毛蕊异黄酮调节SIRT3/SOD2信号通路对小鼠气道上皮细胞损伤的改善作用

Ameliorating effect of calycosin regulating SIRT3/SOD2 signaling pathway on airway epithelial cell damage in mice

目的探讨毛蕊异黄酮(CA)对香烟烟雾(CS)诱导的小鼠气道上皮细胞损伤及沉默蛋白3/超氧化物歧化酶2(SIRT3/SOD2)信号通路的影响。方法将90只雄性BALB/c小鼠随机分为对照(Control)组、CS组、CA低剂量处理组(CA-L组)、CA高剂量处理组(CA-H组)、CA高剂量处理+SIRT3抑制剂3-TYP组(CA-H+3-TYP组),每组18只。采用小动物全身体积描记检测系统检测肺功能指标潮气量(TV)、呼气峰流速(PEF);酶联免疫吸附试验(ELISA)检测血清炎性因子[白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α]及氧化应激[活性氧(ROS)、SOD]水平;HE染色观察肺组织气道上皮细胞损伤;免疫组化检测气道上皮细胞屏障相关蛋白[闭合蛋白(OCLN)、闭锁小带蛋白1(ZO-1)]表达;蛋白免疫印迹检测SIRT3/SOD2信号通路相关蛋白表达。结果与Control组相比,CS组TV、PEF、MAN和SOD水平及OCLN、ZO-1、SIRT3、SOD2蛋白表达水平降低,MLI及IL-6、TNF-α、ROS水平升高(P<0.05);CS组较Control组肺组织结构明显破坏,肺泡增大明显,肺泡周围伴有炎性细胞浸润,气道上皮细胞脱落明显。不同剂量CA均可减轻肺组织破坏,改善肺泡结构,减轻炎性细胞浸润,减少气道上皮细胞脱落,升高TV、PEF、MAN和SOD水平及OCLN、ZO-1、SIRT3、SOD2蛋白表达水平,降低MLI及IL-6、TNF-α、ROS水平,且高剂量CA的作用较低剂量CA显著(P<0.05);SIRT3/SOD2信号通路抑制剂3-TYP可逆转CA对CS诱导的小鼠气道上皮细胞损伤的改善作用。结论CA可改善CS诱导的小鼠气道上皮细胞损伤,其作用机制与激活SIRT3/SOD2信号通路有关。

Objective To investigate effects of calycosin(CA)on cigarette smoke(CS)induced airway epithelial cell damage in mice and the sirtuin 3/superoxide dismutase 2(SIRT3/SOD2)signaling pathway in mice.Methods A total of 90 mice were randomly separated into the control group,the cigarette smoke(CS)group,the CA low-dose treatment group(CA-L group),the CA high-dose treatment group(CA-H group)and the CA high-dose treatment plus SIRT3 inhibitor 3-TYP group(CA-H+3-TYP group),with 18 mice in each group.Tidal volume(TV)and peak expiratory flow rate(PEF)of lung function were detected by whole body plethysmography system.Serum levels of inflammatory factors[interleukin(IL)-6,tumor necrosis factor(TNF)-α]and oxidative stress indicators[reactive oxygen species(ROS),SOD]were detected by enzyme-linked immunosorbent assay(ELISA).The injury of airway epithelial cells in lung tissue was observed by HE staining.The expression levels of barrier related proteins(OCLN and ZO-1)in airway epithelial cells were detected by immunohistochemistry.Immunoblotting was applied to detect the expression of SIRT3/SOD2 signaling pathway related proteins.Results Compared with the control group,levels of TV,PEF,MAN and SOD and the expression levels of OCLN,ZO-1,SIRT3 and SOD2 were decreased in the CS group,while the levels of MLI,IL-6,TNF-αand ROS were increased(P<0.05).Compared with the control group,the lung tissue structure was significantly damaged,the alveolar enlargement was obvious,the surrounding alveolar was accompanied by inflammatory cell infiltration,and the airway epithelial cells were obviously shed in the CS group.Different doses of CA alleviated lung tissue destruction,improved alveolar structure,reduced inflammatory cell infiltration,reduced airway epithelial cell shedding,increased levels of TV,PEF,MAN,SOD and OCLN,ZO-1,SIRT3 and SOD2,and decreased levels of MLI,IL-6,TNF-αand ROS.The effect of high dose CA was more significant than that of low dose CA(P<0.05).SIRT3/SOD2 signaling pathway inhibitor 3-TYP partially reversed the ameliorative effect of CA on CS induced airway epithelial cell injury in mice.Conclusion CA can ameliorate CS induced airway epithelial cell damage in mice,and its mechanism is related to the activation of the SIRT3/SOD2 signaling pathway.