Role Of Exosomes Derived From Bone Marrow Mesenchymal Stem Cell In Rat Hepatic Ischemia And Reperfusion Injury

Objectives:Hepatic Ischemia Reperfusion Injury (HIRI) refers to the phenomenon that liver dysfunctional and structural damage caused by Ischemia and Hypoxia will be more serious when blood reperfuse. HIRI commonly happen in liver surgery, hypovolemic shock, and especially difficult to avoid in liver transplantation. At present, HIRI has received widespread attention in the clinic, but there are no ideal measures to deal with it.Recently, we found that exosomes derived from bone-marrow mesenchymal stem cells can activate the mechanism of cells’ self protection and endogenous regeneration and it can also regulate the immune response to repair the damaged cells. It can protect the organs function by transferring the protein, lipid, mRNA and mircoRNA with biological activity.The purpose of this study is to investigate the effects and the possible mechanisms of BMSCs-exosomes that treat HIRI on the rat model, provide a theory for further research of BMSCs-exosomes.Methods:(1) Rat bone marrow mesenchymal stem cells were harvested under aseptic condition, cultured and passaged to 3-6 generation, obtain stable bone-marrow mesenchymal stem cells.(2) Collect the supernatant of bone marrow mesenchymal stem cell, purified exosomes from the supernatant by ExoQuick-TC. Observed the extracts by Transmission Electron Microscopy (TEM) to determine whether they are exosomes.(3) 32 healthy adult male SD rats were randomly divided into four groups, each group had 8 rats (group sham, group PBS, group BMSCs, group exosomes). By establishing the model of rat HIRI, comparing the expression level of ALT, TNF-α, IL-1β, IL-10, Bcl-2, bax between the groups, at the same time observed the pathological changes and apoptosis of liver cells between the groups.Results:(1) Part of primitive cells had sticked the wall of culture bottle after 48 hours. It had formed a fusiform or polygon after 9-10 days,14 days latter the cells had full the culture bottle. BMSCs had become a unified shuttle shape after 3 generation passage.(2) According to the result of transmission electron microscopy, we found that the extracts were micro-capsule structures with spherical or ellipsoidal shapes, the diameter is 30-100nm, had a complete capsule, and some low density material inside it.(3) HIRI model successfully established, The changes of each group are as follows:①ALT levelsGroup PBS were significantly higher than group sham (P<0.05), group BMSCs and group exosomes was significantly lower than group PBS (P<0.05). The difference between the group BMSCs and group exosomes had no statistical significance (P> 0.05).②TNF-α、IL-1β and IL-10 levelsThe levels of TNF-α and IL-1β in group PBS were significantly higher than group sham (P<0.01); group BMSCs and group exosomes were significantly lower than group PBS(P<0.05). The difference between group BMSCs and group exosomes have no statistical significance (P>0.05).The levels of IL-10 are low both in the group PBS and group sham, but it was significantly higher in group BMSCs and group exosomes (P<0.05). But the difference between group BMSCs and group exosomes have no statistical significance (P>0.05).③ Liver pathologyIn group PBS, we can see liver cells are swelling, Cytoplasmic vacuolization, Sinusoidal congestion and inflammatory cells around. Compared with group PBS, group BMSCs and group exosomes are not so serious. The difference between group BMSCs and group exosomes have no statistical significance.④ Hepatic cell apoptosisThe cells appearing brown are considered TUNEL positive. The group PBS were significantly higher than group sham (P<0.05); group BMSCs and group exosomes were significantly lower than group PBS (P<0.05). The difference between group BMSCs and group exosomes have no statistical significance(P>0.05).⑤the level of bcl-2 and baxThe levels of bcl-2 in group PBS were significantly lower than group sham (P<0.05); group BMSCs and group exosomes were significantly higher than group PBS (P<0.05). The difference between group BMSCs and group exosomes have no statistical significance (P>0.05).The levels of bax in group PBS were significantly higher than group sham (P<0.05); group BMSCs and group exosomes were significantly lower than group PBS (P<0.05). The difference between group BMSCs and group exosomes have no statistical significance (P> 0.05). Conclusion:The results show that BMSCs-exosomes can promote hepatocyte proliferation by the mechanism of inhibiting hepatocellular apoptosis, at the same time, it can reduce inflammatory injury by balance of pro-inflammatory cytokines and anti-inflammatory cytokines to repair the liver ischemia-reperfusion injury. We have also found that the function of BMSCs and BMSCs-exosomes are equivalent in repair HIRI, therefore, the results provide a theory for BMSCs-exosomes can completely replace BMSCs to repair HIRI.