Exosome-encapsulated MicroRNA-140-5p Alleviates Neuronal Injury Following Subarachnoid Hemorrhage Through Regulating IGFBP5-mediated PI3K/AKT Signaling Pathway

Purpose: Extracellular vesicles secreted by adipose tissuederived stromal cells(ADSCs)have shown therapeutic potential in regenerative medicine.Here,we discussed the effects of EV-encapsulated micro RNA(miRNA)-derived from ADSCs on neuronal injury.Methods: Isolation and identification of primary neurons,ADSCs and their exosomal(ADSCs-Exo),DLS analysis of particle size,transmission electron microscope observation of morphology,WB detection of specific markers;electroporation of p EGFP-TDP-43 to establish TDP-43 aggregation neurons;PKH67-ADSCs-Exo and Cy3-miR-140-5p treatment of neurons to detect whether cells internalize ADSCs-Exo transmission of miR-140-5p.ADSCs-Exo and / or miR-140-5p inhibitor treated neurons injured by TDP-43 inclusion bodies,cell viability was detected by CCK8 method,apoptosis was detected by flow cytometry and apoptosis-related proteins were detected by WB;the targeting relationship between miR-140-5p and IGFBP5 was verified by online website prediction and double luciferase report experiment;the viability and apoptosis of neurons were detected after overexpression of IGFBP5 and then treated with ADSCs-Exo.Miltefosine,an inhibitor of PI3K/AKT pathway,was used to detect the effect of PI3K/AKT pathway on neurons injured by TDP-43 inclusion.After ADSCs-Exo treatment,the SAH rat model was treated with Garcia Score,Beam balance testing,water maze and other behavioral experiments,as well as immunohistochemistry and WB to detect the effect of miR-140-5p regulation of PI3K/AKT pathway on TDP-43 inclusion body injured neurons.Results: Successful isolation of primary neurons,ADSCs and ADSCs-Exo;ADSCs-Exo with diameter of 30-110 nm,cup-shaped or spherical,expression of CD63 and TSG101,no expression of Calnexin;successfully established the model of neuronal injury induced by TDP-43 inclusion.ADSCs-Exo transmission of miR-140-5p promotes the viability and inhibits apoptosis of neurons injured by TDP-43 inclusion.IGFBP5 is the direct target gene of miR-140-5p.MiR-140-5p can reduce the neuronal damage caused by TDP-43 inclusion through targeted inhibition of IGFBP5,and inhibition of PI3K/AKT signal pathway can aggravate the neuronal damage caused by TDP-43 inclusion bodies.Exocrine miR-140-5p derived from ADSC can inhibit the activation of PI3K/AKT signal pathway by IGFBP5 in SAH rat model,inhibit TDP-43 inclusion,improve long-term neural functions such as memory impairment caused by nerve injury,and reduce neuronal injury.Conclusions: ADSCs-derived exosome miR-140-5p can inhibit neuronal damage caused by TDP-43 inclusion by activating PI3K/AKT signal pathway by targeting IGFBP5.