Synergistic Effect of Sesamin and γ-Tocotrienol on Promoting Osteoblast Differentiation via AMPK Signaling

Cell Culture and Treatment

The procedure of cell culture was conducted as previously reported ¹³ . Human bone marrow mesenchymal stem cells (hBMSCs), purchased from Nanjing Saigen Biotechnology Co., Ltd, were cultured in α-Minimum essential medium containing 10% fetal bovine serum, 100 mg/L streptomycin and 100 U/L penicillin α-Minimum essential medium in a humid environment at 37 ° C and 5% CO₂. For osteogenic differentiation, when cells reached 80 to 90% confluence, 10 mM β-Glycerophosphate, 100 nM dexamethasone and 200 μM ascorbic acid (osteogenic inducer) were added to the medium and incubated for 14 days, and osteogenic induction medium (OM) was changed every 3 days. Control cells were cultured in the medium without osteogenic inducer.

Sesamin (S9314;Sigma-Aldrich LLC., USA) was dissovled in DMSO. γ-T3 was procured from Sigma Chemical Co (St. Louis, MO, USA). Cells were exposed to 1, 2.5, 5, 10 mg/mL doses of sesamin and 1, 5, 10 mM doses of γ-T3 AMPK inhibitor compound C (CC), which was bought from Calbiochem (Nottingham, UK), was used to pretreat the cells for 2 h.

Cell Counting Kit-8 (CCK-8)

Cell viability was detected using CCK-8 (Beyotime Institute of Biotechnology). Cells were first plated in 96-well plates at a density of 5 × 10⁴ cells/well. Subsequently, 10 µl CCK-8 solution was added and incubated at 37 °C for 4 h. The optical density of each well was determined at 450 nm by a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Western Blot

Total proteins were extracted from the cells in lysis buffer (Cell Signaling Technologies, USA). Quantification of protein concentration was conducted by a bicinchoninic acid (BCA) assay kit (Beyotime, China). Equal amounts of protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in 0.1 M Tris-buffered saline-0.1% Tween-20 buffer (TBST) for 2 h. Then, the membranes were incubated with the primary antibodies and horseradish peroxidase-conjugated secondary antibody for 1 h. Blots were developed with an enhanced chemiluminescence reagent (Amersham Biosciences, USA) and quantified by densitometric scanning and analyses using an ImageQuant LAS 4000 system (Fujifilm, Tokyo, Japan).

Detection of Alkaline Phosphatase (ALP) Activity

The ALP activity was measured at 14 days following cell culture. Cells were incubated in 96-well plates and washed by PBS for three times, followed by the addition and sonication of distilled water to each well. Total protein was then extracted and quantified by BCA protein assay reagent (Pierce, Rockford, IL). Alkaline phosphatase (ALP) activity was assayed by a fluorescence detection kit per the manufacturer's protocol. A standard curve was prepared by p-nitrophenol. Each value was normalized to the protein concentration.

Alizarin Red S (ARS) Staining

14 days after treatment, cell were fixed with 70% ice-cold ethanol for 1 h. After washed three times with PBS, cells were incubated with 20 mM of alizarin red solution for 15 min at room temperature (RT). The stained cells were photographed after washed with PBS three times. Then, 10% cetylpyridinium chloride was added to extract the stained cells. Absorbance was recorded at 562 nm using a spectrophotometer (NanoDrop Technologies, USA) and analyzed using Image J software (National Institutes of Health).

Reverse-Transcription Polymerase Chain Reaction (RT-qPCR)

Total RNA was isolated from the cells using the TRIzol reagent (Invitrogen, Grand Island, NY). After isolation, the purified RNA (500 ng) was reverse transcribed to cDNA using the SuperScript ™ First-strand synthesis system (Invitrogen, Grand Island, NY). RT was also performed without reverse transcriptase to ensure no contamination of genomic DNA in the preparation of total RNA. PCR was conducted in a DNA Engine (ABi 7500) using SYBR GREENER qPCR UNIVERSAL (Invitrogen) according to the manufacturer's protocol. The band intensities of these digital images were determined using ImageJ software. mRNA level was normalized against GAPDH.

Statistical Analysis

Data processed by GraphPad software version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) were expressed as the mean ± standard deviations. Student’s t test or one-way analysis of variance (ANOVA) was utilized to analyze statistically significant differences between two or more groups. P < 0.05 was considered to mean statistical significance.

The Effects of Sesamin and γ-T3 on the Cell Viability of hBMSCs

To examine the effects of sesamin and γ-T3 on osteoporosis, we first utilized different doses of sesamin and γ-T3 to determined their effects on cell viability of hBMSCs. As exhibited in Figure 1C, the slightly reduced viability of hBMSCs was observed when the cells were exposed to 10 µg/mL sesamin, while no changes were found upon the exposure to low doses of sesamin (1, 2.5, and 5 µg/mL). As shown in Figure 1D, exposure to 5 and 10 mg/mL doses of γ-T3 led to significant damage of hBMSC viability. Thus, we chose 5 µg/mL sesamin and 1 µM γ-T3 for the subsequent assays.

Sesamin and γ-T3 Activates the AMPK Signaling

Then, the involvement of sesamin and γ-T3 in AMPK signaling was measured by detection of the protein levels of AMPK signaling-related factors. Notably, respective treatment with sesamin and γ-T3 induced high levels of phosphorylated (p)-AMPK and p-Samd-1, while co-treatment with sesamin and γ-T3 could trigger more increments in their protein levels, as compared to OM group (Figure 2). Further results that pretreatment with 1 µmol/L CC, the AMPK inhibitor, dramatically reduced the phosphorylation of AMPK and Smad-1 indicated that co-treatment with sesamin and γ-T3 activated the AMPK signaling. In summary, sesamin and γ-T3 served as a stimulator of AMPK signaling.

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Figure 2.

Sesamin and γ-T3 activates the AMPK signaling. Western blot analyzed the protein levels of p-AMPK and p-Samd1 in CC-pretreated hBMSCs induced by sesamin and γ-T3. *p < 0.05, ***p < 0.001 vs control. ^$$$p < 0.001 vs OM group. ^###p < 0.001 vs OM + sesamin group. ⁺⁺⁺p < 0.001 vs OM + γ-T3 group. ^&&&p < 0.001 vs OM + sesamin + γ-T3 group.

The Combined Effect of Sesamin and γ-T3 on the Proliferation of hBMSCs via AMPK Signaling

As we demonstrated that co-treatment with sesamin and γ-T3 activated the AMPK signaling, we next wondered whether combined sesamin and γ-T3 could exert better stimulative effect on the proliferation of osteoblasts than treatment of sesamin or γ-T3 alone and whether this effect was mediated via the AMPK pathway. It was indicated by CCK-8 assay that sesamin or γ-T3 treatment alone heightened the cell proliferation, whereas combined sesamin and γ-T3 triggered higher proliferation rate (Figure 3). Nevertheless, relative to the OM + sesamin + γ-T3 group, pretreatment with CC reduced the proliferation stimulated by the synergistic effects of sesamin and γ-T3. On the whole, these results suggested that co-treatment with sesamin and γ-T3 exhibited enhanced proliferation of osteoblasts via AMPK signaling.

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Figure 3.

The combined effect of sesamin and γ-T3 on the proliferation of hBMSCs via AMPK signaling. CCK-8 assay determined the viability of hBMSCs under different treatments. *p < 0.05, ***p < 0.001 vs control. ^$$$p < 0.001 vs OM group. ^###p < 0.001 vs OM + sesamin group. ⁺⁺⁺p < 0.001 vs OM + γ-T3 group. ^&&&p < 0.001 vs OM + sesamin + γ-T3 group.

The Combined Effect of Sesamin and γ-T3 on the Osteoblast Differentiation of hBMSCs via AMPK Signaling

Osteoblast differentiation is conducive to the bone remodeling and treatment of osteoporosis, and thus we conducted ALP assay to validate the combine effects of sesamin and γ-T3 on osteoblast differentiation. It turned out that sesamin and γ-T3 respectively elevated the ALP enzyme activity, albeit not to the extent where co-treatment with sesamin and γ-T3 did (Figure 4A-B). Intriguingly, the enhanced osteoblast differentiation as a consequence of joint effects of sesamin and γ-T3 was crippled by CC pretreatment. Concomitantly, co-treatment of sesamin and γ-T3 displayed more obvious mineral deposition than treatment of sesamin or γ-T3 alone, whereas CC pretreatment abated the potential of hBMSCs to form mineralized nodules (Figure 4C-D). hBMSCs were cultured in OM for 14 days, the markers of osteoblasts, OPN, OCN, Runx2, and Osterix, exhibited similar trends to the osteoblast differentiation and mineralization that had been displayed (Figure 5A-B). Taken together, sesamin and γ-T3 mediated the AMPK signaling to contribute to hBMSCs osteoblast differentiation.

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Figure 4.

The combined effect of sesamin and γ-T3 on the osteoblast differentiation of hBMSCs via AMPK signaling. (A-B) The ALP activity and (C-D) mineralization of hBMSCs in sesamin and γ-T3-induced osteoblasts upon CC pretreatment. **p < 0.01, ***p < 0.001 vs control. ^$$$p < 0.001 vs OM group. ^#p < 0.05, ^##p < 0.01 vs OM + sesamin group. ⁺p < 0.05, ⁺⁺p < 0.01 vs OM + γ-T3 group. ^&&&p < 0.001 vs OM + sesamin + γ-T3 group.

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Figure 5.

The biomarkers of osteoblasts upon CC pretreatment with sesamin andγ-T3 induction. (A) RT-qPCR and (B) western blot analyzed OPN, OCN, Runx2 and Osterix expression in sesamin and γ-T3-induced osteoblasts upon CC pretreatment. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. ^$$$p < 0.001 vs OM group. ^##p < 0.01, ^###p < 0.001 vs OM + sesamin group. ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs OM + γ-T3 group. ^&&&p < 0.001 vs OM + sesamin + γ-T3 group.