VX-765 enhances autophagy of human umbilical cord mesenchymal stem cells against stroke-induced apoptosis and inflammatory responses via AMPK/mTOR signaling pathway.

introductionTo investigate the protective effect of VX-765 on human umbilical mesenchymal stem cells (HUMSCs) in stroke and its mechanism. MATERIALS AND

methodsMouse models of ischemic stroke were established using the distal middle cerebral artery occlusion (dMCAO) method. The dMCAO mice were accordingly transplanted with HUMSCs, VX-765-treated HUMSCs, or VX-765 + MHY185-treated HUMSCs. The HUMSCs were inserted with green fluorescent protein (GFP) for measurement of transplantation efficiency which was determined by immunofluorescence assay. Oxygen-glucose deprivation (OGD) was applied to mimic ischemic environment in vitro experiments, and the HUMSCs herein were transfected with AMPK inhibitor Compound C or autophagy inhibitor 3-MA. MTT assay was used to test the toxicity of VX-765. TUNEL staining and ELISA were applied to measure the levels of apoptosis and inflammatory cytokines (IL-1β, IL-6, and IL-10), respectively. The expressions of autophagy-associated proteins, AMPK, and mTOR were detected by Western blotting. TTC staining was applied to reveal the infarct lesions in the brain of dMCAO mice.

resultsThe pro-inflammatory cytokines, TUNEL-positive cells, and p-mTOR were decreased while the anti-inflammatory cytokine, autophagy-related proteins, and p-AMPK were increased in HUMSCs treated with VX-765 under OGD condition. Different expression patterns were found with the above factors after transfection of 3-MA or Compound C. The pro-inflammatory cytokines, TUNEL-positive cells, and infarct sections were decreased while the anti-inflammatory cytokine and autophagy-related proteins were increased in dMCAO mice transplanted with VX-765-treated HUMSCs compared to those transplanted with HUMSCs only. The autophagy was inhibited while p-mTOR was up-regulated after transfection of MHY.

conclusionVX-765 protects HUMSCs against stroke-induced apoptosis and inflammatory responses by activating autophagy via the AMPK/mTOR signaling pathway in vivo and in vitro.