| Background:Nucleus pulposus cells(NPC)play an important role in the onset and development of intervertebral disc degeneration(IVDD).Ferroptosis is an iron-dependent cellregulated death caused by massive lipid peroxidation-mediated membrane damage.Recent studies have shown that ferroptosis and lipid peroxidation are associated with IVDD,and inhibition of ferroptosis in NPC may be a potential target for the treatment of IVDD.Curcumin(Cur)has various biological activities such as antioxidant and antiinflammatory,and is used to treat a variety of degenerative diseases.However,the effects and mechanisms of curcumin on NPC are still unclear.In this study,we investigated the therapeutic effects of curcumin on the IVDD model established by caudal acupuncture in rats and the inhibition of ferroptosis in NPC by curcumin and its mechanism,which provides a novel potential drug for the treatment of IVDD.Methods:A rat IVDD model was established by acupuncture,and curcumin dissolved in corn oil(50 mg/kg)was administered to rats by gavage immediately after surgery,and the signal intensities of intervertebral discs with different degrees of degeneration were different in magnetic resonance T2-weighted images,and the successful modeling was confirmed by magnetic resonance imaging(MRI)2 weeks after surgery,and hematoxylin-eosin(HE)staining,safranin o-solid green(Safranin O)staining,Masson staining,and observation of disc changes in rats were performed 6 weeks after surgery.Degenerated nucleus pulposus tissue(mild(Mild): grade II or III;severe(Severe):grade IV or V)was collected from patients with lumbar disc herniation(Lumbar disc herniation,LDH)or lumbar spinal stenosis(LSS)according to Pfirrmann grading of MRI images.HE and Safranin O staining were performed to observe the tissue morphology,and the expression levels of ferroptosis marker proteins were detected in mild and severe lumbar nucleus pulposus degeneration tissues using immunohistochemistry and Western Blot to analyze the levels of ferroptosis in nucleus pulposus tissues with different degrees of degeneration.Relevant data were collected from the Gene Expression Omnibus(GEO)database and the ferroptosis database,and the expression levels of ferroptosis-related genes in normal and degenerated medullary tissues were analyzed differently using heat maps.Nucleus pulposus cells were extracted from mildly degenerated lumbar nucleus pulposus tissues for culture and used for experiments when passed to the 2nd generation to determine the effect of different concentrations of curcumin and ferroptosis inducer(1S,3R)-RSL3(RSL3)on nucleus pulposus cell viability.The nucleus pulposus cells were treated with curcumin(10 μM)and examined for changes in relevant protein levels using Western Blot and immunofluorescence assays.The viability and survival of nucleus pulposus nuclei after treatment with different intervention groups(normal group,0.5 μM RSL3-induced group,10 μM curcumin-treated group,150 μM(Deferoxamine,DFO)treated group)were determined by CCK-8 assay,crystalline violet staining,and live-dead staining assay.Intracellular lipid peroxidation levels were detected by C11 BODIPY fluorescent probe,and intracellular reactive oxygen species(ROS)levels were detected by flow cytometry.Western Blot and immunofluorescence experiments were performed according to the above intervention groups to investigate the possible mechanisms of curcumin inhibition of nucleus pulposus cell injury.The nuclear factor erythroid 2-related factor 2(Nrf2)and SQSTM1(P62)genes were knocked down using small interfering RNA(si RNA),and the experimental subgroups were set up after confirming the interference efficiency to further verify the specific pathway of curcumin inhibition of ferroptosis in nucleus pulposus cells.Results:MRI at 2 weeks postoperatively suggested successful IVDD modeling in rats.The results of MRI,HE,Safranin O and Masson staining at 6 weeks postoperatively indicated that the curcumin-treated group had reduced damage to the nucleus pulposus in the rat IVDD model established by caudal pinning compared with the surgical-only group.HE and Safranin O staining showed a significant decrease in the number of nucleus pulposus cells and severe tissue defects in severe nucleus pulposus degenerated tissues compared with those in mild nucleus pulposus degenerated tissues.Immunohistochemistry and Western Blot experiments showed that the expression levels of ferroptosis marker proteins differed in myelinated tissues with different degrees of degeneration.The data collected by thermogram analysis suggested differential expression of ferroptosis-related genes in normal and degenerated nucleus pulposus tissues.Curcumin promoted nucleus pulposus cell viability in a range of concentrations,and RSL3 had a significant damaging effect on nucleus pulposus cells.The levels of Nrf2 and its downstream proteins were increased in curcumin-treated nucleus pulposus cells,and the results of CCK-8 assay,crystal violet staining,and live-dead staining demonstrated that curcumin could significantly inhibit RSL3-induced damage to nucleus pulposus cells.The C11 BODIPY probe assay showed that curcumin could inhibit RSL3-induced lipid peroxidation,while the flow cytometry assay also showed that curcumin could also inhibit RSL3-induced increase in ROS levels.We performed experiments after knocking down Nrf2 and P62 genes,and found that the protein levels of Nrf2,P62,and heme oxygenase-1(HO-1)were restored after curcumin treatment compared with the knockdown group.Conclusion:Curcumin delays intervertebral disc degeneration in rats.Ferroptosis is associated with the progression of IVDD,and appropriate concentrations of curcumin can promote nucleus pulposus cell viability.Curcumin has an activating effect on Nrf2 and its downstream proteins in nucleus pulposus cells,which can inhibit RSL3-induced ferroptosis in nucleus pulposus cells.Curcumin could resist the sensitivity of nucleus pulposus cells to ferroptosis by activating the Nrf2/HO-1 pathway,while possibly inhibiting the disruption of iron homeostasis caused by RSL3 induction.Curcumin may be a potentially effective drug for the prevention and treatment of IVDD by inhibiting ferroptosis in the nucleus pulposus. |
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