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

Abstract

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.

Keywords: VX-765; autophagy; human umbilical mesenchymal stem cells; stroke.

FIGURE 1

VX‐765 protects HUMSCs from OGD‐induced apoptosis and inflammatory responses. Notes: A, Molecular structure of VX‐765. B, MTT tested cell viability. C, TUNEL‐positive cells after 12 h of OGD. D, TUNEL‐positive cells after 24 h of OGD. E, ELISA measured levels of IL‐1β, IL‐6, and IL‐10 after 12 h of OGD. F, ELISA measured levels of IL‐1β, IL‐6, and IL‐10 after 24 h of OGD. (n = 4), *P < .05 **P < .01. Scale bars: 50 μm; HUMSC, human umbilical mesenchymal stem cell; OGD, oxygen‐glucose deprivation

FIGURE 2

VX‐765 promotes autophagy through AMPK/mTOR signaling in HUMSCs exposed to OGD. Notes: A, B, Western blot tested the expressions of autophagy‐associated proteins. C, D, Western blot tested expressions of proteins involved in AMPK/mTOR signaling pathway. E, TUNEL staining after transfection of 3‐MA or compound C. *P < .05, **P < .01. Scale bars: 50 μm; HUMSC, human umbilical mesenchymal stem cell; AMPK/mTOR, AMP‐activated rapamycin protein kinase/mammalian target protein; OGD, oxygen‐glucose deprivation

FIGURE 3

VX‐765 reduces apoptosis and inflammation in HUMSC‐transplanted dMCAO rats. Notes: A, IFA tested the intensity of GFP to verify the transplantation of HUMSCs. B, TTC measured infarcted areas of brain tissue. C, TUNEL tested apoptosis in brain tissues. D, Western blot detected apoptosis‐related proteins. E, ELISA tested the expressions of inflammatory factors. Scale bars: 50 μm; HUMSC, human umbilical mesenchymal stem cell; dMCAO, distal middle cerebral artery occlusion; IFA, Immunofluorescence assay

FIGURE 4

VX‐765 promotes cell autophagy in HUMSC‐transplanted dMCAO rats. Notes: A, B, Western blot tested the expressions of autophagy‐associated proteins. (n = 6). C, Western blot tested mTOR protein expression. (n = 6). *P < .05, **P < .01; HUMSC, human umbilical mesenchymal stem cell; dMCAO, distal middle cerebral artery occlusion