当归黄芪超滤物通过调控Nrf2/xCT/GPX4信号通路抑制铁死亡改善放射性大鼠肺纤维化

Ultrafiltration of Angelicae Sinensis Radix and Astragali Radix inhibits ferroptosis and improves pulmonary fibrosis in rats by regulating Nrf2/xCT/GPX4 signaling pathway

旨在探讨核因子E2相关因子2(Nrf2)/溶质载体家族成员SLC7A11(xCT)/谷胱甘肽过氧化物酶4(GPX4)信号通路介导的铁死亡在放射性肺纤维化中的作用机制及当归黄芪超滤物的干预作用。将50只Wistar大鼠随机分为5组,每组10只。除空白组不予辐射外,其余各组大鼠均经麻醉后接受40 Gy X射线单次局部胸部照射1次,建立放射性肺纤维化大鼠模型。辐射后,当归黄芪超滤物各干预组以0.12、0.24、0.48 g·kg^(-1)剂量分别灌胃,每日1次,连续给药30 d。连续给药30 d后,比色法检测各组肺组织氧化应激指标超氧化物歧化酶(SOD)活性,还原型谷胱甘肽(GSH)、丙二醛(MDA)及Fe^(2+)水平;免疫荧光检测肺组织中活性氧(ROS)荧光表达;苏木素-伊红(HE)、Masson染色观察肺组织病理变化;免疫组化及Western blot检测肺组织Nrf2/xCT/GPX4信号通路及纤维化蛋白表达水平。结果显示,与空白组比较,模型组大鼠Fe^(2+)、MDA水平显著上升,SOD活性、GSH水平显著下降,ROS水平显著上升;肺组织结构遭受严重破坏,肺间质明显增生,肺泡塌陷且实变严重,有较多的炎性细胞聚集和胶原纤维沉积;肺组织损伤程度相对较高,受损线粒体增多、变小、排列紊乱,形态不规则,基质变浅,线粒体嵴大多断裂、变短,轻微扩张,个别线粒体基质电子密度增高,部分线粒体外膜破裂,出现铁死亡特异性线粒体特征性改变;肺组织中转铁蛋白受体1(TFR1)表达显著升高,GPX4、铁蛋白重链1(FTH1)、Nrf2及xCT表达显著降低;α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(collagenⅠ)蛋白表达显著升高。与模型组比较,当归黄芪超滤物干预可明显改善大鼠脂质过氧化、抗氧化相关指标,Fe^(2+)水平显著降低;纤维化程度减轻;肺组织中TFR1、α-SMA、collagenⅠ蛋白表达显著下降,GPX4、FTH1、Nrf2、xCT蛋白表达显著上升。综上所述,当归黄芪超滤物对放射性肺纤维化具有改善作用,其机制可能是通过调控Nrf2/xCT/GPX4信号通路抑制铁死亡发挥作用。

This study aims to investigate the mechanism of ferroptosis mediated by the nuclear factor-E2-related factor 2(Nrf2)/solute carrier family 7 member 11(SLC7A11,also known as xCT)/glutathione peroxidase 4(GPX4)signaling pathway in radiation-induced pulmonary fibrosis and the intervention effect of Angelicae Sinensis Radix(ASR)and Astragali Radix(AR)ultrafiltration extract.Fifty Wistar rats were randomly divided into five groups,with 10 rats in each group.Except for the blank group without radiation,the rats in each group were anesthetized and subjected to a single local chest irradiation of 40 Gy X-rays once to establish a rat model of radiation-induced pulmonary fibrosis.After radiation,the rats in the intervention groups were orally administered with ASR-AR ultrafiltration extract at doses of 0.12,0.24,and 0.48 g·kg^(-1),respectively,once a day for 30 days.After 30 days of continuous administration,the levels of oxidative stress indicators superoxide dismutase(SOD)activity,reduced glutathione(GSH),malondialdehyde(MDA),and ferrous ion(Fe^(2+))in lung tissues of each group were detected by colorimetry.Immunofluorescence was used to detect reactive oxygen species(ROS)fluorescence expression in lung tissues.Hematoxylin-eosin(HE)and Masson staining were performed to observe pathological changes in lung tissues.Immunohistochemistry and Western blot were used to detect the expression levels of Nrf2/xCT/GPX4 signaling pathway and fibrotic proteins in lung tissues.The results showed that compared with the results in the blank group,the levels of Fe^(2+)and MDA in the model group increased,while SOD activity and GSH levels decreased,and ROS levels increased.HE and Masson staining results showed that the structure of lung tissue was seriously damaged,the pulmonary interstitium was significantly proliferated,the alveoli collapsed and consolidated severely,and there were more inflammatory cell aggregates and collagen fiber deposits.Transmission electron microscopy showed that the degree of lung tissue damage in the model group was relatively high,with increased,smaller,and disorganized damaged mitochondria,irregular morphology,shallow matrix,most mitochondria ruptured and shortened,mildly expanded,some mitochondria with increased electron density of the matrix,partial mitochondrial outer membrane rupture,and characteristic changes of ferroptosis-specific mitochondria.Immunohistochemistry showed that the expression of transferrin receptor protein 1(TFR1)in lung tissues was significantly increased,while the expression of GPX4,ferritin heavy chain 1(FTH1),Nrf2,and xCT was significantly decreased.Western blot showed that the expression ofα-smooth muscle actin(α-SMA)and collagenⅠprotein increased.Compared with the model group,the intervention group with ASR-AR ultrafiltration extract significantly improved lipid peroxidation and antioxidant-related indicators,decreased Fe^(2+)levels,alleviated fibrosis,and decreased the expression of TFR1,α-SMA,and collagenⅠproteins in lung tissues,while increased the expression of GPX4,FTH1,Nrf2,and xCT proteins.In summary,ASR-AR ultrafiltration extract has an ameliorative effect on radiation-induced pulmonary fibrosis,and its mechanism may involve the inhibition of ferroptosis by regulating the Nrf2/xCT/GPX4 signaling pathway.