Mechanism Of Arctigenin Inhibits Lung Fibrosis Based On Antioxidant And Antiinflammation

Pulmonary fibrosis(PF)is a common chronic,progressive,and irreversible interstitial lung disease.Once diagnosed,the 5-year average survival rate is only20%～30%.At present,the treatment of most interstitial lung diseases is limited,and few drugs are available.Globally,pirfenidone and nintedanib are the leading drugs for the treatment of PF.Although both have been clinically verified to effectively inhibit PF,they have potential side effects,such as gastrointestinal reaction,liver injury,and bleeding,and their efficacy in a variety of fibrotic lung diseases is unclear.Therefore,it is urgent to develop safe and effective drugs for PF.Arctigenin(ATG)is a lignin compound isolated from Fructus Arctii,which has certain therapeutic effects on liver and renal fibrosis.Based on the theory that different organ fibrosis may have similar mechanisms and drug action targets,we proposed a scientific hypothesis that "Arctigenin could inhibit pulmonary fibrosis ".In order to verify this hypothesis,in this study,both in vitro and in vivo experiments were used.The main research contents are as follows:1.The regulatory effect of arctigenin on lung function in PF miceA pulmonary fibrosis model was established in C57BL/6 mice induced by bleomycin.The improvement effect of arctigenin on pulmonary fibrosis in mice was evaluated by H&E,Masson,and Sirius red staining.The results showed that in the arctigenin treatment group,the pathological changes,inflammatory infiltration,and structural destruction of lung tissue in mice had been improved.The effect of arctigenin on the expression of fibrosis markers in lung tissue of mice with pulmonary fibrosis was evaluated by immunohistochemistry and Western blot.It was found that arctigenin can significantly reduce the expression of fibrosis markers Collagen I,Fibronectin,and α-SMA in lung tissue of mice with pulmonary fibrosis.2.Arctigenin exerts an inhibitory effect on pulmonary fibrosis by regulating oxidative stress in the lung tissue of PF miceThe level of SOD、MDA、GSH in lung issue and the concentration of 8-isoproterenol2α(8-iso-PGF2α)in alveolar lavage fluid were detected by ELISA.It was showed that arctigenin could significantly increase the level of SOD and GSH in the lung tissue of mice with bleomycin induced pulmonary fibrosis and greatly lower the level of MDA and8-iso-PGF2α,which indicates that it has antioxidant effects on lung tissue of pulmonary fibrosis mice.Furthermore,immunofluorescence and immunohistochemistry were used to detect the leve of reactive oxygen species(ROS)in the lung tissue of PF mice,and it was discovered that arctigenin can also significantly reduce the level of ROS in the lung tissue of PF mice.Immunohistochemistry and Western blot were further adopted to detect the expression of Nrf-2,HO-1,and NQO1 in lung tissue.It was revealed that arctigenin is able to increase significantly he expression of Nrf-2,HO-1,and NQO1 in lung tissue,which shows that arctigenin inhibits pulmonary fibrosis in mice based on its antioxidant effect.3.Inhibitory effect of Arctigenin through Signal Mediator TGF-β/ Akt on Pulmonary FibrosisTo further investigate the mechanism of the inhibitory effect of arctigenin on pulmonary fibrosis in mice,immunohistochemistry was used to detect the distribution of TGF-β1,Akt,p-Akt protein in the lung tissue of PF mice;it was discovered that arctigenin can significantly reduce the expression of TGF-β1 and p-Akt in lung tissue,and the inhibitory effect of high-dose arctigenin on TGF-β1 and p-Akt is similar to that of the Nidamib group.Western blot was further employed to detect the protein expression of TGF-β1,Akt and p-Akt in lung tissue,it was revealed that arctigenin has certain inhibitory effect on the protein expression of TGF-β1,Akt and p-Akt.Explanation,which indicates that arctigennin exerts an inhibitory effect on pulmonary fibrosis through influencing TGF-β/Akt signal.4.The regulatory effect of arctigenin on transdifferentiation of A549 cells induced by TGF-β1The influence of arctigenin on the gene expression of markers of fibrosis,such as Collagen I,Fibronectin,and α-SMA,in A549 cells was studied by using q RT-PCR,which revealed that arctigenin can significantly inhibit the gene expression of Collagen I,Fibronectin,and α-SMA in A549 induced by TGF-β1.The influence of arctigenin on the protein expression of Collagen I,Fibronectin,and α-SMA was conducted by adopting Western blot,and it was found that arctigenin can significantly inhibit the protein expression of Fibronectin in A549 cells induced by TGF-β1.The results show that arctigenin can inhibit the transdifferentiation of A549 cells by inhibiting the gene expression or protein expression of Collagen I,Fibronectin,and α-SMA.5.Inhibitory effect of arctigenin on transdifferentiation of A549 cell through oxidation resistance and anti-inflammationThe techniques of q RT-PCR and Western blot were adopted to study the effect of arctigenin on the gene and protein expression of oxidative stress regulatory factors Nrf-2,HO-1,NQO-1 in A549 cells induced by TGF-β1.It was revealed that arctigenin can significantly increase both the gene expression and protein expression of Nrf-2 and NQO-1 in A549 cells,and increase protein expression of HO-1.The method of ELISA was employed to evaluate the level of oxidative stress indicators GSH,MDA,SOD,and inflammatory cytokine IFN-γ,IL-6,TNF-α in A549 cells,and it was discovered that arctigenin is able to increase significantly the level of GSH in A549 cells and reduce the level of MDA;it can also significantly lower the level of inflammatory cytokines of IL-6and TNF-α,while increases that of IFN-γ.In summary,both in vivo and in vitro experiments have confirmed that arctigenin can inhibit pulmonary fibrosis through antioxidant/anti-inflammatory effects.The results of this study provide a scientific theoretical basis for the basic research of arctigenin and its subsequent development as a therapeutic drug for pulmonary fibrosis.