Damage To Human Lumbar Cartilage Endplate And The Protective Effect Of Nrf2 Signaling Pathway On Cartilage Endplate Degeneration

Part 1: Damage to the human lumbar cartilage endplate and its clinical implicationsObjective: The cartilage endplate(CEP)is a thin layer of hyaline cartilage,and its degeneration or damage/defect can accelerate or result in intervertebral disc degeneration.However,the structural features of damaged CEPs have not been well characterized,and this hinders our understanding of the etiology of disc degeneration and pain.We present the structural features of micro-damaged cartilage endplates in the patients with disc degeneration and low back pain(LBP)that might be even regarded as an initial factor for disc degeneration.Material and methods: Human lumbar CEPs were excised from 35 patients(mean age 60.91 yrs)who had disc degeneration and LBP.Control tissue was obtained from 15 patients(mean age 54.67 yrs)with lumbar vertebral burst fractures.LBP and disability were assessed clinically,and all patients underwent anterior vertebral body fusion surgery.CEPs,together with some adjacent nucleus pulposus(NP),were sectioned at 4μm,and stained using H&E,Safranin O/Fast Green,and Alcian Blue.Immunostaining and PCR were used to identify various markers of degeneration,innervation,and inflammation.Results: Histology demonstrated physical micro-damage in 14/35 CEPs from the disc degeneration group.Six major types of damage could be distinguished: fissure,node,vascular mimicry,inclusion of NP tissue within the CEP,inclusion of bone,and inclusion of NP and bone.Pain and disability scores were significantly higher in those with microdamaged CEPs(n=14)than in those with non-damaged CEPs(n=21)(ODI: p=0.0190;JOA: p=0.0205;JOABPEQ: p=0.0034).CEP damage was significantly associated with elevated MMP3(p=0.043),MMP13(p=0.0191),ADAMTS5(p=0.0253),TNF-α(p=0.0011)and Substance P(p=0.0028),and with reduced Sox9(p=0.0212),aggrecan(p=0.0127)and type II collagen(p=0.0139).Conclusion: We present a new classification of human lumbar micro-damaged CEPs.Further,we verify disc degeneration,innervation and discogenic pain in micro-damaged CEPs.Part 2: The expression of Nrf2 in cartilage endplate and its association with cartilage endplate degenerationObjective: CEP degeneration is the main early manifestation of intervertebral disc degeneration(IVDD)and is closely related to oxidative stress and inflammation.Nrf2(nuclear factor E2-related factor 2,NFE2L2)is an important transcription factor for cellular antioxidant and anti-inflammatory response,but the role of Nrf2 in CEP degeneration is still unclear.This part aims to preliminarily clarify the protective effect of Nrf2 signaling pathway on cartilage endplate cells.Material and methods: CEP specimens from the patients with the lumbar spine burst fracture were collected as a control group.These specimens showed no obvious degeneration on MRI images,and received anterior vertebral resection and fusion((n=6,male/female=3/3,age=53.84±10.51 years(27-64 years),Pfirrmann grade I or II).We collected the degenerative CEP specimens from LBP patients as degenerative group((n=21,male/female=9/12,age=58.96±8.43 years(38-79 years),Pfirrmann III,IV or V).The samples were fixed in 4% paraformaldehyde and immersed in 12.5% EDTA for decalcification for 1-2 weeks.After the tissues were were dehydrated with a graded series of ethanol,embedded in paraffin,and then sliced into 4μm sections.And H&E staining,Safranin O/Fast green and Alcian Blue staining were conducted.RT-q PCR and immunostaining(MMP3,MMP13,type Ⅱ collagen,Sox9,aggrecan,Nrf2)are used to identify cartilage endplate degeneration and Nrf2 protein level.In addition,the primary rat cartilage endplate cells were extracted for morphological identification;Nrf2 was overexpressed to detect the expression level of anabolic and catabolic genes of cartilage endplate cells with or without the stimulation of pro-inflammatory factor LPS.Results: Human CEP samples were divided into four groups: control group(Pfirrmann grade I/II,n=6),mild degenerative group(Pfirrmann grade III,n=7),moderate degenerative group(Pfirrmann grade IV,n=8),severe degenerative group(Pfirrmann grade V,n=6).There was no significant statistical difference in the age among the four groups.RT-q PCR results showed that the anabolism-related genes Sox9,aggrecan and type Ⅱ collagen were negatively correlated with the degree of CEP degeneration,while the catabolic genes MMP3 and MMP13 were positively correlated with the degree of CEP degeneration.The results of immunohistochemistry and immunofluorescence showed that there were significantly fewer type II collagen-positive cells in the severely degenerated CEP tissue,and the expression of Nrf2 in the severely degenerated CEP tissue was also significantly reduced,compared with the control group.In addition,Western blotting confirmed that severely degenerated CEP had very little Nrf2 protein.In vitro results showed that overexpression of Nrf2 significantly activated its downstream signaling pathway(Nqo1),and also significantly inhibited LPS-induced MMP13 expression,and promoted the anabolic gene Sox9 expression in the cartilage endplate cells.Conclusion: The level of Nrf2 protein is significantly negatively correlated with the degree of cartilage endplate degeneration,and the protective effect of Nrf2 signaling pathway on cartilage endplate cells is initially verified.Part 3: Activation of Nrf2 signaling by 4-octyl itaconate attenuates the cartilage endplate degenerationObjective: Nrf2(Nuclear Factor E2-related Factor 2,NFE2L2)is an important transcription factor for cellular antioxidant and anti-inflammatory responses.4-octyl itaconate(4OI)can stabilize Nrf2 protein and avoid its degradation,but the role of 4OI in intervertebral disc degeneration is still unclear.This part aims to clarify whether 4OI activating Nrf2 signaling pathway can delay the degeneration of the intervertebral disc by inhibiting macrophage-related inflammation and cartilage endplate catabolism.Material and methods: In vitro,rat cartilage endplate cells were stimulated by the proinflammatory factor lipopolysaccharide(LPS),and then treated with 4OI to detect the levels of ROS,and anabolic and catabolic-related genes(SOX9/Type Ⅱ collagen/Aggrecan,ADAMTS5 /MMP3/MMP13).Through the co-culture experiment of macrophages and cartilage endplate cells,the effect of 4OI on the degeneration of cartilage endplate cells induced by LPS and macrophage-related inflammation was detected.By overexpression and knockdown experiments,the anti-oxidation and anticatabolism effects of Nrf2 on the cartilage endplate cells with LPS were verified;Using the Co-IP and Western blotting experiments,it was analyzed whether Nrf2 ubiquitination and degradation depend on ubiquitinated ligase ZNF598.In vitro and in vivo experiments(rat tail disc puncture model)were to explore the role of 4OI in the intervertebral disc degeneration(IVDD).Results: In vitro experiments showed that 4OI can inhibit the ROS accumulation and catabolism in rat cartilage endplate cells induced by the pro-inflammatory factor LPS.Immunofluorescence results showed that 4OI can significantly inhibit the catabolism of cartilage endplate cells and macrophage-related inflammation.Mechanically,4OI activates the Nrf2 signaling pathway to inhibit the secretion of inflammatory factors(IL-1β)from macrophages induced by LPS,thereby inhibiting the catabolism of inflammation-related cartilage endplate cells.It is further clarified that 4OI inhibits the ZNF598-mediated ubiquitination of Nrf2 in rat cartilage endplate cells.For the rat tail disc degeneration model,we found 4OI significantly improved the progression of intervertebral disc degeneration through imaging(MRI)and histology(Safranin O/Fast Green,Alcian Blue,Sirius Red staining)analysis.Conclusion: 4OI may alleviate the intervertebral disc degeneration through suppressing cartilage endplate degeneration and macrophage-associated inflammation.4OI can be regarded as an alternative therapy for cartilage endplate/intervertebral disc degeneration.