鸡内金提取物通过调节肺泡巨噬细胞自噬水平减轻尘肺大鼠肺间质纤维化的机制

Mechanism of Gallus gallus domesticus extract reducing pulmonary interstitial fibrosis in pneumoconiosis rats by regulating the autophagy level of alveolar macrophages

目的探究鸡内金提取物通过调节肺泡巨噬细胞自噬水平减轻尘肺大鼠肺间质纤维化的机制。方法选择30只雄性SD大鼠根据随机数字表法分为3组,每组10只。对照组为没有粉尘接触的健康SD大鼠;模型组采用一次性除尘方法建立大鼠矽肺模型;治疗组采用一次性除尘方法建立大鼠矽肺模型,每天灌服鸡内金提取物0.2 g。定量逆转录PCR(RT-qPCR)分析自噬相关基因(Beclin、LC3-Ⅱ和LC3-Ⅰ)和凋亡相关基因(Bcl-2)的mRNA表达。培养后6、12、24 h,MTT检测巨噬细胞的活力。培养后12、24 h,通过划痕实验测定细胞的划痕间隙距离。酶联免疫吸附试验法检测促炎因子白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平。蛋白质印迹检测大鼠肺部纤连蛋白、Ⅰ型胶原蛋白、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶-12(MMP-12)、诱导型一氧化氮合酶(iNOS)和重组人精氨酸酶1(Arg1)的蛋白表达。结果模型组大鼠Beclin、LC3-Ⅱ/LC3-ⅠmRNA表达、IL-1β和TNF-α水平以及纤连蛋白、Ⅰ型胶原蛋白、MMP-9、MMP-12蛋白和iNOS蛋白表达水平均较对照组显著升高,Bcl-2 mRNA和Arg1蛋白表达水平均较对照组显著降低(P<0.05),治疗组大鼠Beclin、LC3-Ⅱ/LC3-ⅠmRNA表达、IL-1β和TNF-α水平以及纤连蛋白、Ⅰ型胶原蛋白、MMP-9、MMP-12蛋白和iNOS蛋白表达水平均较模型组显著降低,Bcl-2 mRNA和Arg1蛋白表达水平均较模型组显著升高(P<0.05)。培养后6、12、24 h,模型组较对照组细胞活力降低(P<0.05),治疗组大鼠较模型组细胞活力升高(P<0.05)。培养后12、24 h,模型组大鼠细胞划痕间隙距离较对照组降低(P<0.05),治疗组大鼠细胞划痕间隙距离较模型组升高(P<0.05)。结论鸡内金提取物通过诱导肺泡巨噬细胞自噬而发挥抗炎和抗纤维化作用。自噬通过抑制纤连蛋白、Ⅰ型胶原蛋白、MMP-9和MMP-12的表达,来保护肺泡巨噬细胞免受二氧化硅诱导的细胞凋亡,从而进一步减少由二氧化硅颗粒引起的炎症因子的分泌和最终的纤维化。

Objective To explore the mechanism of Gallus gallus domesticus extract reducing pulmonary interstitial fibrosis in pneumoconiosis rats by regulating the autophagy level of alveolar macrophages.Methods A total of 30 male Sprague-Dawley rats were selected and divided into 3 groups according to the random number table method,each group 10 rats.The control group was healthy SD rats without dust exposure;the model group established a rat silicosis model using a one-time dust removal method;the treatment group established a rat silicosis model using a one-time dust removal method,and was administered 0.2 g of Gallus gallus domesticus extract daily.The mRNA expression of autophagy-related genes(Beclin,LC3-Ⅱand LC3-Ⅰ)and apoptosis-related genes(Bcl-2)was analyzed by RT-qPCR;The viability of macrophages was detected by MTT at 6,12,24 h after culture.The scratch gap distance of different groups of cells was compared by scratch experiment at 12,24 h after culture;The protein expression levels of pro-inflammatory factors interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay.Western blotting was used to detect the expression of rat lung fibronectin,typeⅠcollagen,matrix metalloproteinase-9(MMP-9),matrix metalloproteinase-12(MMP-12),inducible nitric oxide synthase(iNOS)and Arginase-1(Arg1).Results Compared with the control group,Beclin,LC3-Ⅱ/LC3-ⅠmRNA expression,IL-1βand TNF-αlevels,and fibronectin,typeⅠcollagen,MMP-9,MMP-12 and iNOS protein expression levels in the model group were significantly higher,while Bcl-2 mRNA and Arg1 protein expression levels were significantly reduced(P<0.05);Compared with the model group,Beclin,LC3-Ⅱ/LC3-ⅠmRNA expression,IL-1βand TNF-αlevels,as well as fibronectin,typeⅠcollagen,MMP-9,MMP-12 protein and iNOS protein expression levels in the treatment group were significantly reduced,while the expression levels of Bcl-2 mRNA and Arg1 protein increased significantly(P<0.05).At 6,12,24 h after culture,the cell viability of the model group was lower than that of the control group(P<0.05),and the cell viability of the treatment group was higher than that of the model group(P<0.05).The distance between scratches in the model group at 12,24 h was lower than that of the control(P<0.05),and the distance between cell scratches in the treatment group was higher than that in the model group(P<0.05).Conclusion Gallus gallus domesticus extract exerts anti-inflammatory and anti-fibrotic effects by inducing autophagy in alveolar macrophages.Autophagy protects alveolar macrophages from silica-induced apoptosis by inhibiting the expression of fibronectin,typeⅠcollagen,MMP-9 and MMP-12,thereby further reducing the secretion of inflammatory factors caused by silica particles And final fibrosis.