Exosome Mediated MiR-383-3p Promote Neuronal Necroptosis Via TNF-α-RIP1/3-MLKL Pathway

Background and Objective:Intracerebral hemorrhage(ICH)accounted for 10-20%of all stroke,with high mortality and poor prognosis.In China,Particularly,ICH have imposed a heavy health burden in public with a higher proportion of all strokes.At present,there is no effective treatment method except surgery for ICH,so it is particularly important to find a new treatment approach.ICH can cause primary and secondary injury,among which the secondary injury includes necrosis,apoptosis,autophagy,necroptosis and other mechanisms.Necroptosis is a discovered injury mechanism in ICH in recent years.Necroptosis is similar to necrosis,but a kind of programmed death.Micro ribonucleic acid(micro RNA),as one of the exosome loads,regulates a variety of physiological and pathological processes.It is reported that exosomes,as a signal transduction carrier,exist in a variety of cells,Such as Microglia,macrophages,mesenchymal stem cells and so on.They can carry micro RNA(mi R)to transmit information between cells and regulate the function of target cells.Currently,it has not been clear whether exosome micro RNAs can regulate necroptosis in ICH.We explored the effect of micro RNA derived from exosomes on necroptosis in ICH,in order to provide a reliable idea for finding a new treatment plan of ICH.Methods:1.Propidium iodide(PI)staining was performed on ICH rats at different periods.Western Blot(WB)was used to detect the expression of receptor-interacting protein 1(RIP1)and receptor-interacting protein 3(RIP3)in ICH rats at different periods.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the expression levels of various micro RNAs in ICH rats after 3 days and the expression levels of mi R-383-3p in ICH rats at different stages.2.Transmission electron microscopy and dynamic light scattering were used to determine the morphological characteristics and size of exosomes,and the isolated exosomes were identified by WB.The relationship between exosomes and neurons was demonstrated by immunofluorescence.Flow cytometry was used to detect neuronal necroptosis in the co-culture system of microglia exosomes,activated microglia negative control exosomes,and activated microglia mi R-383-3p knockdown(KD)exosomes respectively.The expression levels of RIP1,RIP3and mixed lineage kinase domain-like protein(MLKL)in microglia exosomes,activated microglia exosomes,and activated microglia mi R-383-3p KD exosomes respectively were detected respectively by WB.3.The level of mi R-383-3p in Sham,ICH and ICH+mi R-383-3p inhibitor was detected by RT-q PCR.PI staining was used to observe the degree of necroptosis of ICH cells with in Sham,ICH and ICH+mi R-383-3p inhibitor respectively.The expression levels of transcription factor 4(ATF4),RIP1,RIP3 and MLKL in Sham,ICH and ICH+mi R-383-3p inhibitor were detected by WB.The expression levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in Sham,ICH and mi R-383-3p KD ICH were detected by enzyme-linked immunosorbent assay(ELISA).Result:1.In ICH rat,the expression of RIP1 and RIP3 and the number of PI~+cells gradually increased,reached the peak on day 3,and then decreased gradually.The ICH expression of mi R-383-3p,mi R-181a-5p,and mi R-375 at 3 days were all increased(P<0.05),and the expression difference of mi R-383-3p was the most obvious.Further detection of the expression of mi R-383-3p showed that it reached a peak in 3 days and then gradually decreased.2.Exosomes can be taken up by neurons.Besides,in vitro,exosome-mediated mi R-383-3p can increase the expression of RIP1,RIP3 and MLKL,and increase the number of neuronal necroptosis.3.In vivo,mi R-383-3p can increase the number of PI-positive cells,the expression of IL-1βand TNF-α,the expression of RIP1,RIP3,MLKL,and decrease the expression of ATF4.Conclusion:In ICH model,the expression of mi R-383-3p was increased,and mi R-383-3p can up-regulate the expression of TNF-αvia exosomes as vectors,so as to enhance the pathological process of necroptosis and aggravate the injury after ICH.Therefore,mi R-383-3p plays an important role in ICH,suggesting that exosome-mediated mi R-383-3p may be a potential pathway for the treatment of ICH.