NGF receptors and PI3K/AKT pathway involved in glucose fluctuation-induced damage to neurons and α-lipoic acid treatment

Abstract

Background: Glucose fluctuation promotes neuronal apoptosis, which plays a central role in diabetic encephalopathy (DE). Nerve growth factor (NGF), and its interaction with high-affinity (TrkA) and low-affinity (p75NTR) receptors, are involved in neuronal survival. NGF/TrkA contributes to the activation of the PI3K/AKT pathway, which is beneficial for neuronal survival, and α-Lipoic acid (ALA) exerts clinically favorable neuroprotective effects in the periphery. Whether NGF receptors and the PI3K/AKT pathway are involved in glucose fluctuation-induced neuronal damage, as well as the potential molecular mechanism of ALA in protecting glucose fluctuation-induced neuronal damage, remain unclear.

Results: The results indicated that constant high glucose (CHG) and intermittent high glucose (IHG) significantly increased the expression of Bax and caspase-3, and decreased the expression of TrkA/p75NTR and p-AKT/AKT, while ALA stimulation reversed the above proteins in PC12 cells. IHG stimulates apoptosis more effectively than CHG in PC12 cells, which is related to the PI3K/AKT pathway but not to the TrkA/p75NTR. Furthermore, neuronal apoptosis induced by IHG was aggravated by the TrkA inhibitor K252a or the PI3K/AKT inhibitor LY294002, but this effect was alleviated by the p75NTR inhibitor TAT-pep5.

Conclusion: Glucose fluctuation induced cell apoptosis by regulating the TrkA/p75NTR and PI3K/AKT pathway, meanwhile ALA exhibited neuroprotective effects in response to IHG and CHG. These observations indicated that the PI3K/AKT pathway and the balance of TrkA/p75NTR are likely to serve as potential therapeutic targets for DE. In addition, ALA could be a possible therapeutic drug for DE.

Keywords: Cell apoptosis; Diabetic encephalopathy; Intermittent high glucose; PI3K/AKT; TrkA; p75NTR.

Fig. 1

a Effect of glucose on PC12 cell viability. b Effects of ALA on PC12 cell viability under normal glucose concentration. c Effects of ALA on PC12 cell viability under glucose fluctuation. Results are presented as the mean ± SD (n = 3). ***P < 0.001, **P < 0.01,*P < 0.05, compared to the (a) control or (b, c) ALA (0 µmol/L) group. ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 versus the ALA (100 µmol/L) group

Fig. 2

ALA treatment attenuated IHG induced cell injuries in PC12 cells. a, c Effects of IHG on PC12 cell viability and ALA treatment. Cells were observed by microscope, and viability was determined by MTT assay. b, d Effects of IHG on PC12 cell apoptosis and ALA treatment. Apoptosis was detected by flow cytometry. Results are shown as the mean ± SD (n = 3). ***P < 0.001, **P < 0.01,*P < 0.05 versus the control; ^#P < 0.05 compared to the CHG group; and ⁺P < 0.05 versus the IHG + ALA group

Fig. 3

ALA treatment inhibited IHG-induced apoptosis in PC12 cells. The density of (a) Bax, Bcl-2, and Caspase-3 were measured by Western blot. Quantitative data of (b) Bax, (c) Bcl-2, (d) Bax/Bcl-2, and (e) Caspase-3 are presented. Data are shown as mean ± SD (n = 3). ***P < 0.05, **P < 0.01, *P < 0.05 versus the control; ##P < 0.01, #P < 0.05 compared to the CHG group; and ⁺⁺P < 0.01, ⁺P < 0.05 versus the IHG + ALA group

Fig. 4

ALA treatment affected IHG-induced NGF receptorsin PC12 cells. a The density of TrkA and p75NTR were determined by Western blot. Quantitative data of (b) TrkA, c p75NTR, and d TrkA/p75NTR are presented. Data are presented as mean ± SD (n = 3). **P < 0.01,*P < 0.05 compared to the control; ^##P < 0.01 versus the CHG group; and ⁺⁺P < 0.01, ⁺P < 0.05 compared to the IHG + ALA group

Fig. 5

ALA treatment altered the IHG-inhibited PI3K/AKT pathway in PC12 cells. a The density of AKT and p-AKT were measured by Western blot. Quantitative data of b p-AKT, c AKT, and d p-AKT/AKT are presented. Data are shown as mean ± SD (n = 3). **P < 0.01 compared to the control; ^#P < 0.05 compared to the CHG group; and ⁺⁺P < 0.01 versus the IHG + ALA group

Fig. 6

ALA protected IHG-induced cell apoptosis through NGF receptors and the PI3K/AKT pathway in PC12 cells. Bax/Bcl-2 and Caspase-3 levels were evaluated in the presence or absence of K252a (a–c), TAT-pep5 (d-f), and LY294002 (g–i), respectively. Data are shown as the mean ± SD (n = 3). ^##P < 0.01, ^#P < 0.05 versus the IHG group; ⁺⁺P < 0.01, ⁺P < 0.05 compared to the IHG + ALA group; and *P < 0.05 compared to the IHG + inhibitor (K252a, TAT-pep5, or LY294002)