Molecular Mechanism Of Ferroptosis Regulating The Development Of Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic pulmonary fibrosis（IPF）is a kind of fibrotic lung disease characterized by progressive decline in lung function.The main pathological features of IPF are excessive proliferation of fibroblasts and accumulation of extracellular matrix（ECM）.The prognosis of IPF is poor,and the median survival time after diagnosis is 2-3 years.In recent decades,the prevalence and incidence of IPF have been increasing year by year.At present,the etiology of IPF is unknown,and the pathogenesis is the abnormal pulmonary trauma repair process caused by alveolar epithelial injury,and it is believed that the main injury form of alveolar epithelial cells is apoptosis.The transformation and excessive proliferation of fibroblasts into myofibroblasts is the main cause of collagen accumulation in lung and the formation of scar tissue,which leads to pulmonary ventilation dysfunction and pulmonary fibrosis.Although two marketed drugs,Nidanib and pirfenidone,have been approved for clinical treatment of IPF,they can only delay the decline of lung function,but cannot prevent the progression of the disease,and have certain side effects.Ferroptosis is an important new form of cell death that has been discovered in recent years.It is mainly characterized by the accumulation of iron and the production of lipid peroxides,and it is associated with a variety of diseases.According to previous studies,dysregulation of iron metabolism is associated with a variety of lung diseases,including IPF,but there has been little research on the relationship between ferroptosis and pulmonary fibrosis.Therefore,exploring the possible relationship and molecular mechanism between ferroptosis and IPF can provide new treatment strategies and solutions for idiopathic pulmonary fibrosis.AimsTo explore the molecular mechanism and role of ferroptosis in the process of IPF,and to find out the possible pathway that regulates the progression of pulmonary fibrosis based on ferroptosis.1.To explore the possible relationship and molecular mechanism between ferroptosis and the occurrence and development of IPF through in vitro and in vivo experiments.2.To explore the possible ways of inhibiting the progression and symptoms of IPF based on ferroptosis in vivo model.Methods and Results1.Lipid peroxidation and iron overload were observed in lung tissues of bleomycin-induced pulmonary fibrosis mice1)After injecting bleomycin into the lungs of mice,HE staining and Masson staining showed the thickening of alveolar wall and increase of collagen deposition,which proved that the IPF model was established successfully.Immunohistochemical staining showed that the expression ofα-SMA,FAPαand TGF-βin mouse lung tissue increased firstly and then decreased with time.Multicolor immunofluorescence staining showed that the expression ofα-SMA,TGF-βand macrophage marker F4/80 in mouse lung tissue was increased at 14 days after bleomycin induction.2)Western blot and flow cytometry showed that the levels of lipid peroxide marker4-hydroxynonenal（4-HNE）and intracellular Fe²⁺in the lung tissues of bleomycin-induced mice increased at 7 and 14 days compared with the control group,and both changes were greater at 7 days than at 14 days.2.Bleomycin and lipopolysaccharide（LPS）induced lipid peroxidation and ferroptosis in lung epithelial cells BEAS-2B in vitro1)Flow cytometry showed that bleomycin and LPS induced the increase of ROS,Lipid ROS and Fe²⁺contents in BEAS-2B cells,and Ferrostatin-1（Fer-1）,a ferroptosis inhibitor,could inhibit the content of Lipid ROS and Fe²⁺.2)Nucleic acid staining of dead/live cells confirmed that bleomycin and LPS could induce cell death in BEAS-2B cells and Fer-1 could partially inhibit cell death.3.Liproxstatin-1（Lip-1）,another ferroptotic inhibitor,inhibited bleomycin-induced ferroptosis of type II alveolar epithelia in vivo,and alleviated bleomycin-and LPS-induced lung injury and fibrosis,while DFO alleviated bleomycin-induced pulmonary fibrosis1)Multicolor immunofluorescence staining showed that bleomycin induced ferroptosis of type II alveolar epithelial cells in lung tissue,and Lip-1 inhibited this phenomenon.2)HE staining and Western blot showed that Lip-1 could alleviate the damage of bleomycin and LPS induced lung tissue structure and partially reduce the accumulation of lipid peroxides in lung tissue.HE staining confirmed that DFO could partially alleviate bleomycin induced lung tissue structure damage.3)Masson staining and immunohistochemical staining demonstrated that Lip-1inhibited bleomycin-induced collagen deposition and expression ofα-SMA,FAPαand TGF-βin lung tissues of mice,while DFO inhibited this phenomenon.4.TGF-βis involved in fibroblasts activation by promoting intracellular iron accumulation1)Flow cytometry showed that bleomycin induced the increase of Fe²⁺level in pulmonary fibroblasts.Fluorescence microscopy showed that TGF-βand Ferric ammonium citrate（FAC）stimulation could increase the content of Fe²⁺in MRC-5 cells.TGF-β-blocker SB431542 and iron chelating agent deoxamine（DFO）could inhibit the increase of Fe²⁺.2)CCK8 and Western blot showed that low concentration of iron promoted the proliferation and activation of MRC-5,and DFO could inhibit the proliferation and activation of MRC-5 under this condition.q PCR,Western blot and Transwell illustrated that TGF-βcould promote the activation,invasion and migration of fibroblasts.Correspondingly,Western blot,immunofluorescence and Transwell showed that SB431542 and DFO could inhibit these phenomena.5.TGF-βup-regulated TFRC expression through the TAZ-TEAD4 pathway to promote iron accumulation and activation in fibroblasts1)Western blot,q PCR,flow cytometry and tissue immunofluorescence were used to confirm the expression of TFRC in fibroblasts stimulated by TGF-βin vivo and in vitro.After Si RNA knockdown and lentivirus overexpression of TFRC,Western blot and fluorescence microscopy verified that fibroblasts activation was enhanced by up-regulation of TFRC and further increase of intracellular Fe²⁺level.2)Western blot,immunofluorescence and dual luciferase reporting system showed that TGF-βpromoted TFRC expression by activating the formation and nuclear localization of TAZ-TEAD4 complex.6.Moderate iron accumulation induces fibroblasts activation while initiating the regulation of ferroptotic defense pathway1)Flow cytometry and nucleic acid staining of dead/live cells showed that low concentration of iron accumulation caused the increase of lipid ROS but did not trigger the ferroptosis of fibroblasts.2)RNA sequencing results of fibroblast cell lines stimulated by low concentration of FAC showed that low level of FAC could activate fatty acid metabolism and lipid metabolism pathways in fibroblasts,and reduce the sensitivity of cells to ferroptosis.7.Fibroblasts specific TFRC knockout mice showed reduced symptoms in bleomycin-induced pulmonary fibrosis model1)Intraperitoneal injection of tamoxifen could induce specific knockout of TFRC in FSP1-Cre ERT;TFRC^flox/flox transgenic mouse fibroblasts.Multicolor immunofluorescence staining demonstrated that FSP1 positive cells in FSP1 specific TFRC knockout mice lack TFRC.2)HE staining and Masson staining demonstrated that fibroblasts TFRC knockout mice had lower lung structure destruction and less collagen deposition compared with the control group.Immunohistochemical staining also confirmed that protein expression ofα-SMA,FAPαand TGF-βin lung tissues of knockout mice was decreased.ConclusionIn the early inflammatory stage of pulmonary fibrosis,the inducers bleomycin and LPS can induce ferroptosis in alveolar epithelial cells,while the ferroptoic inhibitor Lip-1can inhibit cell death and inflammatory response,and thus alleviate the symptoms of bleomycin-and LPS-induced pulmonary fibrosis.Meanwhile,the iron chelating agent DFO can also inhibit bleomycin-induced pulmonary fibrosis.In the late stage of fibrosis,TGF-βinduces the increase of intracellular Fe²⁺by up-regulating the expression of TFRC in fibroblasts,thus promoting the transformation of fibroblasts into myofibroblasts and accelerating pulmonary fibrosis.Mechanistically,TGF-βpromoted the expression and nuclear localization of TAZ,and the binding of TAZ to TEAD-4 enhanced and promoted TFRC transcription.At the same time,low Fe²⁺level affected fibroblasts lipid metabolism and other related pathways,thus reducing the sensitivity of cells to ferroptosis and fibroblasts escape from ferroptosis.In transgenic mice,specific knockout of TFRC in fibroblasts alleviates bleomycin-induced pulmonary fibrosis in mice.In conclusion,ferroptosis and iron homeostasis are involved in and regulate pulmonary fibrosis during inflammation and fibrosis.Meanwhile,ferrototic inhibitors and iron chelating agents may also be potential strategies for IPF treatment.