Quercetin Ameliorates Oxidative Stress-induced Senescence In Nucleus Pulposus-derived Mesenchymal Stem Cells Via The MiR-34a-5p/SIRT1 Axis

Objective: Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Several previous studies have indicated that oxidative stress and reactive oxygen species(ROS)are highly associated with the progression of IDD,and the progression of IDD is considered to be the result of disc cell senescence caused by ROS accumulation.Nucleus pulposus-derived mesenchymal stem cells(NPMSC)are present in the intervertebral disc,which play a significant role in the repair and regeneration of degenerative intervertebral disc.ROS not only causes oxidative damage to the extracellular matrix in the intervertebral disc,but also induces oxidative damage to DNA,proteins and mitochondria,which in turn leads to NPMSC senescence.The endogenous repair ability of NPMSC in degenerative IVD is achieved by differentiation into NP cells and inhibiting cell apoptosis and/or senescence.The differentiation potential and self-renewal ability of NPMSC are reduced when ROS induces NPMSC senescence,and their number and function are correspondingly reduced,which in turn leads to the failure of endogenous repair of IDD and exacerbates the progress of IDD.Therefore,reducing oxidative stress-induced NPMSC senescence may be important for delaying the progress of IDD.Quercetin,a natural flavonoid widely present in various plants,has antioxidative stress,anti-inflammatory,and antiaging properties.Quercetin has been used as a senolytic in oxidative stress associated degenerative diseases.The present study aimed to investigate the role of Quercetin in oxidative stress-induced NPMSC damage and its possible mechanisms.Methods: NPMSCs were obtained from SD rats,and immunofluorescent staining was used to detect the CD markers of stem cells described by ISCT.CCK-8 assay was used to evaluate the effect of Quercetin and Tert-butyl hydroperoxide(TBHP)on NPMSC viability,the effect of Quercetin on NPMSC proliferation was detected by Ed U staining,SA-β-Gal staining was used to evaluate the cellular senescence,flow cytometry was used to detect cell cycle,JC-1 assay was used to measure MMP,and the expression level of ROS were detected by ROS assay kit.Lentivirus and small interfering RNA were transfected into NPMSC cells.Dual-luciferase reporter assay was used to identify the relationship between miR-34a-5p and SIRT1.The content of miR-34a-5p was detected by q PCR.The expression level of p16、p21、p53、IL-1β、IL-6、MMP13and SIRT1 were determined by western blot.15 male SD rats were randomly divided into the following three groups: control group,IDD group,and Quercetin group.The rats were anesthetized with pentobarbital,and the tail skin was sterilized by povidone iodine.A 21 G needle was used to puncture the coccygeal intervertebral disc(Co 6-7),and the needle was rotated 180° and kept in the disc for 5 s at a depth of 5 mm.After surgery,the Quercetin group was treated with Quercetin(100 mg/kg,)every other day by intragastric administration for 4 weeks.The control group and IDD group were treated with 0.5% sodium carboxymethyl cellulose solution for 4 weeks.At 4 weeks postinjury,all rats were placed in a prone position after anesthetization with pentobarbital,and X-ray images were acquired.Hematoxylin-eosin(HE),Alcian blue,Safranin O-fast Green,and immunofluorescent staining were used to evaluate the intervertebral disc.Result: NPMSC were successful obtained from SD rats and correspond to the standards of stem cells described by ISCT.The concentration of 20 μM Que and 100 μM TBHP for 24 h were selected for use in subsequent experiments.The results showed that Quercetin promoted cell proliferation,decreased β-galactosidase expression and ROS production.Quercetin can decrease the expression of p16,p21,p53,IL-1β,IL-6 and MMP-13,while increase the expression of SIRT1.Further studies revealed that oxidative stress increased miR-34a-5p expression in NPMSC,while Quercetin treatment decreased miR-34a-5p expression.Meanwhile,dual luciferase assay demonstrated that SIRT1 is a target gene of miR-34a-5p.After overexpression and silencing of SIRT1 in NPMSC,the expression of p16,p21,p53,IL-1β,IL-6 and MMP-13 were increased and the expression of SIRT1 was decreased,while Quercetin treatment could partially reverse this result.Meanwhile,animal experiment showed that Quercetin treatment can reduce the loss of intervertebral space height.The results of alcian blue,Safranin O-fast Green,and immunofluorescent staining showed that Quercetin could delay the degradation of extracellular matrix(ECM)including aggrecan and type II collagen.Conclusion: In summary,the present study demonstrated that Quercetin prevents oxidative stress-induced senescence of NPMSC via the miR-34a/SIRT1 signaling pathway,which in turn delays the progress of IDD.