Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

Insights of stem cell-based endogenous repair of intervertebral disc degeneration

Low back pain has become more prevalent in recent years,causing enormous economic burden for society and government.Common therapies used in clinics including conservative treatment and surgery can only relieve pain.Subsequent cell-based treatment such as mesenchymal stem cell transplantation poses problems such as short duration of therapeutic effect and tumorigenesis.Recently,the discovery and identification of stem cell niche and stem/progenitor cells in intervertebral disc bring increased attention to endogenous repair strategy.Therefore,we review the studies involving endogenous repair strategy and present the characteristics and current status of this treatment.Meanwhile,we also discuss the strategy and perspective of endogenous repair strategy in future.

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy

BACKGROUND To date,there has been no effective treatment for intervertebral disc degeneration(IDD).Nucleus pulposus-derived mesenchymal stem cells(NPMSCs)showed encouraging results in IDD treatment,but the overexpression of reactive oxygen species(ROS)impaired the endogenous repair abilities of NPMSCs.6-gingerol(6-GIN)is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.AIM To investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.METHODS The cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN.ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis.Matrix metalloproteinase(MMP)was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay.TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate.Additionally,autophagy-related proteins(Beclin-1,LC-3,and p62),apoptosisassociated proteins(Bcl-2,Bax,and caspase-3),and PI3K/Akt signaling pathwayrelated proteins(PI3K and Akt)were evaluated by Western blot analysis.Autophagosomes were detected by transmission electron microscopy in NPMSCs.LC-3 was also detected by immunofluorescence.The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction(RT-PCR),and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.RESULTS 6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs,decreased hydrogen peroxide-induced intracellular ROS levels,and inhibited cell apoptosis.6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression.The MMP,Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide.6-GIN treatment promoted extracellular matrix(ECM)expression by reducing the oxidative stress injury-induced increase in MMP-13 expression.6-GIN activated autophagy by increasing the expression of autophagy-related markers(Beclin-1 and LC-3)and decreasing the expression of p62.Autophagosomes were visualized by transmission electron microscopy.Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux.The PI3K/Akt pathway was also found to be activated by 6-GIN.6-GIN inhibited NPMSC apoptosis and ECM degeneration,in which autophagy and the PI3K/Akt pathway were involved.CONCLUSION 6-GIN efficiently decreases ROS levels,attenuates hydrogen peroxide-induced NPMSCs apoptosis,and protects the ECM from degeneration.6-GIN is a promising candidate for treating IDD.

Urolithin a alleviates oxidative stress-induced senescence in nucleus pulposus-derived mesenchymal stem cells through SIRT1/PGC-1α pathway

BACKGROUND In degenerative intervertebral disc(IVD),an unfavorable IVD environment leads to increased senescence of nucleus pulposus(NP)-derived mesenchymal stem cells(NPMSCs)and the inability to complete the differentiation from NPMSCs to NP cells,leading to further aggravation of IVD degeneration(IDD).Urolithin A(UA)has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress.AIM To explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism.METHODS In vitro,we harvested NPMSCs from rat tails,and divided NPMSCs into four groups:the control group,H2O2 group,H2O2+UA group,and H2O2+UA+SR-18292 group.Senescence-associatedβ-Galactosidase(SA-β-Gal)activity,cell cycle,cell proliferation ability,and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α(SIRT1/PGC-1α)pathway-related proteins and mRNA were used to evaluate the protective effects of UA.In vivo,an animal model of IDD was constructed,and Xrays,magnetic resonance imaging,and histological analysis were used to assess whether UA could alleviate IDD in vivo.RESULTS We found that H2O2 can cause NPMSCs senescence changes,such as cell cycle arrest,reduced cell proliferation ability,increased SA-β-Gal activity,and increased expression of senescence-related proteins and mRNA.After UA pretreatment,the abovementioned senescence indicators were significantly alleviated.To further demonstrate the mechanism of UA,we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1αpathway that regulates mitochondrial function.UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1αpathway.In vivo,we found that UA treatment alleviated an animal model of IDD by assessing the disc height index,Pfirrmann grade and the histological score.CONCLUSION In summary,UA could activate the SIRT1/PGC-1αsignaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.