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, 9 (1), 18424

The Role and Mechanism of SIRT1 in Resveratrol-regulated Osteoblast Autophagy in Osteoporosis Rats

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The Role and Mechanism of SIRT1 in Resveratrol-regulated Osteoblast Autophagy in Osteoporosis Rats

Xuhao Yang et al. Sci Rep.

Abstract

Osteoporosis is widely regarded as one of the typical aging-related diseases due to the impairment of bone remodeling. The silent information regulator of transcription1 (SIRT1) is a vital regulator of cell survival and life-span. SIRT1 has been shown to be activated by resveratrol treatment, and also has been proved to prevent aging-related diseases such as osteoporosis. However, the role of SIRT1 about autophagy or mitophagy of osteoblasts in resveratrol-regulated osteoporotic rats remains unclear. This study seeks to investigate the role of SIRT1 about autophagy or mitophagy in osteoblasts through PI3K/Akt signaling pathway in resveratrol-regulated osteoporotic rats. The vivo experiment results have revealed that resveratrol treatment significantly improved bone quality and reduced the levels of serum alkaline phosphatase and osteocalcin in osteoporotic rats. Moreover, Western bolt analysis showed that expression of SIRT1, LC3, and Beclin-1 in osteoblasts increased, while p-AKT and p-mTOR were downregulated in osteoporosis rats with high dose resveratrol treatment. On the other hand, resveratrol treatment increased the SIRT1 activity, LC3 and Beclin-1 mRNA expression in the dexamethasone (DEX)-treated osteoblasts. More mitophagosomes were observed in the DEX-treated osteoblasts with resveratrol. Meanwhile, the TOM20, Hsp60, p-Akt and p-mTOR activities were decreased in the DEX-treated osteoblasts with resveratrol. Resveratrol treatment did not change the p-p38 and p-JNK activities in the osteoblasts. These results revealed that resveratrol treatment protected osteoblasts in osteoporosis rats by enhancing mitophagy by mediating SIRT1 and PI3K/AKT/mTOR signaling pathway.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Bone mineral density (BMD) and femoral porosity based on the pixel intensity of proximal epiphysis and serum bio-marker [alkaline phosphatase (ALP) and osteocalcin (OC)] changes in osteoporosis and resveratrol treated groups. (A) Resveratrol treatment restored BMD reduction in osteoporosis rats. (B) Femoral porosity based on the pixel intensity of proximal epiphysis changes in control, osteoporosis and resveratrol treated groups. (C) ALP changes in control, osteoporosis and resveratrol treated groups. (D) OC changes in control, osteoporosis and resveratrol treated groups. LD, low-dose; HD, high-dose. *P < 0.05 vs. control group; #P < 0.05 vs. Ost group.
Figure 2
Figure 2
The expression of osteocalcin and SIRT1 in the rats’ femur of different groups by using immunohistochemical staining. Ost, osteoporosis; RES, resveratrol; LD, low-dose; HD, high-dose.
Figure 3
Figure 3
Western blot showing expression of the SIRT1, LC3, Beclin-1, p-mTOR and p-AKT in osteoblasts among the groups. (A) Western blot shows the expression of SIRT1 expression in osteoblasts of different groups. (B) Western blot shows the expression of LC-3 in osteoblasts of different groups. (C) Western blot shows the expression of Beclin-1 in osteoblasts of different groups. (D) Western blot shows the expression of p-mTOR in osteoblasts of different groups. (E) Western blot shows the expression of p-AKT in osteoblasts of different groups. *P < 0.05 vs. control group; #P < 0.05 vs. Ost group. Ost, osteoporosis; RES, resveratrol; LD, low-dose; HD, high-dose.
Figure 4
Figure 4
Expression of the SIRT1 in osteoblasts of different groups. (A) Expression of SIRT1 mRNA in osteoblasts treated with increasing concentrations of resveratrol (0 M, 10−8 M, 10−7 M, 10−6 M), as assessed with quantitative real-time polymerase chain reaction (RT-PCR). (B) Expression of SIRT1 in osteoblasts treated with resveratrol (50 µM) for 0, 5, 30, 60, 120 min, as assessed with Western blot. (C) Confocal immunofluorescence (IF) staining of osteoblasts with or without resveratrol stained for SIRT1 (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The intensity of SIRT1 was higher in serum-starved osteoblasts cells cultured in serum-free medium with resveratrol. (D) Expression of SIRT1 protein in osteoblasts among different groups, as assessed with Western blot. (E) BrdU and MTT assays for proliferation and viability of osteoblasts that were treated with or without dexamethasone, with or without resveratrol and NAM. Analysis of the proliferation and viability of osteoblasts with different treatments as assessed with the BrdU assays and the MTT assay. The figures represent three independent experiments. Asterisks indicate significant differences compared to the control group (*P < 0.05).
Figure 5
Figure 5
Confocal microscope analysis was performed to evaluate autophagy in cells treated with DEX, pre-incubated with RES or pre-incubated with NAM. Representative images of fluorescent LC3 puncta are shown.
Figure 6
Figure 6
Assessment of autophagy in osteoblast induced by DEX and RES. (A) The LC3 mRNA levels were detected using RT-PCR assay. (B) The Beclin-1 mRNA levels were detected using RT-PCR assay. (C) The LC3 activity of treated cells were detected using Western blot assay. β-actin was used as an reference gene. (D) Results of densitometric analysis for Western blot.
Figure 7
Figure 7
Transmission electron microscopy (TEM) showing mitophagosomes in osteoblasts. Transmission electron microscopy images indicating double membrane vacuoles in osteoblasts treated with10−6 M DEX with or without the presence of RES for 24 h. The vacuoles are indicated with arrows.
Figure 8
Figure 8
Assessment of autophagy in osteoblast induced by DEX and RES. The Beclin-1 (A), Atg7 (B), TOM20 (C) and Hsp60 (D) protein levels of treated cells were detected using Western blot assay.
Figure 9
Figure 9
Western blot shows expression of p-mTOR, p-AKT, p-p38, and p-JNK in osteoblasts with different treatments. (A) Western blot shows the expression of p-mTOR in osteoblasts among different groups. (B) Western blot shows the expression of p-AKT in osteoblasts among different groups. (C) Western blot shows the expression of p-p38 in osteoblasts among different groups. (D) Western blot shows the expression of p-JNK in osteoblasts among different groups. Asterisks indicate significant differences compared to control (*P < 0.05).

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