Nrf2 Suppresses Apoptosis And Senescence Of Nucleus Pulposus Cells Induced By Oxidative Stress

Objectives:Low back pain is an age-related degenerative disease as well as the most common musculoskeletal disorder.It often leads to the loss of labor force,reduces the quality of life,and brings about great social and economic burdens.At present,the key cause of low back pain is thought to be the intervertebral disc degeneration(IDD),of which the main pathological changes include excessive apoptosis and senescence of nucleus pulposus cells(NPCs),oxidative stress,matrix degradation and so on.However,the specific pathological mechanism has not been fully elucidated,and the effective therapeutic drugs are limited.Nrf2 is a key transcription factor involved in maintaining intracellular homeostasis by evoking multifaceted responses to stresses via regulating the expression of target genes.In recent years,due to the important regulatory functions of Nrf2 in oxidative stress,inflammatory response,cell metabolism and other aspects,more and more researches focus on its roles in the occurrence and development of diseases,including tumors and chronic diseases,and take Nrf2 as the potential target for the prevention and treatment of these diseases.However,there are few reports on the relationship between Nrf2 and IDD,and its role in apoptosis and senescence of NPCs.Previous studies have shown that Kinsenoside has obvious anti-inflammatory and anti-apoptotic effects,but its impacts on the regulation of Nrf2 and the apoptosis and senescence of NPCs under oxidative stress are not clear.Therefore,the purpose of this study is to explore the expression changes and significance of Nrf2 in human nucleus pulposus tissues of intervertebral discs with different degenerative degrees,as well as its role in oxidative stress-induced injuries of NPCs,and to analyze the effect of Kinsenoside on Nrf2 signaling pathway and conduct the relevant in vitro experiments,ultimately providing new insights into the mechanism of IDD and also a new target for the prevention and treatment of this disease.Methods:1.According to the preoperative MRI and Pfirrmann grading criteria,the specimens of nucleus pulposus tissues with different degrees of degeneration were collected.The expression level of Nrf2,Cleaved-caspase3(C-caspase3)and p16 in nucleus pulposus tissues was detected by immunohistochemistry and Western blot.Then the obtained data were analyzed statistically.2.The research object in this study is rat NPCs that were treated with TBHP to establish the cell model of apoptosis and senescence under oxidative stress in vitro.The expression level of Nrf2 was overexpressed or knocked down in NPCs via lentivirus transfection.Then the effects of Nrf2 on apoptosis and senescence of NPCs induced by oxidative stress were analyzed by using ROS detection,flow cytometry,?-galactosidase staining and Western blot.3.NPCs were pretreated with Kinsenoside,then the impacts of Kinsenoside on apoptosis,senescence and mitochondrial homeostasis were evaluated by CCK-8,flow cytometry,EdU staining,MitoSOX staining and Western blot.The expression level of Nrf2 and its downstream antioxidant proteins in Kinsenoside-treated NPCs was detected by Western blot,and the changes in upstream AKT and MAPK signaling pathway were also detected.In addition,the relationship between Nrf2 and Kinsenoside-mediated cytoprotection was determined in Nrf2-deficient NPCs using JC-1 staining,TUNEL assay,?-galactosidase staining and Western blot.Results:1.The protein level of Nrf2 and the Nrf2 immunopositive cells decreased in human nucleus pulposus tissues with the aggravation of disc degeneration.The correlation analysis indicated that the expression level of Nrf2 in human nucleus pulposus tissues was negatively correlated with the degree of disc degeneration.Besides,the expression levels of C-caspase3 and p16 were increased in degenerated nucleus pulposus tissues.2.Under TBHP-induced oxidative stress,the protein levels of Cleaved-caspase3(C-caspase3)and p16 in NPCs were increased in a concentration-and time-dependent manner.However,under oxidative stress,the lentivirus-mediated Nrf2 knockdown increased the content of ROS,the number of apoptotic cells and ?-galactosidase positive cells,as well as the protein levels of C-caspase3 and p16,comparing to the control group.On the contrary,when Nrf2 was overexpressed in NPCs,the above changes were significantly inhibited.3.After the NPCs were pretreated with Kinsenoside,the oxidative stress-induced apoptosis and senescence were suppressed,and the mitochondrial homeostasis was better maintained.In addition,Kinsenoside could activate the upstream AKT and ERK1/2 pathways of Nrf2 and promote the expression of Nrf2 and its downstream antioxidant proteins,including SOD2 and NQO1,in NPCs.However,the Kinsenoside-mediated activation of Nrf2 could be respectively blocked by the inhibitors of AKT and ERK1/2 pathway.Moreover,Nrf2 knockdown could comprise the cytoprotection of Kinsenoside in NPCs under oxidative stress.Conclusions:This study demonstrated that the expression of Nrf2 in human nucleus pulposus tissues of discs decreased with the degree of degeneration,and there was a negative correlation between them,suggesting that Nrf2 might play an important role in the occurrence and development of IDD.As a key antioxidant transcription factor,Nrf2 could reduce the intracellular oxidative stress level and inhibit the apoptosis and senescence of NPCs under oxidative stress.It is worth noting that Kinsenoside can activate Nrf2 through the Akt and ERK1/2 pathways and promote the expression of antioxidant proteins,including SOD2 and NQO1,in NPCs,inhibiting the apoptosis and senescence of NPCs under oxidative stress.Moreover,this cytoprotection is closely associated with the activation of Nrf2 in NPCs.To sum up,this study suggests that Nrf2 may be involved in disc degeneration,providing new insights into the pathogenesis of IDD as well as the theoretical basis for the future therapeutic strategies targeting Nrf2.