Autophagy alleviates the decrease in proliferation of amyloid β1‑42‑treated bone marrow mesenchymal stem cells via the AKT/mTOR signaling pathway

Abstract

Alzheimer's disease (AD) and osteoporosis (OP) are 2 common progressive age‑associated diseases, primarily affecting the elderly worldwide. Accumulating evidence has demonstrated that patients with AD are more likely to suffer from bone mass loss and even OP, but whether it is a pathological feature of AD or secondary to motor dysfunction remains poorly understood. The present study aimed to investigate whether amyloid‑β1‑42 (Aβ1‑42), the typical pathological product of AD, exhibited a negative effect on the proliferation of bone marrow mesenchymal stem cells (BMSCs) and the role of autophagy. The proliferation of BMSCs was measured using a Cell Counting Kit‑8 assay, cell cycle analysis and 5‑ethynyl‑2'‑deoxyuridine (EdU) staining. The autophagy‑associated proteins microtubule‑associated proteins 1A/1B light chain 3B and sequestosome 1 (p62) were evaluated by western blot analysis and autophagosomes were detected by transmission electron microscopy and immunofluorescence. The activity of the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was measured using western blot analysis, and the autophagy inducer rapamycin (RAPA), inhibitor 3‑methyladenine (3‑MA) and the AKT activator SC79 were also used to investigate the role of AKT/mTOR signaling pathway and autophagy in the proliferation of BMSCs. The results suggested that the proliferation of BMSCs treated with Aβ1‑42 was inhibited, with the autophagy level increasing following treatment with Aβ1‑42 in a dose‑dependent manner, while the AKT/mTOR signaling pathway participated in the regulation of the autophagy level. Activation of autophagy using RAPA inhibited the decrease in proliferation of BMSCs, while suppression of autophagy by 3‑MA and activation of the AKT/mTOR signaling pathway increased the decrease in proliferation of BMSCs caused by Aβ1‑42. It was concluded that Aβ1‑42, as an external stimulus, suppressed the proliferation of BMSCs directly and that the AKT/mTOR signaling pathway participated in the regulation of the level of autophagy. Concomitantly, autophagy may serve as a resistance mechanism in inhibiting the decreased proliferation of BMSCs treated with Aβ1‑42.

Figure 1.

Effect of Aβ_1–42 on the proliferation of BMSCs. BMSCs were treated with Aβ_1–42 at various concentrations for 48 h. (A) Cell cycle was examined by flow cytometry. (B) Quantification of cell cycle flow cytometry data. **P<0.01 vs. the control group. ^##P<0.01. n=10 per group. (C) Cell viability was estimated using a Cell Counting Kit-8 assay. **P<0.01 vs. the control group. ^#P<0.05 and ^##P<0.01. n=10 per group. (D) Cell proliferation was additionally detected by EdU staining. Scale bar=50 µM. (E) Quantification of the EdU staining data. **P<0.01 vs. the control group. ^##P<0.01. n=10 per group. All values are presented as the mean ± standard error of the mean from 3 independent experiments. Aβ, amyloid β; BMSC, bone mesenchymal stem cells; EdU, 5-ethynyl-2′-deoxyuridine.

Figure 2.

Autophagy is involved in the effect of Aβ_1–42-treated BMSCs. (A) Western blot analysis demonstrating protein levels of LC3 and p62 following treatment for 48 h with different concentrations of Aβ_1–42. (B) Quantification of the western blot analysis data. *P<0.05, **P<0.01 and ***P<0.001 vs. the control group. ^#P<0.05, ^##P<0.01 and ^###P<0.001. n=10 per group. (C) Fluorescence microscopy demonstrating LC3 puncta with green fluorescence in BMSCs treated with Aβ_1–42 at various concentrations. (D) Quantification of the fluorescence microscopy data. *P<0.05 and **P<0.01 vs. the control group, ^##P<0.01 and ^###P<0.001. n=10 per group. (E) TEM images indicating the difference in autophagosomes (arrows) between the control and 5 µM/l Aβ_1–42 groups. (F) Quantification of the TEM data. **P<0.01 vs. the control group. n=10 per group. All values are presented as the mean ± standard error of the mean from 3 independent experiments. Aβ, amyloid β; BMSC, bone mesenchymal stem cells; LC3, microtubule-associated proteins 1A/1B light chain 3B; LC3 II, lipid-modified LC3; p62, sequestosome 1; TEM, transmission electron microscopy.

Figure 3.

Autophagy is mediated via the AKT/mTOR signaling pathway in Aβ_1–42-treated BMSCs. (A) Western blot analysis demonstrating the protein levels of LC3, p62, AKT, p-AKT, mTOR and p-mTOR following treatment with Aβ_1–42, 3-MA, SC79 and SC79 + RAPA for 48 h, and the expression of the AKT/mTOR signaling pathway-associated proteins of AKT, p-AKT, mTOR and p-mTOR following treatment with Aβ_1–42, 3-MA, SC79 and SC79 + RAPA for 48 h. (B) Quantification of the AKT, p-AKT, mTOR and p-mTOR western blot analysis data. **P<0.01 vs. the control group. ^#P<0.05 and ^##P<0.01 vs. the Aβ_1–42 group, ^&P<0.05 vs. the Aβ_1–42 group. ^{^^}P<0.01 vs. the Aβ_1–42 + SC79 group. n=10 per group. (C) Quantification of the western blot analysis data demonstrating the expression of the AKT/mTOR signaling pathway-associated proteins AKT, p-AKT, mTOR and p-mTOR. **P<0.01 vs. the control group. ^##P<0.01 vs. the Aβ_1–42 group. ^&P<0.05 and ^&&P<0.01 vs. the Aβ_1–42 group. ^{^^}P<0.01 vs. the Aβ_1–42 + SC79 group. n=10 per group. All values are presented as the mean ± standard error of the mean from 3 independent experiments. Aβ, amyloid β; BMSC, bone mesenchymal stem cells; mTOR, mammalian target of rapamycin; AKT, protein kinase B; p, phosphorylated; RAPA, rapamycin; LC3, microtubule-associated proteins 1A/1B light chain 3B; LC3 II, lipid-modified LC3; p62, sequestosome 1; 3-MA, 3-methyladenine.

Figure 4.

Autophagy serves a protective role against the decrease of proliferation induced by Aβ_1–42. (A) BMSCs were treated with Aβ_1–42, 3-MA, SC79 and SC79 + RAPA for 48 h and the cell cycle analysis was performed using flow cytometry. (B) Quantification of cell cycle flow cytometry data. **P<0.01 vs. the control group. ^#P<0.05 vs. the Aβ_1–42 group. ^&P<0.05 vs. the Aβ_1–42 group. ^{^}P<0.05 vs. the Aβ_1–42 + SC79 group. n=10 per group. (C) Cell viability was evaluated by Cell Counting Kit-8 assay. **P<0.01 vs. the control group. ^#P<0.05 vs. the Aβ_1–42 group. ^&P<0.05 vs. the Aβ_1–42 group. ^{^^}P<0.01 vs. the Aβ_1–42 + SC79 group. n=10 per group. (D) EdU staining was used for detection of cell proliferation. (E) Quantification of the EdU staining data. **P<0.01 vs. the control group. ^#P<0.05 vs. the Aβ_1–42 group. ^&P<0.05 vs. the Aβ_1–42 group. ^{^}P<0.05 vs. the Aβ_1–42 + SC79 group. n=10 per group. All values are presented as the mean ± standard error of the mean from 3 independent experiments. Aβ, amyloid β; BMSC, bone mesenchymal stem cells; 3-MA, 3-methyladenine; RAPA, rapamycin; EdU, 5-ethynyl-2′-deoxyuridine.