阿魏酸钠对NALP3炎性复合体的调节及在肺纤维化中的作用

发布时间：2019-07-05 13:57

【摘要】：目的探讨NALP3炎性复合体在肺纤维化过程中的活化情况,并观察阿魏酸钠(sodium ferulate,SF)在肺纤维化过程中对炎性复合体的影响。方法体内实验以博莱霉素(BLM)气管滴注构建小鼠肺纤维化模型(BLM组),治疗组在用BLM建模后第2天以SF(100mg/kg)每日1次腹腔注射(SF组),对照组(不建模)和BLM组注射等量磷酸盐缓充液,建模后第21天取肺组织行Masson染色,观察肺组织胶原沉积(Ashcroft评分)情况;以碱水解法测定肺组织羟脯氨酸(HYP)含量;体外实验以H_2O_2刺激小鼠胚胎成纤维细胞NIH-3T3以构建成纤维细胞氧化应激模型,干预组以SF 400μg/mL预处理2h,再用H_2O_2培养细胞,采用Real time-PCR检测小鼠肺组织及体外实验分组处理细胞相关炎性细胞因子NALP3、caspase-1、凋亡相关斑点样蛋白(ASC)、collagen-1、α平滑肌肌动蛋白(α-SMA)mRNA,Western blot检测小鼠肺组织NALP3蛋白,分组处理细胞NALP3、collagen-1、α-SMA蛋白的表达;ELISA测定肺组织上清及细胞上清中IL-1β质量浓度。结果体内实验SF处理后,肺纤维化模型小鼠Ashcroft评分及HYP含量较BLM组有所减少(P0.05);肺组织NALP3炎性复合体NALP3、ASC、caspase-1mRNA,NALP3蛋白的表达和IL-1β的合成较BLM组均有减少(P0.05)。体外实验显示H_2O_2能有效刺激NALP3炎性复合体NALP3、ASC、caspase-1mRNA的表达(P0.05)与IL-1β的分泌(P0.05),并且能有效促进成纤维细胞NALP3蛋白、collagen-1和α-SMA的表达(P0.05);SF预处理能在基因水平抑制NALP3、ASC、caspase-1、collagen-1和α-SMA的表达(P0.05),且在蛋白水平减少NALP3、collagen-1、α-SMA的表达(P0.05),降低IL-1β水平(P0.05)。结论阿魏酸钠可能通过抑制氧化应激诱导的NALP3炎症复合体活化,从而抑制成纤维细胞活化,展现其抗纤维化作用。
[Abstract]:Objective to investigate the activation of NALP3 inflammatory complex in the process of pulmonary fibrosis and to observe the effect of sodium ferulate (sodium ferulate,SF) on inflammatory complex during pulmonary fibrosis. Methods the mouse pulmonary fibrosis model (BLM group) was established by intratracheal drip of bleomycin (BLM) in vivo. SF (100mg/kg) was injected intravenously once a day in the treatment group (SF group) on the second day after BLM modeling. The control group (no modeling) and BLM group were injected with the same amount of phosphate buffer solution. On the 21st day after modeling, the lung tissue was stained with Masson to observe the collagen deposition (Ashcroft score) in the lung tissue. The content of hydroxyproline (HYP) in lung tissue was determined by alkali hydrolysis. Mouse embryonic fibroblasts NIH-3T3 were stimulated by H_2O_2 in vitro to establish the oxidative stress model of fibroblasts. In the intervention group, the cells were pretreated with SF 400 渭 g / mL for 2 h, and then cultured with H_2O_2. The lung tissue of mice was detected by Real time-PCR and the apoptosis-related spotted protein (ASC), collagen-1, of NALP3,caspase-1, was treated in vitro. The expression of NALP3 protein in lung tissue of mice was detected by 伪-smooth muscle actin (伪-SMA) mRNA,Western blot, and the expression of NALP3,collagen-1, 伪-SMA protein in cells treated with 伪-smooth muscle actin (伪-smooth muscle actin) was detected. ELISA was used to determine the concentration of IL-1 尾 in lung tissue culture and cell culture. Results after SF treatment in vivo, the Ashcroft score and HYP content of pulmonary fibrosis model mice were lower than those of BLM group (P 0.05), and the expression of NALP3 inflammatory complex NALP3,ASC,caspase-1mRNA,NALP3 protein and the synthesis of IL-1 尾 in lung tissue were lower than those in BLM group (P 0.05). In vitro experiments showed that H_2O_2 could effectively stimulate the expression of NALP3,ASC,caspase-1mRNA in NALP3 inflammatory complex (P 0.05) and the secretion of IL-1 尾 (P 0.05), and effectively promote the expression of NALP3 protein, collagen-1 and 伪-SMA in fibroblasts (P 0.05). SF pretreatment could inhibit the expression of NALP3,ASC,caspase-1,collagen-1 and 伪-SMA at gene level (P 0.05), and decrease the expression of NALP3,collagen-1, 伪-SMA (P 0.05) and IL-1 尾 level (P 0.05) at protein level. Conclusion Sodium ferulate may inhibit the activation of fibroblasts and show its anti-fibrosis effect by inhibiting the activation of NALP3 inflammatory complex induced by oxidative stress.
【作者单位】：四川大学华西医院呼吸与危重症医学科;
【基金】：四川省科技厅科技支撑计划项目(No.0040205301A53)资助
【分类号】：R563

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