The Mechanism Of TRPV4-mediated Calcium Influx Induced Ferroptosis In Lung Epithelial Cells Affecting Acute Lung Injury

Background:Acute lung injury（ALI）is a common pulmonary disease in critically ill patients,which often leads to the development of multiple organ dysfunction,high mortality and poor prognosis.Although great progress has been made in the research of the pathogenesis of ALI in the past decades,the clinical therapies that can be used to effectively intervene in ALI are still limited.So far,no drug has been proved to be effective in curing ALI.As a second messenger,Ca²⁺is widely involved in many physiological processes such as cell proliferation,muscle contraction,and enzyme regulation.Intracellular Ca²⁺homeostasis plays a key role in maintaining the integrity of lung epithelial cells.Elevated intracellular Ca²⁺promotes increased permeability of lung epithelial cells,leading to ALI.Ferroptosis,a form of nonapoptotic cell death,is characterized by the accumulation of intracellular iron and lipid peroxidation.Ferroptosis is involved in the regulation oflung injury.Many studies have shown that lung injury can be improved by inhibiting ferroptosis.How to inhibit ferroptosis may become a research trend in the treatment of ALI.Tansient receptor potential vanilloid 4（TRPV4）is a non-selective calcium permeable protein that is functionally expressed in endothelial and epithelial cells of the respiratory system.Previous studies have shown that TRPV4 can affect pulmonary edema by acting in lung epithelial cells.In addition,studies have shown that the cell receptor TRPV4 ion channel has a relationship with ferroptosis.However,whether TRPV4 channel mediates ferroptosis to regulate ALI and its regulatory mechanism is still unclear.Part I Calcium influx and ferroptosis in acute lung injury miceObjective:We aimed to explore the Ca²⁺and ferroptosis in ALI animal model and cell model.Methods:In this study,the LPS-induced ALI mouse model was established.HE staining was used to detect the pathological changes of lung tissue.Immunofluorescence double staining was used to detect the expression and co-localization of GPX4,SLC7A11 and TRPV4,respectively.The human normal lung epithelial cell line BEAS-2B was selected for in cell experiments to construct LPS induced ALI cell model.The cytoplasmic ROS was detected by DCFH-DA reactive oxygen species detection kit,the lipid oxidation was detected by MDA content detection kit,and the expressions of GPX4,SLC7A11 and TRPV4 were detected by WB.Calcium ion concentration assay kit cytosolic calcium ion concentration.Results:1.Compared with the control group,the lung tissue of LPS-induced ALI mice showed obvious edema,congestion and a large number of inflammatory cell infiltration,and the fluorescence of GPX4 and SLC7A11 was weak,while TRPV4 showed hyperfluorescence,and TRPV4 could co-localize with GPX4 and SLC7A11.2.Compared with the control cells,ROS and MDA levels were significantly increased,GPX4 and SLC7A11 protein expression was significantly decreased,and cell viability was significantly decreased in the LPS-induced ALI cell model.However,Fer-1,an iron death inhibitor,can promote the viability of ALI cells.3.TRPV4 protein expression and intracellular Ca²⁺concentration were up-regulated in LPS-induced ALI cell model compared with control cells.Part Ⅱ Molecular mechanism of TRPV4 regulating calcium influx and ferroptosis in lung epithelial cellsObjective:We aimed to investigate the molecular mechanism of TRPV4-mediated Ca²⁺influx and ferroptosis in lung epithelial cells to regulate ALI.Methods:In this study,LPS was used to induce lung epithelial cells to construct a ALI cell model.To understand the role of TRPV4 in ALI,we treated lung epithelial cells with TRPV4 antagonist（GSK2193874）and TRPV4 agonist（GSK1016790A）to investigate the effect of TRPV4 on Ca²⁺influx and ferroptosis in lung epithelial cells.The downstream target genes of TRPV4 were screened by transcriptome sequencing,and the expression of candidate target genes was verified by q PCR.Reversion experiments were used to verify that TRPV4 regulated Ca²⁺and ferroptosis through target genes.Results:1.Inhibition of TRPV4 activity blocked Ca²⁺influx in ALI cell model and alleviated ferroptosis of ALI cells compared with ALI group.2.Inhibition of TRPV4 caused the expression of 246 m RNAs changed in ALI+GSK2193874 group,among which 89 m RNAs were up-regulated and 158 mRNAs were down-regulated.The genes regulated by TRPV4 were mainly involved in signaling pathways such as"activation of transmembrane receptor protein tyrosine kinase activity","HIF-1 signaling pathway"and"endocrine factor regulation of calcium reabsorption".3.q PCR showed that compared with ALI group,COL1A2 expression was down-regulated only in ALI+GSK2193874 group.4.Compared with ALI group,TRPV4 antagonist GSK2193874 inhibited Ca²⁺influx and ferroptosis,and this inhibitory effect was reversed by COL1A2 overexpression.Part Ⅲ Therapeutic effects of targeted inhibition of TRPV4 and ferroptosis on acute lung injuryObjective:This part was designed to explore the therapeutic effect of inhibiting TRPV4 and ferroptosis on ALI animal model.Methods:In this study,the LPS-induced ALI mouse model was constructed and treated with TRPV4 inhibitor GSK2193874 and ferroptosis inhibitor Fer-1.The pathological changes of the lung tissue of the mice were detected by HE staining,and the lung edema was detected by dry and wet weight of the lung.The MDA content detection kit was used to detect the MDA level of mice,and the GPX4 expression was detected by Western Blot and IHC.Results:1.Compared with the control group,the ALI group had more severe lung tissue structural damage,alveolar hemorrhage,interstitial edema and alveolar edema,while the ALI+GSK2193874 treatment group and ALI+Fer-1 treatment group had less damage.2.Compared with the control group,the ALI group had the highest level of MDA and the lowest level of GPX4,while ALI+GSK2193874 treatment group and ALI+Fer-1 treatment group could effectively alleviate ferroptosis in ALI.Conclusions:1.In ALI mouse model and LPS-induced ALI cell model,the intracellular Ca²⁺ concentration and ferroptosis level of lung epithelial cells were increased.2.The expression of TRPV4 protein was increased in ALI,and inhibition of TRPV4 activity could block Ca²⁺influx and alleviate ALI induced ferroptosis in lung epithelial cells.3.TRPV4 promoted the expression of COL1A2,and overexpression of COL1A2 reversed the regulatory effect of TRPV4 on calcium influx and ferroptosis in lung epithelial cells.4.Targeted inhibition of TRPV4 reduced ferroptosis and improved pulmonary edema in ALI mice.