| Background:Acute lung injury(ALI)is an acute clinical syndrome characterized by severe hypoxemia,airway dysfunction and uncontrolled inflammation of the lungs.More and more studies have found that the activation of macrophages and the transformation of M1 phenotype play an important role in the development of lung inflammation.As an organelle closely related to energy metabolism,mitochondria are closely related to their functions,whether it is cell survival or apoptosis.Studies have suggested that when lipopolysaccharide(LPS)and IFN-y are used to stimulate macrophages,the mitochondria of macrophages will undergo significant metabolic reprogramming,which is essential for the immune function mediated by macrophages.Cell therapy based on Mesenchymal stem cells(MSCs)has gradually become a promising candidate for the treatment of ALI.While MSCs regulate the inflammatory response of macrophages,macrophages also It can regulate the self-renewal of stem cells and mobilize the recruitment of stem cells to inflammation sites,thereby enhancing the negative regulation of inflammation of MSCs.Recent studies have shown that in various disease models,MSCs and MSCs-derived exosomes have the potential to reduce lung injury and inflammation.Exosomes can interact with other cells through paracrine mechanisms,mediate communication between regulatory genes between cells,or be internalized through intercellular interactions,and lead to biological responses through the ligand-receptor pathway.Compared with stem cell transplantation,exosomes have the advantages of being more stable and storable,without the risk of aneuploidy and the possibility of immune rejection,and have the potential to constitute a safe and effective cell-free therapy.Many studies have focused on the effects of MSCs exosomes on immune cells,including macrophages,T lymphocytes,dendritic cells,and natural killer cells;however,regarding fat-derived MSCs(Adipose Mesenchymal Stem Cells,AdMSCs).The crosstalk between exosomes and macrophages is poorly understood,and the interaction mechanism between the two is worthy of further exploration.Objectives:To verify the therapeutic effect of exosomes derived from adipose-derived mesenchymal stem cells on acute lung injury,and to explore the effector molecular mechanism of exosomes by regulating the mitochondrial metabolism of macrophages to enhance the anti-inflammatory effect of macrophages.To provide reliable scientific evidence for the treatment of immune-inflammatory diseases by exosomes,with a view to finding new research ideas and programs for the treatment of immune-related inflammatory diseases by exosomes.Methods:1)Separate and expand AdMSCs from human adipose tissue,and collect AdMSC-exos from the cell culture supernatant by differential centrifugation.30 male C57BL/6 mice were randomly divided into three groups:normal control group(Ctrl group),model control group(PBS group),and AdMSC-exos transplantation group.Except for the Ctrl group,the other two groups induced acute lung injury models in mice by intratracheal instillation of LPS solution,and transplanted PBS or AdMSC-exos through the tail vein 4 hours after modeling.After 24 hours,lung tissue was obtained for H&E staining,and the bronchoalveolar lavage fluid(BALF)was detected by flow cytometry.Enzyme linked immunosorbent assay(ELISA)and qPCR were used to observe the expression levels of IL-1?,IL-6 and other inflammatory factors in blood and lung tissues.At the same time,MH-S(alveolar macrophage cell line)cells were used for in vitro experiments,and qPCR was used to observe the IL-1?,IL-6,TNF-? and IL-10 in MH-S cells after LPS stimulation by AdMSC-exos The expression level of inflammatory factors and the influence of macrophage polarization;Western blot was used to detect the changes of NF-?B,MAPKs and other inflammatory activation-related signal pathways in MH-S cells under the action of AdMSC-exos after LPS stimulation.2)Using MH-S cells to carry out in vitro experiments,AdMSC-exos was added to the culture supernatant of MH-S cells in advance,and LPS was added half an hour later to induce MH-S inflammation.Observe the effect of AdMSC-exos on the mitochondrial function of MH-S cells stimulated by LPS.The ultrastructure changes of mitochondria of MH-S cells were observed by transmission electron microscope.To detect the effects of AdMSC-exos on the proliferation and apoptosis of MH-S,as well as the effects of mtDNA copy number,ATP content,cellular ROS production and mitochondrial function in cell mitochondria,the hippocampal cell energy/bioenergy metabolism real-time analyzer(Seahorse XF Extracellular Flux Analyzers)analyze the oxygen consumption of cells and the rate of extracellular acid production.At the same time,in vivo experiments were carried out.First,30 male C57BL/6 mice were randomly divided into three groups:normal control group(Ctrl group),model control group(PBS group),and AdMSC-exos transplantation group.Except for the Ctrl group,the other two groups induced acute lung injury models in mice by intratracheal instillation of LPS solution,and injected PBS and AdMSC-exos through the tail vein 4 hours after modeling.After 24 hours of modeling,observe the distribution of fluorescently labeled AdMSC-exos in the lungs of the mouse using a confocal microscope.The immune cells in the alveoli were obtained by alveolar lavage,and they were cultured adherently for 2 hours to obtain the primary mouse.Alveolar macrophages(AMs)were used to further detect mtDNA copy number and ATP content in AMs of each group of mice.Flow cytometry was used to detect cellular ROS production and functional mitochondrial damage in AMs.Then use chlorophosphate liposomes to clear alveolar macrophages,and observe the protective effect of AdMSC-exos on the lungs of mice after alveolar macrophages are depleted.Through adoptive transfer of Bone marrow-derived macrophages(BMDM)treated with AdMSC-exos,the effect of exosomes on the mitochondrial metabolism of macrophages was further observed.Animal experiment results also show that AdMSC-exos carrying stem cell mitochondria can be internalized into mouse lung tissue macrophages.AdMSC-exos can increase the copy number of mtDNA in mouse AMs cells,and the low ATP content of cells after LPS stimulation is effectively reversed.Flow cytometry analysis results show that AdMSC-exos can reduce the ROS production of AMs cells after LPS stimulation,and the number of damaged mitochondrial functions is also greatly reduced.Western blot results showed that AdMSC-exos can increase the expression of AMs mitochondrial biosynthesis and transcription-related molecular proteins.For ALI model mice depleted of AMs,the protective effect of AdMSC-exos on lung tissue injury and inflammation inhibition was significantly weakened.The adoptive transfer of BMDM cells pretreated with AdMSC-exos can also effectively inhibit LPS-induced lung injury and inflammation.3)The expression of mitochondrial respiratory chain complex in exosomes was detected by qPCR and Western blot.Use totipotent nuclease to degrade the nucleic acid components in AdMSC-exos,and observe the inhibitory effect of degraded AdMSC-exos on MH-S inflammation.The effect of AdMSC-exos on mitochondrial biosynthesis and transport of mouse alveolar macrophages was observed by Western blot.In in vitro experiments,AdMSC-exos was added to the culture supernatant of cultured MH-S cells in advance,and LPS was added half an hour later to induce MH-S inflammation.The effects of AdMSC-exos on LPS stimulation were observed by qPCR and western blot.The effect of mitochondrial respiratory chain complex in MH-S cells.4)Observe the effects of curcumin pretreatment on AdMSCs and their derived exosomes after 24 hours.Use CCK8 method to detect the viability of AdMSCs,flow cytometry to detect stem cell surface markers CD 105,CD90,CD45,CD34 and stem cell apoptosis,and observe the effect of curcumin on stem cell differentiation.The exosomes in the culture supernatant of AdMSCs before and after curcumin pretreatment were collected by differential centrifugation.The expressions of commonly used exosomes markers CD9,CD63,TSG101 were detected by western blot,observe the characterization form and concentration of exosomes by transmission electron microscope and NTA particle size analyzer.The effects of AdMSC-exos on the inflammatory response of MH-S cells induced by LPS and the expression of NF-?B and MAPKs signal pathway proteins were detected by qPCR and Western blot.Results:1)After treatment with adipose-derived mesenchymal stem cell exosomes,the symptoms of lung injury in ALI model mice were effectively relieved,the inflammatory response in the lungs and the whole body was reduced,the levels of pro-inflammatory cytokines in lung tissues and BALF decreased,and the levels of anti-inflammatory cytokines were reduced.Increased expression level.AdMSC-exos transplantation can also increase the number of mouse primary alveolar macrophages(Alveolar macrophages,AMs)in the alveolar lavage fluid,and reduce the number of neutrophils and monocyte macrophages.In in vitro experiments,AdMSC-exos can promote the differentiation of MH-S cells into M2 type macrophages,inhibit the phosphorylation level of related proteins on inflammatory signaling pathways NF-kB and MAPKs,enhance the ability of macrophages to resist inflammation,and reduce the cell Inflammation.2)AdMSC-exos transfers mitochondria to MH-S cells and co-localizes with the inner membrane of MH-S mitochondria.The transfer of exosomes through mitochondria has a good promotion effect on the mitochondrial function of MH-S cells.AdMSC-exos pretreatment can improve the destruction of mitochondria after LPS stimulation,increase the number of mitochondrial cristae,restore their normal structure,and increase the mitochondrial mtDNA copy number of MH-S cells,increase the production of mitochondrial ATP,and impair mitochondrial function.The degree is reduced,which further promotes cell proliferation and reduces apoptosis,and also improves the oxidative phosphorylation and glycolysis capacity of macrophages.Animal experiments also show that AdMSC-exos carrying stem cell mitochondria can be internalized into mouse lung tissue macrophages,AdMSC-exos can increase the copy number of mtDNA in mouse AMs cells,and the low ATP content of cells after LPS stimulation is effectively reversed.Flow cytometry analysis results show that AdMSC-exos can reduce the ROS production of AMs cells after LPS stimulation,and the number of damaged mitochondrial functions is also greatly reduced.For ALI model mice depleted of AMs,the protective effect of AdMSC-exos on lung tissue injury and inflammation inhibition was significantly weakened.The adoptive transfer of BMDM cells pretreated with AdMSC-exos can also effectively inhibit LPS-induced lung injury and inflammation.3)The experiment found that mitochondrial complex I subunit NDUFV2 exists in AdMSCs exosomes in the form of nucleic acid and protein.Further experimental results found that when the nucleic acid in AdMSC-exos is degraded,its ability to regulate the inflammatory response of receptor macrophages decreases.qPCR and Western blot results show that LPS stimulation can lead to the loss of subunits I to V on the cellular mitochondrial respiratory chain complex,especially the down-regulation of the complex I NDUFV2,and the administration of AdMSC-exos can effectively increase the cellular mitochondrial complex expression.qPCR,flow cytometry and other experimental results show that when the mitochondrial respiratory chain complex INDUFV2 is knocked down,AdMSC-exos's ability to inhibit macrophages immunosuppression and improve mitochondrial metabolism is weakened.4)Curcumin pretreatment has no effect on the growth,morphology,and phenotype of stem cells,nor does it change the characteristics of their derived exosomes.In addition,curcumin can significantly increase the amount of exosomes secreted by stem cells after treating AdMSCs.In terms of inhibiting inflammation,the use of curcumin to pretreat AdMSCs can enhance the effect of AdMSC-exos in reducing the phosphorylation level of related proteins on inflammatory signaling pathways NF-?B and MAPKs,and effectively reduce cell inflammation.Conclusions:Human adipose-derived mesenchymal stem cell-derived exosomes can inhibit inflammation by regulating NF-?B and MAPKs signaling pathways,regulate the immune function of macrophages,and promote the activation of macrophages from pro-inflammatory M1 to anti-inflammatory M2 Polarization,effectively alleviating acute lung injury in mice induced by LPS;AdMSC-exos transfers mitochondria to macrophages in a dose-dependent manner.The transfer of mitochondria can improve the structural integrity and biogenetics of mitochondria of macrophages,improve the ability of cell oxidative phosphorylation and glycolysis,promote cell proliferation and reduce cell apoptosis.The improvement of mitochondrial function of alveolar macrophages further promotes the functional shift of endotoxin-stressed macrophages to an anti-inflammatory phenotype,thereby reducing the damage and inflammation of lung tissue in ALI model mice.The protective effect of AdMSC-exos is to a large extent The above depends on mitochondrial transfer and the metabolic reprogramming of alveolar macrophages;AdMSC-exos contains the NDUFV2 fragment of the mitochondrial complex in AdMSCs cells,and is located on the mitochondrial inner membrane of recipient macrophages through transfer to adjust LPS Mitochondrial dysfunction caused by the lack of mitochondrial complexes in the recipient cells after stimulation,then plays a role in inhibiting the inflammatory response.At the same time,curcumin can increase the production of stem cell exosomes after pretreatment of AdMSCs,greatly improve the efficiency of extracting exosomes,and can also improve the ability of AdMSC-exos to resist macrophage inflammation.In general,the treatment of exosomes represents a promising acellular therapy.Exosomes including the transfer of mitochondrial fragments also provide new insights for the clinical treatment of diseases related to mitochondrial dysfunction.Traditional Chinese medicine and stem cell therapy Combined use can play a complementary role,and this idea also provides more options for the clinical treatment of a variety of immune inflammatory diseases. |
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