Matrix Stiffness Promotes Lung Fibroblasts Mitochondrial Fission To Mediate Lung Fibrosis

BackgroundIdiopathic pulmonary fibrosis(IPF)is a chronic fatal disease with progressive development.Excessive deposition,remodeling,and cross-linking of extracellular matrix(ECM)can lead to increased stiffness of ECM,which is an important feature in the process of IPF.Mitochondrial fission of pulmonary fibroblasts may be involved in the development of pulmonary fibrosis.In this study,the soft gel and stiff gel were constructed to simulate the ECM stiffness of normal and IPF patients’lung tissues in vitro,and to explore whether the increase of matrix stiffness can promote the occurrence and development of pulmonary fibrosis by regulating the mitochondrion fission of pulmonary fibroblasts,thereby regulating the migration of pulmonary fibroblasts.Method1.In vitro experiment:IPF patients’pulmonary fibroblasts were cultured on soft gel and stiff gel for 48 hours respectively.The morphology of mitochondria was analyzed by confocal microscope and the parameters were analyzed.The expression of DRP1,MFF,FIS1 was detected by Real-time PCR and Western blot;By knocking down or overexpressing the expression of DRP1 in lung fibroblasts and conducting Transwell migration experiment,it was determined whether mitochondrial fission regulated the migration of lung fibroblasts;The gradient gel was constructed to simulate the stiffness of fibrotic lung tissue,to confirm the durotaxis of lung fibroblasts and explore the role of mitochondrial fission and adenosine triphosphate(ATP)production during this process;Through the interference of polypeptide P259 with the effect of DRP1/MFF,we can reduce the mitochondrion fission,and make it clear that the mitochondrion fission regulated by DRP1/MFF can promote the durotaxis of pulmonary fibroblasts.2.In vivo experiment:bleomycin was used to construct the pulmonary fibrosis model.The expression of DRP1 and MFF of the was detected by immunofluorescence and Western blot;Frozen sections of lung tissues from IPF patients and healthy controls were obtained,and the differential expressions of DRP1 and MFF in lung tissues of the two groups were detected by immunofluorescence.Result1.Compared with soft gel,the morphology of mitochondria of lung fibroblasts on stiff gel tends to be fission,with lower average area of mitochondria(10.81±1.98μm~2vs 7.17±2.22μm~2),shorter average perimeter(19.22±2.78μm vs 11.64±1.95μm),lower form factor(3.14±0.39 vs 1.99±0.17),lower aspect ratio(2.32±0.15 vs 2.12±0.10),fewer branches(2.31±0.26 vs 1.82±0.31),fewer branch junfctions(0.57±0.13vs 0.35±0.13),shorter branch length(12.19±2.47μm vs 7.40±2.36μm),and shorter average branch length(5.34±0.60μm vs 3.87±0.62μm),with statistically significant differences(P<0.0001);The relative expression of DRP1 and MFF protein and m RNA increased significantly(P<0.05).2.Human lung fibroblasts were transfected with control si RNA(si con)and DRP1 si RNA(si-DRP1)respectively for Transwell experiment.Compared with cells on soft gel,the number of migrating cells on hard glue increased(4047±68.61 vs 2471±279.1,P=0.0007).After down-regulation of DRP1 expression,the number of migrated cells on hard gel was reduced(4047±68.61 vs 2382±304.8,P=0.0008).3.Compared with cells on soft or hard gel,the distribution of mitochondria in the pre nuclear and post nuclear regions of lung fibroblasts on gradient gel was statistically significant.The pre nuclear distribution of mitochondria was higher than the post nuclear distribution(69.13%±6.63%vs 30.87%±6.63%,P<0.0001).Compared with the cells on soft gel,the relative ATP production of cells cultured on hard gel was significantly increased(P<0.0001).4.Compared with the control group,cell migration distance in P259group was significantly reduced(636.1±117.9μm vs 97.7±23.06μm,P=0.0015).5.Compared with the control group,DRP1 and MFF protein increased significantly on bleomycin lung fibrosis model(P<0.05).ConclusionThe increase of matrix stiffness promotes the mitochondrial fission of lung fibroblasts.The mitochondrial fission regulated by DRP1 plays an important role in the migration of lung fibroblasts regulated by matrix stiffness.There is a polarized distribution of mitochondria during the durotaxis of lung fibroblasts.Mitochondria are mainly distributed at the front end of the cells,and this distribution mode is related to production of ATP,Polypeptide P259 can inhibit the mitochondrial fission regulated by DRP1/MFF of pulmonary fibroblasts,thereby inhibiting their durotaxis.