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Resveratrol protects against apoptosis induced by interleukin-1? in nucleus pulposus cells via activating mTOR/caspase-3 and GSK-3?/caspase-3 pathways.

ABSTRACT: OBJECTIVE:The purpose of the present study was to investigate the specific downstream signaling pathway mediated by PI3K/Akt in resveratrol (RES) anti-apoptosis of nucleus pulposus cells (NPCs). MATERIALS AND METHODS:Human NPCs were cultured and divided into six groups. Interleukin (IL)-1? was used to induce apoptosis and RES to inhibit apoptosis. Fluorescence-activated cell sorting (FACS) analysis was used to test apoptotic incidence of NPCs, cell counting kit-8 (CCK-8) assay was performed to detect cell viability, The expression level of caspase-3 mRNA was detected by RT-qPCR, and protein levels were determined by Western blot. RESULTS:Flow cytometry analysis showed that IL-1? increased the apoptosis rate of NPCs in each group, and RES significantly decreased the apoptosis rate, while rapamycin (RAPA) and SB216763 inhibited the effect of RES and increased the apoptosis rate again. Similarly, CCK-8 showed that IL-1? decreased activity of NPCs in each group, while RES increased cell activity, RAPA and SB216763 inhibited the effect of RES and decreased cell activity. RT-qPCR results showed IL-1? significantly increased the level of caspase-3 expression, but it was significantly decreased by using RES, RAPA and SB216763 respectively attenuated effects of RES. Western blot results showed that activated caspase-3 was inhibited by RES effect, and was up-regulated again after the addition of RAPA and SB216763. In addition, p-mTOR and p-GSK-3? were up-regulated by RES and down-regulated by RAPA and SB216763. CONCLUSION:RES can inhibit apoptosis induced by IL-1? in human NPCs. PI3K/Akt/mTOR/caspase-3 and PI3K/Akt/GSK-3?/caspase-3 pathways are potential mechanisms underlying this process.

SUBMITTER: Guo X

PROVIDER: S-EPMC7391128 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Resveratrol protects against apoptosis induced by interleukin-1β in nucleus pulposus cells via activating mTOR/caspase-3 and GSK-3β/caspase-3 pathways.

Guo Xiaohui X Bai Xiaoliang X Zhang Feng F Zheng Long L Ding Wenyuan W Yang Sidong S

Bioscience reports 20200701 7

<h4>Objective</h4>The purpose of the present study was to investigate the specific downstream signaling pathway mediated by PI3K/Akt in resveratrol (RES) anti-apoptosis of nucleus pulposus cells (NPCs).<h4>Materials and methods</h4>Human NPCs were cultured and divided into six groups. Interleukin (IL)-1β was used to induce apoptosis and RES to inhibit apoptosis. Fluorescence-activated cell sorting (FACS) analysis was used to test apoptotic incidence of NPCs, cell counting kit-8 (CCK-8) assay was ...[more]

PMID: 32696949

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Project description:BackgroundIntervertebral disc degeneration results from a variety of etiologies, including inflammation and aging. Degenerated intervertebral discs feature down-regulated extracellular matrix synthesis, resulting in losing their ability to retain water and absorb compression. Celecoxib is a well-known selective cyclooxygenase-2 inhibitor for treating arthritis and relieving pain. Nevertheless, the mechanism of Celecoxib for treating inflammation-related intervertebral disc degeneration has not yet been clarified.MethodProtein synthesis was analyzed by western blot. Fluorescent probes DCFH-DA and MitoSox Red detected reactive oxygen species and were measured by flow cytometry. The activity of the kinase pathway was evaluated by protein phosphorylation. Autophagy was monitored by mRFP-GFP-LC3 transfection and LC3 analysis. Mitochondrial apoptotic proteins were analyzed by western blot and cell membrane integrity was measured by flow cytometry. The autophagic gene was silenced by siRNA.ResultsIn this study, interleukin-1β stimulation reduced the synthesis of aggrecan, type I and II collagen and caused excessive production of reactive oxygen species. We looked for a therapeutic window of Celecoxib for nucleus pulposus cells to regain extracellular matrix synthesis and reduce oxidative stress. To look into nucleus pulposus cells in response to stimuli, enhancement of autophagy was achieved by Celecoxib, confirmed by mRFP-GFP-LC3 transfection and LC3 analysis. The mammalian target of rapamycin and a panel of downstream proteins responded to Celecoxib and propelled autophagy machinery to stabilize homeostasis. Ultimately, inhibition of autophagy by silencing autophagy protein 5 disrupted the protective effects of Celecoxib, culminating in apoptosis.ConclusionIn summary, we have demonstrated a new use for the old drug Celecoxib that treats intervertebral disc degeneration by enhancing autophagy in nucleus pulposus cells and opening a door for treating other degenerative diseases.

Dataset Information

Resveratrol protects against apoptosis induced by interleukin-1? in nucleus pulposus cells via activating mTOR/caspase-3 and GSK-3?/caspase-3 pathways.

Publications

Resveratrol protects against apoptosis induced by interleukin-1β in nucleus pulposus cells via activating mTOR/caspase-3 and GSK-3β/caspase-3 pathways.

Similar Datasets

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