Protective effect of human umbilical cord mesenchymal stem cell-derived exosomes on rat retinal neurons in hyperglycemia through the brain-derived neurotrophic factor/TrkB pathway

Abstract

Aim: To explore whether human umbilical cord mesenchymal stem cell (hUCMSC)-derived exosomes (hUCMSC-Exos) protect rat retinal neurons in high-glucose (HG) conditions by activating the brain-derived neurotrophic factor (BDNF)-TrkB pathway.

Methods: hUCMSC-Exos were collected with differential ultracentrifugation methods and observed by transmission electron microscopy. Enzyme-linked immunosorbent assays (ELISAs) was used to quantify BDNF in hUCMSC-Exos, and Western blot was used to identify surface markers of hUCMSC-Exos. Rat retinal neurons were divided into 4 groups. Furthermore, cell viability, cell apoptosis, and TrkB protein expression were measured in retinal neurons.

Results: hUCMSCs and isolated hUCMSC-Exos were successfully cultured. All hUCMSC-Exos showed a diameter of 30 to 150 nm and had a phospholipid bimolecular membrane structure, as observed by transmission electron microscopy. ELISA showed the BDNF concentration of hUCMSCs-Exos was 2483.16±281.75. hUCMSCs-Exos effectively reduced the apoptosis of retinal neuron rate and improved neuron survival rate, meanwhile, the results of immunofluorescence verified the fluorescence intensity of TrKB in neurons increased. And all above effects were reduced by treated hUCMSCs-Exos with BDNF inhibitors. hUCMSC-Exos effectively reduced the apoptosis rate of retinal neurons by activating the BDNF-TrkB pathway in a HG environment.

Conclusion: In the HG environment, hUCMSC-Exos could carry BDNF into rat retinal neurons, inhibiting neuronal apoptosis by activating the BDNF-TrkB pathway.

Keywords: BDNF-TrkB pathway; diabetic retinopathy; exosomes; human umbilical cord mesenchymal stem cells; retinal neurons.

Figure 1. Observation of hUCMSC

A: Observation under an inverted microscope showed vortex-like growth (magnification 40×). Scale bars: 200 µm. B: Under high magnification, most hUCMSCs have long fusiform shapes, and a few are polygonal (magnification 100×). Scale bars: 50 µm.

Figure 2. Identification of hUCMSC-Exos

A: Transmission electron microscopy showed the teacup-like vesicular structure of hUCMSC-Exos (marked with arrows); these exosomes measured approximately 30-150 nm in diameter; B: hUCMSC-Exos were positive for the surface marker proteins CD9 and CD63 and negative for Calnexin; C: Analysis of the size distribution of hUCMSC-Exos with a NanoSight analysis system; the peak size is 117.5 nm. Scale bars: 100 nm.

Figure 3. The morphology and vitality of rat retinal neurons

A: Observation of rat retinal neurons on an inverted microscope 5d after treatment; B: Rat retinal neurons were observed under a high-power microscope 5d later. Scale bars: 50 µm. C: The mean OD values of rat retinal neurons in all groups; ^aP<0.05. Magnification 100×.

Figure 4. Annexin V-FITC/PI double-staining flow cytometry showed the proportions of apoptotic neurons

The apoptosis rate has been marked in each figure.

Figure 5. Immunocytochemical staining of TrkB in neurons

A: The cell nuclei were stained with DAPI, and TrkB was stained as red; B: The mean TrkB expression of rat retinal neurons as determined by immunostaining; ^aP<0.05. Scale bars: 50 µm.