Autophagy Inhibits Inflammation-Mediated Endplate Degeneration By Enhancing Nrf2/Keap1 Signalling Of Cartilage Endplate Stem Cells

Background:Cartilage endplate(CEP)degeneration inhibits the transport of metabolites and nutrients in the intervertebral disc and is an important initiating factor of intervertebral disc degeneration(IDD).Early degeneration of CEP,which is referred to as Modic type ? change,involves the microfracture of CEP and subchondral bone with adjacent bone marrow edema.Under the stimulation of inflammatory and physiochemical factors,CEP degeneration is further aggravated and eventually progresses to Modic type ?,which involves the sclerosis of CEP and subchondral bone.Calcified CEP impedes the transport of nutrients and metabolic wastes,ultimately cause or aggravate the degeneration of the IVD.CEP degeneration leads to aging of cartilage endplate stem cells(CESCs),loss of cell phenotype and differentiation function,degradation of the extracellular matrix,which finally resulting in degeneration of the CEP.However,the mechanisms that regulate inflammation-induced CEP degeneration remain unclear.Autophagy insufficiency is considered to play an important role in the development of IDD.Inflammatory factors such as tumor necrosis factor alpha(TNF-?)and interleukin-1 beta(IL1-?)are highly expressed in the CEP of Modic changes.The nuclear factor-erythroid-2-related factor 2(Nrf2)/Kelch-like ECH-associated protein 1(Keap1)pathway plays a central role in cellular resistance to oxidative stress by controlling the expression of antioxidant proteins.In the presence of reactive oxygen species(ROS)or inflammatory factors,the Nrf2/Keap1 complex is disrupted,and newly synthesized Nrf2 translocates to the nucleus and activates the transcription of antioxidant proteins.During aging,both Nrf2 and basal autophagy levels decrease,resulting in increased vulnerability of CESCs to inflammatory and physiochemical factors.To investigate the role of autophagy and Nrf2/Keap1 signalling and their relationship in the degeneration of CEP are beneficial for the prevention of disease and for the development of clinical therapies.Methods:Part 1:Establishment of mouse sub-endplate fracture model and investigate the role of autophagy in the degeneration of CEP.Fractures were made at a point 1 mm below the endplates of the caudal discs,later,chloroquine(CQ)and rapamycin(Rap)were injected intraperitoneally every 3 days for 30 days.The caudal vertebrae were separated,and sliced for histology and immunohistochemistry analyses.Part 2:Investigate the role and mechanism of autophagy in the TNF-? induced CESCs damage.CESCs were isolated from mouse CEP and characterized by flow cytometry and osteogenic,adipogenic and chondrogenic differentiation.CESCs were treated with inflammatory cytokine TNF-?,and autophagy was inhibited by chloroquine or activated by rapamycin.The cell senescence was measured by Senescence-associated ?-galactosidase(SA-?-gal)staining,and oxidative stress was detected by flow cytometry and Western-Blot.Expression of matrix metallopeptidase was tested by immunohistochemical staining,PCR and Western-Blot.Differentiation potential alteration was investigated by PCR,Western-Blot and differentiation induction.Part 3:Investigate the mechanism of autophagy in enhancing Nrf2/Keap1 signalling in CESCs and inhibits inflammation-mediated CEP degeneration.The expression of Nrf2 after treated with TNF-? and autophagy inhibitor chloroquine or activator rapamycin was determined by immunohistochemical staining,immunofluorescence staining,PCR and Western-Blot.To investigate the relationship of autophagy and Nrf2/ Keap1 signalling pathway,Nrf2 was over-expressed by using lentivirus.Results:Part 1:1.The expression of TNF-? increased while LC3 decreased after sub-endplate fracture on mouse caudal vertebral.2.Autophagy inhibited by CQ aggravates CEP degeneration after sub-endplate fracture,while activated by Rap protects CEP.Part 2:1.CESCs isolated from mouse CEP exhibits MSC-like characteristics.2.TNF-? activate autophagy of CESCs,and it could be inhibited by CQ or activated by Rap.3.After treated with TNF-?,oxidative stress and cell senescence of CESCs increased;inhibition of autophagy aggravated these effects,while activation of autophagy alleviated them.4.After treated with TNF-?,the expression of matrix metallopeptidases MMP13 and ADAMTS5 of CESCs increased;inhibition of autophagy further promoted their expression while activation of autophagy decreased them.5.After treated with TNF-?,the expression of osteogenic genes(Runx2 and Col?)and osteogenic differentiation increased,and the expression of chondrogenic genes(Sox9 and Col?)and chondrogenic differentiation decreased;inhibition of autophagy further aggravated these effects while activation of autophagy alleviated them.Part 3:1.Autophagy activation promoted the expression of Nrf2 and inhibited the expression of Keap1 of CESCs after treated with TNF-?,whereas when autophagy was inhibited,the effect was reversed.2.Autophagy activation promoted the nuclear translocation of Nrf2,thus enhanced the expression of down-stream antioxidant proteins expression.3.Nrf2 over-expression protected CESCs from TNF-? induced damge and was regulated by autophagy.Conclusion:On the basis of successful construction of a CEP degeneration mouse model,we found that autophagy could protect CESCs from chronic inflammation.Its effects were achieved by promoting antioxidant protein expression,scavenging ROS,reducing cell senescence and maintaining the differentiation potential of CESCs.There is a close relationship between autophagy and Nrf2/Keap1 signalling pathway.Inhibition or activation of autophagy affects Nrf2 expression and nuclear translocation,and then mediate inflammation induced CEP degeneration by regulating the expression of antioxidant proteins.