Liproxstatin-1 Alleviates Bleomycin Induced Pulmonary Fibrosis And Its Associated Mechanisms

Background:Idiopathic pulmonary fibrosis(IPF),a terminal disease with undetermined etiology and limited treatment option is extremely lethal,with an average life expectancy of only 2-3 years after diagnosis.Both genetic susceptibility and environmental factors or damage contribute to the recurrent injury and abnormal repair of epithelial cells,cause chronic inflammatory infiltration,induce the abnormal activation of fibroblast/myofibroblast,promote superabundant extracellular matrix(ECM)protein deposition,finally result in progressive and irreversible fibrosis.Previous studies found that redox imbalance(increased oxidative stress and compromised antioxidant ability)played an important role in the development of IPF.Not only the IPF samples(serum,bronchus alveolar lavage fluid,and lung tissue)but also the the atmosphere were demonstrated to fill with oxidized proteins and lipids.In view of the crucial role of redox imbalance in IPF development,the maintainence of redox homeostasis was deemed to be a potential therapeutic target to IPF for years.Variety studies found that the maintainence of antioxidant function of mouse can alleviate the progression of fibrosis in experimental animal model.However,several antioxidant including N-acetylcysteine(NAC)were prove to have no antfibrotic funtion in clinical trials.Thus,the way of applying antioxidants to treat pulmonary fibrosis still long and full of thorns.As a radical-trapping antioxidant,Liproxstatin-1(Lip-1)is a potent lipid autoxidation inhibitor which can directly repress the radical chain propagation.Lip-1 was proved to alleviate acute radiation induced mice lung injury and inhibit cigarette smoke extract caused bronchial epithelial cells death.Based on the protective function of Lip-1 in previous researches,it hypothesizes that Lip-1 might suppress pulmonary fibrosis by reshaping redox equilibrium.Objective:This study aim to investigate whether and how Lip-1 regulates bleomycin(BLM)induced pulmonary fibrosis both in vivo and in vitro.Methods:1.Eight-week-old male C57BL/6 mice weighed about 23g were induced by BLM intratracheally to performe the pulmonary fibrosis model.All the mice were randomly divided into four groups:control group(Con),bleomycin treated group(BLM:3.5mg/kg),Liproxstatin-1 treated group(Lip-1:10mg/kg)and bleomycin plus Liproxstatin-1 co-treated group(BLM+Lip-1).After 21 days,the weight of mice and lungs,as well as the macroscopic structure of lungs were recorded;the serum and bronchoalveolar lavage fluid(BALF)were prepared to calculate the level of proinflammatory cytokine;the 4%paraformaldehyde-fixed,paraffin-embedded lung tissues were sliced to investigate the pathologic change and production of collagen by performing hematoxylin-eosin solution(HE)staining,Masson’s Trichrome staining and Sirius Red staining;Tunel staining was carried out to evaluate cell apoptosis in the fibrosis lung;Immunofluorescence staining was implemented to show the amount and distribution of alveolar epithelial cell;transmission electron microscope(TEM)was applied to observe the ultra microstructure of cells;the level of oxidative products including reactive oxygen species(ROS)and malondialdehyde(MDA),the activity of various antioxidant glutathione(GSH),catalase(CAT),and total superoxide dismutase(T-SOD)were tested using protein lysate;oxidative stress related profibrotic pathways(ROS/p53)were analyzed by Western blot and Immunofluorescence staining.2.The viability of A549 cells treated with different concentration of BLM(0,10,40,80,160,320,640 and 800ug/ml)for 24 h were performed by MTT assay;the optimum time of BLM in inducing A549 cells injury and fibrosis was evaluated by Western blot.3.In the AECs injury model induced by BLM,the A549 cells were divided into four groups:control group(Con),BLM treated group(BLM:40ug/ml),Lip-1 treated group(Lip-1:2UM)and BLM plus Lip-1 co-treated group(BLM+Lip-1).MTT assay was performed to assess the cell viability;lactate dehydrogenase(LDH)assay was carried out to measure the cellular damage;a DCFH-DA probe was used to estimate the level of ROS;the amount of MDA and the activity of various antioxidant(GSH,CAT,T-SOD)were tested using protein lysate;Western blot and Immunofluorescence staining were adopted to calculate the expression of p53 and its possible targets including BCL2-Associated X(BAX),a-smooth muscle actin(a-SMA),and p21,to evaluate the function of Lip-1 in alleviating pulmonary fibrosis.Result:In vivo:1.In mice model of pulmonary fibrosis,HE staining confirmed that BLM could induce inflammation and destroy the structure in pulmonary,while the addition of Lip-1 alleviated such inflammatory infiltration and structure damage.2.Lip-1 can also mitigate the expression of transforming growth factor β1,interleukin-6(IL-6),interleukin-10(IL-10),and(TGF-β1),tumour necrosis factor α(TNF-α)in BALF induced by BLM.3.Masson’s trichrome staining,Sirius red staining,and hydroxyproline(HYP)test found that Lip-1 could reduce the generation of collagen induced by BLM in mice.4.The tests to evaluate oxidative stress identified that BLM promoted the produce of ROS and MDA,inhibited the activity of GSH,CAT,and T-SOD,while the addition of Lip-1 reversed these alterations in mice model.5.Lip-1 can relieve the cell apoptosis,alveolar epithelial cells(AECs)reduction and mitochondria damage induced by BLM in mice lung.6.Lip-1 can attenuate ROS/p53 pathway related to redox which was activated by BLM in mice.In vitro:1.The cell viability in A549 cells was reduced with increasing BLM concentration in a concentration-dependent manner.2.The addition of BLM resulted in A549 cells mesenchymal like changes in a time-dependent manner.3.Lip-1 can recover the cell viability and decreased LDH relaese,all which were disordered by BLM in A549 cells.4.Lip-1 can alleviate the oxidative damage(ROS and MDA)in A549 cells induced by BLM.5.Lip-1 can restore the reductive ability in A549 cell damaged by BLM through enhancing GSH expression,with little effect on CAT and T-SOD activity.6.Lip-1 can attenuate ROS/p53 related pathways including BAX(apoptosis associated)and α-SMA(fibrosis associated)pathways,but did not affect the p21(senescence associated)pathway,all which are activated by BLM.Conclusion:1.The inflammatory infiltration was extensive,redox balance was disturbed,collagen secretion was increased,apoptosis was increased,and AECs were destroyed and decreased in BLM induced mice pulmonary fibrosis model.2.Lip-1 can improve the inflammatory response,inhibit collagen secretion,reshape redox balance,and protect AECs in BLM induced mice pulmonary fibrosis model.3.Lip-1 can improve BLM induced A549 cells destruction,reduce oxidative damage and enhance antioxidant capacity.4.Lip-1 alleviates bleomycin-induced pulmonary fibrosis in mice and A549 cells damage,which may relate to the regulation of ROS/p53 pathway(apoptosis and fibrosis associated,other than senescence associated).