The Mechanism Of Adipose-derived Mesenchymal Stem Cells Derived Exosomes Alleviating Macrophage Pyroptosis And PMVECs Ferroptosis To Alleviate Acute Lung Injury In Sepsis

BackgroundSepsis is a common disease with high morbidity,complex pathogenesis,critical condition and high mortality rate,which is one of the most challenging diseases faced with medical staff.Acute lung injury(ALI)is one of the most common and serious complications of sepsis,which occurs the earliest in the development of sepsis.Previous studies have shown that macrophages would be activated by invasive pathogen during sepsis.Activated macrophages can produce excessive inflammatory cytokines and reactive oxygen species to remove pathogens.However,these productions also lead to injury of the pulmonary microvascular endothelial cells(PMVECs),resulting in barrier dysfunction and infiltration of more inflammatory cells in the lungs,such as neutrophils and macrophages.Infiltrated inflammatory cells in the lung tissue lead to increased inflammatory response,resulting in continuous injury to PMVECs.Consequently,macrophage pyroptosis and PMVECs ferroptosis play a key role in the process of ALI in sepsis,while attenuating macrophage pyroptosis and protecting PMVECs from ferroptosis caused by excessive inflammatory response are the effective therapeutic strategies for ALI in sepsis.Adipose-derived mesenchymal stem cells(ADSCs)exosome,an important means of intercellular communication,plays an important role in tissue repair and regeneration by regulating a variety of cellular functions.ADSCs exosome could exert protective effect in inflammatory diseases,such as: attenuating macrophages induced excessive inflammatory response in sepsis.Our previous study showed that ADSCs exosomes could exert a protective effect in sepsis by regulating HO-1 expression in macrophage to attenuate excessive oxidative stress.However,the specific regulatory mechanism underlying protective effect of ADSCs exosome on sepsis ALI needs to be further explored,especially regulation effect and mechanism underlying macrophage pyroptosis and PMVECs ferroptosis.PurposeThe aim of this study is to explore the protective effect and mechanism of ADSCs exosomes on sepsis ALI.Observe the protective effect and specific mechanism of ADSCs exosomes on macrophage pyroptosis by constructing macrophage pyroptosis model and excessive inflammatory induced PMVECs ferroptosis model.Thereby,providing a theoretical basis for the clinical application of ADSCs exosomes in the treatment of sepsis ALI and a new strategy for the treatment of sepsis ALI.Methods1.Collect adipose tissues from patients undergoing abdominal surgery at Department of Burns and Cutaneous Surgery,Xijing Hospital.Primary ADSCs were extracted from adipose tissues,and characterized with lipogenic and osteogenesis induction as well as flow cytometry.Supernatant culture medium of ADSCs was collected,while exosome in it was extracted by ultracentrifugation.The extracted exosomes were further characterized by TEM,NTA and Western blotting.2.Cecal ligation and puncture(CLP)was conducted in BALB/C mice to generate sepsis ALI,while ADSCs exosomes were intravenously injected through tail vein.Observe the mortality rate of mice in different treatment groups within 48 hours.Collect serum and alveolar lavage fluid of different mice to detect the expression of various cytokines.And collect the hearts,livers,lungs and kidneys to observe organ damage.Protein and RNA in lung tissue were extracted to detect the indicators related to cellular pyroptosis and ferroptosis.Lung tissues were immunohistochemically stained with ROS and 8OHd G to observe the oxidative stress damage in the lung tissues,while immunofluorescent staining for MPO and macrophage specific markers and pyroptosis-related molecules was conducted to observe macrophage polarization and pyroptosis.And alveolar macrophages were collected by lung lavage to observe the pyroptosis and macrophage polarization.Immunofluorescence and immunohistochemical staining of lung tissues for 4HNE,GPX4 and Nrf2 were performed to observe the damage of PMVECs and protective effect of ADSCs exosomes on lung tissues of septic mice.3.Lipopolysaccharide(LPS)-stimulated RAW264.7 macrophages were used to construct a macrophage pyroptosis model,while ADSCs exosomes were added to observed their effect on inflammation.The expression of various inflammatory factors in the supernatant was detected by ELISA and Bio Legend LEGENDplex.The expressions of M1/M2 macrophage-specific markers were detected by flow cytometry and Western blottingt.Western blotting and immunofluorescence staining were conducted to detect the expression of molecules related to pyroptosis.Next,expression of NLRP3 was inhibited by transfection of NLRP3-specific si RNA,and effect of ADSCs exosomes on macrophage pyroptosis was further observed when NLRP3 was inhibited.Non-coding RNA sequencing of ADSCs exosomes was performed to screen out miRNAs that might regulate macrophage pyroptosis.And the regulatory effect of miR-24-3p,which was highly expressed in ADSCs exosomes,on NLRP3 was detected by dual luciferase reporter gene.4.PMVECs were extracted from BALB/C mouse and verified by CD31 immunofluorescence staining.The supernatants of LPS-stimulated RAW264.7 cells were collected to stimulate PMVECs so as to construct excessive inflammatory injury model on PMVECs,while ADSCs exosomes were added to observe the protective effect on PMVECs.The proliferation,migration and tube-forming ability of PMVECs were detected by CCK8,scratch and tube-forming assay,while the oxidative stress injury of PMVECs were observed by immunohistochemical staining of ROS,8OHd G,JC-1 and lipid peroxidation.Expression of ferroptosis-related indexes was detected by GSH,FRAP,SOD and CAT.The protective effect of ADSCs exosomes and expression of ferroptosis-related molecules on PMVECs was further detected by western blotting and immunofluorescence staining.The regulation effect of ADSCs exosomes on PMVECs ferroptosis was observed after GPX4 inhibited by RSL3.Nuclear factor-erythroid2-related factor 2(Nrf2)and Kelch-like epichlorohydrinassociated protein 1(Keap1),upstream regulators of GPX4,were detected by western blotting and PCR.And the regulatory role of Keap1 by miR-125b-5p,highly expressed in exosomes of ADSCs,was detected by a dual luciferase reporter gene.Results1.ADSCs were successfully extracted,lipogenic and osteogenic induction of which confirmed multi-differentiation potential,while flow cytometry confirmed the characteristics of stem cells.The exosomes in ADSCs supernatants were successfully extracted by differential ultracentrifugation.The extracted exosomes were identified by NTA,western blotting and TEM,the results confirmed that the ADSCs exosomes were successfully extracted.2.In CLP-induced sepsis model,ADSCs exosomes reduced the expression of inflammatory factors in serum and lung tissues,attenuated organ damage,and lowered the mortality of septic mice.ADSCs exosomes could significantly attenuate the injury of septic lung tissues.Further analysis showed that ADSCs exosomes reduced the proportion of M1-macrophages and polarized macrophages towards M2-type in lung tissues,while reducing the expression of pyroptosis-related molecules.ADSCs exosomes also attenuated the damage of PMVECs caused by excessive inflammatory response in septic lung tissues and improved PMVECs ferroptosis.ADSCs exosomes reduced the expression of 4HNE and increased the expression of anti-ferroptosis(GPX4 and Nrf2)molecules in lung tissues.3.Macrophage pyroptosis model was successfully established by LPS stimulation,and ADSCs exosome treatment attenuated LPS-induced inflammatory factor expression,decreased iNOS and CD86 expression,increased Arg-1 and CD206 expression,and polarized macrophages toward the M2 type,while attenuating ROS accumulation.Further pyroptosis-related molecule assays confirmed that ADSCs exosome attenuated macrophage pyroptosis-related molecule expression.The anti-inflammatory effect of ADSCs exosomes was dependent on NLRP3 via NLRP3 inhibition assay.And it was verified by dual luciferase reporter gene that ADSCs exosomes attenuated macrophage pyroptosis response by delivering miR-24-3b,highly expressed in ADSCs exosomes,to modulate the NLRP3-related pathway.4.PMVECs injury model was successfully constructed by stimulating PMVECs with supernatants from LPS-treated RAW264.7 cells.ADSCs exosomes could attenuate the damage of excessive inflammatory response on the proliferation,migration,and tubeforming ability of PMVECs,while attenuated the oxidative stress damage of PMVECs by increase the antioxidant capacity.Meanwhile,ADSCs exosomes could attenuate excessive inflammation-induced ferroptosis through the increase of expression of anti-ferroptosis molecules,such as GPX4 and Nrf2.Further GPX4 inhibition experiments showed that ADSCs exosomes attenuated inflammatory response-induced ferroptosis by upregulating GPX4.Meanwhile,ADSCs exosomes decreased Keap1 expression and increased Nrf2 expression and nuclear translocation.miRNA analysis and further inhibition experiments confirmed that delivery of miR-125b-5p by ADSCs exosomes inhibited Keap1 and attenuated ferroptosis in PMVECs.ConclusionsADSCs exosomes could attenuate the inflammatory response and lung injury in CLPinduced sepsis mice,hence reduced the mortality of mice.ADSCs exosomes could attenuate macrophage pyroptosis and excessive inflammatory response by inhibiting the expression of NLRP3 in macrophages through miR-24-3b.It could also attenuate the expression of Keap1 in PMVECs caused by excessive inflammatory response,promote the expression and nuclear translocation of Nrf2,and up-regulate the expression of the anti-ferroptosis protein GPX4 by delivery of miR125b-5p.This study confirms that ADSCs exosomes attenuate septic ALI by delivering highly-expressed miRNAs to macrophages and PMVECs to attenuate macrophage pyroptosis and inflammatory response as well as ferroptosis in PMVECs,which provides a theoretical basis for the treatment of septic ALI by ADSCs exosome,and provides a new possible clinical strategy for the treatment of sepsis.