Abstract:

Objective To investigate the protective effect of human bone marrow mesenchymal stem cell-derived exosomes （hBMSCs-Exo） on neurological injury after intracerebral hemorrhage （ICH） in rats， and to elucidate the regulatory mechanism of autophagy in this process. Methods ?For in vivo experiments，thirty male SD rats were randomly divided into sham operation group， ICH group， and ICH+Exo group， with 10 rats in each group. Collagenase injection method was used to establish the rat ICH model in ICH group and ICH+Exo group. The rats in sham operation group were only drilled and injected with the same volume of normal saline. At 24 h after modeling， the rats in ICH+Exo group were injected with 20 μg hBMSCs-Exo into the brain tissue， and the rats in ICH group were only injected with phosphate-buffered saline （PBS）. The modified neurological severity scores （mNSS） of the rats in various groups were evaluated on days 1， 3， 7， 14， and 21 post-surgery. At day 7 post-surgery， Nissl staining method was used to detect the numbers of Nissl-positive neurons in the brain tissue of the rats in various groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL）method was used to detect the percentages of TUNEL-positive cells in the brain tissue of the rats in various groups. Immunofluorescence staining method was used to observe the expressions of neuronal nuclear protein （NeuN）， glial fibrillary acidic protein （GFAP）， and ionized calcium-binding adapter molecule 1 （Iba1） proteins in the brain tissue of the rats in various groups. Immunohistochemistry method was used to detect the expressions of autophagy-related proteins including microtubule-associated protein light chain 3 （LC3）， Beclin-1， P62， and autophagy-related protein 7 （ATG7） in the brain tissue of the rats in various groups. For in vitro experiments， 1-methyl-4-phenylpyridinium ion （MPP?） was used to induce oxidative stress in SH-SY5Y cells. The cells were divided into control group， MPP? group， MPP?+Exo group， and MPP⁺+Exo+3-methyladenine （3-MA） group. Methylthiazolyldiphenyl-tetrazolium bromide（MTT） method was used to detect the cell activities in various groups. Acridine orange （AO） staining method was used to evaluate the levels of autophagy of the cells in various groups. Results The mNSS score results showed that compared with sham operation group， the mNSS scores of the rats in ICH group were significantly increased at all time points（P<0.05）；compared with ICH group，the mNSS scores of the rats in ICH+Exo group were significantly decreased at all time points （P<0.05）. The Nissl staining results showed that compared with sham operation group，the number of Nissl-positive neurons in the brain tissue of the rats in ICH group was significantly decreased （P<0.001）；compared with ICH group，the number of Nissl-positive neurons in the brain tissue of the rats in ICH+Exo group was significantly increased （P<0.01）. The TUNEL staining results showed that compared with sham operation group，the percentage of TUNEL-positive cells in the brain tissue of the rats in ICH group was significantly increased （P<0.05） ；compared with ICH group，the percentage of TUNEL-positive cells in the brain tissue of the rats in ICH+Exo group was significantly decreased （P<0.05）. The immunofluorescence staining results showed that compared with sham operation group，the number of NeuN-positive cells in the brain tissue of the rats in ICH group was significantly decreased （P<0.05），and no statistically significant difference was observed in the percentages of positive area of GFAP and Iba1 in the brain tissue （P>0.05） ；compared with ICH group，the number of NeuN-positive cells in the brain tissue of the rats in ICH+Exo group was significantly increased （P<0.05）. The immunohistochemistry method results showed that compared with sham operation group，the percentages of positive area of LC3，Beclin-1，P62，and ATG7 proteins in the brain tissue of the rats in ICH group were significantly increased （P<0.05）；compared with ICH group，the percentage of positive area of P62 protein in the brain tissue of the rats in ICH+Exo group was significantly decreased （P<0.05）， while the percentages of positive area of LC3 and ATG7 proteins in the brain tissue of the rats were significantly increased （P<0.05 or P<0.01）. The in vitro MTT assay results showed that compared with MPP? group，the cell activity of SH-SY5Y cells in MPP?+Exo group was significantly increased （P<0.05）； compared with MPP?+Exo group， the cell activity of SH-SY5Y cells in MPP?+Exo+3-MA group was significantly decreased （P<0.05）. The AO staining method results showed that compared with MPP? group， the autophagy level of SH-SY5Y cells in MPP?+Exo group was significantly increased （P<0.05）； compared with MPP?+Exo group， the autophagy level of SH-SY5Y cells in MPP?+Exo+3-MA group was significantly decreased （P<0.05）. Conclusion ?hBMSCs-Exo alleviates neurological injury after ICH and improves neurological function in rats， and its effects are associated with promoting autophagic flux and inhibiting neuronal apoptosis. Autophagy regulation may be a key mechanism underlying its neuroprotective effect.

Key words: Exosomes, Intracerebral hemorrhage, Autophagy, Mesenchymal stem cells, Neuroprotection