水飞蓟宾抑制Fas/FasL信号通路对肺结核大鼠炎症损伤及巨噬细胞凋亡的影响

Effects of Silybin inhibiting Fas/FasL signaling pathway on inflammatory injury and macrophage apoptosis in pulmonary tuberculosis rats

目的:探究水飞蓟宾对肺结核(TB)大鼠肺部炎症损伤、巨噬细胞凋亡的影响,及对死亡受体Fas及其配体FasL的调控作用。方法:采用尾静脉注射结核分枝杆菌(Mtb)法制备TB大鼠模型,并随机分为模型组、水飞蓟宾组、Fas过表达重组蛋白(pcDNA-Fas)组、pcDNA-Fas阴性对照(pcDNA-NC)组、水飞蓟宾+pcDNA-Fas组,每组15只,另取15只大鼠为正常对照组。抗酸染色检测肺组织感染情况;HE染色观察肺组织病理变化;ELISA法检测肺组织TNF-α、IL-6表达;Annexin VFITC/PI双染结合流式细胞术检测肺泡巨噬细胞凋亡率;Western blot检测Fas、FasL、caspase8、caspase3、巨噬细胞炎症蛋白-2(MIP-2)表达水平。结果:与正常对照组相比,模型组大鼠肺组织炎症因子表达、肺泡巨噬细胞凋亡率升高,肺组织Mtb感染及干酪样坏死严重,Fas/FasL介导的caspase8/3凋亡途径活化(P<0.05)。与模型组相比,水飞蓟宾组大鼠肺组织炎症因子表达、肺泡巨噬细胞凋亡率降低,肺组织Mtb感染及干酪样坏死缓解,Fas/FasL介导的caspase8/3凋亡途径活性降低(P<0.05)。pc-DNA-Fas可进一步激活Fas/FasL介导的caspase8/3凋亡途径活化,加重肺组织Mtb感染及干酪样坏死,促进肺组织炎症损伤及巨噬细胞凋亡,并减弱水飞蓟宾发挥的抗Fas/FasL活化、抗炎及抗巨噬细胞凋亡作用(P<0.05)。结论:水飞蓟宾可能通过抑制Fas/FasL信号通路发挥抗Mtb感染、抗肺组织巨噬细胞凋亡及抗肺部炎症损伤作用。

Objective:To explore the effects of Silybin on pulmonary inflammatory injury and macrophage apoptosis in pulmo-nary tuberculosis(TB)rats,and its regulation on death receptor Fas and its ligand FasL.Methods:TB rat model was prepared by tail vein injection of Mycobacterium tuberculosis(Mtb).Rats were randomly separated into model group,Silybin group,Fas overexpres-sion recombinant protein(pcDNA-Fas)group,pcDNA-Fas negative control(pcDNA-NC)group and Silybin+pcDNA-Fas group,with 15 rats in each group,and another 15 rats were selected as normal control group.Acid-fast staining was used to measure infection of lung tissue;HE staining was performed to observe pathological changes of lung tissue;expressions of TNF-αand IL-6 in lung tissue were detected by ELISA;apoptosis rate of alveolar macrophages was detected by flow cytometry combined with Annexin V-FITC/PI staining;expression levels of Fas,FasL,caspase8,caspase3 and macrophage inflammatory protein-2(MIP-2)were detected by Western blot.Results:Compared with normal control group,expressions of inflammatory factors in lung tissue and apoptotic rate of alveolar macrophages were increased in model group,Mtb infection and caseous necrosis in lung tissue were severe,and Fas/FasLmediated caspase8/3 apoptotic pathway was activated(P<0.05).Compared with model group,expressions of inflammatory factors in lung tissue and apoptosis rate of alveolar macrophages in Silybin group were reduced,Mtb infection and caseous necrosis in lung tissue were alleviated,and the activity of Fas/FasL-mediated caspase8/3 apoptosis pathway decreased(P<0.05).pcDNA-Fas was able to further activate Fas/FasL-mediated caspase8/3 apoptotic pathway,aggravate lung tissue Mtb infection and caseous necrosis,promote inflammatory damage in lung tissue and macrophage apoptosis,and weaken the anti-Fas/FasL activation,anti-inflammatory and anti-apoptotic effects of Silybin(P<0.05).Conclusion:Silybin may play an anti-Mtb infection,anti-apoptosis of lung tissue macro-phages and anti-inflammatory effects by inhibiting the Fas/FasL signaling pathway.