NO-induced Exosome Secreted By Human Placenta-derived Mesenchymal Stem Cells Rich In MiR-126 For The Treatment Of Sepsis In Rats

Background The global incidence of sepsis is about 27-30 million per year,and the death toll is still as high as 7-9 million.Sepsis still lacks effective treatment.In recent years,with the deepening of stem cell research,stem cells have achieved good results in the treatment of sepsis.However,its mechanism of action is not to replace necrotic cells by differentiation and proliferation;it relies on its paracrine function to correct the imbalance of immune function through immune regulation,thereby exerting therapeutic effects.On the other hand,stem cell treatment has the disadvantages of carcinogenic risk,missed optimal treatment window,and low number of cells reaching the affected area.Regeneration studies have shown that exosomes are important material carriers for stem cells to exert their paracrine functions.The exosomes use their contents to reprogram the target cells,thereby exerting their regulatory effects.Among them,mi RNA is one of the main pharmacodynamic molecules of exosomes.Recently,studies have shown that NO-induced mi R-126 rich placental mesenchymal stem cell exosomes can promote the regeneration and repair of vascular injury.However,vascular endothelial injury and DIC are important pathological mechanisms of sepsis.Therefore,it is hypothesized that NO-induced mi R-126 rich placental mesenchymal stem cell exosomes have a role in the treatment of sepsis.This experiment attempts to verify the therapeutic effects of exosomes by constructing a rat model of sepsis and explore its possible pharmacological mechanisms.Purpose1.The first part: By separating and culturing placental mesenchymal stem cells,they were NO induced.The exosomes were further enriched from the conditioned medium for identification and comparison.which provided materials for subsequent experiments.2.The second part: A rat sepsis model was established.To study the therapeutic effect of exosome rich in mi R-126 on septic rats and explore its possible pharmacological mechanisms.Experimental methods1.The first part: Get a healthy placenta clinically.Culture and passage of human placental mesenchymal stem cells(hp-MSC)were carried out by enzymatic digestion.The fourth generation of hp-MSC was used to induced and cultured with NO,mi R-126 inhibitor and null control.Three exosomes(N-Exo,I-Exo and Exo)were obtained by ultracentrifugation.The morphology of the exosomes was observed and compared by transmission electron microscopy.Western blotting was used to detect exosome surface marker molecules.The contents of three exosomes mi R-126-3p and-5p were detected and compared by q RT-PCR.Thereby comprehensively identify exosomes and compare the differences between the three.2.The second part: A rat model of sepsis was established and divided into four groups:control group(C group,sham operation + PBS),sepsis group(S group,CLP + PBS),Exo treatment group(N group,CLP + N-Exo),I-Exo treatment group(group I,CLP+I-Exo).At 48 hours after modeling,Blood liver and kidney function index(ALT,AST,BUN)and inflammatory factors(IL-6,IL-10,TNF-?,IFN-? and IL-1?)were measured in each group.The lungs and kidneys of the rats were taken for HE staining and pathological scores were performed.The ratio of vascular microleakage and dry/wet weight in the lungs and kidneys of each group of rats was measured.The contents of mi R-126-3p and-5p in the kidney of each group were detected by q PCR.Western blot was used to detect the contents of HMGB1,RAGE,TRL-2,TRL-4 and p-p65 in the kidney of each group.Finally,the 7-day survival rate of each group of rats was observed and counted.Results1.The first part: All three exosomes were round membrane vesicles,and there was no significant difference in morphology.The average particle size of N-Exo was 70.9±5.3nm;Exo was 71.5±4.4 nm,and I-Exo was 72.3±5.1 nm.There was no significant statistical difference between the three(P>0.05).All three exosomes express high levels of CD9 and CD63 and hardly express Tubulin.There was a statistically significant difference in the content of mi R-126 among the three exosomes(P<0.05),I-Exo < Exo< N-Exo.2.2.The second part:(1)Seven-day survival rate of each group of rats: There was no significant difference between the S group and the I group(P>0.05),but both were significantly lower than the N group and the C group(P<0.05).(2)At 48 hours after operation,the ratio of liver and kidney function and inflammatory factors,lung and renal pathological scores,vascular microleakage and dry/wet weight: no statistically significant difference between group S and group I,group N and group C(P >0.05),but group S and group I were significantly higher than group N and group C(P<0.05).(3)The content of mi R-126-3p and-5p in the kidneys of each group: There was no significant difference between the S group and the I group,N group and C group(P>0.05),but the S group and the I group were significantly lower than the N group.And group C(P<0.05).(4)The contents of HMGB1,RAGE,TRL-2,TRL-4,and p-p65 in the kidneys of each group: There was no significant difference between the S group and the I group,N group and C group(P>0.05),but the S group and Group I was significantly higher than group N and group C(P<0.05).Conclusion1.This study successfully cultured and induced three different exosomes of placental mesenchymal stem cells,identified and compared them.NO can obviously increase mi R-126 content in stem cell exosomes.2.N-Exo may reduce the level of HMGB1 in septic rats by infusion of mi R-126,and thereby inhibit the activation of NF-?B through signal transduction of TLR-2,TLR-4and RAGE receptors.In addition,it inhibits the release of various inflammatory factors and alleviates the inflammatory response,thereby reducing vascular endothelial cell damage and vascular microleakage,protecting the functions of important organs,and ultimately improving the survival rate of septic rats.