Astaxanthin Suppresses Oxidative Stress And Calcification In Vertebral Cartilage Endplate Via Activating Nrf2/HO-1 Signaling Pathway

Background:Cartilage endplate(CEP)degeneration is an important initiating factor leading to intervertebral disc degeneration(IVDD).Astaxanthin(AST)is a natural lipid-soluble and red-orange carotenoid which possesses various biological activities,including antioxidant,anti-inflammatory,and anti-aging effects in multiple organisms.However,the effects and mechanism of AST on endplate chondrocytes remain largely unknown.The objective of the current study was to investigate the effects and of AST on CEP degeneration and its underlying molecular mechanisms.Methods:Tert-butyl hydroperoxide(TBHP)was used to mimic the IVDD pathological environment.Immunofluorescence,Western blot,flow cytometry,alizarin red staining and alkaline phosphatase staining were used to detect the regulatory effect of AST on Nrf2 signaling pathway and damage-related events,including oxidative stress,ferroptosis,mitophagy,apoptosis and degeneration of cartilage endplate.Chondrocytes were transfected with specific small interfering RNA(siRNA)targeting mouse Nrf2 gene,and Nrf2 expression level was detected by Western blot to evaluate the transfection efficiency.The L4/5 bilateral lateral joint,supraspinous,and interspinous ligaments were transected according to a previously reported methodology to establish the IVDD model.Finally,the effect of astaxanthin on cartilage endplate degeneration was verified by Micro-CT analysis,hematoxylin-eosin staining,and immunohistochemical observation.Results:CCK8 cell viability assays showed that AST at concentrations ranging from 0 to 80μM had no significant effect on endplate chondrocyte viability in vitro.Western blot and immunofluorescence results showed that AST significantly promoted Nrf2 protein expression and nuclear translocation.DCFH-DA staining and flow cytometry showed that AST inhibited the TBHP-induced increase in ROS production.At the same time,AST treatment reduced the TBHP-induced increase in green fluorescence intensity of JC-1 monomer and increased red JC-1 aggregates to inhibit the collapse of mitochondrial membrane potential.In addition,Western blot and immunofluorescence showed that AST ameliorated the process of mitophagy,inhibited ferroptosis,ameliorated calcification,apoptosis and extracellular matrix(ECM)degradation of endplate chondrocytes.However,knockdown of Nrf2 using siRNA reversed AST-induced mitophagy,ferroptosis and its protective effects.In addition,AST successfully inhibited the NF-κB activity induced by oxidative stimulation and alleviated the inflammatory response.In vivo experiments,Micro-CT,three-dimensional reconstruction,and HE staining showed that AST alleviated the improvement of intervertebral space height,smoother endplate cartilage surface,and reduced endochondral osteogenesis.Immunohistochemistry results also confirmed that AST successfully activated Nrf2 signaling pathway and alleviated cartilage calcification and degeneration and extracellular matrix degradation.Conclusions:1.AST not only has strong antioxidant and anti-inflammatory ability,but also has high biological safety.2.TBHP intervention disrupted the redox balance of endplate cartilage,which mediated mitochondrial dysfunction and eventually led to calcification and degeneration of endplate cartilage.3.AST could protect vertebral endplate cartilage from oxidative stress and degeneration by activating Nrf2/HO-1 pathway in vitro and in vivo.Our results imply that AST may serve as a potential therapeutic agent for IVDD progression and treatment.