The Mechanism Of Optineurin Alleviating The Degeneration Of Intervertebral Disc Induced By Oxidative Stress By Activating Mitophagy

Background and objectiveWith the aging global population,the prevalence of age-related degenerative diseases is increasing.Lower back pain（LBP）,mainly caused by intervertebral disc degeneration（IVDD）,has become a global health problem that severely affects people’s quality of life,causes disability,and imposes a significant socioeconomic burden.senescence of nucleus pulpous cells（NPCs）and degradation of the extracellular matrix are important molecular mechanisms of IVDD.With the aging and degeneration of intervertebral discs,aging NPCs are accumulated continuously,and the level of matrix anabolism related proteins decreases,leading to the gradual loss of the ability of NP to maintain matrix homeostasis and self-renewal.Oxidative stress is a pathological factor associated with a variety of degenerative diseases,such as glaucoma,osteoarthritis,and neurodegenerative diseases.In normal intervertebral discs,reactive oxygen species（ROS）participate in physiological processes as signal molecules,but in degenerated human intervertebral discs,abnormally high ROS levels can lead to intervertebral disc lesions.Therefore,inhibiting oxidative stress in NPCs and restoring redox homeostasis may be an effective measure to slow down IVDD.Mitochondria are not only the most important source of energy in the cell,but also a major source of intracellular ROS.Mitochondrial dysfunction causes excessive ROS production,which in turn attacks and damages the mitochondria themselves,leading to further production of mitochondrial ROS（mt ROS）,creating a vicious cycle that leads to cellular dysfunction.As a selective form of autophagy,mitophagy maintains the dynamic homeostasis of the mitochondrial network by eliminating damaged or abnormal mitochondria.Insufficient mitophagy will lead to the failure of timely clearance of damaged mitochondria in cells,thus increasing the production of mt ROS.Abnormal mitophagy is associated with a variety of diseases,including Parkinson’s disease,heart disease and osteoporosis.Mitophagy has recently been shown to play an important role in slowing the progression of IVDD.Several studies have demonstrated that mitophagy is deficient in degenerating discs and that reactive oxygen species are elevated in degenerating discs causing oxidative stress damage to NPCs in the disc.Due to the degeneration of intervertebral disc and the increase of reactive oxygen species,mitochondria in NPCs are damaged by oxidative stress,resulting in the activation of PINK1-Parkin mitophagy pathway,which enables cells to clear damaged mitochondria to fight oxidative stress damage.Considering that oxidative stress and mitophagy are related to the pathogenesis of IVDD,enhancing mitophagy may have beneficial effects on IVDD and delay its progression.Optineurin（OPTN）is a highly conserved protein that plays an important role in vesicle trafficking and NF-k B signal transduction.Importantly,OPTN was identified as a mitophagy receptor and found to contain a ubiquitin binding domain that binds polyubiquitinated substrates and transports them to autophagosomes,and finally to lysosomes through the LC3interaction domain.However,little is known about the role of OPTN in IVDD.We infer that OPTN and basal mitophagy levels in NPCs decrease during intervertebral disc degeneration,resulting in their vulnerability to oxidative stress and reduced ability to aging and matrix synthesis.Investigating the role of OPTN and mitophagy in disc degeneration may help to improve our understanding the mechanisms of disc degeneration and provide new directions for its prevention.MethodsPart1:collection and detection of human and rat intervertebral disc tissues and establishment of oxidative stress-induced aging model of NPCs.1.Collection and detection of specimens:according to the different Pfirrmann grades,human intervertebral disc nucleus pulposus（NP）tissues with grade II-V degeneration and rat caudal intervertebral disc nucleus pulpous at the age of 1 and 20 months were collected.After lysis,the protein was collected for WB test to detect the expression of OPTN.2.Establishment of oxidative stress injury model of nucleus pulposus cells and detection of OPTN expression:NPCs were extracted from the nucleus pulpous tissue of 2-month-old rat caudal intervertebral disc according to the previous method of our group,and different concentrations of hydrogen peroxide were used to induce the senescence of nucleus pulposus cells.The proliferation activity of cells was detected using CCK-8 kit.Through senescence relatedβGalactosidase（SA-β-Gal）stanning to detect cell senescence.The expression of senescence related proteins,matrix related proteins and OPTN were detected by Western blot.3.Establishment of rat model of intervertebral disc degeneration and detection of the expression of OPTN in sections by immunofluorescence staining.Part 2:Study on the role of OPTN in oxidative stress injury of nucleus pulposus cells1.According to the sequence characteristics of OPTN,lentivirus was constructed to observe its role in oxidative stress injury of NPCs through overexpression and knockdown of OPTN expression in NPCs.The expression of senescence-associated proteins and matrix-associated proteins was detected using Western-Blot;senescence associatedβ-galactosidase（SA-β-gal）was used to detect cellular senescence.2.Eight-week-old SD rats were anesthetized with 2%（w/v）pentobarbital sodium（40mg/kg）,and the caudal disc（Co4/5）was positioned and marked by palpation.After sterilization,the puncture needle（27g）was vertically passed through the back skin into the annulus fibrosus（AF）,with a depth of 4 mm,rotated 360°and stayed in the intervertebral disc for 1 minute.Empty or overexpressing viruses were injected according to different groups.The changes of intervertebral disc signal were observed by magnetic resonance.X-ray was used to detect the change of intervertebral space height.He and safranin fast green staining were used to observe the histological changes of intervertebral discs and to score the histological changes.Part 3:Mechanism of OPTN alleviating oxidative stress injury of NPCs through mitophagy.1.NPCs were treated with different combinations by using Cs A,an inhibitor of mitophagy,CCCP,an agonist,and Mito TEMPO,a mitochondrial ROS（mt ROS）scavenger.The expression of senescence related proteins and matrix related proteins were detected by Western blot;senescence associatedβ-galactosidase（SA-β-gal）was used to detect cell senescence.Mitosox staining was used to observe the production level of mt ROS in cells.The level of intracellular mitophagy was observed by using the lentivirus transfected mitolysosome tagged virus m Cherry-Egfp-Fis1.2.NPCs were treated with different combinations by using mitophagy inhibitor CSA,mitochondrial ROS（mt ROS）scavenger Mito TEMPO and OPTN overexpressing virus.The expression of senescence related proteins and matrix related proteins were detected by Western blot.Senescence associatedβ-galactosidase（SA-β-gal）was used to detect cell senescence.Mitosox staining was used to observe the production level of mt ROS in cells.The level of intracellular mitophagy was observed by using the lentivirus transfected mitolysosome tagged virus m Cherry-e GFP-FIS1.ResultsPart 1.1.The expression level of OPTN in human and rat intervertebral discs decreased with the aggravation of degeneration.2.After hydrogen peroxide treatment,reactive oxygen species in rat NPCs increased,and the proportion of aging cells increased.The expression OPTN was elevated.Mitophagy was elevated.3.The animal model confirmed that the expression of OPTN in degenerated rat intervertebral discs was reduced.Part 2.1.Overexpression of OPTN significantly inhibited oxidative stress-induced senescence in NPCs,promoted matrix synthesis,and inhibited matrix degradation.knockdown of OPTN promoted senescence,inhibited matrix synthesis,and promoted matrix degradation in NPCs.2.In vivo experiments confirmed that the overexpression of OPTN virus group significantly increased the signal intensity of T2 image of intervertebral disc magnetic resonance compared with the empty virus group.X-ray examination showed that the height of intervertebral space also increased.He and safranin fast green staining showed that the histological score of the intervertebral disc was significantly better than that of the empty vector group.Part 3.1.After activation of mitophagy,mitophagy in NPCs increased,senescent cells decreased,matrix synthesis increased,and degradation decreased.After inhibiting and activating mitophagy,mitophagy in NPCs decreased,aging cells increased,matrix synthesis decreased,and degradation increased.2.After activating mitophagy,the mitochondrial ROS in cells was significantly lower than that in hydrogen peroxide treatment,while after inhibiting mitophagy,the mitochondrial ROS in cells was significantly higher than that in hydrogen peroxide treatment.3.After Mito TEMPO was added,compared with the hydrogen peroxide+Cs A group,the senescence of NPCs was significantly reduced,matrix synthesis increased,and matrix degradation decreased.4.After overexpression of OPTN,mitolysosomes increased significantly in hydrogen peroxide treated cells,but decreased significantly after Cs A was added.5.After overexpression of OPTN,the mitochondrial ROS in NPCs was significantly decreased compared with the hydrogen peroxide group and the hydrogen peroxide+empty vector group.However,after Cs A was added,mitochondrial ROS increased significantly,and after Mito TEMPO was added,mt ROS decreased significantly.ConclusionOur study shows that OPTN is differentially expressed in normal and degenerated NP tissue in human and rats,and in H₂O₂-treated rat NPC.Further studies suggest that OPTN can resist oxidative stress-induced myeloid senescence and matrix degradation,and that OPTN inhibits cellular senescence and restores matrix homeostasis by enhancing mitochondrial autophagy to remove excess mt ROS caused by oxidative stress injury,thereby alleviating oxidative stress-induced disc degeneration.