Role Of NF-кB Signaling Pathway In Mesenchymal Stem Cells Regulated Macrophage Function In The Treatment Of Burns Sepsis

The mechanism and treatment of burn sepsis has been a difficult research focus.Mesenchymal stem cells (MSCs) are multipotent cells that are being clinically explored asa new therapeutic for treating a variety of immune-mediated diseases. The alternativefunctions are now being characterized in the context of MSC transplantation, wherebyparacrine interactions between MSCs and host cells of both the innate and adaptiveimmune systems have been shown to relate directly to the therapeutic activity of MSCs. Inrecent years, it has pioneered studies to investigate systemic administration of MSCs as atherapy for steroid-refractive graft-versus-host disease (GvHD), Crohn’s disease, type Idiabetes mellitus (IDDM), myocardial infarction (MI), and chronic obstructive pulmonarydisease (COPD) and other diseases. Because early has been in GVHD, systemic lupuserythematosus, rheumatoid arthritis, acute lung injury, acute renal dysfunction, acuteautoimmune liver damage, adipose mesenchymal stem cells made significant therapeuticeffect. However, there is still little research about MSC therapeutic on burn sepsis. And its therapeutic mechanism is still not very clear.First, it is a very important to establish an stable, high replicated burn sepsis modeland harvest a large mount of mesenchymal stem cells. In the first experiment, weestablished a mouse model of burn sepsis with a III°burn wound of about10%ofTBSA, followed by inoculating with2×105CFU Pseudomonas aeruginosa in femaleC57BL/6mice. The mice with burn sepsis developed SIRS and MODS and had a50%-80%high rate of mortality,, which were reproducible. This novel mouse model of burnsepsis assembled the pathogenesis and clinical characteristics of human with burn sepsis.In the second part of the experiment, it aim to harvest Adipose-derived stem cells ofC57mice in vitro. ADSCs were isolated by enzyme digestion. And ADSCs within3passages were used to verify the mult-potential differentiation capacity and FACS analysis.It showed us the cells had typical morphological characters of stem cells and were positivein the ADSCs’markers CD90, CD105, CD44, negative in CD34, CD45.In the third experiment，it aim to investigate ADSCs therapeutic function on burnsepsis. After administration of2×105ADSCs, we obeseved clinical severity score forevaluation of the clinical status and tested mortality, organ bacteria quantitation, liver andkidney function, as well as changes in proinflammatory/anti-inflammatory cytokines inorgans or blood. Experimental results show that:1, the general condition of mice,spontaneous activity, and the reaction of the outside has improved significantly; After24h,48h to72h, organs bacteria quantitation were significantly reduced;3, TNF-, IL-12expression decreased significantly and anti-inflammatory cytokines IL-10expression wassignificantly elevated in liver and lung mRNA. The serum TNF-, IL-6and otherproinflammatory cytokines and anti-inflammatory cytokines IL-10changes have the sameresults.4, HE staining of liver, lung and kidney organs is shown the infiltration ofinflammatory cells significantly reduce, cell necrosis and apoptosis was significantlyreduced, improved organ function. These results suggest that intravenous administration ofADSCs improves SIRS and MODS with burn sepsis. These demonstrated that thefeasibility and effectiveness of ADSCs administration for experimental burn sepsis,providing the basis for development of a potentially effective immunomodulatory strategy to decrease morbidity and mortality in clinical sepsis.In the fourth experiment, it aims to investigate interaction of ADSCs withRaw264.7macrophages and interaction of ADSCs with NF-кB signaling pathway in theirantiinfammatory/immune modulatory effects. We co-culture ADSCs with Raw264.7macrophages after stimulated by LPS. And We used cell surface antigen CD206expression and intracellular cytokine expression patterns to study the immunophenotypeof macrophages at the end of this coculture period in vitro. They were divided into fourgroups: normal control group, LPS stimulation group Transwell group, mixed group.Experimental results show that:1, Macrophages cocultured with ADSCs consistentlyshowed high-level expression of CD206, a marker of alternatively activated macrophages.However, macrophages cocultured with MSCs also expressed high levels of TGF-b,COX-2and low levels of tumor necrosis factor alpha (TNF-a) and IL-6compared tocontrols.2, ADSCs co-cultured group, nuclear translocation of P65cells and its upstreammolecular p-IKB-a, p-of IKK-phosphorylation significantly inhibited. The experimentalresults show that the ADSCs may be adjusted not only by paracrine role of macrophages,but also by contact between macrophages play a regulating role. PGE2and NF-кBsignaling pathway may be key factors, it is also the key point in therapy for sepsis withADSCs.