Study Of The Mechanism That CISD2 Affects Intervertebral Disc Degeneration By Regulating The Level Of Autophagy In Nucleus Pulposus Cells

Objective:When the intervertebral disc is overly damaged by continuous mechanical stress,trauma,and reactive oxygen species(ROS),it is highly likely to cause the occurrence of excessive autophagy of cells within the disc,leading to abnormal death of nucleus pulposus cells and reduction of extracellular matrix(ECM),which eventually triggers intervertebral disc degeneration(IVDD).In this study,we observed the cellular and tissue changes in the degenerated discs and investigated the role of CISD2,a negative regulator of autophagy,on IVDD.Methods:(1)The nucleus pulposus tissues of patients with different degrees of intervertebral disc degeneration were selected,and the changes in the location and content of CISD2,the changes in the content of proteoglycan in the nucleus pulposus tissues of each group were detected by immunohistochemistry,and the changes in the content of cartilage components in the nucleus pulposus tissues of each group were detected by the method of Arsenic Blue staining.(2)vitro part of animal experiment:Ten young C57 mice were selected to extract the nucleus pulposus tissue o f the rat caudal intervertebral disc under a microscope,and the primary nucleus pulposus cells of the intervertebral disc were obtained after treatment with collagenase,and the cells were passaged and cultured to the second generation before performing the following experiments.first,the cells were first divided into three groups in the first part of the experiment: Normal group(N group),IL-1 group(IL-1group),curcumin intervention group(IL-1 + C group).After each group of cells was treated with different conditions,the changes of CISD2 expression and the changes of secretory factors,matrix metalloproteinase 3(MMP3)and proteoglycan(Aggrecan),related to disc degeneration,were detected by Western blot(WB)and cell crawling in each group of cells.In the second part of the experiment,cells were divided into 2 groups:experimental group(CISD2-Si RNA+IL-1+C group)and control group(Con-Si RNA+IL-1+C group),and after knocking down the CISD2 gene with small interference,we use WB to detected these molecules above again to verify the retarding effect of curcumin on intervertebral disc cell degeneration by agonizing CISD2 production.(3)vivo part of animal experiments: a mouse rat tail model of intervertebral disc degeneration was constructed,and the animals were divided into normal group(NC group),simple acupuncture group(experimental group),and CISD2 agonist(curcumin)group,the change of intervertebral disc height and signal in each group were detected by imaging performance to verify the effect of CISD2 on intervertebral disc degeneration.Results:(1)According to the immunohistochemical staining,CISD2 was mainly expressed in the nucleus pulposus of human intervertebral disc tissues,and the expression of CISD2 decreased with the increasing degeneration of human intervertebral disc tissues(grade III,IV,and V),which proved the existence and significance of CISD2 in the nucleus pulposus of human intervertebral discs.(2)Using WB,and cell crawl,we detected a significant decrease in the expression of CISD2 protein in mice with degenerated nucleus pulposus cells(IL-1 group)compared to the normal group(N group),accompanied by an increase in the expression of inflammatory factors associated with disc cell degeneration(e.g.,MMP3,MMP13)and a decrease in the expression of secreted factors in normal cells and important molecules in the matrix(e.g.,Aggrecan,etc.),it indicates that CISD2 expression is reduced after in vitro-induced disc cell degeneration in mice,and it can be used as one of the markers of disc degeneration;Using WB,cell crawling we also found that degenerating medullary cells treated with curcumin(IL-1+C group)resulted in increased expression of CISD2 accompanied by a significant decrease in expression of inflammatory factors,an increase in expression of normal components and an improvement in intervertebral disc degeneration;In addition,with WB we also found that the degeneration of intervertebral discs did not significantly improve after Si-CISD2 treatment of degenerated nucleus pulposus cells even with curcumin(IL-1+C+Si-CISD2).These results demonstrate that curcumin can increase the expression of CISD2 in intervertebral disc cells,and CISD2 as a regulator can affect the process of degeneration of intervertebral disc cells in mice.(3)Construction of an intervertebral disc degeneration model in mice: C urcumin was injected into the intervertebral disc after acupuncture,and after 2 weeks,we found that the intervertebral disc height was significantly reduced in the acupuncture group compared with the normal group,while the reduction in intervertebral disc height was significantly reduced after curcumin injection;we also found that the intervertebral disc injected with curcumin had higher signal,more water content in the nucleus pulposus and less degeneration than the acupuncture group,so curcumin could slow down the degeneration of the intervertebral disc.Conclusion:CISD2 content in degenerated disc tissue will decrease,it correlates with the degree of degeneration.the progression of disc degeneration can be influenced by regulating CISD2 content,this ability may be achieved by regulating autophagy in cell.