Combined effect of 17β-estradiol and resveratrol against apoptosis induced by interleukin-1β in rat nucleus pulposus cells via PI3K/Akt/caspase-3 pathway

Abstract

Background: In previous studies, both 17β-estradiol (E2) and resveratrol (RES) were reported to protect intervertebral disc cells against aberrant apoptosis. Given that E2 has a better anti-apoptotic effect with more cancer risk and RES has an anti-apoptotic effect with less cancer risk, the combined use of E2 with RES is promising in developing clinical therapies to treat apoptosis-related diseases such as intervertebral disc degeneration in the future.

Objective: The purpose of this study was to explore the combined effect of E2 with RES on rat nucleus pulposus cells and the underlying mechanisms.

Methods: TUNEL assay and FACS analysis were used to determine apoptotic incidence of nucleus pulposus cells. MTS assay was used to determine cell viability, and cellular binding assay was used to determine cell-ECM (extracellular matrix) ability. Real-time quantitative RT-PCR was to determine mRNA level of target genes. And Western blot was used to determine the protein level.

Results: Both E2 and RES decreased apoptotic incidence when used singly; interestingly, they decreased apoptosis more efficiently when used combinedly. Meanwhile, E2 and RES combined together against the decrease of cell viability and binding ability resulting from IL-1β cytotoxicity. As well, activated caspase-3 was suppressed by the combined effect. Furthermore, IL-1β downregulated expression level of type II collagen and aggrecan (standing for anabolism), while upregulated MMP-3 and MMP-13 (standing for catabolism). However, the combined use of E2 with RES effectively abolished the above negative effects caused by IL-1β, better than either single use. Finally, it turned out to be that E2 and RES combined together against apoptosis via the activation of PI3K/Akt/caspase-3 pathway.

Conclusion: This study presented that IL-1β induced aberrant apoptosis, which was efficiently resisted by the combined use of E2 with RES via PI3K/Akt/caspase-3 pathway.

Keywords: 17β-estradiol; Apoptosis; Intervertebral disc degeneration; Nucleus pulposus; PI3K/Akt; Resveratrol.

Figure 1. FACS analysis for apoptotic incidence.

(A) NPCs were plated into 6-well plates at a density of 2 × 10⁵ cells per well and divided into ten groups as presented in the figure. All groups were incubated for 24 h in the serum-free medium without phenol red. Apoptotic cells were detected using an Annexin V-FITC/PI kit (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Apoptotic cells, stained positive for annexin V-FITC, negative for PI, or double positive, were counted. (B) Data are represented as a percentage of the total cell count. Data analysis was determined by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. * p < 0.05 by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; RSV, resveratrol; mean ± SD (standard deviation); n = 6.

Figure 2. TUNEL assay for apoptosis.

NPCs were divided into five groups. As a control, group A was treated with vehicle mixture (ethanol and DMSO, <0.1%; ethanol was the solvent of E2 and DMSO was the solvent of RSV). Group B was treated with 75 ng/ml IL-1β. Group C was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 for 30 min. Group D was treated with 75 ng/ml IL-1β with the pretreatment of 200 μM RSV for 30 min. Group E was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 and 200 μM RSV for 30 min. All groups were incubated for 24 h in the serum-free medium without phenol red. All cells were stained red by PI, and apoptotic cells presented green. (A) Scale bar, 200 μm; (B) Scale bar, 50 μm; NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; RSV, resveratrol; PI, prodium iodide; n = 6.

Figure 3. MTS assay for cell viability.

NPCs were treated in five groups as presented in the figure and then seeded into 96-well plates. All groups were incubated for 24 h in the serum-free medium without phenol red. Cell viability was determined by MTS assay using CellTiter 96® AQueous MTS Reagent Solution (Promega, Madison, WI, USA) according to manufacturer’s instruction. The optical density was measured at 492 nm with a microplate reader (Shimadzu, Kyoto, Japan) and cell viability was normalized as a percentage of control. * p < 0.05, by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; RSV, resveratrol; mean ± SD (standard deviation); n = 6.

Figure 4. Cellular binding ability to type II collagen.

NPCs were divided into five groups. As a control, group A was treated with vehicle mixture (ethanol and DMSO, <0.1%; ethanol was the solvent of E2 and DMSO was the solvent of RSV). Group B was treated with 75 ng/ml IL-1β. Group C was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 for 30 min. Group D was treated with 75 ng/ml IL-1β with the pretreatment of 200 μM RSV for 30 min. Group E was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 and 200 μM RSV for 30 min. All groups were incubated for 24 h in the serum-free medium without phenol red. * p < 0.05, by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; RSV, resveratrol; mean ± SD (standard deviation); n = 6.

Figure 5. Active caspase-3 activity assay.

NPCs were divided into five groups. As a control, group A was treated with vehicle mixture (ethanol and DMSO, <0.1%; ethanol was the solvent of E2 and DMSO was the solvent of RSV). Group B was treated with 75 ng/ml IL-1β. Group C was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 for 30 min. Group D was treated with 75 ng/ml IL-1β with the pretreatment of 200 μM RSV for 30 min. Group E was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 and 200 μM RSV for 30 min. All groups were incubated for 24 h in the serum-free medium without phenol red. Caspase-3 activity was determined using a caspase-3 activity kit (Beyotime, Shanghai, China). Caspase-3 activity is expressed as the fold change in enzyme activity over control. * p < 0.05, by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; RSV, resveratrol; mean ± SD (standard deviation); n = 6.

Figure 6. RT-qPCR analysis.

NPCs were divided into five groups. As a control, group A was treated with vehicle mixture (ethanol and DMSO, <0.1%; ethanol was the solvent of E2 and DMSO was the solvent of RSV). Group B was treated with 75 ng/ml IL-1β. Group C was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 for 30 min. Group D was treated with 75 ng/ml IL-1β with the pretreatment of 200 μM RSV for 30 min. Group E was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 and 200 μM RSV for 30 min. All groups were incubated for 24 h in the serum-free medium without phenol red. * p < 0.05, by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; RSV, resveratrol; RT-qPCR, reverse transcription and real-time quantitative polymerase chain reaction; MMP, matrix metalloproteinase; mean ± SD (standard deviation); n = 6.

Figure 7. Protein levels of Akt, p-Akt(Ser473) and active caspase-3.

NPCs were divided into four groups. Group A was treated with 75 ng/ml IL-1β. Group B was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2 and 200 μM RSV for 30 min. Group C was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2, 200 μM RSV, and 1 μM ICI for 30 min. Group D was treated with 75 ng/ml IL-1β with the pretreatment of 1 μM E2, 200 μM RSV and 50 μM LY294002(PI3K/Akt inhibitor). All groups were incubated for 24 h in the serum-free medium without phenol red. * p < 0.05, by one-way analysis of variance (ANOVA) accompanied by pairwise comparison using SNK-q test. NPCs, nucleus pulposus cells; IL-1β, interleukin-1β; E2, 17β-estradiol; ICI, ICI182780; RSV, resveratrol; LY, LY294002, 50 μM; mean ± SD (standard deviation); n = 6.