibrosis (PF), and evaluate the effect of sodium ferulate (SF) in the relationship of NALP3 and PF.

METHODS

Establishing PF experimental model via bleomycin (BLM) intratracheal injection (BLM group, SF group), treated with SF daily (SF group) or PBS [BLM group, control (CON) group] and mice were executed on day 21. Ashcroft score was used to assess lung fibrosis in mice PF model. The content of hydroxyproline (HYP) in lung tissue was determined by alkaline hydrolysis. Fibroblast NIH-3T3 was treated with H₂O₂ to trigger cell oxidative stress experiments (H₂O₂group). Cell was pre-administrated with SF 2 h before H₂O₂ stimulation in H₂O₂+SF group. Blank group without any treatments, was set as control. Real time-PCR was used to investigate the expressions of three elements of inflammasome[, caspase-1, apoptosis-associated speck-like protein ()], collagen-1 and α smooth muscle actin () mRNA in both lung tissue and fibroblast. Western blot was used to detect protein level of NALP3 in mice lung tissue and collagen-1, in fibroblast as well. Meanwhile, IL-1β content in lung tissue and cell supernatant was measured by ELISA.

RESULTS

experiment, SF treated mice showed lower Ashcroft score and HYP content and decreased caspase-1 mRNA expressions and IL-1β production, NALP3 protein level compared with BLM group (<0.05). experiment, H₂O₂ increased NALP3 (<0.05), ASC (<0.01), caspase-1 (<0.05) expressions and IL-1β releasing (<0.05)and promoted the expressions of collagen-1 and in both gene and protein levels (<0.05) in NIH-3T3. NALP3 activation was partly inhibited in H₂O₂+SF group (<0.05). The mRNA expression levels of collagen-1 and were reduced in H₂O₂+SF group (<0.05) and the protein expressions of and collagen-1 were decreased (<0.05) compared with those of H₂O₂ group.

CONCLUSION

Sodium ferulate may suppress oxidative stress mediated NALP3 activation to inhibit fibroblast activation in the anti-fibrosis effect.

摘要

目的

探讨实验性肺纤维化（PF）过程中NALP3炎性小体的激活情况，并评估阿魏酸钠（SF）在NALP3与PF关系中的作用。

方法

通过气管内注射博来霉素（BLM）建立PF实验模型（BLM组、SF组），SF组每日给予SF处理，BLM组和对照组（CON组）给予PBS处理，于第21天处死小鼠。采用Ashcroft评分评估小鼠PF模型中的肺纤维化情况。通过碱性水解法测定肺组织中羟脯氨酸（HYP）的含量。用H₂O₂处理成纤维细胞NIH-3T3以进行细胞氧化应激实验（H₂O₂组）。在H₂O₂+SF组中，细胞在H₂O₂刺激前2小时预先给予SF。设置未进行任何处理的空白组作为对照。采用实时定量聚合酶链反应（Real time-PCR）检测肺组织和成纤维细胞中炎性小体的三个元件[半胱天冬酶-1（caspase-1）、凋亡相关斑点样蛋白（ASC）]、Ⅰ型胶原（collagen-1）和α平滑肌肌动蛋白（α-SMA）的mRNA表达。采用蛋白质免疫印迹法（Western blot）检测小鼠肺组织中NALP3的蛋白水平以及成纤维细胞中collagen-1、α-SMA的蛋白水平。同时，采用酶联免疫吸附测定法（ELISA）检测肺组织和细胞上清液中白细胞介素-1β（IL-1β）的含量。

结果

实验中，与BLM组相比，SF处理的小鼠Ashcroft评分和HYP含量降低，caspase-1 mRNA表达、IL-1β产生及NALP3蛋白水平均下降（P<0.05）。实验中，H₂O₂增加了NIH-3T3细胞中NALP3（P<0.05）、ASC（P<0.01）、caspase-1（P<0.05）的表达以及IL-1β的释放（P<0.05），并在基因和蛋白水平上促进了collagen-1和α-SMA的表达（P<0.05）。在H₂O₂+SF组中，NALP3的激活受到部分抑制（P<0.05）。与H₂O₂组相比，H₂O₂+SF组中collagen-1和α-SMA的mRNA表达水平降低（P<0.05），α-SMA和collagen-1的蛋白表达下降（P<0.05）。