安五脂素对小鼠肺纤维化的改善作用及其机制研究

Effect of anwulignan on pulmonary fibrosis in mice and its mechanism

目的探讨安五脂素对肺纤维化的干预作用及其机制。方法雄性ICR小鼠65只,随机分成对照组、BLM模型组、安五脂素低剂量组(1 mg/kg)、安五脂素高剂量组(4 mg/kg)与N-乙酰半胱氨酸组(150 mg/kg),每组13只。采用气管灌注5 mg/kg博来霉素(BLM)诱导小鼠肺纤维化模型。收集各组小鼠血清和肺组织,采用HE染色和Masson三色染色观察肺组织病理学变化并进行肺纤维化评分;采用试剂盒测定血清氧化应激指标及肺组织羟脯氨酸(HYP)含量;采用Western blotting及qRT-PCR检测铁死亡通路相关基因的表达水平。取对数生长期HFL-1细胞:(1)设置对照组与安五脂素给药组(0.3125、0.625、1.25、2.5、5、10、20、40、80μmol/L),采用CCK-8法检测安五脂素对HFL-1细胞的毒性作用。(2)设置对照组、转化生长因子(TGF)-β_(1)模型组、安五脂素低剂量组(5μmol/L)与安五脂素高剂量组(10μmol/L),以TGF-β_(1)诱导肺纤维化细胞模型,采用试剂盒测定氧化应激指标及活性氧(ROS)水平,采用Western blotting及qRT-PCR检测铁死亡通路相关基因的表达水平。结果HE染色、Masson染色结果及肺组织HYP含量测定结果均提示BLM诱导的小鼠肺纤维化模型构建成功,给予安五脂素后肺纤维化表型明显改善。CCK-8检测结果显示,当安五脂素浓度<20μmol/L时对HFL-1细胞的增殖活性无明显影响(P>0.05)。与BLM或TGF-β_(1)模型组比较,给予安五脂素可明显提高小鼠血清及HFL-1细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性及谷胱甘肽(GSH)水平,明显降低丙二醛(MDA)含量(P<0.01或P<0.001)。与TGF-β_(1)模型组比较,给予10μmol/L安五脂素可明显降低HFL-1细胞中ROS水平(P<0.01)。Westernblotting和qRT-PCR检测结果显示,给予安五脂素可明显上调小鼠肺组织及HFL-1细胞中铁死亡通路中谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)、转铁蛋白(TF)的表达水平,下调转铁蛋白受体1(TFR1)的表达水平(P<0.05、P<0.01或P<0.001)。结论安五脂素可通过抑制氧化应激和铁死亡而改善肺纤维化,为开发肺纤维化的治疗药物提供了一定的理论基础。

Objective To investigate the interventional effect of anwulignan on pulmonary fibrosis and the underlying mechanism.Methods Sixty-five male ICR mice were randomly divided into control group,bleomycin(BLM)model group,anwuzhisu low dose group(1 mg/kg),anwuzhisu high dose group(4 mg/kg)and N-acetylcysteine group(150 mg/kg),with 13 mice in each group.Mouse pulmonary fibrosis model was induced by intratracheal perfusion of 5 mg/kg BLM.The serum and lung tissues were collected,and the pathological changes of lung tissues were observed by HE staining and Masson trichrome staining;serum oxidative stress index and hydroxyproline(HYP)content in lung tissue were measured by kit;the expression level of iron death pathway related genes was detected by Western blotting and qRT-PCR.Take the logarithmic growth phase HFL-1 cells,(1)set the control group and anwuzhisu administration group(0.3125,0.625,1.25,2.5,5,10,20,40,80μmol/L),and CCK-8 method was used to detect the toxic effect of anwuzhi on HFL-1 cells.(2)Set the control group,transforming growth factor(TGF)-β_(1) model group,anwuzhisu low dose group(5μmol/L)and anwuzhisu high dose group(10μmol/L),with TGF-β_(1) induced pulmonary fibrosis cell model.The oxidative stress index and reactive oxygen species(ROS)level were measured by kit;the expression level of iron death pathway related genes was detected by Western blotting and qRT-PCR.Results HE staining,Masson trichrome staining and the increase of HYP content indicated that the BLMinduced pulmonar y fibrosis model was successfully constructed,and the pulmonar y fibrosis phenotype was significantly improved after the administration of anwulignan.CCK-8 assay showed that the concentration of anwulignan<20μmol/L had no significant effect on the proliferation activity of HFL-1 cells(P>0.05).Compared with model group,the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and the levels of glutathione(GSH)in lung tissues of mice and HFL-1 cells were significantly increased after the administration of anwulignan,while the levels of malondialdehyde(MDA)were significantly decreased(P<0.01 or P<0.001).Compared with TGF-β_(1) model group,10μmol/L anwulignan could decrease the level of ROS in HFL-1 cells(P<0.01).Western blotting and qRT-PCR results showed that anwulignan could significantly up-regulate the expressions of ferroptosis pathway related genes in lung tissues of mice and HFL-1 cells including glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and transferrin(TF),and significantly downregulate the expression of transferrin receptor 1(TFR1)(P<0.05,P<0.01 or P<0.001).Conclusion Anwulignan can improve pulmonary fibrosis by inhibiting oxidative stress and ferroptosis,laying a foundation for the development of clinical drugs for pulmonary fibrosis.