Ozone Exposure Induces Metabolic Disorders And NAD+ Depletion Through PARP1 Activation In Spinal Cord Neurons

BackgroundOzone is an inorganic molecule and allotrope of oxygen with strong oxidizing capacity.It is a highly reactive agent consisting of three oxygen atoms bonded in a V-like shape.It occurs both naturally in the atmosphere and as a man-made product by industrial activities.Ozone is the major photochemical constitute of polluted air,which induces a dose dependent oxidative stress in tissues due to its strong capacity to produce free radicals from different interactions including protein oxidation,enzymatic inactivation,lipoperoxidation of cell membranes,DNA destruction,and cell apoptosis.Despite of these interactions,ozone has been widely used for different musculoskeletal disorders.During the last decade,ozone therapy alone or in combination with other modalities,has been extensively used in clinical practice for treatment of herniated discs,low back pain(LBP)and other chronic pains.Further translational and clinical trials have shown the therapeutic efficacy and safety of ozone for other disorders including degenerative disorders,vascular and immune diseases.The main administrations routes of ozone in the ozone therapy are percutaneous,intradiscal,and intramuscular routes.Current evidence shows the potential therapeutic efficacy of ozone therapy in herniated discs and for pain management in LBP.However,ozone therapy has been associated with toxic effects on the respiratory,endocrine,cardiovascular systems as well as nervous system because of its strong oxidizing property and inducing systemic inflammation.Ginanneschi et al.reported that transcutaneous intradiscal injection of ozone for L4-L5 disk herniation resulted in ventral and dorsal root injury.In this regard,some studies have investigated the effects and mechanisms of action of ozone exposure on metabolic disorders and reported the associations between ozone exposure and metabolic disorders.Although many studies have been conducted on the cytotoxic effects of ozone,the underlying signaling pathways and molecular mechanisms of susceptibility and the disorder are not fully understood.However,evidence from human and animal studies suggests that ozone-induced neuroinflammation,oxidative stress,microglial activation,cerebrovascular dysfunction,and alterations in the blood-brain barrier are the main mechanisms of ozone induced cytotoxicity in central nervous system.Defining the cytotoxicity and mechanisms of action of ozone exposure particularly in spinal cord neurons(SCNs)is necessary to develop efficient ozone therapy as well as new protective strategies for individuals at risk.In this regard,a better understanding of the mediators and involved signaling pathways is of prime importance.The nuclear enzyme poly(ADP)-ribose polymerase-1(PARP1)is the primary subtype of a protein family,which contains polyadenosine diphosphate ribose and polymerase activity.PARP1 is a key moderator for cell death in oxidative stress,ischemia,and excitotoxicity.PARP1 utilizes oxidized nicotinamide adenine dinucleotide(NAD+)as a substrate to catalyze the covalent attachment of ADP-ribose units onto various target proteins,such as aspartate,glutamate,lysine,tyrosine,and serine.Moreover,PARP1 catalyzes the addition of NAD+of poly(ADP)-ribose(PAR)onto itself in response to oxidative DNA damage.Recently,researchers have employed metabolomics analysis to investigate the roles and molecular pathways involved in cellular metabolism disorders under oxidative stress.Oxidized NAD(NAD+)and reduced NAD(NADH)as metabolic cofactors play vital role in cellular energy metabolism and are also involved in calcium homeostasis,mitochondrial function,oxidative stress,gene expression,aging and apoptosis.DNA damage-induced PARP activation leads to depletion of NAD+which subsequently impedes cellular energy metabolism.Moreover,PARP1 activation could hinder hexokinase(HK),which is a crucial enzyme in the glycolysis pathway through PARylation process and subsequently leading to ATP deprivation and cell death called id parthanatos.The signaling pathways involved in the neuronal death induced by PARP 1 activation are not yet fully determined.Different studies have been conducted on this regard.Enrichment analysis revealed NAD+-related metabolic disorders induced by excessive activation of PARP1 after DNA damage.Activating PARP1 would lead to cytosolic NAD+depletion and mitochondrial release of apoptosis-inducing factor(AIF),and different studies have investigated the causal relationships between PARP1 activation and NAD+depletion.Strong evidence shows that NAD+depletion is a causal process in PARP1-mediated cell death so that NAD+depletion and glycolytic failure result in mitochondrial AIF release.Conrad et al.showed that NAD+depletion is necessary and sufficient for PARP1-mediated neuronal death.They used extracellular NAD+to restore neuronal NAD+levels after PARP1 activation.Exogenous NAD+used P2X-gated channels to enter neurons to restore cytosolic NAD+that subsequently inhibited excess PARP1 activation and prevented the AIF translocation,glycolytic inhibition,mitochondrial failure,and neuron death.They used metabolic substrates,such as pyruvate,hydroxybutyrate,or acetoacetate to circumvent the glycolytic inhibition and then prevented mitochondrial failure and neuron death.Other finding of this group was that using NAD+glycohydrolase to deplete intracellular cytosolic NAD+lead to blockage of the glycolysis inhibition,AIF translocation,mitochondrial depolarization,and neuron death,and the process was independent of PARP1 activation.NAD+is an important coenzyme in redox reaction of cells and plays significant roles in the process of cell tricarboxylic acid cycle(TCA),fat b oxidation,glucose metabolism,and amino acid metabolism.Recent studies have confirmed that excessive PARP1 activation could promote NAD+depletion,which could affect cell energy metabolism and reduce ATP levels leading to cell necrosis.Some studies have investigated the causal relationship of PARP activation and subsequent NAD+depletion and cell death and demonstrated direct evidence on causal relationship between PARP activation,NAD+depletion,and cell death.Heller et al.used islet cells from mice with a disrupted and inactivated PARP gene(PARP-/-mice)to investigate the effects of DNA-damaging radicals and relationship between PARP activation,NAD+depletion,and cell death.They reported that mutant islet cells showed more resistant to the toxicity of DNA-damaging radicals and did not show NAD+depletion after exposure to the DNA-damaging radicals.This finding indicates that most of NAD+depletion following the exposure to oxidative factors is due to PARP activation.Furthermore,Eliasson et al.demonstrated that neural cells of PARP-/-mice show significant protection against different oxidants inducing glutamate-mediated ischemic injuries indicating the involvement of PARP activation in neuronal damage following focal cerebral ischemia.The resistance of inactivated PARP gene to different oxidative factors has been reported in different diseases including diabetes.For instance,Burkart et al.showed PARP-/-mice are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin.The findings of the previous studies have suggested that PARP1 inhibitors might have protective effects against oxidative stress-induced cell necrosis.This study aimed to investigate the effects and possible mechanisms of action of ozone on SCNs metabolism using metabolomics analysis.The findings show that NAD+depletion is caused by excessive activation of PARP1.Moreover,we found that ozone-induced DNA damage could be one of the main causes of ozone-induced metabolic disorders of SCNs.Using PARP1 inhibitors can prevent NAD+depletion and promote cell viability during ozone exposure.Part ? The study of the effect of ozone on the metabolic state of rat spinal cord neurons and the related mechanismObjectiveMetabolome analysis was used to detect the changes of the metabolic status of primary spinal cord nerve cells after medical ozone treatment,and to verify the differential expression of a series of metabolic molecules in primary spinal cord nerve cells caused by ozone.The possible molecular mechanisms and pathways of neurotoxicity of medical ozone were studied by means of information biology analysis and statistical methods.Methods1.Extraction and in vitro culture of primary rat spinal cord nerve cells.2.Cell immunofluorescence was used to determine the purity of the cells by detecting NF200 and DNA in the nucleus.3.The experimental groups were as follows:40?g/mL medical ozone treatment group(40?g/mL medical ozone treatment for 1h)and control group(no medical ozone treatment).Each group was set with 6 replicates.4.The LC-MS samples of primary spinal cord nerve cells were processed and extracted by cold methanol quenching method.5.The effect of medical ozone on the expression level of metabolic molecules in primary spinal cord nerve cells was detected by liquid chromatography-mass spectrometry.6.Denoising,baseline correction,peak alignment,data standardization and data conversion were performed on the original data of the metabolome analysis results.7.Multivariate Analysis of pre-processed data was carried out by information biology technology,and principle component analysis(PCA)was used to identify the quality of the test data.Metaboalalyst software was used for univariate analysis to obtain the metabolic molecular differences of primary spinal cord nerve cells in rats after 40?g/mL medical ozone exposure.8.After the screening of metabolites,the Metlin database was used for biomarker identification,and the degree,distribution and related metabolic pathways of metabolic molecular differences were observed by generating volcanic maps,cluster heat maps and enrichment analysis maps.Results1.Extraction,culture and morphological observation of primary spinal cord nerve cells in ratsThe results showed that the primary spinal nerve cells cultured in vitro were round and transparent,with good refractive ability and strong stereoscopic sense.Some large nuclei with distinct nucleoli were observed.On the 7th day of culture,the nerve cells and their protrusion were connected with each other and formed a dense network,forming a relatively complete network.2.Immunofluorescence identification of primary rat spinal cord nerve cells cultured in vitroNF200 immunofluorescence assay was performed on the primary spinal cord nerve cells of rats cultured to the 7th day,and NF200 expression was positive in more than 90%of the cells.The purity of the cultured primary spinal cord nerve cells met the experimental requirements of this study.3.Total ion flow chromatographic comparison of QC samplesThe total ion flow chromatograms of QC samples at each stage basically overlapped,and the retention time of the instrument was good.The instrument test result is stable.4.Difference change of total ion flow chromatogram in medical ozone treatment groupThe metabolic molecules in primary spinal cord nerve cells of rats treated with 40?g/mL medical ozone were significantly changed compared with the control group,and the total peak area in the ozone exposure group was significantly lower than that in the control group under ESI+mode.5.PCA analysisIn the positive mode,PC1 contained 46.0%cumulative variance,followed by PC213.9%.In the negative mode,two principal components were obtained,among which PC1 contained 43.6%cumulative variance,followed by PC2 15.3%6.PLS-DA analysisIn the positive mode,there are two principal components,R2X=0.419,R2Y=0.993,Q2=0.886;In negative mode,R2X=0.469,R2Y=0.994,Q2=0.945.7.Identification of metabolites of intracellular differences after ozone treatmentThe volcanic map shows the distribution of metabolic molecules with different expression levels between the 40?g/mL medical ozone treatment group and the control group.By combining Fold change(FC)with FDR-adjusted p-values(FDR-adjusted p-values),The VIP(Variable Importance in the Projection)value of the OPLS-DA model(threshold>;1)To find the corresponding metabolic molecules.A total of 104 metabolic molecules were determined to be differentially expressed,among which 58 metabolic molecules had increased intracellular concentrations and 46 metabolic molecules had decreased intracellular concentrations.Small molecule metabolites in each group were sorted out for cluster statistics,and 25 of them were found to change the intra-group and inter-group differences by thermal map.These differentially expressed metabolic molecules are mainly involved in fatty acids,tricarboxylic acid cycle,purine metabolism,niacin and nicotinamide metabolism,amino acid metabolism,lipid metabolism,and riboflavin metabolism pathway.8.Enrichment analysisEnrichment analysis showed that the metabolites differentially expressed were mainly involved in fatty acid,tricarboxylic acid cycle,purine metabolism,niacin and nicotinamide metabolism,amino acid,lipid metabolism,and riboflavin metabolism.ConclusionThe expression level of metabolic molecules in spinal cord of primary rats treated with 40?g/mL medical ozone was significantly different from that of control group.The levels of ADP,AMP,aconic acid,UDP-glucose,NAM,L-glutamine,riboflavin and other metabolic molecules were differentially changed,mainly involving fatty acid,tricarboxylic acid cycle,purine metabolism,niacin and niacinamide metabolism,amino acid,lipid metabolism,riboflavin metabolism pathway.Part ? Ozone exposure induces NAD+depletion through PARPl activation in spinal cord neuronsObjectiveThe second stage will conduct specific studies on the changes of important metabolic molecules based on the data obtained in the previous part,verify whether ozone exposure causes DNA damage in spinal nerve cells,and explore whether the NAD depletion caused by PARP1 overactivation induced by DNA single strand break can cause energy metabolism disorders in spinal neurons.To observe the effects of PARP1 inhibitor on the levels of NAD,ATP and cell survival of spinal cord nerve cells.To determine whether vereliparib(ABT-888)could inhibit AMPK activation of medical ozone.Methods1.CCK-8 assay was used to determine the effect of 0-60?g/mL medical ozone on the activity of primary spinal cord nerve cells,and whether PARP1 inhibitors had protective effects on cells.2.The effects of 0,30 and 40?g/mL medical ozone treatment on the level of ATP in primary spinal cord nerve cells of rats were determined by luciferin-luciferase assay.3.The expression levels of NAD and NADH in primary spinal cord nerve cells of rats were determined by colorimetry,and the depletion of NAD in cells under different concentrations of ozone and PARP1 activation were observed.4.Western blotting was used to determine whether medical ozone could cause DNA damage in primary spinal cord nerve cells and further lead to PARP1 overactivation4.1 The changes of yH2AX expression level in primary spinal cord nerve cells treated with medical ozone were determined by Western blotting to observe whether there was DNA damage in the cells.4.2 Western blotting was used to determine the changes of PARP1 expression in primary spinal cord nerve cells after medical ozone treatment.4.3 Western blotting was used to determine the change of PAR expression level in primary spinal cord nerve cells after medical ozone treatment to observe the activation level of PARP1 in cells.4.4 Western blotting was used to observe whether PARP1 inhibitor could affect the expression levels of yH2AX,PARP1 and its activation product PAR.5.The effect of PARP1 inhibitor on the activation of medical ozone AMPK was detected by Western blotting.Results1.The toxicity of different concentrations of medical ozone on primary spinal cord nerve cellsMedical ozone exposure after 1 h,10,20,30 mu g/ml survival rate and the control group no significant difference between ozone treatment group,40,50,60 mu g/ml spinal cord neurons survival of ozone treatment group than the control group significantly decreased(P<0.05),40 mu g/ml spinal cord neurons survival rate was 70%,the ozone treatment group and 50 and 60 mu g/ml ozone treatment group of spinal cord neurons survival rate were 52%and 34%respectively.2.Medical ozone treatment caused a decrease in ATP concentration in primary spinal cord nerve cellsThe level of intracellular ATP decreased after treatment with 30 and 40?g/mL medical ozone for 10min(P<0.05).At 60min of medical ozone exposure,the ATP level of 40?g/mL ozone exposure group was significantly lower than that of control group and 30?g/mL ozone exposure group(P<0.05).3.Medical ozone causes cellular DNA damage,overactivation of PARP-1 and induction of NAD depletionThe level of DNA damage marker protein in ozone treatment group at 30?g/mL had no significant change compared with the control group,and PARP1 and its activation product PAR had no significant difference compared with the control group.The level of yH2AX in 40?g/mL ozone treatment group was significantly increased(P<0.05),the level of PARP1 was significantly increased(P<0.05),the level of intracellular NAD in 40?g/mL medical ozone treatment group was significantly decreased(P<0.05),and the level of ATP was significantly lower than that in control group and 30?g/mL medical ozone treatment group(P<0.05).4.PARP1 inhibitors exert a protective effect by preventing ozone-induced NAD depletion of spinal neuronsAfter treatment with ABT-888 for 2h,there was no significant difference in the level of PAR in spinal cord neurons under 30 and 40?g/mL ozone exposure compared with the control group,and there was no significant decrease in NAD and ATP levels.When treated with 40 and 50?g/mL medical ozone,cell survival rate of ABT-888 drug intervention group was significantly higher than that of non-drug intervention group(P<0.05).After the treatment with ABT-888,the intracellular ATP level in the 40?g/mL medical ozone treatment group was still lower than that in the control group(P<0.05),and there was no significant difference in the level of ATP in the primary spinal cord nerve cells between different medical ozone treatment time.5.The effect of PARP1 inhibitor on medical ozone activation of AMPKAfter treatment with 40?g/mL medical ozone for 1h,the intracellular PAMPK/AMPK value was significantly increased compared with the control group(P<0.05).After treatment with ABT-888 for 2h,the intracellular PAMPK/AMPK value of the group treated with 40?g/mL medical ozone was significantly higher than that of the control group(P<0.05),and there was no significant difference between the group treated with 40?g/mL medical ozone and the group without ABT-888 intervention.ConclusionMedical ozone can cause DNA damage of spinal cord nerve cells and lead to disorder of energy metabolism of spinal cord nerve cells.ABT-888 plays a neuroprotective role by avoiding NAD depletion caused by PARP1 overactivation during 40?g/mL ozone treatment.