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

Introduction: To investigate the protective effect of VX-765 on human umbilical mesenchymal stem cells (HUMSCs) in stroke and its mechanism.

Materials and methods: Mouse 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.

Results: The 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.

Conclusion: VX-765 protects HUMSCs against stroke-induced Apoptosis and inflammatory responses by activating Autophagy via the AMPK/mTOR signaling pathway in vivo and in vitro.