The Mechanisms Of Nucleus Pulposus Matrix Sclerosis Weakening Glycolysis And Promoting Intervertebral Disc Degeneration By Activating MRTF-A

Objective: Intervertebral disc degeneration(IVDD)is a chronic and progressive process that leads to a range of pathological changes,such as nucleus pulposus(NP)tissue fibrosis,matrix degeneration,annulus rupture,and nerve compression.NP tissue is normally located in a closed and anoxic environment,and its energy source is mainly related to glycolysis.However,the energy metabolism disorder and dysfunction of NP cells attribute to various pathological factors are the key factors for the onset and progression of IVDD.Matrix stiffness signal can affect cell function and energy metabolism in various diseases.Therefore,NP tissue sclerosis may affect the energy metabolism of NP cells and further aggravate the progression of IVDD,forming a vicious cycle.This study was aimed to explore the potential relationship between NP tissue matrix sclerosis and cell energy metabolism,and to provide new ideas for the treatment of IVDD.Methods: In vitro experiments,NP cells were used as cell models.The effects of matrix stiffness on the cells were evaluated by culturing the cells in hydrogels of different stiffness.Subsequently,the metabolic and glycolysis function of NP cells were verified by a series of basic experiments after receiving myocardin-related transcription factors-A(MRTF-A)overexpressed adenovirus/inhibitor(CCG)treatment.The target proteins regulated by MRTF-A were analyzed by RNA sequencing of NP cells that cultured with different stiffness,and the regulatory relationship between MRTF-A and glycolysis-related proteins was verified.In vivo experiments,the animal IVDD model was established by acupuncture of the tail intervertebral disc(IVD)of rats.In the IVDD model,MRTF-A overexpressed adenovirus/CCG was injected into the tail IVD to explore the effect of MRTF-A on IVDD progression in vivo.The X-ray,micro computed tomography(Mirco-CT)and magnetic resonance imaging(MRI)were performed at four and eight weeks after surgery.At the endpoint,the corresponding IVD tissues were fixed and embedded for further histological staining.Results: Increased matrix stiffness led to metabolic dysfunction and decreased glycolysis function of NP cells.Overexpression of MRTF-A in NP cells will downregulate the expression of kinase D interacting substrate of 220 k Da(Kidins220)and inhibit the phosphorylation of adenosine 5’-monophosphate-activated protein kinase(AMPK),resulting in the degeneration of NP cells and the decline of glycolysis.On the contrary,inhibition of MRTF-A can up-regulate Kinins220 expression and promote AMPK phosphorylation,thus activating the glycolysis function of NP cells.In addition,injecting MRTF-A overexpressed adenovirus into the rat tail IVD induced disc degeneration and reduced glycolysis function,whereas CCG application alleviated IVDD caused by acupuncture and partially restored glycolysis function.Conclusions: Matrix sclerosis can affect the normal function and glycolysis of NP cells by regulating the MRTF-A/Kidins220/AMPK pathway,thus aggravating the progression of IVDD.Part Ⅰ: Nucleus Pulposus Matrix Sclerosis Promotes Intervertebral Disc Degeneration by Activating MRTF-AObjective: MRTF-A is a key factor that can respond to mechanical stress and stiffness(rigidity)signals,and can be activated into the nucleus to exert functions.With the progression of IVDD,the degree of NP tissue stiffness is increasing,resulting in a series of adverse consequences such as NP tissue prominence and annulus fibrosus rupture.However,the regulator relationship between stiffness and MRTF-A as well as the mechanism and effect of MRTF-A in the process of NP tissue stiffness remain to be explored.This part aims to explore the effect of NP matrix stiffness on MRTF-A factor and its role in the course of IVDD.Methods: In this part,NP cells were used as cell models.NP cells were cultured with hydrogels of different stiffness to evaluate the effect of stiffness on the cells.The metabolic function of NP cells was verified by a series of basic experiments after the intervention of MRTF-A overexpressed-adenovirus/CCG.In this part,in vivo experiments were divided into two parts: 1.The IVDD model was constructed by acupuncture of the rat tail IVD.CCG was injected into the surgery area in treatment group,and followed by weekly intraperitoneal injection of CCG.2.Control and MRTF-A overexpressed adenovirus were injected separately into the rat tail IVD.Radiographic assessments(X-ray,Mirco-CT,and MRI)were performed at the injective segment at week 4 and 8,and the corresponding disc was fixed and embedded for histologically related staining in the end.Results: Matrix sclerosis stimulated the upregulation of degenerative phenotypic protein expression and promoted the activation of MRTF-A in NP cells.The overexpression of MRTF-A contributed to the NP cells synthetic dysfunction and degeneration.On contrary,inhibition of MRTF-A activity in sclerotic matrix can restore partial anabolic function of NP cells.Moreover,disc injection of MRTF-A overexpressed adenovirus induces disc degeneration in rats,whereas IVDD can be effectively alleviated by the application of CCG after disc acupuncture.Conclusions: With the increase of NP matrix stiffness,MRTF-A can enter the nucleus and promote the degeneration of NP cells,thus aggravating the progression of IVDD.Moreover,MRTF-A overexpression in the IVD of rats can induce the development of IVDD,while inhibition of MRTF-A activity can alleviate the progression of intervertebral disc degeneration in the rat model.Part Ⅱ: Activation of MRTF-A Downregulates Glycolysis by Inhibiting Kidins220/AMPK PathwayObjective: AMPK is a classical pathway that regulates glucose uptake and glycolysis,which is a key process of energy uptake in NP cells.With the progression of IVDD,the decrease of glycolysis function caused by various factors is one of the main causes of degeneration and dysfunction of NP cells.In a variety of diseases,the stiffness of the extracellular matrix(ECM)can affect cell function and metabolism,but the effect of NP tissue sclerosis on the glycolysis process of IVDD progression is still not explored.The purpose of this part is to explore the potential correlation between NP matrix stiffness and decreased glycolysis function and the specific mechanism of their interaction.Methods: Firstly,NP cells under different stiffness were collected for RNA sequencing,and a list of related proteins that could catalyse downstream protein phosphorylation was screened out through functional analysis.The target proteins regulated by MRTF-A were verified by PCR after MRTF-A overexpressed adenovirus/CCG treatment.To explore changes in AMPK pathway,and expression of downstream glycolytic enzymes such as phosphofructokinase-1(PFKM),6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3)and phospholipase D1(PLD1),different combinations of intervention(MRTF-A overexpressed adenovirus/inhibitor,Kidins220 overexpressed/knockdown adenovirus,and AMPK agonist/inhibitor)were used to stimuli NP cells.The above pathway relationships were also verified by Co-Immunoprecipitation(Co-IP).The IVD sections of the first part were extracted for further immunohistochemistry(IHC)assay(Kidins220,PFKM,PFKFB3 and PLD1)and analysis.Results: The increase of matrix stiffness caused 3954 differential genes in NP cells.Through functional selection and PCR verification,it was found that Kidins220 gene expression was regulated by MRTF-A.Moreover,the binding and regulatory relationship of Kidins220 with AMPK was verified by Co-IP and Kidins220 positive/negative regulation.CCG,Kidins220 overexpressed adenovirus and AMPK agonist can improve the glycolysis function of NP cells,whereas MRTF-A overexpressed adenovirus,Kidins220 knockout adenovirus and AMPK inhibitor reduce glycolysis.In vivo,IVDD model induced by acupuncture of the tail IVD or single injection of MRTF-A overexpressed adenovirus can decrease Kidins220,PFKM,PFKFB3,and PLD1 expression,whereas CCG injection can reverse this decreased glycolysis function.Conclusions: Matrix sclerosis activates MRTF-A,downregulates Kidins220 expression and inhibits AMPK activation,thereby reducing the glucose uptake and glycolysis function of NP cells.In addition,MRTF-A overexpression in the IVD resulted in decreased glycolysis function,while inhibition of MRTF-A activity partially restored glycolysis function in the IVDD model.