Mechanism Of Cadmium Chloride Induced Senescence Of Annulus Fibrosus Cells In Intervertebral Disc Degeneration Through JNK/p53 Pathway

Background:Low back pain(LBP)is widespread worldwide,and its high incidence rate has brought greate conomic and health burdens to society and individuals.Research has found that intervertebral disc degeneration(IVDD)is the main reason of chronic LBP,while advanced age is considered to be the main risk factor for IVDD.Aging is caused by the accumulation of long-term molecular and cellular damage level,which leads to the damage of tissue homeostasis and the decline of physiological function.In humans,senescence is associated with an increased incidence of IVDD,including abnormal expression-of collagen and proteoglycan,degeneration and calcification.It is reported that decreased production of extracellular matrix(ECM),increased production of degrading enzymes and up-regulated expression of inflammatory cytokines will contribute to the loss of structural integrity of intervertebral disc tissue and accelerate IVDD,but the pathological mechanism of IVDD has not been fully clarified.Therefore,the senescence of annulus fibrosus cell may be closely related to IVDD.Cadmium(Cd)is a common heavy metal pollutant,which can accumulate in bones and other tissues and organs,and cause serious health and social problems,and Cd exposure can significantly induce senescence of bone marrow mesenchymal stem cells(BMMSCs)and nerve cells.The study found that the concentration of Cd in the blood of patients with spinal osteoarthritis was significantly higher than that of the normal patients,indicating that Cd toxicity is closely related to the occurrence of spinal diseases.Objective:The purpose of this study was to explore the effect of cadmium chloride(CdCl2)on annulus fibrosus cells and its possible mechanism,so as to provide a feasible target for the prevention-of IVDD induced by Cd exposure.Methods:Annulus fibrosus cells of intervertebral disc of SD rats were obtained.The thirdgeneration annulus fibrosus cells were treated with different concentrations of CdCl2.The cell viability and proliferation were observed by CCK-8 method.The annulus fibrosus cells were treated with OμM,1μM and 5μM CdCl2,and the effect of CdCl2 on annulus fibrosus cells was observed by EdU method,senescence-associeted β-galactosidase staining and Western blot analysis.Then transcriptome sequencing and bioinformatics analysis of Kyoto encyclopedia of genes and genomes were performed on annulus fibrosus cells with or without CdCl2.The third-generation annulus fibrosus cells were divided into control group,CdCl2 group and SP600125(inhibitor of JNK pathway)group.Differential genes and related pathways were identified by mRNA transcriptome sequencing,protein expression of related genes was detected by Western blot and immunofluorescence analysis,the number of positive cells was detected by senescence-associated β-galactosidase staining,cell cycle was observed by flow cytometry,transcriptional levels of related genes were verified by quantitative real-time polymerase chain reaction(RT-PCR),mitochondrial membrane potential was determined by JC-1 kit,the level of intracellular reactive oxygen species(ROS)was detected by DHE fluorescence-probe,and the level of intracellular adenosine triphosphate(ATP)was demonstrated by ATP detection kit.Results:1.CdCl2 can inhibit the vitality and proliferation of annulus fibrosus cells and promote cell senescence in a time-dependent and-concentration-dependent manner.2.The mRNA transcriptome sequencing and bioinformatics analysis of Kyoto Encyclopedia of Gene and Genome show that the main signal transduction pathways are MAPK signal pathway,cell cycle signal pathway,p53 signal pathway and so on,which are related to cell proliferation and senescence.Immunofluorescence and Western blot analysis showed that the gene expression of JNK pathway,an critical branch of MAPK signal pathway,was distinctly up-regulated in CdCl2 treated group compared with the control group.After the use of JNK signal pathway inhibitor SP600125,the positive rate of annulus fibrosus cells senescence,the proportion of JNK phosphorylation,the expression of p16,p21,p53 and senescenceassociated secretion phenotype(SASP)IL-1β and IL-6 were significantly down-regulated.This suggests that CdCl2 may promote annulus fibrosus cell senescence by activating JNK signal pathway.3.CdCl2 can increase the content of ROS and decrease the level of mitochondrial membrane potential and intracellular ATP in annulus fibrosus cell.It suggests that CdCl2 may participate in annulus fibrosus cell senescence through mitochondrial pathway.Conclusion:CdCl2 can inhibit the vitality and proliferation of annulus fibrosus cells and promote cell senescence.At the same time,Cd exposure can promote annulus fibrosus cell senescence by activating JNK signal pathway.In addition,mitochondrial dysfunction is also involved in annulus fibrosus cell senescence induced by Cd exposure.