Fig 2: miR-140-5p shuttled by MSC-derived EVs represses brain injury and microglial M1 activation in mice after subarachnoid hemorrhage (SAH). A, The expression of miR-140-5p in EVs from MSCs transfected with miR-140-5p mimic or miR-140-5p inhibitor detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). *P <.05 vs EVs from mimic-NC-transfected MSCs. #P <.05 vs EVs from inhibitor-NC-transfected MSCs. Sham-operated mice were used as control, and SAH mice were treated or not treated with EVs or EVs-miR-140-5p. B, Quantification of neurological function score (n = 6). C, TdT-mediated dUTP-biotin nick end labeling with NeuN (TUNEL/NeuN) immunofluorescence staining of the cerebral cortex (scale bar = 100 µm; DAPI: blue; TUNEL: green; NeuN: red) and the number of the TUNEL/NeuN positive cells (n = 6). D, The expression of IL-6, IL-1ß, and TNF-a in serum detected by enzyme-linked immunosorbent assay (ELISA) (n = 6). E, Representative immunofluorescence micrographs of microglia polarization the cerebral cortex and the number of CD16-positive M1 microglia and CD206-positive M2 microglia (scale bar = 25 µm; CD16/CD206: red, Iba 1: green, DAPI: blue). F, The expression of miR-140-5p in SAH mice detected by RT-qPCR. G, Western blot analysis of the expression of ALK5 and NADPH oxidase 2 (NOX2) in SAH mice. *P <.05 vs SAH mice; #P <.05 vs SAH mice treated with EVs. The data were measurement data, which were expressed by mean ± standard deviation. Comparison among multiple groups was analyzed by one-way variance of analysis. n = 6..

Fig 3: Activin-like kinase 5 (ALK5) silencing attenuates neurological dysfunction and neuroinflammation in subarachnoid hemorrhage (SAH) mice. Sham-operated mice were used as control, and SAH mice were treated or not treated with shRNA negative control (sh-NC) or shRNA ALK5 (sh-ALK5). A, The expression of ALK5 determined by western blot analysis (n = 6). B, Quantification of the severity of subarachnoid hemorrhage evaluated by SAH grading (n = 6). C, Quantification of neurological score (n = 6). D, TdT-mediated dUTP-biotin nick end labeling with NeuN (TUNEL/NeuN) immunofluorescence staining (scale bar = 100 µm; DAPI: blue; TUNEL: green; NeuN: red) and the number of the TUNEL/NeuN positive cells (n = 6). E, The expression of IL-6, IL-1ß, and TNF-a in serum detected by enzyme-linked immunosorbent assay (ELISA) (n = 6). *P < .05 vs sham-operated mice; #P < .05 vs SAH mice treated with sh-NC. The data were measurement data, which were expressed by mean ± standard deviation. Comparison among multiple groups was analyzed by one-way variance of analysis..

Fig 4: Activin-like kinase 5 (ALK5) is negatively targeted by miR-140-5p in BV2 cells. A, TargetScan website predicting specific binding sites between ALK5 and miR-140-5p. B, The targeting relationship between ALK5 and miR-140-5p evaluated by dual luciferase reporter assay. C, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection of the expression of miR-140-5p in BV2 cells transfected with miR-140-5p mimic. D, RT-qPCR detection of the mRNA expression of ALK5 and NADPH oxidase 2 (NOX2) in BV2 cells transfected with miR-140-5p mimic. E, Western blot analysis of the protein expression of ALK5 and NOX2 in BV2 cells transfected with miR-140-5p mimic. * P <.05 vs BV2 cells transfected with mimic-NC. The data were measurement data, which were expressed by mean ± standard deviation. The comparison between the two groups was analyzed by unpaired t-test. The experiment was repeated three times.

Fig 5: Mechanism. MSC-EVs transferred miR-140-5p into microglia, where miR-140-5p diminished the expression of Activin-like kinase 5 (ALK5), and thereby abolished the promoting effect of ALK5 on NADPH oxidase 2 (NOX2) expression, ultimately repressing M1 microglia activation and inflammatory response in subarachnoid hemorrhage (SAH).