Mesenchymal Stem Cell Conditioned Medium Relieves Acute Lung Injury Through KGF And Its Mechanism

Objective: Acute lung injury(ALI)is an acute diffuse inflammatory lung injury caused by a variety of pathogenic factors and the mortality rate is as high as 30-60%.ALI can lead to atelectasis or even acute respiratory distress syndrome(ARDS).Its pathogenesis includes imbalances of inflammatory/anti-inflammatory reaction,redox,and apoptosis.The pathological characteristics usually include severe hypoxemia,pulmonary edema and excessive accumulation of neutrophils in the lung.Lipopolysaccharides(LPS)as an important inflammatory inducer,pulmonary infection caused by which is one of the most common causes of ALI.In this study,we established the model of ALI induced by LPS,and explored its therapeutic methods and mechanisms.Mesenchymal stem cell(MSC)has natural regenerative and anti-inflammatory properties,and shows great therapeutic potential in lung diseases,including ALI.In recent years,with increasing attention to MSC-related adverse reactions,research emphasis has turned to MSC-derived exosomes or MSC conditioned medium(MSC-CM)for simpler strategy with fewer potential limitations.Keratinocyte growth factor(KGF)is mainly produced by MSC and may ameliorate LPS-induced ALI,possibly by acting on alveolar type II(ATII)cells,while the specific mechanism remains to be further studied.Organoids are derived from stem cells or organ-specific progenitor cells,which structure and function are consistent with in vivo organs,and can be widely used in disease modeling and regenerative medicine.Notably,the lung organoids derived from ATII cells can express epithelial sodium channel(ENa C),which is in charge of sodium water transport and critical to the clearance of alveolar edema fluid in ALI patients.In this study,we aim to explore the effects of MSC-CM secreted KGF on the proliferation and differentiation of ATII cells by establishing mouse and alveolar organoid models,and to investigate the involvement of ENa C regulation,which helps to provide an extensive clinical application of MSC-CM in the treatment of ALI.Methods:To clarify the therapeutic effect of MSC-CM and KGF on LPS-induced ALI model in mice:1.The mouse ALI model induced by LPS was established,and MSC-CM was injected into the tail vein for treatment.The morphological changes of lung tissue in mice were observed;The alveolar fluid clearance,lung wet-dry weight ratio,total lung water content,lung coefficient and other related indicators of ALI were measured.2.Inflammatory associated protein TNF-α and IL-1β/6 in bronchoalveolar lavage fluid were detected by ELISA.3.Bioinformatics analysis showed that KGF was closely related to ALI,inflammation and cell proliferation.The concentration of KGF in MSC-CM was detected by ELISA kit.4.LPS-induced mouse ALI model was treated by intraperitoneal injection of KGF,and then the lung water clearance rate and lung wet-dry weight ratio were measured to determine the effect of KGF on lung edema in mice.Establish 3D alveolar organoids model and clarify the effect of MSC-CM and KGF on this model:1.Mouse ATII cells were extracted and purified,and the cell purity was detected by flow cytometry,and 3D alveolar organoids were establishted by 3D culture technology,and identified by immunofluorescence,HE staining and other experiments.2.The effects of MSC-CM and KGF on the formation of 3D alveolar organoids were explored through the formation efficiency of alveolar organoids.3.The morphological changes of 3D alveolar organoids were observed through optical microscope and HE staining to determine whether MSC-CM and KGF can recover the damage of 3D alveolar organoids induced by LPS.4.Inflammatory associated protein TNF-α and IL-1β/6 in alveolar organoids conditioned medium were detected by ELISA.5.KGF in MSC-CM was neutralized with KGF neutralizing antibody,and then the effects of MSC-CM and KGF on the proliferation and differentiation of ATII cells in 3D alveolar organs were determined by Ed U staining and immunofluorescence.6.Combined with KGF neutralizing antibody,immunofluorescence and quantitative realtime polymerase chain reaction(q RT-PCR)technology,the expression of ENa C in 3D alveolar organs was detected.Exploration of the relevant mechanisms of MSC-CM and KGF on LPS induced ALI in ATII cells:1.Western blot was used to detect the protein expression of the protein domain-binding motif(TAZ)in the Hippo signal pathway related to cell proliferation and organ development,and to clarify the effect of MSC-CM and KGF on the proliferation of ATII cells.2.ALI/ARDS related genes and signal pathways were analyzed by analyzing single cell sequencing data and other bioinformatics technologies.3.The effect of MSC-CM and KGF on the expression of inflammatory related nuclear factor κB(NF-κB)signal pathway nucleoprotein in ATII cells was determined by Western blot.4.The relationship between the NF-κB and ENa C was explored by blocking the activation of the pathway with inhibitors,and then detecting the expression of ENa C with Western blot.5.Western blot was used to detect the protein expression of ENa C after LPS,MSC-CM and KGF treatment of ATII cells.6.After neutralizing KGF in MSC-CM,the effect of MSC-CM on the expression of TAZ which is associated with cell proliferation,ENa C,and NF-κB protein was detected by Western blot.Results:MSC-CM and KGF had therapeutic effects on LPS-induced ALI model in mice:1.We observed the morphological changes of the lung in mice after treatment.The lung tissues of Control group and CM group were uniformly pink,with intact capsule and sharp edge.However,the appearance of LPS-induced lungs was swollen and hyperemic,which was improved after MSC-CM administration.2.MSC-CM could reverse the enhanced expression levels of IL-1β,IL-6,and TNF-α in bronchoalveolar lavage fluid of LPS-treated mice.3.MSC-CM could partially restore the LPS-inhibited alveolar fluid clearance,an indicator for lung fluid transport.4.Compared with the control group,the lung wet dry weight ratio,lung index,and total lung water content in LPS group increased,whereas decreased after MSC-CM treatment,indicating the edema of lung tissue was improved significantly.5.It was found that KGF was closely related to inflammation and cell proliferation by using bioinformatics technology,and the concentration of KGF in MSC-CM was 30.29pg/m L by using ELISA kit.6.After treating ALI mouse model with KGF,its effects on alveolar fluid clearance and lung wet/dry weight ratio in mice were similar to those of MSC-CM.MSC-CM and KGF could recover LPS-induced 3D alveolar organoids damage:1.After LPS treatment,the size of 3D alveolar organoids decreased significantly,while MSC-CM had a reversal effect.2.MSC-CM could reverse the enhanced expression levels of IL-1β,IL-6,and TNF-α in conditioned meidium of alveolar organoids.3.The organoid forming efficiency in the condition of both MSC-CM and KGF was significantly higher than that in Control group,suggesting the formation and maturation of alveolar organoids may be improved.4.HE staining results showed that the morphological structure of organoids was significantly impaired after exposure to LPS,which partially recovered by the administration of MSC-CM and KGF.5.Ed U staining test and immunofluorescence test confirmed that MSC-CM and KGF can promote the proliferation of ATII cells in 3D alveolar organoids and affect their differentiation;after neutralizing KGF in MSC-CM,the effect is weakened.6.The immunofluorescence assay results showed that α-ENa C expression in alveolar organoids was enhanced by MSC-CM and KGF after LPS administration.Furthermore,the q RT-PCR results verified that MSC-CM and KGF could also increase α,β,and γ-ENa C expression at m RNA levels;the effect of MSC-CM weakened after neutralizing KGF.TAZ protein and NF-κB signal pathway participated in the therapeutic effect of KGF on ALI in MSC-CM:1.MSC-CM up-regulated the expression of TAZ protein related to cell proliferation.2.The results of bioinformatics analysis showed that ALI/ARDS was closely related to NF-κB signal pathway.3.The nucleoprotein expression of NF-κB p65 was increased by LPS treatment,and decreased by MSC-CM and KGF treatment.4.The inhibition of NF-κB signal pathway could up-regulate the protein expression of α-ENa C.5.MSC-CM and KGF could enhance LPS-inhibited the protein expression of α-ENa C.6.After neutralizing KGF in MSC-CM,the effect of MSC-CM on the expression of TAZ,ENa C,and NF-κB protein was weakened.Conclusion: Both MSC-CM and KGF have therapeutic effects on LPS-induced mouse ALI model,promote the formation of 3D alveolar organoids,partially recover the morphological damage caused by LPS,and promote the repair of damaged alveolar epithelium.The mechanism may be related to the promotion of ATII cell proliferation by up-regulating the expression of TAZ protein,in addition,they may reduce inflammation and up-regulate the expression of ENa C by inhibiting the NF-κB signal pathway.The therapeutic effect of MSC-CM on ALI is closely related to soluble cytokine KGF.This result provides theoretical and experimental basis for MSC-CM to be more widely used in clinical treatment of ALI.