| Objectives To investigate the therapeutic effect of astrocyte-derived exosomes(ADEVs)on intracerebral hemorrhage in rats and whether the mechanism is through c-myc affecting ER stress and autophagy.Methods 1 250 g-270 g healthy male SD rats were randomly divided into four groups,sham operation group(Sham group),model group(ICH group),vehicle control group(PBS group)and astrocyte-derived exosomes treatment group(ADEVs group),with 15 rats in each group.In the ICH group,a rat model of intracerebral hemorrhage was prepared by injection(50 μL)into the basal ganglia of autologous blood,and the ADEVs group was given intracerebroventricular injection of 20 μg of exosomes 6 hours after intracerebral hemorrhage modeling;animal experiments were performed at five different time points: 1day,3 days,7 days,14 days,and 21 days in each group.2 Astrocytes were extracted and cultured from the whole young brain of 1 to 3 d SD rats,and the cells were identified using immunofluorescence.3 Exosomes were extracted from astrocyte supernatants by differential centrifugation and identified by transmission electron microscopy,particle size detection,and Western blotting.4 Behavioral scoring was performed by Longa scoring rats;learning and memory function changes were detected by water maze in rats with intracerebral hemorrhage.5 Histopathological changes around intracerebral hemorrhage in rats were observed by HE staining;pathological changes around intracerebral hemorrhage lesions were observed under a microscope using Nissl staining and undamaged neurons were counted.6 The positive expression levels of GRP78,ATF-6,LC3,Beclin-1 and c-myc in the brain tissues of each group by immunohistochemistry.The protein expression levels of GRP78,ATF-6,LC3,Beclin-1 and c-myc in the brain tissues of each group were detected by Western blot.Results 1 Compared with the sham group,the Longa scores of rats in the ICH group at 1day,3 days,7 days,14 days,and 21 days were significantly higher(P<0.05),and different degrees of reduced activity,inflexible limb movement,and leftward circle or leftward inclination during walking occurred at each observation time point,and the Longa scores of rats decreased after ADEVs intervention compared with the ICH model group(P<0.05).2The results of water maze test showed that compared with the sham group,the time to find the platform of rats in each ICH group was significantly prolonged after 7 days,14 days and21 days,and the movement trajectory was complex(P<0.05);after exosome treatment,compared with the ICH group,the time to find the platform of rats at each time point of ADEVs was shortened,and the movement trajectory was relatively simple and tended,no significant difference in ICH PBS group.3 HE staining microscopy showed that the cells in the basal ganglia of the brain tissue of rats in the sham group were arranged neatly,the staining was lighter,and there was no obvious pathological change;compared with the sham group,the nerve cells in the brain tissue around the hemorrhagic foci of the ICH group were disorganized,degenerated,necrotic,swollen,and the intercellular space was widened at 7days after intracerebral hemorrhage;compared with the ICH group,the pathological changes in the brain tissue around the hemorrhagic foci of the ADEVs group were relatively mild at both time points(P<0.05).4 After Nissl staining,the Nissl bodies of neurons in the basal ganglia of the Sham group were abundant,and the number of positive cells was more(P<0.05).Compared with the sham group,the neuronal loss was significant,the staining was lighter,and the number of positive cells was significantly reduced in the ICH group at 7 days after intracerebral hemorrhage in rats(P<0.05).Compared with the ICH group,the number of Nissl staining positive cells in neurons was significantly increased in the ADEVs group,no significant difference in ICH PBS group.5 Immunohistochemical results showed that only weak positive expression of GRP78,ATF-6,LC3,Beclin-1 and c-myc could be detected in sham group;compared with sham group,the positive expression of GRP78 and ATF-6 protein in perihematomal brain tissue of rats at 7 days after ICH was significantly increased(P<0.05);after ADEVs treatment,compared with ICH,the positive expression of c-myc,GRP78,ATF-6,LC3 and Beclin1 protein in perihematomal brain tissue of rats in ADEVs group was significantly decreased(P<0.05),and the staining was relatively lighter,but still higher than that in sham group.6 The results of immunoblotting showed that the contents of Sham GRP78,ATF-6,LC3,Beclin-1 and c-myc were low and at physiological levels.Compared with the sham group,GRP78,ATF-6,LC3,Beclin-1 and c-Myc protein expression were significantly increased in the ICH model group,and GRP78,ATF-6,LC3,Beclin-1 and c-Myc expression were reduced after ADEVs intervention(P<0.05).Conclusions 1 Autophagy and ER stress involved in the pathological process of secondary injury in rat intracerebral hemorrhage.2 ADEVs play an important neuroprotective role by regulating ER stress and autophagy activation after intracerebral hemorrhage.The neuroprotective effect of 3ADEVs may be achieved by inhibiting c-myc expression to regulate ER stress and autophagy.Figure17;Table4;Reference 130... |
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