Effect Of Exosomes Derived From Mesenchymal Stem Cells On Lipopolysaccharide-induced Acute Lung Injury Model

Objective:To observed the mechanism of the disease and the effects on the derived exosomes originated from human mesenchymal stem cells of the umbilical cord in the trachea on inflammation relief,antioxidant stress and macrophage activation in the lipopolysaccharide-induced model of acute lung injury in C57BL/6 mice.Method:(1)Isolation and identification of neonatal umbilical cord mesenchymal stem cells derived exosomes: the normal delivery of umbilical cord was collected,and then isolated the Walton gum tissue,and the cells were cultured in the special medium for mesenchymal stem cells with the method of tissue block sticking.The cells were passed on when the density of which was 70-80%.The supernatant of MSCs culture was collected in the 5th generation,and exocrine was extracted by ultracentrifugation at 4 ℃and the membrane protein antigen(CD9,TSG101,etc.)was detected by Western blot(WB),and the size of vesicles was analyzed by electron microscopy.(2)In vitro,the activation of MSC-EVs on the RAW264.7 cells induced by lipopolysaccharide(LPS)of gram negative bacilli was studied.The cells were randomly divided into control group,LPS group and LPS + MSC-EVs group.The LPS +MSC-EVs group was pretreated with MSC-EVs(10g/ml)in serum-free medium for 12 hours to ensure uptake.Then,the macrophages in LPS group and LPS + MSC-EVs group were stimulated with 100ng/ml LPS for 12 h.Then,the level of TLR4,TNF-α and IL-1 β protein in the cells of each group was detected by Western blot.(3)Constructing animal models of acute lung injury: Elected 45 healthy small and black C57BL/6 mice,randomly divided into sham operation group,ALI model group and ALI+ MSC-EVs treatment group.The normal saline,LPS and MSC-EVs were injected into the lungs via endotracheal intubation of mice according to the group conditions by intratracheal administration.After the model was established,the overall situation of the mice was carefully monitored.Mice were sacrificed 24 hours,4 days,14 days after successful model sleep and perfusion,and mouse lung tissue was collected.(4)The left lungs of mice at 24 hours,4 days,and 14 days were stained with he to determine pathological lung changes and to determine whether MSC-EVs play a therapeutic role in ALI.(5)According to the preliminary judgment of HE staining results,the right lung tissues of mice were selected for m RNA sequencing in 4 days,and the go enrichment and KEGG enrichment were improved according to the selected genes.The therapeutic effect of the biological effects of MSC-EVs on ALI was determined.(6)Combined with HE staining and m RNA sequencing,the expression of Nrf2,Keap1,HO-1,TLR4,NF-κB p65,i NOS and Arg1 were detected by Western blot in the right lung tissues of mice on 4 and 14 days.Result:(1)The isolated MSc EVs were observed as oval vesicles under transmission electron microscope.The expression of CD9 and TSG101 was detected by Western blot method compared with MSC.It indicated that the collected exocrine was in accordance with the identification standard and could be used for subsequent experiments.(2)In vitro,Raw264.7 cells were able to take up exosomes.Compared with the control group,the expression levels of TLR4,IL-1 β,TNF-α in LPS group were significantly higher(p < 0.05),while the expression levels of factors in LPS + MSC EVs group were significantly lower than that in LPS group(p < 0.05).(3)In vivo experiments,HE staining of each group can be found that the control group has thiner alveolar wall,no widening of alveolar interval,congestion,and no inflammatory exudation.The bronchiectasis of alveoli was the most serious in ALI group,and the alveolar wall was congested and there were a large number of inflammatory cells in the alveoli.The MSC-EVs treatment group was between the control group and the acute lung injury group.Compared with the control group,the lung tissue injury score of the mice in the acute lung injury group was significantly increased,and the score of lung tissue injury in MSC-EVs treatment group was significantly lower than that in ALI group,and the above symptoms were the most obvious on the fourth day.(4)The results showed that:(1)the absolute value of Fold Change was ≥ 2 and p< 0.05 was used as the standard to screen the differential expression m RNA.A total of154 up regulated m RNA and 68 down regulated m RNA were selected.The selected differential genes included a large number of genes related to immune regulation mechanism and redox mechanism,such as TLR4,Arg1,HO-1,etc.;(2)through the enrichment and analysis of GO function,biological process,cellular component,molecular function were selected.The results esposed that the genes were enriched in 55 go items.There are many go items in biological process,including positive regulation of immune response and acute inflammatory reaction.Organelles,cell membrane,vesicles and vesicles in cell components are enriched,while on molecular function level,they are mainly concentrated in chemokine activity and cysteine peptidase activity;(3)after enrichment analysis of KEGG pathway,it is found that differential expression genes are mainly concentrated in The immune mechanism and 151 KEGG pathways in signal transduction mechanism,including toll like receptor signaling pathway,influenza A virus pathway,nod like receptor signaling pathway,etc.(5)Western blot showed that the expression of Nrf2,HO-1 and Arg1 in lung tissue of ALI model mice increased on the fourth day(p < 0.05),and the expression of these molecules was significantly higher in ALI + MSC-EVs group than in ALI model group.The expression of NF-κB p65 and i NOS in lung tissue of ALI model mice was significantly increased(p< 0.05),and the expression of these four molecules decreased after the treatment of exosomes(p < 0.05),and the difference was statistically significant.However,at 14 days after the disease model established,except Nrf2,the other molecular differences were not statistically significant.It is suggested that the disease course of lung injury model constructed in vivo is self limited.Conclusion:The experimental results demonstrated that the mesenchymal stem cells derived exosome mainly depended on the M2 activation of macrophages.The anti-oxidation and anti-inflammatory effects of LPS induced C57bl/6 mice were exerted by transcription factor E2 related factor signaling pathway and Toll like receptor 4/ NF-κB signaling pathway.