The Effect And Mechanism Study Of Peptide DR3penA On Alleviating Silica- And Paraquat-induced Pulmonary Fibrosis

Interstitial lung disease（ILD）is a group of restrictive lung diseases characterized by interstitial inflammation and fibrosis,with patients eventually dying of respiratory failure.Among them,the incidence of idiopathic pulmonary fibrosis（IPF）and ILDs associated with multi-hazard exposure is increasing yearly.However,available small molecule therapeutic agents only mitigate disease progression and cannot effectively reverse pulmonary fibrosis with the disadvantages of significant side effects and high cost.Therefore,there is a low five-year survival rate and poor prognosis for patients with these diseases.It is of great importance to develop new safe and effective against pulmonary fibrosis drugs.Our group developed a novel and low toxicity peptide DR3penA with significantly improved serum stability by designing and modifying the antioxidant peptide DR8,and confirmed that the DR3penA has good antifibrotic activity in bleomycin murine model,with potential for further development.Since a single animal model is often limited in predicting efficacy in human,studies using multiple models can evaluate efficacy from various perspectives and thus promote the development of lead compounds into drugs.Therefore,in view of the urgency of developing effective drugs against silicosis and paraquat lung and the need for preclinical efficacy evaluation of DR3penA in multiple models,this study evaluated the efficacy of DR3penA in other two pulmonary fibrosis models induced by silica（SiO₂）and paraquat（PQ）respectively.The experimental results showed that the peptide DR3penA significantly inhibited the abnormally high expression of fibrosis markersα-SMA,Fibronectin and Collagen I in the SiO₂-induced A549 cell model and the TGF-β1-induced BEAS-2B cell model.The antifibrotic activity of DR3penA was tested by multiple methods including H&E and Masson staining,immunohistochemistry,western blot and RT-q PCR in SiO₂-and PQ-induced lung fibrosis model.The results suggested that DR3penA（0.5,1.6 and 5.0mg/kg）reduced the abnormally high expression of the fibrosis markers Fibronectin and Collagen I in silicosis mice and alleviate the extent of pulmonary fibrosis,and its anti-pulmonary fibrosis effect in mice was not significantly different from that of the positive control drug pirfenidone（PFD）（100 mg/kg）.In the PQ-induced rat lung fibrosis model,DR3penA（0.200,0.632 and 2.000 mg/kg）decreased the excessive deposition of extracellular matrix（ECM）and mitigated the extent of interstitial lung solidification,and its inhibitory effect on Fibronectin and Collagen I was not significantly different compared to the positive control drug PFD（100 mg/kg）.As both SiO₂ and PQ can contribute to the progression of lung fibrosis by inducing oxidative stress and triggering the Epithelial-mesenchymal transition（EMT）process in organisms.Therefore,we examined the expression of oxidative stress markers nicotinamide adenine dinucleotide phosphate oxidases 4（NOX4）and EMT markers（E-Cadherin and N-Cadherin）both in vitro and in vivo,as well as the production of reactive oxygen species（ROS）in cells and the levels of antioxidant glutathione（GSH）and lipid peroxidation product malondialdehyde（MDA）and the catalase（CAT）activity in the serum of the animals.The results suggested that DR3penA depressed SiO₂-and PQ-induced oxidative stress and EMT processes obviously.The preliminary mechanistic study revealed that in silicosis,DR3penA negatively regulated PI3K/AKT pathway by inhibiting oxidative stress with reducing ROS production and thus hindered the EMT process to ultimately fight pulmonary fibrosis.In PQ-induced pulmonary fibrosis,DR3penA was able to reduce the phosphorylation levels of ERK and p38 by inhibiting oxidative stress and abnormally high expression of TGF-β,thereby impeding the EMT process and thus exerting an anti-pulmonary fibrosis effect.In summary,this study found that DR3penA could effectively inhibit the progression of SiO₂-and PQ-induced pulmonary fibrosis.And through the conversion of drug bioavailability under different routes of administration,DR3penA exerts antifibrotic activity equivalent to that of the positive control drug pirfenidone（PFD）at lower doses.The mechanistic studies suggested that DR3penA can exert anti-pulmonary fibrosis effects by inhibiting the activation of PI3K/AKT and MAPK signaling pathways.Therefore,DR3penA can be used as a candidate compound for the treatment of fibrotic ILDs to evaluate in greater depth on medicinal properties.This study provides a research basis for the development of peptide drugs with independent intellectual property rights for the treatment of fibrotic diseases.