A Study On The Effect And Mechanism Of Sonic Hedgehog/Gli1 Signaling Cascade On Oxidative Stress Of Nucleus Pulposus Cells And Corresponding Intervertebral Disc Degeneration

Background and objectives:Chronic low back pain(CLBP)is a leading cause of disability and labor loss and affects approximately 700 million people worldwide,which causes a serious reduction in quality of life and leads to significant medical costs.The etiology of CLBP is complex and includes biological,psychological and social factors.Intervertebral disc degeneration(IDD)is the main cause of CLBP.It is important to understand that IDD is influenced by age,gender,genetics,mechanical factors,and environmental factors.Nucleus pulposus(NP)cells are important functional cells to maintain the homeostasis of intervertebral disc(IVDs),and decreased numbers of NP cells leads to the disturbance of IVD homeostasis,thus driving the pathological process of IDD.Oxidative stress inhibits extracellular matrix synthesis in NP cells,and enhances non-physiological death of NP cells,leading to disruption of IVD homeostasis.To elucidate the regulation mechanism of oxidative stress in NP cells and IVD homeostasis maintenance will contribute the development of new treatments for preventing and retarding IDD.Sonic Hedgehog/Glioma-associated oncogene1(Shh/Gli1)signaling cascade plays an important role in IVD development.And the activate or close of Shh/Gli1 signaling cascade in different time and space with each other to regulate the development,growth and maturation of IVDs.Recently,several studies have reported that the Shh/Gli1 signaling cascade promotes the transcription and activation of antioxidant genes to exert antioxidant effects.However,the role of Shh/Gli1 signaling cascade in oxidative stress of NP cells and maintenance of IVD homeostasis remains unclear.Glutathione peroxidases 4(GPX4)provides a specific target of significance for new pharmacological,precision,and other therapeutic approaches.As the main cellular defense system against oxidative stress injury,GPX4 plays a key role in the prevention and treatment of diseases characterized by oxidative stress injury.Recent studies suggest that enhanced GPX4 methylation induces oxidative stress and ferroptosis of NP cells in IDD.However,there are still lack of the regulation mechanism of GPX4 at the transcription level of NP cells and its effect on oxidative stress and corresponding IDD.The objective of this study was to determine the role of Shh/Gli1 signaling cascades in the regulation of IDD.In addition,we will explore the regulatory role and mechanism of Shh/Gli1 signaling cascade on oxidative stress in NP cells,and whether GPX4 mediates the regulation of Shh/Gli1 signaling cascade on oxidative stress in NP cells.This project aims to observe the spatial and temporal distribution and dynamic changes of Shh,Gli1 and GPX4 in IVDs.The multi-scale detection technique was used to systematically study the role of Shh/Gli1 signaling cascade in regulating oxidative stress in NP cells and corresponding IDD from the whole to the cellular level,and clarify the role of GPX4 in the regulation process.Materials and Methods:Part 1: Establishment of IDD model induced by endogenous and exogenous factors.1.IVD tissues of aging related IDD model were obtained from human and rats of different ages,and percutaneous IVD puncture was performed to create a IDD model induced by exogenous factors.2.Magnetic resonance imaging(MRI),paraffin-embedded section hematoxylin-eosin(H&E)staining and safranin O-Fast Green staining were used to study the morphology of IVDs and NP in IDD models.3.Immunofluorescence staining and western blot were used to detect the expression of ACAN,collagen Ⅱ and MMP13 in IDD models.4.The enrichment of differentially expressed genes(DEGs)and functional pathways in NP tissues of rats of different ages was detected by transcriptome sequencing.Part 2: Expression of Shh/Gli1 signaling cascade and oxidative stress in IDD.1.Immunofluorescence staining and western blot were used to study the trend of Shh/Gli1 signaling cascades during aging related IDD in humans and rats.2.Lipofuscin staining in paraffin-embedded sections,lipid oxidation,total SOD activity,GPX activity,western blot and apoptosis kit were used to study the expression of oxidative stress in aging related IDD in rats.3.Extract,isolate,culture and identify rat NP cells.Part 3: To explore the regulation effect of Shh/Gli1 signaling cascade on oxidative stress and corresponding IDD.1.The distribution of fluorescence tracer compound indocyanine green in rats after intraperitoneal injection was observed with small animal imaging apparatus.2.Intraperitoneal injection of recombinant Shh protein(r Shh)to active the Shh/Gli1 signaling cascade,while intraperitoneal injection of GANT61 to suppress the Shh/Gli1 signaling cascade.3.MRI,paraffin-embedded H&E staining and safranin O-Fast Green staining were used to study the morphology of IVDs and NP in different groups in vivo.4.Lipofuscin staining in paraffin-embedded sections,lipid oxidation,total SOD activity and GPX activity were used to study the expression of oxidative stress in different groups in vivo.5.Western blot was used to study the expression of ACAN,collagen Ⅱ,MMP13,4HNE,GPX4 and SOD2 in different groups in vivo.Part 4: The regulation and mechanism of Shh/Gli1 signaling cascade on oxidative stress in NP cells.1.The expression of Shh/Gli1 signaling cascade in NP cells was regulated by lentivirus(sh-Gli1,Lv-Gli1)and inhibitors(GANT61,r Shh).2.Morphological changes of NP cells under different treatment were observed by transmission electron microscopy.3.Lipid oxidation,total SOD activity,GPX activity,DCFH-DA probe,cell apoptosis and mitochondrial membrane potential were used to investigate the expression of oxidative stress in different groups in vitro.4.Transcriptome sequencing was used to detect the enrichment of DEGs and functional pathways in NP cells that inhibited/activated Shh/Gli1 signaling pathway and the control group.Part 5: To explore the role and mechanism of GPX4 in mediating Shh/Gli1 signaling cascade.1.Immunofluorescence staining and western blot were used to study the trend of GPX4 in aging related IDD in humans and rats.2.Jaspar database(https://jaspar.genereg.net/)was used to predict the binding site of Gli1 in the GPX4 promoter region,chromatin immunocoprecipitation method(Ch IP-q PCR)was used to verify the four specific fragments found in the GPX4 promoter region as primer sequences.3.Si-GPX4 suppressed GPX4 in rat NP cells,and the role of GPX4 in mediating Shh/Gli1 signaling cascade was observed based on TBHP-induced oxidative stress model in NP cells.Results:Part 1: Endogenous and exogenous IDD models were established successfully.1.IDD in rats and humans was increased with age.Percutaneous IVD puncture significantly induced IDD in rats.2.With IDD,The MRI-T2 signal intensity,area of the NP and height of the intervertebral space decreased.The vacuolated and reticular-shaped NP cells were transformed into chondrocyte-like nested NP cells.The number of NP cells gradually decreased.The number of cracks between cells increased,and the cells were surrounded by more extracellular matrix.3.The expressions of ACAN and collagen Ⅱ were decreased,while the expressions of MMP13 were increased in IDD.4.Transcriptome analysis of rats NP tissues of different ages indicated that DEGs were mainly concentrated in biological processes and signaling pathways closely related to IVD homeostasis and cell senescence regulation.Part 2: In the process of IDD,Shh/Gli1 signaling cascades decreased gradually and oxidative stress increased gradually.1.The expressions of Shh/Gli1 signaling cascade decreased gradually in aging related IDD in rats and human.2.The expressions of oxidative stress increased gradually in aging related IDD in rats and human.3.Primary NP cells were obtained and identified to be consistent with the expression characteristics of NP cell markers.Part 3: Shh/Gli1 signaling cascade regulates oxidative stress of IVDs and affects the occurrence and progression of IDD.1.Indocyanine green distribution in rat IVDs was detected by imaging system,suggesting that intraperitoneal injection has a direct effect on IVDs.2.The activation of Shh/Gli1 signaling cascade by r Shh effectively relieved the progression of IDD,while GANT61 inhibits Shh/Gli1 signaling cascade caused IDD and accelerated the progression of IDD.3.Shh/Gli1 signaling cascade activation effectively relieved the oxidative stress of IVDs,and Shh/Gli1 signaling cascade inhibition significantly increased oxidative stress of IVDs.Part 4: Shh/Gli1 signaling cascade regulates oxidative stress in NP cells.1.Shh/Gli1 signaling cascade inhibition significantly increased oxidative stress in NP cells.2.Activation of Shh/Gli1 signaling cascade reduced TBHP-induced oxidative stress in NP cells.3.DEGs analysis of m RNA-seq showed that the Shh/Gli1 signaling cascade regulated GPX4 expression.Part 5: GPX4 mediates Shh/Gli1 signaling cascade to regulate oxidative stress in NP cells.1.The expressions of GPX4 decreased gradually in aging related IDD in rats and human.2.Gli1 bound to the GPX4 promoter region and may be involved in GPX4 transcriptional regulation.3.Suppression of GPX4 significantly reduced the protective effect of Shh/Gli1 signaling cascade on oxidative stress.Conclusions:In this study,the mechanism of Shh/Gli1 signaling cascade on oxidative stress in NP cells and the progress of IDD was investigated in vitro and in vivo.In addition,the key mechanism of the regulation of antioxidative enzyme GPX4 by Shh/Gli1 signaling cascade was explored at the molecular level.The possible mechanism of GPX4 mediated Shh/Gli1 signaling cascade regulating oxidative stress in NP cells was revealed.This study provides a new perspective on the regulatory mechanism of oxidative stress in NP cells,and a new potential target for the prevention,remission and treatment of IDD.Here are the main conclusions:1.The IDD models of rats induced by endogenous factors age and exogenous factors puncture were established successfully.And the imaging,histological and molecular biological changes during IDD in rats were confirmed.2.Shh/Gli1 signaling cascade was gradually downregulated and the oxidative stress was significantly increased during the process of age-related IDD in rats,suggesting that the Shh/Gli1 signaling cascade had a potential function of regulating oxidative stress and IDD.3.In vivo,the key regulatory role of Shh/Gli1 signaling cascade in oxidative stress of IDD was confirmed,and we found that Shh/Gli1 signaling cascade plays a key role in the occurrence and progression of IDD by affecting IVD homeostasis.4.Shh/Gli1 signaling cascade inhibition caused increase of oxidative stress in NP cells,and activation of Shh/Gli1 signaling cascade effectively protected TBHP-induced oxidative stress in NP cells.Meanwhile,transcription factor Gli1 regulated GPX4 expression and GPX enzyme activity.5.Shh/Gli1 signaling cascade transcription factor Gli1 binds and may activate GPX4 promoter,thus activating GPX4 transcription activity to increase GPX4 expression and GPX enzyme activity,maintain the stability of REDOX system in NP cells and promoting the protection against oxidative stress.